COVID-2019 Alert

The latest information about the 2019 Novel Coronavirus, including vaccine clinics for children ages 6 months and older.

La información más reciente sobre el nuevo Coronavirus de 2019, incluidas las clínicas de vacunación para niños de 6 meses en adelante.


Kathleen Sakamoto, MD

  • “My goal is to cure 100 percent of children with cancer because I believe that's possible.”

I am the first person in my family to graduate from college, much less medical school and graduate school. My mother grew up in Japan during WWII and my father was in a Japanese Internment camp. My parents taught me the importance of a strong work ethic and commitment to excellence.

I am serious about patient care and focused on being compassionate and meeting the needs of my patients, who are going through one of the most difficult experiences of their lives. I get so much pleasure out of my work, and my patients inspire me every day. I am proud to be a member of this extraordinary hospital, where we are committed to taking the best care of children. The more I'm here, the more I realize how great this place is. The quality of the physicians, leaders and scientists is unparalleled.



Work and Education

Professional Education

University of Cincinnati College of Medicine, Cincinnati, OH, 1985


Children's Hospital Los Angeles, Los Angeles, CA, 1988


Children's Hospital Los Angeles, Los Angeles, CA, 1991

Board Certifications

Pediatric Hematology-Oncology, American Board of Pediatrics

Pediatrics, American Board of Pediatrics

Conditions Treated

Bone Marrow Failure Disorders

Pediatric Cancers

All Publications

Diamond Blackfan anemia is mediated by hyperactive Nemo-like kinase. Nature communications Wilkes, M. C., Siva, K., Chen, J., Varetti, G., Youn, M. Y., Chae, H., Ek, F., Olsson, R., Lundback, T., Dever, D. P., Nishimura, T., Narla, A., Glader, B., Nakauchi, H., Porteus, M. H., Repellin, C. E., Gazda, H. T., Lin, S., Serrano, M., Flygare, J., Sakamoto, K. M. 2020; 11 (1): 3344


Diamond Blackfan Anemia (DBA) is a congenital bone marrow failure syndrome associated with ribosomal gene mutations that lead to ribosomal insufficiency. DBA is characterized by anemia, congenital anomalies, and cancer predisposition. Treatment for DBA is associated with significant morbidity. Here, we report the identification of Nemo-like kinase (NLK) as a potential target for DBA therapy. To identify new DBA targets, we screen for small molecules that increase erythroid expansion in mouse models of DBA. This screen identified a compound that inhibits NLK. Chemical and genetic inhibition of NLK increases erythroid expansion in mouse and human progenitors, including bone marrow cells from DBA patients. In DBA models and patient samples, aberrant NLK activation is initiated at the Megakaryocyte/Erythroid Progenitor (MEP) stage of differentiation and is not observed in non-erythroid hematopoietic lineages or healthy erythroblasts. We propose that NLK mediates aberrant erythropoiesis in DBA and is a potential target for therapy.

View details for DOI 10.1038/s41467-020-17100-z

View details for PubMedID 32620751

MMP9 inhibition increases erythropoiesis in RPS14-deficient del(5q) MDS models through suppression of TGF-beta pathways. Blood advances Youn, M., Huang, H., Chen, C., Kam, S., Wilkes, M. C., Chae, H., Sridhar, K. J., Greenberg, P. L., Glader, B., Narla, A., Lin, S., Sakamoto, K. M. 2019; 3 (18): 275163


The del(5q) myelodysplastic syndrome (MDS) is a distinct subtype of MDS, associated with deletion of the ribosomal protein S14 (RPS14) gene that results in macrocytic anemia. This study sought to identify novel targets for the treatment of patients with del(5q) MDS by performing an in vivo drug screen using an rps14-deficient zebrafish model. From this, we identified the secreted gelatinase matrix metalloproteinase 9 (MMP9). MMP9 inhibitors significantly improved the erythroid defect in rps14-deficient zebrafish. Similarly, treatment with MMP9 inhibitors increased the number ofcolony forming unit-erythroid colonies and the CD71+erythroid population from RPS14 knockdown human BMCD34+cells. Importantly, we found that MMP9 expression is upregulated in RPS14-deficient cells by monocyte chemoattractant protein 1. Double knockdown of MMP9 and RPS14 increased the CD71+population compared with RPS14 single knockdown, suggesting that increased expression of MMP9 contributes to the erythroid defect observed in RPS14-deficient cells. In addition, transforming growth factorbeta(TGF-beta) signaling is activated in RPS14 knockdown cells, and treatment with SB431542, a TGF-betainhibitor, improved the defective erythroid development of RPS14-deficient models. We found that recombinant MMP9 treatment decreases the CD71+population through increased SMAD2/3 phosphorylation, suggesting that MMP9 directly activates TGF-betasignaling in RPS14-deficient cells. Finally, we confirmed that MMP9 inhibitors reduce SMAD2/3 phosphorylation in RPS14-deficient cells to rescue the erythroid defect. In summary, these study results support a novel role for MMP9 in the pathogenesis of del(5q) MDS and the potential for the clinical use of MMP9 inhibitors in the treatment of patients with del(5q) MDS.

View details for DOI 10.1182/bloodadvances.2019000537

View details for PubMedID 31540902

Innate immune system activation in zebrafish and cellular models of Diamond Blackfan Anemia SCIENTIFIC REPORTS Danilova, N., Wilkes, M., Bibikova, E., Youn, M., Sakamoto, K. M., Lin, S. 2018; 8: 5165


Deficiency of ribosomal proteins (RPs) leads to Diamond Blackfan Anemia (DBA) associated with anemia, congenital defects, and cancer. While p53 activation is responsible for many features of DBA, the role of immune system is less defined. The Innate immune system can be activated by endogenous nucleic acids from non-processed pre-rRNAs, DNA damage, and apoptosis that occurs in DBA. Recognition by toll like receptors (TLRs) and Mda5-like sensors induces interferons (IFNs) and inflammation. Dying cells can also activate complement system. Therefore we analyzed the status of these pathways in RP-deficient zebrafish and found upregulation of interferon, inflammatory cytokines and mediators, and complement. We also found upregulation of receptors signaling to IFNs including Mda5, Tlr3, and Tlr9. TGFb family member activin was also upregulated in RP-deficient zebrafish and in RPS19-deficient human cells, which include a lymphoid cell line from a DBA patient, and fetal liver cells and K562 cells transduced with RPS19 shRNA. Treatment of RP-deficient zebrafish with a TLR3 inhibitor decreased IFNs activation, acute phase response, and apoptosis and improved their hematopoiesis and morphology. Inhibitors of complement and activin also had beneficial effects. Our studies suggest that innate immune system contributes to the phenotype of RPS19-deficient zebrafish and human cells.

View details for PubMedID 29581525

Niclosamide suppresses acute myeloid leukemia cell proliferation through inhibition of CREB-dependent signaling pathways ONCOTARGET Chae, H., Cox, N., Dahl, G. V., Lacayo, N. J., Davis, K. L., Capolicchio, S., Smith, M., Sakamoto, K. M. 2018; 9 (4): 430117


CREB (cAMP Response Element Binding protein) is a transcription factor that is overexpressed in primary acute myeloid leukemia (AML) cells and associated with a decreased event-free survival and increased risk of relapse. We recently reported a small molecule inhibitor of CREB, XX-650-23, which inhibits CREB activity in AML cells. Structure-activity relationship analysis for chemical compounds with structures similar to XX-650-23 led to the identification of the anthelminthic drug niclosamide as a potent anti-leukemic agent that suppresses cell viability of AML cell lines and primary AML cells without a significant decrease in colony forming activity of normal bone marrow cells. Niclosamide significantly inhibited CREB function and CREB-mediated gene expression in cells, leading to apoptosis and G1/S cell cycle arrest with reduced phosphorylated CREB levels. CREB knockdown protected cells from niclosamide treatment-mediated cytotoxic effects. Furthermore, treatment with a combination of niclosamide and CREB inhibitor XX-650-23 showed an additive anti-proliferative effect, consistent with the hypothesis that niclosamide and XX-650-23 regulate the same targets or pathways to inhibit proliferation and survival of AML cells. Niclosamide significantly inhibited the progression of disease in AML patient-derived xenograft (PDX) mice, and prolonged survival of PDX mice. Niclosamide also showed synergistic effects with chemotherapy drugs to inhibit AML cell proliferation. While chemotherapy antagonized the cytotoxic potential of niclosamide, pretreatment with niclosamide sensitized cells to chemotherapeutic drugs, cytarabine, daunorubicin, and vincristine. Therefore, our results demonstrate niclosamide as a potential drug to treat AML by inducing apoptosis and cell cycle arrest through inhibition of CREB-dependent pathways in AML cells.

View details for PubMedID 29435104

Small molecule inhibition of cAMP response element binding protein in human acute myeloid leukemia cells. Leukemia Mitton, B., Chae, H., Hsu, K., Dutta, R., Aldana-Masangkay, G., FERRARI, R., Davis, K., Tiu, B. C., Kaul, A., Lacayo, N., Dahl, G., Xie, F., Li, B. X., Breese, M. R., Landaw, E. M., Nolan, G., Pellegrini, M., Romanov, S., Xiao, X., Sakamoto, K. M. 2016


The transcription factor CREB (cAMP Response-Element Binding Protein) is overexpressed in the majority of acute myeloid leukemia (AML) patients, and this is associated with a worse prognosis. Previous work revealed that CREB overexpression augmented AML cell growth, while CREB knockdown disrupted key AML cell functions in vitro. In contrast, CREB knockdown had no effect on long-term hematopoietic stem cell activity in mouse transduction/transplantation assays. Together, these studies position CREB as a promising drug target for AML. To test this concept, a small molecule inhibitor of CREB, XX-650-23, was developed. This molecule blocks a critical interaction between CREB and its required co-activator CBP (CREB Binding Protein), leading to disruption of CREB-driven gene expression. Inhibition of CBP-CREB interaction induced apoptosis and cell-cycle arrest in AML cells, and prolonged survival in vivo in mice injected with human AML cells. XX-650-23 had little toxicity on normal human hematopoietic cells and tissues in mice. To understand the mechanism of XX-650-23, we performed RNA-seq, ChIP-seq and Cytometry Time of Flight with human AML cells. Our results demonstrate that small molecule inhibition of CBP-CREB interaction mostly affects apoptotic, cell-cycle and survival pathways, which may represent a novel approach for AML therapy.

View details for DOI 10.1038/leu.2016.139

View details for PubMedID 27211267

Small molecule screen for inhibitors of expression from canonical CREB response element-containing promoters. Oncotarget Mitton, B., Hsu, K., Dutta, R., Tiu, B. C., Cox, N., McLure, K. G., Chae, H., Smith, M., Eklund, E. A., Solow-Cordero, D. E., Sakamoto, K. M. 2016; 7 (8): 8653-8662


The transcription factor CREB (cAMP Response Element Binding Protein) is an important determinant in the growth of Acute Myeloid Leukemia (AML) cells. CREB overexpression increases AML cell growth by driving the expression of key regulators of apoptosis and the cell cycle. Conversely, CREB knockdown inhibits proliferation and survival of AML cells but not normal hematopoietic cells. Thus, CREB represents a promising drug target for the treatment of AML, which carries a poor prognosis. In this study, we performed a high-throughput small molecule screen to identify compounds that disrupt CREB function in AML cells. We screened ~114,000 candidate compounds from Stanford University's small molecule library, and identified 5 molecules that inhibit CREB function at micromolar concentrations, but are non-toxic to normal hematopoietic cells. This study suggests that targeting CREB function using small molecules could provide alternative approaches to treat AML.

View details for DOI 10.18632/oncotarget.7085

View details for PubMedID 26840025

View details for PubMedCentralID PMC4890994

Biology of the bone marrow microenvironment and myelodysplastic syndromes. Molecular genetics and metabolism Rankin, E. B., Narla, A., Park, J. K., Lin, S., Sakamoto, K. M. 2015; 116 (1-2): 24-28


Myelodysplastic syndromes (MDS) are characterized by cytopenias resulting from ineffective hematopoiesis with a predisposition to transform to acute myeloid leukemia (AML). Recent evidence suggests that the hematopoietic stem cell microenvironment contributes to the pathogenesis of MDS. Inflammation and hypoxia within the bone marrow are key regulators of hematopoietic stem and progenitor cells that can lead to several bone marrow failure syndromes, including MDS. In this brief review, we provide an overview of the clinical and molecular features of MDS, the bone marrow microenvironment, and specific pathways that lead to abnormal blood cell development in MDS. Characterization of key steps in the pathogenesis of MDS will lead to new approaches to treat patients with this disease.

View details for DOI 10.1016/j.ymgme.2015.07.004

View details for PubMedID 26210353

View details for PubMedCentralID PMC4618471

Replication factor C3 is a CREB target gene that regulates cell cycle progression through the modulation of chromatin loading of PCNA LEUKEMIA Chae, H., Mitton, B., Lacayo, N. J., Sakamoto, K. M. 2015; 29 (6): 1379-1389


CREB (cyclic AMP response element-binding protein) is a transcription factor overexpressed in normal and neoplastic myelopoiesis and regulates cell cycle progression, although its oncogenic mechanism has not been well characterized. Replication factor C3 (RFC3) is required for chromatin loading of proliferating cell nuclear antigen (PCNA) which is a sliding clamp platform for recruiting numerous proteins in the DNA metabolism. CREB1 expression, which was activated by E2F, was coupled with RFC3 expression during the G1/S progression in the KG-1 acute myeloid leukemia (AML) cell line. There was also a direct correlation between the expression of RFC3 and CREB1 in human AML cell lines as well as in the AML cells from the patients. CREB interacted directly with the CRE site in RFC3 promoter region. CREB-knockdown inhibited primarily G1/S cell cycle transition by decreasing the expression of RFC3 as well as PCNA loading onto the chromatin. Exogenous expression of RFC3 was sufficient to rescue the impaired G1/S progression and PCNA chromatin loading caused by CREB knockdown. These studies suggest that RFC3 may have a role in neoplastic myelopoiesis by promoting the G1/S progression and its expression is regulated by CREB.

View details for DOI 10.1038/leu.2014.350

View details for Web of Science ID 000355615000017

View details for PubMedID 25541153

View details for PubMedCentralID PMC4456282

Targeting novel signaling pathways for resistant acute myeloid leukemia MOLECULAR GENETICS AND METABOLISM Sakamoto, K. M., Grant, S., Saleiro, D., Crispino, J. D., Hijiya, N., Giles, F., Platanias, L., Eklund, E. A. 2015; 114 (3): 397-402


Acute myeloid leukemia (AML) is a hematologic malignancy that is the most common type of acute leukemia diagnosed in adults and the second most common type in children. The overall survival is poor and treatment is associated with significant complications and even death. In addition, a significant number of patients will not respond to therapy or relapse. In this review, several new signaling proteins aberrantly regulated in AML are described, including CREB, Triad1, Bcl-2 family members, Stat3, and mTOR/MEK. Identifying more effective and less toxic agents will provide novel approaches to treat AML.

View details for DOI 10.1016/j.ymgme.2014.11.017

View details for Web of Science ID 000351191800128

View details for PubMedCentralID PMC4355162

The Multitargeted Receptor Tyrosine Kinase Inhibitor Linifanib (ABT-869) Induces Apoptosis through an Akt and Glycogen Synthase Kinase 3 beta-Dependent Pathway MOLECULAR CANCER THERAPEUTICS Hernandez-Davies, J. E., Zape, J. P., Landaw, E. M., Tan, X., Presnell, A., Griffith, D., Heinrich, M. C., Glaser, K. B., Sakamoto, K. M. 2011; 10 (6): 949-959


The FMS-like receptor tyrosine kinase 3 (FLT3) plays an important role in controlling differentiation and proliferation of hematopoietic cells. Activating mutations in FLT3 occur in patients with acute myeloid leukemia (AML; 15%-35%), resulting in abnormal cell proliferation. Furthermore, both adult and pediatric patients with AML harboring the FLT3 internal tandem duplication (ITD) mutation have a poor prognosis. Several inhibitors have been developed to target mutant FLT3 for the treatment of AML, yet the molecular pathways affected by drug inhibition of the mutated FLT3 receptor alone have not been characterized as yet. Linifanib (ABT-869) is a multitargeted tyrosine kinase receptor inhibitor that suppresses FLT3 signaling. In this article, we show that treatment with linifanib inhibits proliferation and induces apoptosis in ITD mutant cells in vitro and in vivo. We show that treatment with linifanib reduces phosphorylation of Akt and glycogen synthase kinase 3 (GSK3). In addition, we show that inhibition of GSK3 decreases linifanib-induced apoptosis. This study shows the importance of GSK3 as a potential target for AML therapy, particularly in patients with FLT3 ITD mutations.

View details for DOI 10.1158/1535-7163.MCT-10-0904

View details for Web of Science ID 000291428000004

View details for PubMedID 21471285

View details for PubMedCentralID PMC3112478

CREB and leukemogenesis. Critical reviews in oncogenesis Cho, E., Mitton, B., Sakamoto, K. M. 2011; 16 (1-2): 37-46


Acute myeloid leukemia (AML) is one of the most common leukemias with a 20% 5-year event-free survival in adults and 50% overall survival in children, despite aggressive chemotherapy treatment and bone marrow transplantation. The incidence and mortality rates for acute leukemia have only slightly decreased over the last 20 years, and therefore greater understanding of the molecular mechanisms associated with leukemic progression is needed. To this end, a number of transcription factors that appear to play a central role in leukemogenesis are being investigated; among them is the cAMP response element binding protein (CREB). CREB is a transcription factor that can regulate downstream targets involving in various cellular functions including cell proliferation, survival, and differentiation. In several studies, the majority of bone marrow samples from patients with acute lymphoid and myeloid leukemia demonstrate CREB overexpression. Moreover, CREB overexpression is associated with a poor outcome in AML patients. This review summarizes the role of CREB in leukemogenesis.

View details for PubMedID 22150306

View details for PubMedCentralID PMC3243968

Ribosomal protein S19 deficiency in zebrafish leads to developmental abnormalities and defective erythropoiesis through activation of p53 protein family BLOOD Danilova, N., Sakamoto, K. M., Lin, S. 2008; 112 (13): 5228-5237


Mutations in several ribosomal proteins (RPs) lead to Diamond-Blackfan anemia (DBA), a syndrome characterized by defective erythropoiesis, congenital anomalies, and increased frequency of cancer. RPS19 is the most frequently mutated RP in DBA. RPS19 deficiency impairs ribosomal biogenesis, but how this leads to DBA or cancer remains unknown. We have found that rps19 deficiency in ze-brafish results in hematopoietic and developmental abnormalities resembling DBA. Our data suggest that the rps19-deficient phenotype is mediated by dysregulation of deltaNp63 and p53. During gastrulation, deltaNp63 is required for specification of nonneural ectoderm and its up-regulation suppresses neural differentiation, thus contributing to brain/craniofacial defects. In rps19-deficient embryos, deltaNp63 is induced in erythroid progenitors and may contribute to blood defects. We have shown that suppression of p53 and deltaNp63 alleviates the rps19-deficient phenotypes. Mutations in other ribosomal proteins, such as S8, S11, and S18, also lead to up-regulation of p53 pathway, suggesting it is a common response to ribosomal protein deficiency. Our finding provides new insights into pathogenesis of DBA. Ribosomal stress syndromes represent a broader spectrum of human congenital diseases caused by genotoxic stress; therefore, imbalance of p53 family members may become a new target for therapeutics.

View details for DOI 10.1182/blood-2008-01-132290

View details for Web of Science ID 000261513400057

View details for PubMedID 18515656

The role of CREB as a proto-oncogene in hematopoiesis and in acute myeloid leukemia CANCER CELL Shankar, D. B., Cheng, J. C., Kinjo, K., Federman, N., Moore, T. B., Gill, A., Rao, N. P., Landaw, E. M., Sakamoto, K. M. 2005; 7 (4): 351-362


CREB is a transcription factor that functions in glucose homeostasis, growth factor-dependent cell survival, and memory. In this study, we describe a role of CREB in human cancer. CREB overexpression is associated with increased risk of relapse and decreased event-free survival. CREB levels are elevated in blast cells from patients with acute myeloid leukemia. To understand the role of CREB in leukemogenesis, we studied the biological consequences of CREB overexpression in primary human leukemia cells, leukemia cell lines, and transgenic mice. Our results demonstrate that CREB promotes abnormal proliferation and survival of myeloid cells in vitro and in vivo through upregulation of specific target genes. Thus, we report that CREB is implicated in myeloid cell transformation.

View details for Web of Science ID 000228741700010

View details for PubMedID 15837624

Ubistatins inhibit proteasome-dependent degradation by binding the ubiquitin chain SCIENCE Verma, R., Peters, N. R., D'onofrio, M., Tochtrop, G. P., Sakamoto, K. M., Varadan, R., Zhang, M. S., Coffino, P., Fushman, D., Deshaies, R. J., King, R. W. 2004; 306 (5693): 117-120


To identify previously unknown small molecules that inhibit cell cycle machinery, we performed a chemical genetic screen in Xenopus extracts. One class of inhibitors, termed ubistatins, blocked cell cycle progression by inhibiting cyclin B proteolysis and inhibited degradation of ubiquitinated Sic1 by purified proteasomes. Ubistatins blocked the binding of ubiquitinated substrates to the proteasome by targeting the ubiquitin-ubiquitin interface of Lys(48)-linked chains. The same interface is recognized by ubiquitin-chain receptors of the proteasome, indicating that ubistatins act by disrupting a critical protein-protein interaction in the ubiquitin-proteasome system.

View details for Web of Science ID 000224304000050

View details for PubMedID 15459393

Development of protacs to target cancer-promoting proteins for ubiquitination and degradation MOLECULAR & CELLULAR PROTEOMICS Sakamoto, K. M., Kim, K. B., Verma, R., Ransick, A., Stein, B., Crews, C. M., Deshaies, R. J. 2003; 2 (12): 1350-1358


The proteome contains hundreds of proteins that in theory could be excellent therapeutic targets for the treatment of human diseases. However, many of these proteins are from functional classes that have never been validated as viable candidates for the development of small molecule inhibitors. Thus, to exploit fully the potential of the Human Genome Project to advance human medicine, there is a need to develop generic methods of inhibiting protein activity that do not rely on the target protein's function. We previously demonstrated that a normally stable protein, methionine aminopeptidase-2 or MetAP-2, could be artificially targeted to an Skp1-Cullin-F-box (SCF) ubiquitin ligase complex for ubiquitination and degradation through a chimeric bridging molecule or Protac (proteolysis targeting chimeric molecule). This Protac consisted of an SCF(beta-TRCP)-binding phosphopeptide derived from IkappaBalpha linked to ovalicin, which covalently binds MetAP-2. In this study, we employed this approach to target two different proteins, the estrogen (ER) and androgen (AR) receptors, which have been implicated in the progression of breast and prostate cancer, respectively. We show here that an estradiol-based Protac can enforce the ubiquitination and degradation of the alpha isoform of ER in vitro, and a dihydroxytestosterone-based Protac introduced into cells promotes the rapid disappearance of AR in a proteasome-dependent manner. Future improvements to this technology may yield a general approach to treat a number of human diseases, including cancer.

View details for DOI 10.1074/mcp.T300009-MCP200

View details for Web of Science ID 000187250900010

View details for PubMedID 14525958

Protacs: Chimeric molecules that target proteins to the Skp1-Cullin-F box complex for ubiquitination and degradation PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Sakamoto, K. M., Kim, K. B., Kumagai, A., Mercurio, F., Crews, C. M., Deshaies, R. J. 2001; 98 (15): 8554-8559


The intracellular levels of many proteins are regulated by ubiquitin-dependent proteolysis. One of the best-characterized enzymes that catalyzes the attachment of ubiquitin to proteins is a ubiquitin ligase complex, Skp1-Cullin-F box complex containing Hrt1 (SCF). We sought to artificially target a protein to the SCF complex for ubiquitination and degradation. To this end, we tested methionine aminopeptidase-2 (MetAP-2), which covalently binds the angiogenesis inhibitor ovalicin. A chimeric compound, protein-targeting chimeric molecule 1 (Protac-1), was synthesized to recruit MetAP-2 to SCF. One domain of Protac-1 contains the I kappa B alpha phosphopeptide that is recognized by the F-box protein beta-TRCP, whereas the other domain is composed of ovalicin. We show that MetAP-2 can be tethered to SCF(beta-TRCP), ubiquitinated, and degraded in a Protac-1-dependent manner. In the future, this approach may be useful for conditional inactivation of proteins, and for targeting disease-causing proteins for destruction.

View details for Web of Science ID 000169967000064

View details for PubMedID 11438690

View details for PubMedCentralID PMC37474

Net1 stimulates RNA polymerase I transcription and regulates nucleolar structure independently of controlling mitotic exit MOLECULAR CELL Shou, W. Y., Sakamoto, K. M., Keener, J., Morimoto, K. W., Traverso, E. E., Azzam, R., Hoppe, G. J., Feldman, R. M., DeModena, J., Moazed, D., Charbonneau, H., Nomura, M., Deshaies, R. J. 2001; 8 (1): 45-55


The budding yeast RENT complex, consisting of at least three proteins (Net1, Cdc14, Sir2), is anchored to the nucleolus by Net1. RENT controls mitotic exit, nucleolar silencing, and nucleolar localization of Nop1. Here, we report two new functions of Net1. First, Net1 directly binds Pol I and stimulates rRNA synthesis both in vitro and in vivo. Second, Net1 modulates nucleolar structure by regulating rDNA morphology and proper localization of multiple nucleolar antigens, including Pol I. Importantly, we show that the nucleolar and previously described cell cycle functions of the RENT complex can be uncoupled by a dominant mutant allele of CDC14. The independent functions of Net1 link a key event in the cell cycle to nucleolar processes that are fundamental to cell growth.

View details for Web of Science ID 000170081900008

View details for PubMedID 11511359

Intrinsic suppression of type I interferon production underlies the therapeutic efficacy of IL-15-producing natural killer cells in B-cell acute lymphoblastic leukemia. Journal for immunotherapy of cancer Kumar, A., Taghi Khani, A., Duault, C., Aramburo, S., Sanchez Ortiz, A., Lee, S. J., Chan, A., McDonald, T., Huang, M., Lacayo, N. J., Sakamoto, K. M., Yu, J., Hurtz, C., Carroll, M., Tasian, S. K., Ghoda, L., Marcucci, G., Gu, Z., Rosen, S. T., Armenian, S., Izraeli, S., Chen, C., Caligiuri, M. A., Forman, S. J., Maecker, H. T., Swaminathan, S. 2023; 11 (5)


BACKGROUND: Type I interferons (IFN-Is), secreted by hematopoietic cells, drive immune surveillance of solid tumors. However, the mechanisms of suppression of IFN-I-driven immune responses in hematopoietic malignancies including B-cell acute lymphoblastic leukemia (B-ALL) are unknown.METHODS: Using high-dimensional cytometry, we delineate the defects in IFN-I production and IFN-I-driven immune responses in high-grade primary human and mouse B-ALLs. We develop natural killer (NK) cells as therapies to counter the intrinsic suppression of IFN-I production in B-ALL.RESULTS: We find that high expression of IFN-I signaling genes predicts favorable clinical outcome in patients with B-ALL, underscoring the importance of the IFN-I pathway in this malignancy. We show that human and mouse B-ALL microenvironments harbor an intrinsic defect in paracrine (plasmacytoid dendritic cell) and/or autocrine (B-cell) IFN-I production and IFN-I-driven immune responses. Reduced IFN-I production is sufficient for suppressing the immune system and promoting leukemia development in mice prone to MYC-driven B-ALL. Among anti-leukemia immune subsets, suppression of IFN-I production most markedly lowers the transcription of IL-15 and reduces NK-cell number and effector maturation in B-ALL microenvironments. Adoptive transfer of healthy NK cells significantly prolongs survival of overt ALL-bearing transgenic mice. Administration of IFN-Is to B-ALL-prone mice reduces leukemia progression and increases the frequencies of total NK and NK-cell effectors in circulation. Ex vivo treatment of malignant and non-malignant immune cells in primary mouse B-ALL microenvironments with IFN-Is fully restores proximal IFN-I signaling and partially restores IL-15 production. In B-ALL patients, the suppression of IL-15 is the most severe in difficult-to-treat subtypes with MYC overexpression. MYC overexpression promotes sensitivity of B-ALL to NK cell-mediated killing. To counter the suppressed IFN-I-induced IL-15 production in MYChigh human B-ALL, we CRISPRa-engineered a novel human NK-cell line that secretes IL-15. CRISPRa IL-15-secreting human NK cells kill high-grade human B-ALL in vitro and block leukemia progression in vivo more effectively than NK cells that do not produce IL-15.CONCLUSION: We find that restoration of the intrinsically suppressed IFN-I production in B-ALL underlies the therapeutic efficacy of IL-15-producing NK cells and that such NK cells represent an attractive therapeutic solution for the problem of drugging MYC in high-grade B-ALL.

View details for DOI 10.1136/jitc-2022-006649

View details for PubMedID 37217248

SATB1 chromatin loops regulate Megakaryocyte/Erythroid Progenitor Expansion by facilitating HSP70 and GATA1 induction. Stem cells (Dayton, Ohio) Wilkes, M. C., Chae, H. D., Scanlon, V., Cepika, A. M., Wentworth, E. P., Saxena, M., Eskin, A., Chen, Z., Glader, B., Roncarolo, M. G., Nelson, S. F., Sakamoto, K. M. 2023


Diamond Blackfan Anemia (DBA) is an inherited bone marrow failure syndrome associated with severe anemia, congenital malformations and increased risk of developing cancer. The chromatin-binding SATB1 is downregulated in Megakaryocyte/Erythroid Progenitors (MEPs) in patients and cell models of DBA, leading to a reduction in MEP expansion. Here we demonstrate that SATB1 expression is required for the upregulation of the critical erythroid factors HSP70 and GATA1 that accompanies MEP differentiation. SATB1 binding to specific sites surrounding the HSP70 genes, promotes chromatin loops that are required for induction of HSP70, which in turn promotes GATA1 induction. This demonstrates that SATB1, although gradually downregulated during myelopoiesis, maintains a biological function in early myeloid progenitors.

View details for DOI 10.1093/stmcls/sxad025

View details for PubMedID 36987811

Can you hear me now? Tools for cultivating a culture of respect, value, and appreciation within pediatric hematology, oncology, and cellular therapy. Pediatric blood & cancer Tal, A., Moerdler, S., Fernandez, C. R., Dome, J. S., Sakamoto, K. M. 2022: e30127


The American Society of Pediatric Hematology/Oncology (ASPHO) conducted a workshop "Can you hear me now? Cultivating a culture of respect, value, and appreciation within pediatric hematology/oncology" at their annual meeting in May 2022 in hopes of exploring how the members can enhance wellness in a climate of increasing diversity. Initiatives in the past have focused on personal care, but it has been widely shown that administrative and institutional driven initiatives are essential to create an environment of wellness. In this interactive workshop, we discovered that 22% of participants felt their institution does not instill a culture of respect. We offered tools to the audience on multiple levels: graceful self-promotion, diversity and inclusion, and leadership perspective on creating a culture of respect to address the individual, local community, and top-down leadership approaches. Here, we offer a summary on the content of the workshop, and expand upon many of the discussion points that were raised during the workshop. We bring forth novel information on each topic individually from diverse points of view, specific to the field of pediatric hematology/oncology (PHO). We aim to highlight the importance of creating a diverse and respectful work environment in PHO in hopes of ensuring motivated, satisfied, and fulfilled healthcare providers who feel appreciated and valued.

View details for DOI 10.1002/pbc.30127

View details for PubMedID 36495252

Development of clinical pathways to improve multidisciplinary care of high-risk pediatric oncology patients. Frontiers in oncology Reschke, A., Richards, R. M., Smith, S. M., Long, A. H., Marks, L. J., Schultz, L., Kamens, J. L., Aftandilian, C., Davis, K. L., Gruber, T., Sakamoto, K. M. 2022; 12: 1033993


Clinical pathways are evidence-based tools that have been integrated into many aspects of pediatric hospital medicine and have proven effective at reducing in-hospital complications from a variety of diseases. Adaptation of similar tools for specific, high-risk patient populations in pediatric oncology has been slower, in part due to patient complexities and variations in management strategies. There are few published studies of clinical pathways for pediatric oncology patients. Pediatric patients with a new diagnosis of leukemia or lymphoma often present with one or more "oncologic emergencies" that require urgent intervention and deliberate multidisciplinary care to prevent significant consequences. Here, we present two clinical pathways that have recently been developed using a multidisciplinary approach at a single institution, intended for the care of patients who present with hyperleukocytosis or an anterior mediastinal mass. These clinical care pathways have provided a critical framework for the immediate care of these patients who are often admitted to the pediatric intensive care unit for initial management. The goal of the pathways is to facilitate multidisciplinary collaborations, expedite diagnosis, and streamline timely treatment initiation. Standardizing the care of high-risk pediatric oncology patients will ultimately decrease morbidity and mortality associated with these diseases to increase the potential for excellent outcomes.

View details for DOI 10.3389/fonc.2022.1033993

View details for PubMedID 36523979

View details for PubMedCentralID PMC9744920

Animal models of Diamond-Blackfan anemia: updates and challenges. Haematologica Liu, Y. L., Shibuya, A., Glader, B., Wilkes, M. C., Barna, M., Sakamoto, K. M. 2022


Diamond-Blackfan anemia (DBA) is a ribosomopathy that is characterized by macrocytic anemia, congenital malformations, and early onset during childhood. Genetic studies have demonstrated that most patients carry mutations in one of the 20 related genes, most of which encode ribosomal proteins (RP). Treatment of DBA includes corticosteroid therapy, chronic red blood cell transfusion, and other forms of immunosuppression. Currently, hematopoietic stem cell transplantation is the only cure for DBA. Interestingly, spontaneous remissions occur in 10-20% of transfusion-dependent DBA patients. However, there is no consistent association between specific mutations and clinical manifestations. In the past decades, researchers have made significant progress in understanding the pathogenesis of DBA, but it remains unclear how the ubiquitous RP haploinsufficiency causes the erythroid-specific defect in hematopoiesis in DBA patients, and why there is a difference in penetrance and spontaneous remission among individuals who carry identical mutations. In this paper, we provide a comprehensive review of the development of DBA animal models and discuss the future research directions for these important experimental systems.

View details for DOI 10.3324/haematol.2022.282042

View details for PubMedID 36384250

Novel Small Molecule and Peptide Inhibitors of CREB in Leukemia Cells Dalloul, J., Youn, M., Mark, K., Powers, A., Dror, R., Wakatsuki, S., Sakamoto, K. M. AMER SOC HEMATOLOGY. 2022: 4949-4950
Niclosamide Inhibits Proliferation of Leukemia Cells and Synergizes with Chemotherapy Mark, K., Robbins, M., Gamble, A., Chae, H., Bassik, M., Han, K., Sakamoto, K. M. AMER SOC HEMATOLOGY. 2022: 8870-8871
Management of Chronic Myeloid Leukemia in Children and Young Adults. Current hematologic malignancy reports Ford, M., Mauro, M., Aftandilian, C., Sakamoto, K. M., Hijiya, N. 2022


PURPOSE OF REVIEW: Due to lack of pediatric-specific data, the management of chronic myeloid leukemia (CML) in pediatric, adolescents, and young adults is guided by adult CML evidence-based recommendations. Pediatric CML presents differently than adult CML and is often a more aggressive disease with different biological and host factors, yet there is sparse literature on how to address those differences.RECENT FINDINGS: Over the past two decades, tyrosine kinase inhibitors (TKIs) have changed the way CML is treated. There are currently three FDA-approved TKIs (imatinib, dasatinib, and nilotinib) for pediatric patients. When choosing which TKI to begin treatment with, there are many factors that should be considered on a case-to-case basis to obtain optimal outcomes. The safety profiles for long-term TKI use in pediatrics require further study. Unlike adults, children are still actively growing during TKI use, and the effect on development can be detrimental. TKI therapy is not recommended during pregnancy with variable but significant risk of fetal abnormalities and miscarriage, warranting counseling for young female patients prior to beginning TKIs. Attempts for treatment-free remission (TFR) by planned TKI cessation in eligible adult patients in deep and sustained molecular remission are now done as a standard of practice. However, data is sparse in the pediatric population. There is currently an ongoing Children's Oncology Group (COG) study to determine the feasibility of TFR as a treatment goal. Further research and additional pediatric trials are needed to characterize the unique aspects of CML in children and adolescents and optimize outcomes.

View details for DOI 10.1007/s11899-022-00673-5

View details for PubMedID 35920965

Downregulation of SATB1 by miRNAs Reduces Megakaryocyte/Erythroid Progenitor Expansion in pre-clinical models of Diamond Blackfan Anemia. Experimental hematology Wilkes, M. C., Scanlon, V., Shibuya, A., Celika, A. M., Eskin, A., Chen, Z., Narla, A., Glader, B., Roncarolo, M. G., Nelson, S. F., Sakamoto, K. M. 2022


Diamond Blackfan Anemia (DBA) is an inherited bone marrow failure syndrome that is associated with anemia, congenital anomalies, and cancer predisposition. It is categorized as a ribosomopathy, because over 80% or patients have haploinsufficiency of either a small or large subunit-associated ribosomal protein (RP). The erythroid pathology is predominantly due to a block and delay in early committed erythropoiesis with reduced Megakaryocyte/Erythroid Progenitors (MEPs). To understand the molecular pathways leading to pathogenesis of DBA, we performed RNA-seq on mRNA and miRNA from RPS19-deficient human hematopoietic stem and progenitor cells (HSPCs) and compared an existing database documenting transcript fluctuations across stages of early normal erythropoiesis. We determined the chromatin regulator, SATB1 was prematurely downregulated through the coordinated action of upregulated miR-34 and miR-30 during differentiation in ribosomal-insufficiency. Restoration of SATB1 rescued MEP expansion, leading to a modest improvement in erythroid and megakaryocyte expansion in RPS19-insufficiency. However, SATB1 expression did not impact expansion of committed erythroid progenitors, indicating ribosomal insufficiency impacts multiple stages during erythroid differentiation.

View details for DOI 10.1016/j.exphem.2022.04.005

View details for PubMedID 35460833

Cryo-EM, Protein Engineering, and Simulation Enable the Development of Peptide Therapeutics against Acute Myeloid Leukemia. ACS central science Zhang, K., Horikoshi, N., Li, S., Powers, A. S., Hameedi, M. A., Pintilie, G. D., Chae, H., Khan, Y. A., Suomivuori, C., Dror, R. O., Sakamoto, K. M., Chiu, W., Wakatsuki, S. 2022; 8 (2): 214-222


Cryogenic electron microscopy (cryo-EM) has emerged as a viable structural tool for molecular therapeutics development against human diseases. However, it remains a challenge to determine structures of proteins that are flexible and smaller than 30 kDa. The 11 kDa KIX domain of CREB-binding protein (CBP), a potential therapeutic target for acute myeloid leukemia and other cancers, is a protein which has defied structure-based inhibitor design. Here, we develop an experimental approach to overcome the size limitation by engineering a protein double-shell to sandwich the KIX domain between apoferritin as the inner shell and maltose-binding protein as the outer shell. To assist homogeneous orientations of the target, disulfide bonds are introduced at the target-apoferritin interface, resulting in a cryo-EM structure at 2.6 A resolution. We used molecular dynamics simulations to design peptides that block the interaction of the KIX domain of CBP with the intrinsically disordered pKID domain of CREB. The double-shell design allows for fluorescence polarization assays confirming the binding between the KIX domain in the double-shell and these interacting peptides. Further cryo-EM analysis reveals a helix-helix interaction between a single KIX helix and the best peptide, providing a possible strategy for developments of next-generation inhibitors.

View details for DOI 10.1021/acscentsci.1c01090

View details for PubMedID 35233453

CytofIn enables integrated analysis of public mass cytometry datasets using generalized anchors. Nature communications Lo, Y., Keyes, T. J., Jager, A., Sarno, J., Domizi, P., Majeti, R., Sakamoto, K. M., Lacayo, N., Mullighan, C. G., Waters, J., Sahaf, B., Bendall, S. C., Davis, K. L. 2022; 13 (1): 934


The increasing use of mass cytometry for analyzing clinical samples offers the possibility to perform comparative analyses across public datasets. However, challenges in batch normalization and data integration limit the comparison of datasets not intended to be analyzed together. Here, we present a data integration strategy, CytofIn, using generalized anchors to integrate mass cytometry datasets from the public domain. We show that low-variance controls, such as healthy samples and stable channels, are inherently homogeneous, robust against stimulation, and can serve as generalized anchors for batch correction. Single-cell quantification comparing mass cytometry data from 989 leukemia files pre- and post normalization with CytofIn demonstrates effective batch correction while recapitulating the gold-standard bead normalization. CytofIn integration of public cancer datasets enabled the comparison of immune features across histologies and treatments. We demonstrate the ability to integrate public datasets without necessitating identical control samples or bead standards for fast and robust analysis using CytofIn.

View details for DOI 10.1038/s41467-022-28484-5

View details for PubMedID 35177627

Molecular Systems Architecture of Interactome in the Acute Myeloid Leukemia Microenvironment. Cancers Ayyadurai, V. A., Deonikar, P., McLure, K. G., Sakamoto, K. M. 2022; 14 (3)


A molecular systems architecture is presented for acute myeloid leukemia (AML) to provide a framework for organizing the complexity of biomolecular interactions. AML is a multifactorial disease resulting from impaired differentiation and increased proliferation of hematopoietic precursor cells involving genetic mutations, signaling pathways related to the cancer cell genetics, and molecular interactions between the cancer cell and the tumor microenvironment, including endothelial cells, fibroblasts, myeloid-derived suppressor cells, bone marrow stromal cells, and immune cells (e.g., T-regs, T-helper 1 cells, T-helper 17 cells, T-effector cells, natural killer cells, and dendritic cells). This molecular systems architecture provides a layered understanding of intra- and inter-cellular interactions in the AML cancer cell and the cells in the stromal microenvironment. The molecular systems architecture may be utilized for target identification and the discovery of single and combination therapeutics and strategies to treat AML.

View details for DOI 10.3390/cancers14030756

View details for PubMedID 35159023

GPC2-CAR T cells tuned for low antigen density mediate potent activity against neuroblastoma without toxicity CANCER CELL Heitzeneder, S., Bosse, K. R., Zhu, Z., Zhelev, D., Majzner, R. G., Radosevich, M. T., Dhingra, S., Sotillo, E., Buongervino, S., Pascual-Pasto, G., Garrigan, E., Xu, P., Huang, J., Salzer, B., Delaidelli, A., Raman, S., Cui, H., Martinez, B., Bornheimer, S. J., Sahaf, B., Alag, A., Fetahu, I. S., Hasselblatt, M., Parker, K. R., Anbunathan, H., Hwang, J., Huang, M., Sakamoto, K., Lacayo, N. J., Klysz, D. D., Theruvath, J., Vilches-Moure, J. G., Satpathy, A. T., Chang, H. Y., Lehner, M., Taschner-Mandl, S., Julien, J., Sorensen, P. H., Dimitrov, D. S., Maris, J. M., Mackall, C. L. 2022; 40 (1): 53-+
Downregulation of SATB1 by miRNAs Reduces Megakaryocyte/Erythroid Progenitor Expansion in pre-clinical models of Diamond Blackfan Anemia Experimental Hematology Wilkes, M. C., Scanlon, V., Shibuya, A., Cepika, A., Eskin, A., Chen, Z., Narla, A., Glader, B., Roncarolo, M., Nelson, S. F., Sakamoto, K. M. 2022
GPC2-CAR Tcells tuned for low antigen density mediate potent activity against neuroblastoma without toxicity. Cancer cell Heitzeneder, S., Bosse, K. R., Zhu, Z., Zhelev, D., Majzner, R. G., Radosevich, M. T., Dhingra, S., Sotillo, E., Buongervino, S., Pascual-Pasto, G., Garrigan, E., Xu, P., Huang, J., Salzer, B., Delaidelli, A., Raman, S., Cui, H., Martinez, B., Bornheimer, S. J., Sahaf, B., Alag, A., Fetahu, I. S., Hasselblatt, M., Parker, K. R., Anbunathan, H., Hwang, J., Huang, M., Sakamoto, K., Lacayo, N. J., Klysz, D. D., Theruvath, J., Vilches-Moure, J. G., Satpathy, A. T., Chang, H. Y., Lehner, M., Taschner-Mandl, S., Julien, J., Sorensen, P. H., Dimitrov, D. S., Maris, J. M., Mackall, C. L. 1800


Pediatric cancers often mimic fetal tissues and express proteins normally silenced postnatally that could serve as immune targets. We developed Tcells expressing chimeric antigen receptors (CARs) targeting glypican-2 (GPC2), a fetal antigen expressed on neuroblastoma (NB) and several other solid tumors. CARs engineered using standard designs control NBs with transgenic GPC2 overexpression, but not those expressing clinically relevant GPC2 site density (5,000 molecules/cell, range 1-6* 103). Iterative engineering of transmembrane (TM) and co-stimulatory domains plus overexpression of c-Jun lowered the GPC2-CAR antigen density threshold, enabling potent and durable eradication of NBs expressing clinically relevant GPC2 antigen density, without toxicity. These studies highlight the critical interplay between CAR design and antigen density threshold, demonstrate potent efficacy and safety of a lead GPC2-CAR candidate suitable for clinical testing, and credential oncofetal antigens as a promising class of targets for CAR Tcell therapy of solid tumors.

View details for DOI 10.1016/j.ccell.2021.12.005

View details for PubMedID 34971569

Physician risk perceptions and surveillance practices for tyrosine kinase inhibitor long-term effects in pediatric CML. Pediatric hematology and oncology Smith, S. M., Zhang, S., Sundaram, V., Roth, M., Andolina, J. R., Schapira, L., Sakamoto, K. M., Kolb, E. A., Hijiya, N., Chaudhury, S. 1800: 1-15


Chronic myeloid leukemia (CML) is effectively treated with long-term tyrosine kinase inhibitor (TKI) therapy, yet little is known about risks of prolonged TKI exposure in young patients, and long-term effect monitoring is not standardized. We surveyed North American pediatric oncologists (n=119) to evaluate perceived risk of and surveillance practices for potential toxicities associated with prolonged TKI exposure in children and adolescents/young adults (AYAs) with CML. Survey domains included general and specific risk perceptions and surveillance practices for asymptomatic patients on chronic TKI therapy. We analyzed data descriptively and explored relationships between risk perceptions and surveillance. Risk perceptions varied among oncologists but were similar across six categories (thyroid, cardiac, vascular, metabolic, fertility, psychologic), with less than one-third rating each risk as moderate or high in pediatric and AYA patients. More oncologists perceived moderate or high risk of growth abnormalities in children (62% pediatric, 14% AYA) and financial toxicity in all patients (60% pediatric, 64% AYA). A greater proportion of oncologists with moderate or high perceived risk of thyroid abnormalities reported testing thyroid function compared to those with lower perceived risk; patterns for metabolic risk/lipid tests and cardiac risk/tests were similar. In summary, we found that pediatric oncologists had variable risk perceptions and surveillance practices for potential toxicities associated with prolonged TKI exposure. Standardizing surveillance would help quantify risks and refine recommendations.Supplemental data for this article is available online at .

View details for DOI 10.1080/08880018.2021.2017085

View details for PubMedID 34918996

Comparison of the Transcriptomic Signatures in Pediatric and Adult CML. Cancers Youn, M., Smith, S. M., Lee, A. G., Chae, H., Spiteri, E., Erdmann, J., Galperin, I., Jones, L. M., Donato, M., Abidi, P., Bittencourt, H., Lacayo, N., Dahl, G., Aftandilian, C., Davis, K. L., Matthews, J. A., Kornblau, S. M., Huang, M., Sumarsono, N., Redell, M. S., Fu, C. H., Chen, I., Alonzo, T. A., Eklund, E., Gotlib, J., Khatri, P., Sweet-Cordero, E. A., Hijiya, N., Sakamoto, K. M. 1800; 13 (24)


Children with chronic myeloid leukemia (CML) tend to present with higher white blood counts and larger spleens than adults with CML, suggesting that the biology of pediatric and adult CML may differ. To investigate whether pediatric and adult CML have unique molecular characteristics, we studied the transcriptomic signature of pediatric and adult CML CD34+ cells and healthy pediatric and adult CD34+ control cells. Using high-throughput RNA sequencing, we found 567 genes (207 up- and 360 downregulated) differentially expressed in pediatric CML CD34+ cells compared to pediatric healthy CD34+ cells. Directly comparing pediatric and adult CML CD34+ cells, 398 genes (258 up- and 140 downregulated), including many in the Rho pathway, were differentially expressed in pediatric CML CD34+ cells. Using RT-qPCR to verify differentially expressed genes, VAV2 and ARHGAP27 were significantly upregulated in adult CML CD34+ cells compared to pediatric CML CD34+ cells. NCF1, CYBB, and S100A8 were upregulated in adult CML CD34+ cells but not in pediatric CML CD34+ cells, compared to healthy controls. In contrast, DLC1 was significantly upregulated in pediatric CML CD34+ cells but not in adult CML CD34+ cells, compared to healthy controls. These results demonstrate unique molecular characteristics of pediatric CML, such as dysregulation of the Rho pathway, which may contribute to clinical differences between pediatric and adult patients.

View details for DOI 10.3390/cancers13246263

View details for PubMedID 34944883

The Serine Threonine Kinase Inhibitor Ots-167 Improves Erythropoiesis through Suppression of Nlk Activity in Diamond Blackfan Anemia Models Shibuya, A., Wilkes, M. C., Serrano, M., Flygare, J., Glader, B., Narla, A., Sakamoto, K. M. AMER SOC HEMATOLOGY. 2021
Aberrant Histone Landscape in Juvenile Myelomonocytic Leukemia Sinha, R., Dvorak, M., Niemeyer, C. M., Sakamoto, K. M., Patil, R., Jutz, P., Liu, Y., Kuo, A., Bertaina, A. AMER SOC HEMATOLOGY. 2021
SATB1 Regulates Chromatin Organization and HSP70 Expression in Early Erythropoiesis and Is Downregulated in Models of Diamond Blackfan Anemia Wilkes, M. C., Shibuya, A., Scanlon, V. M., Chae, H., Narla, A., Sakamoto, K. M. AMER SOC HEMATOLOGY. 2021
Nutritional Supplements, Ginseng and Leucine, Increase Erythropoiesis in Diamond Blackfan Anemia Models through Inhibition of Nemo-like Kinase Wilkes, M. C., Shibuya, A., Mercado, J. D., Narla, A., Glader, B., Sakamoto, K. M. AMER SOC HEMATOLOGY. 2021
Signaling Pathways That Regulate Normal and Aberrant Red Blood Cell Development. Genes Wilkes, M. C., Shibuya, A., Sakamoto, K. M. 2021; 12 (10)


Blood cell development is regulated through intrinsic gene regulation and local factors including the microenvironment and cytokines. The differentiation of hematopoietic stem and progenitor cells (HSPCs) into mature erythrocytes is dependent on these cytokines binding to and stimulating their cognate receptors and the signaling cascades they initiate. Many of these pathways include kinases that can diversify signals by phosphorylating multiple substrates and amplify signals by phosphorylating multiple copies of each substrate. Indeed, synthesis of many of these cytokines is regulated by a number of signaling pathways including phosphoinositide 3-kinase (PI3K)-, extracellular signal related kinases (ERK)-, and p38 kinase-dependent pathways. Therefore, kinases act both upstream and downstream of the erythropoiesis-regulating cytokines. While many of the cytokines are well characterized, the nuanced members of the network of kinases responsible for appropriate induction of, and response to, these cytokines remains poorly defined. Here, we will examine the kinase signaling cascades required for erythropoiesis and emphasize the importance, complexity, enormous amount remaining to be characterized, and therapeutic potential that will accompany our comprehensive understanding of the erythroid kinome in both healthy and diseased states.

View details for DOI 10.3390/genes12101646

View details for PubMedID 34681039

The active component of Ginseng, Ginsenoside Rb1, improves erythropoiesis in models of Diamond Blackfan Anemia by targeting Nemo-like Kinase. The Journal of biological chemistry Wilkes, M. C., Jung, K., Lee, B. E., Saxena, M., Sathianathen, R. S., Mercado, J. D., Perez, C., Flygare, J., Narla, A., Glader, B., Sakamoto, K. M. 2021: 100988


Nemo-like kinase (NLK) is a member of the MAPK family of kinases and shares a highly conserved kinase domain with other MAPK family members. The activation of NLK contributes to the pathogenesis of Diamond Blackfan Anemia (DBA), reducing c-myb expression and mTOR activity, and is therefore a potential therapeutic target. Unlike other anemia's, the hematopoietic effects of DBA are largely restricted to the erythroid lineage. Mutations in ribosomal genes induces ribosomal insufficiency and reduced protein translation, dramatically impacting early erythropoiesis in the bone marrow of DBA patients. We sought to identify compounds that suppress NLK and increases erythropoiesis in ribosomal insufficiency. We report that the active component of ginseng, ginsenoside Rb1, suppresses NLK expression and improves erythropoiesis in in vitro models of Diamond Blackfan Anemia. Ginsenoside Rb1-mediated suppression of NLK occurs through the upregulation of miR-208, which binds to the 3'-UTR of NLK mRNA and targets it for degradation. We also compare ginsenoside Rb1-mediated upregulation of miR-208 with metformin-mediated upregulation of miR-26. We conclude that targeting NLK expression through miRNA binding of the unique 3'-UTR is a viable alternative to the challenges of developing small molecule inhibitors to target the highly conserved kinase domain of this specific kinase.

View details for DOI 10.1016/j.jbc.2021.100988

View details for PubMedID 34298020

RSK Isoforms in Acute Myeloid Leukemia. Biomedicines Youn, M., Gomez, J. O., Mark, K., Sakamoto, K. M. 2021; 9 (7)


Ribosomal S6 Kinases (RSKs) are a group of serine/threonine kinases that function downstream of the Ras/Raf/MEK/ERK signaling pathway. Four RSK isoforms are directly activated by ERK1/2 in response to extracellular stimuli including growth factors, hormones, and chemokines. RSKs phosphorylate many cytosolic and nuclear targets resulting in the regulation of diverse cellular processes such as cell proliferation, survival, and motility. In hematological malignancies such as acute myeloid leukemia (AML), RSK isoforms are highly expressed and aberrantly activated resulting in poor outcomes and resistance to chemotherapy. Therefore, understanding RSK function in leukemia could lead to promising therapeutic strategies. This review summarizes the current information on human RSK isoforms and discusses their potential roles in the pathogenesis of AML and mechanism of pharmacological inhibitors.

View details for DOI 10.3390/biomedicines9070726

View details for PubMedID 34202904

Activated Natural Killer Cells Predict Poor Clinical Prognosis in High-risk B- and T- cell Acute Lymphoblastic Leukemia. Blood Duault, C., Kumar, A., Taghi Khani, A., Lee, S. J., Yang, L., Huang, M., Hurtz, C., Manning, B., Ghoda, L. Y., McDonald, T., Lacayo, N. J., Sakamoto, K. M., Carroll, M. P., Tasian, S. K., Marcucci, G., Yu, J., Caligiuri, M. A., Maecker, H. T., Swaminathan, S. 2021


B- and T- cell acute lymphoblastic leukemia (B/T-ALL) may be refractory or recur after therapy by suppressing host anti-cancer immune surveillance mediated specifically by natural killer (NK) cells. We delineated the phenotypic and functional defects in NK cells of high-risk B/T-ALL patients using mass, flow, and in silico cytometry, with the goal of further elucidating the role of NK cells in sustaining ALL regression. We found that, compared to normal counterparts, NK cells in B/T-ALL patients are less cytotoxic, but exhibit an activated signature characterized by high CD56, high CD69, production of activated NK-origin cytokines, and calcium signaling. We demonstrated that defective maturation of NK cells into cytotoxic effectors prevents NK cells of ALL patients from lysing NK-sensitive targets as efficiently as normal NK cells. Additionally, we showed that NK cells in ALL are exhausted, which is likely caused by their chronic activation. We found that increased frequencies of activated cytokine-producing NK cells are associated with increased disease severity and independently predict poor clinical outcome in ALL patients. Our studies highlight the benefits of developing NK cell profiling as a diagnostic tool to predict clinical outcome in patients with ALL and underscore the clinical potential of allogeneic NK infusions to prevent ALL recurrence.

View details for DOI 10.1182/blood.2020009871

View details for PubMedID 34077953

Patterns of surveillance for late effects of BCR-ABL tyrosine kinase inhibitors in survivors of pediatric Philadelphia chromosome positive leukemias. BMC cancer Smith, S. M., Sabnis, H. S., Lewis, R. W., Effinger, K. E., Bergsagel, J., Patterson, B., Mertens, A., Sakamoto, K. M., Schapira, L., Castellino, S. M. 2021; 21 (1): 474


BACKGROUND: Targeted anticancer therapies such as BCR-ABL tyrosine kinase inhibitors (TKIs) have improved outcomes for chronic myeloid leukemia (CML) and Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ALL). However, little is known about long-term risks of TKIs in children. Exposure-based survivorship guidelines do not include TKIs, thus surveillance practices may be variable.METHODS: We retrospectively examined surveillance for cardiac and endocrine late effects in children receiving TKIs for Ph+leukemias, diagnosed at <21years between 2000 and 2018. Frequency of echocardiogram (ECHO), electrocardiogram (EKG), thyroid stimulating hormone (TSH), dual-energy x-ray absorptiometry (DXA), and bone age testing were abstracted. Descriptive statistics were stratified by leukemia type.RESULTS: 66 patients (CML n=44; Ph+ALL n=22) met inclusion criteria. Among patients with CML, 1 evaluation was done: ECHO (50.0%), EKG (48.8%), TSH (43.9%), DXA (2.6%), bone age (7.4%). Among patients with Ph+ALL, 1 evaluation was done: ECHO (86.4%), EKG (68.2%), TSH (59.1%), DXA (63.6%), bone age (44.4%). Over a median 6.3 and 5.7years of observation, respectively, 2% of patients with CML and 57% with Ph+ALL attended a survivorship clinic.CONCLUSIONS: Despite common exposure to TKIs in survivors of Ph+leukemias, patterns of surveillance for late effects differed in CML and Ph+ALL, with the latter receiving more surveillance likely due to concomitant chemotherapy exposures. Targeted therapies such as TKIs are revolutionizing cancer treatment, but surveillance for late effects and referral to survivorship clinics are variable despite the chronicity of exposure. Evidence based guidelines and longer follow-up are needed.

View details for DOI 10.1186/s12885-021-08182-z

View details for PubMedID 33926411

Chronic Myelogenous Leukemia in Childhood. Current oncology reports Smith, S. M., Hijiya, N., Sakamoto, K. M. 2021; 23 (4): 40


PURPOSE OF REVIEW: Chronic myelogenous leukemia (CML) is rare in children, requiring extrapolation from treatment of adults. In this review, we explore similarities and differences between adult and pediatric CML with a focus on therapeutic advances and emerging clinical questions.RECENT FINDINGS: Pediatric CML is effectively treated with long-term targeted therapy using tyrosine kinase inhibitors (TKIs). Newly diagnosed pediatric patients in chronic phase can now be treated with imatinib, dasatinib, or nilotinib without allogeneic hematopoietic stem cell transplantation. While treatment-free remission is possible in adults in chronic phase with optimal response to therapy, data are currently insufficient to support stopping TKI in pediatrics outside of a clinical trial. Knowledge gaps remain regarding long-term and late effects of TKIs in pediatric CML. Targeted therapy has markedly improved outcomes for pediatric CML, while raising a number of clinical questions, including the possibility of treatment-free remission and long-term health implications of prolonged TKI exposure at a young age.

View details for DOI 10.1007/s11912-021-01025-x

View details for PubMedID 33718985

Navigating Uncertain Risks: Physician Risk Perceptions and Surveillance Practices for Long-Term Effects of Tyrosine Kinase Inhibitors in Pediatric Chronic Myeloid Leukemia Smith, S., Roth, M., Andolina, J., Sakamoto, K., Schapira, L., Kolb, E. A., Hijiya, N., Chaudhury, S. WILEY. 2020: S224
Metformin-induced suppression of NLK improves erythropoiesis in pre-clinical models of Diamond Blackfan Anemia through induction of miR-26a. Experimental hematology Wilkes, M. C., Siva, K., Varetti, G., Mercado, J., Wentworth, E. P., Perez, C., Saxena, M., Kam, S., Kapur, S., Chen, J., Narla, A., Glader, B., Lin, S., Serrano, M., Flygare, J., Sakamoto, K. M. 2020


Diamond Blackfan Anemia (DBA) results from haploinsufficiency of ribosomal protein subunits in hematopoietic progenitors in the earliest stages of committed erythropoiesis. Nemo-like kinase (NLK) is chronically hyperactivated in committed erythroid progenitors and precursors in multiple human and murine models of DBA. Inhibition of NLK activity, or suppression of NLK expression, both improve erythroid expansion in these models. Metformin is a well-tolerated drug for type 2 diabetes mellitus with multiple cellular targets. Here we demonstrate that metformin improves erythropoiesis in human and zebrafish models of DBA. Our data shows that the effects of metformin on erythroid proliferation and differentiation is mediated by suppression of NLK expression through induction of miR-26a, which recognizes a binding site within the NLK 3'UTR to facilitate transcript degradation. We propose that induction of miR-26a is a potentially novel approach to treat DBA and could improve anemia in DBA patients without the potentially adverse side effects of metformin in a DBA patient population.

View details for DOI 10.1016/j.exphem.2020.09.187

View details for PubMedID 32926965

Is cancer latency an outdated concept? Lessons from chronic myeloid leukemia. Leukemia Abecasis, M., Cross, N. C., Brito, M., Ferreira, I., Sakamoto, K. M., Hijiya, N., Score, J., Gale, R. P. 2020


Our concept of cancer latency, the interval from when a cancer starts until it is diagnosed, has changed dramatically. A prior widely-used definition was the interval between an exposure to a cancer-causing substance and cancer diagnosis. However, this definition does not accurately reflect current knowledge of how most cancers develop assuming, mostly incorrectly, one exposure is the sole cause of a cancer, ignoring the possibility the cancer being considered would have developed anyway but that the exposure accelerated cancer development and eliding the randomness in when a cancer is diagnosed. We show, using chronic myeloid leukaemia as a model, that defining cancer latency is not as simple as it once seemed. It is difficult or impossible to know at which event or mutation to start to clock to measure cancer latency. It is equally difficult to know when to stop the clock given the stochastic nature of when cancers are diagnosed. Importantly, even in genetically-identical twins with the same driver mutation intervals to develop cancer vary substantially. And we discuss other confonders. Clearly we need a new definition of cancer latency or we need to abandon the concept of cancer latency in the modern era of cancer biology.

View details for DOI 10.1038/s41375-020-0957-z

View details for PubMedID 32632094

Screening practices for late effects in pediatric patients on tyrosine kinase inhibitors. Smith, S. M., Sabnis, H. S., Lewis, R., Effinger, K., Bergsagel, D., Patterson, B., Mertens, A. C., Sakamoto, K., Schapira, L., Castellino, S. M. LIPPINCOTT WILLIAMS & WILKINS. 2020
Metabolomics in acute myeloid leukemia. Molecular genetics and metabolism Wojcicki, A. V., Kasowski, M. M., Sakamoto, K. M., Lacayo, N. 2020


Acute myeloid leukemia (AML) is a complex, heterogenous hematological malignancy caused by mutations in myeloid differentiation and proliferation. Response to therapy and long-term outcomes vary widely based on chromosomal and molecular aberrations. Many platforms have been used to characterize and stratify AML. Metabolomics, the global profiling of small molecules in a biological sample, has emerged in the last decade as an important tool for studying the metabolic dependency of cancer cells. Metabolic reprogramming is not only an important manifestation of AML but clinically relevant for diagnosis, risk stratification and targeted drug development. In this review, we discuss notable metabolic studies of the last decade and their application to novel therapies.

View details for DOI 10.1016/j.ymgme.2020.05.005

View details for PubMedID 32457018

Repurposing Drugs for Acute Myeloid Leukemia: A Worthy Cause or a Futile Pursuit? Cancers Wojcicki, A. V., Kadapakkam, M., Frymoyer, A., Lacayo, N., Chae, H., Sakamoto, K. M. 2020; 12 (2)


Acute myeloid leukemia (AML) is a clinically and genetically heterogenous malignancy of myeloid progenitor cells that affects patients of all ages. Despite decades of research and improvement in overall outcomes, standard therapy remains ineffective for certain subtypes of AML. Current treatment is intensive and leads to a number of secondary effects with varying results by patient population. Due to the high cost of discovery and an unmet need for new targeted therapies that are well tolerated, alternative drug development strategies have become increasingly attractive. Repurposing existing drugs is one approach to identify new therapies with fewer financial and regulatory hurdles. In this review, we provide an overview of previously U.S. Food and Drug Administration (FDA) approved non-chemotherapy drugs under investigation for the treatment of AML.

View details for DOI 10.3390/cancers12020441

View details for PubMedID 32069925

EPIGENETIC TARGETING OF TERT-ASSOCIATED GENE EXPRESSION SIGNATURE IN HUMAN NEUROBLASTOMA WITH TERT OVEREXPRESSION. Cancer research Huang, M. n., Zeki, J. n., Sumarsono, N. n., Coles, G. L., Taylor, J. S., Danzer, E. n., Bruzoni, M. n., Hazard, F. K., Lacayo, N. J., Sakamoto, K. M., Dunn, J. C., Spunt, S. L., Chiu, B. n. 2020


Neuroblastoma is a deadly pediatric solid tumor with infrequent recurrent somatic mutations. Particularly, the pathophysiology of tumors without MYCN amplification remains poorly defined. Utilizing an unbiased approach, we performed gene set enrichment analysis of RNA-seq data from 498 neuroblastoma patients and revealed a differentially overexpressed gene signature in MYCN non-amplified neuroblastomas with telomerase reverse transcriptase (TERT) gene overexpression and coordinated activation of oncogenic signaling pathways, including E2Fs, Wnt, Myc, and the DNA repair pathway. Promoter rearrangement of the TERT gene juxtaposes the coding sequence to strong enhancer elements, leading to TERT overexpression and poor prognosis in neuroblastoma, but TERT-associated oncogenic signaling remains unclear. ChIP-seq analysis of the human CLB-GA neuroblastoma cells harboring TERT rearrangement uncovered genome-wide chromatin co-occupancy of Brd4 and H3K27Ac and robust enrichment of H3K36me3 in TERT and multiple TERT-associated genes. Brd4 and cyclin-dependent kinases (CDKs) had critical regulatory roles in the expression and chromatin activation of TERT and multiple TERT-associated genes. Epigenetically targeting Brd4 or CDKs with their respective inhibitors suppressed the expression of TERT and multiple TERT-associated genes in neuroblastoma with TERT overexpression or MYCN amplification. ChIP-seq and ChIP-qPCR provided evidence that the CDK inhibitor directly inhibited Brd4 recruitment to activate chromatin globally. Therefore, inhibiting Brd4 and CDK concurrently with AZD5153 and dinaciclib would be most effective in tumor growth suppression, which we demonstrated in neuroblastoma cell lines, primary human cells, and xenografts. In summary, we describe a unique mechanism in neuroblastoma with TERT overexpression and an epigenetically targeted novel therapeutic strategy.

View details for DOI 10.1158/0008-5472.CAN-19-2560

View details for PubMedID 31900258

RSK inhibitor BI-D1870 inhibits acute myeloid leukemia cell proliferation by targeting mitotic exit. Oncotarget Chae, H. D., Dutta, R. n., Tiu, B. n., Hoff, F. W., Accordi, B. n., Serafin, V. n., Youn, M. n., Huang, M. n., Sumarsono, N. n., Davis, K. L., Lacayo, N. J., Pigazzi, M. n., Horton, T. M., Kornblau, S. M., Sakamoto, K. M. 2020; 11 (25): 23872403


The 90 kDa Ribosomal S6 Kinase (RSK) drives cell proliferation and survival in cancers, although its oncogenic mechanism has not been well characterized. Phosphorylated level of RSK (T573) was increased in acute myeloid leukemia (AML) patients and associated with poor survival. To examine the role of RSK in AML, we analyzed apoptosis and the cell cycle profile following treatment with BI-D1870, a potent inhibitor of RSK. BI-D1870 treatment increased the G2/M population and induced apoptosis in AML cell lines and patient AML cells. Characterization of mitotic phases showed that the metaphase/anaphase transition was significantly inhibited by BI-D1870. BI-D1870 treatment impeded the association of activator CDC20 with APC/C, but increased binding of inhibitor MAD2 to CDC20, preventing mitotic exit. Moreover, the inactivation of spindle assembly checkpoint or MAD2 knockdown released cells from BI-D1870-induced metaphase arrest. Therefore, we investigated whether BI-D1870 potentiates the anti-leukemic activity of vincristine by targeting mitotic exit. Combination treatment of BI-D1870 and vincristine synergistically increased mitotic arrest and apoptosis in acute leukemia cells. These data show that BI-D1870 induces apoptosis of AML cells alone and in combination with vincristine through blocking mitotic exit, providing a novel approach to overcoming vincristine resistance in AML cells.

View details for DOI 10.18632/oncotarget.27630

View details for PubMedID 32637030

View details for PubMedCentralID PMC7321696

Pharmacological Inhibition of Nemo-like Kinase Rescues mTOR-Mediated Translation and Primes Progenitors for Leucine-Stimulated Erythroid Expansion in Pre-Clinical Models of Diamond Blackfan Anemia Wilkes, M. C., Mercado, J. D., Saxena, M., Chen, J., Siva, K., Chae, H., Youn, M., Wentworth, E., Jung, K., Sathianathen, R., Patel, H., Glader, B., Narla, A., Repellin, C. E., Gazda, H. T., Serrano, M., Flygare, J., Sakamoto, K. M. AMER SOC HEMATOLOGY. 2019
INHIBITION OF NEMO-LIKE KINASE IMPROVES ERYTHROPOIESIS IN MODELS OF DIAMOND BLACKFAN ANEMIA Takasaki, K., Wilkes, M., Chen, J., Siva, K., Varetti, G., Dever, D., Youn, M., Chae, H., Mercado, J., Saxena, M., Narla, A., Glader, B., Porteus, M., Repellin, C., Gazda, H., Serrano, M., Flygare, J., Sakamoto, K. WILEY. 2019
SAR optimization studies on modified salicylamides as a potential treatment for acute myeloid leukemia through inhibition of the CREB pathway. Bioorganic & medicinal chemistry letters Chae, H. D., Cox, N. n., Capolicchio, S. n., Lee, J. W., Horikoshi, N. n., Kam, S. n., Ng, A. A., Edwards, J. n., Butler, T. L., Chan, J. n., Lee, Y. n., Potter, G. n., Capece, M. C., Liu, C. W., Wakatsuki, S. n., Smith, M. n., Sakamoto, K. M. 2019


Disruption of cyclic adenosine monophosphate response element binding protein (CREB) provides a potential new strategy to address acute leukemia, a disease associated with poor prognosis, and for which conventional treatment options often carry a significant risk of morbidity and mortality. We describe the structure-activity relationships (SAR) for a series of XX-650-23 derived from naphthol AS-E phosphate that disrupts binding and activation of CREB by the CREB-binding protein (CBP). Through the development of this series, we identified several salicylamides that are potent inhibitors of acute leukemia cell viability through inhibition of CREB-CBP interaction. Among them, a biphenyl salicylamide, compound 71, was identified as a potent inhibitor of CREB-CBP interaction with improved physicochemical properties relative to previously described derivatives of naphthol AS-E phosphate.

View details for DOI 10.1016/j.bmcl.2019.06.023

View details for PubMedID 31253529

Comparison of the Transcriptomic Signature of Pediatric Vs. Adult CML and Normal Bone Marrow Stem Cells Chae, H., Murphy, L. C., Donato, M., Lee, A. G., Sweet-Cordero, E., Abidi, P., Bittencourt, H., Lacayo, N. J., Dahl, G., Aftandilian, C., Davis, K. L., Huang, M., Sumarsono, N., Redell, M., Fu, C. H., Chen, I. L., Alonzo, T. A., Eklund, E. A., Gotlib, J. R., Khatri, P., Hijiya, N., Sakamoto, K. M. AMER SOC HEMATOLOGY. 2018
Chromatin Organization By SATB1 Regulates HSP70 Induction in Early Erythropoiesis and Lost in Diamond Blackfan Anemia Wilkes, M. C., Takasaki, K., Youn, M., Chae, H., Narla, A., Sakamoto, K. M. AMER SOC HEMATOLOGY. 2018
MMP9 Inhibition Rescues the Erythroid Defect in RPS14-Deficient Del(5q) MDS Models Youn, M., Huang, H., Chen, C., Kam, S., Wilkes, M. C., Chae, H., Narla, A., Lin, S., Sakamoto, K. M. AMER SOC HEMATOLOGY. 2018
Pharmacological Inhibition of Nlk (Nemo-like Kinase) Rescues Erythropoietic Defects in Pre-Clinical Models of Diamond Blackfan Anemia Wilkes, M. C., Chen, J., Siva, K., Veretti, G., Dever, D. P., Youn, M., Chae, H., Mercado, J. D., Saxena, M., Narla, A., Glader, B., Porteus, M., Repellin, C. E., Gazda, H. T., Serrano, M., Flygare, J., Sakamoto, K. M. AMER SOC HEMATOLOGY. 2018
Chromation Remodeling Therapy and Capizzi Methotrexate in Treatment-Related MDS/AML Aftandilian, C., Sakamoto, K. M., Davis, K. L., Dahl, G., Lacayo, N. J. AMER SOC HEMATOLOGY. 2018
CBP Modulates Sensitivity to Dasatinib in Pre-BCR+ Acute Lymphoblastic Leukemia CANCER RESEARCH Duque-Afonso, J., Lin, C., Han, K., Morgens, D. W., Jeng, E. E., Weng, Z., Jeong, J., Wong, S., Zhu, L., Wei, M. C., Chae, H., Schrappe, M., Cario, G., Duyster, J., Xiao, X., Sakamoto, K. M., Bassik, M. C., Cleary, M. L. 2018; 78 (22): 6497-6508
CBP modulates sensitivity to dasatinib in pre-BCR+ acute lymphoblastic leukemia. Cancer research Duque-Afonso, J., Lin, C., Han, K., Morgens, D. W., Jeng, E. E., Weng, Z., Jeong, J., Wong, S. H., Zhu, L., Wei, M. C., Chae, H., Schrappe, M., Cario, G., Duyster, J., Sakamoto, K. M., Bassik, M. C., Cleary, M. L. 2018


Dasatinib is a multi-tyrosine kinase inhibitor approved for treatment of Ph+ acute lymphoblastic leukemia (ALL), but its efficacy is limited by resistance. Recent preclinical studies suggest that dasatinib may be a candidate therapy in additional ALL subtypes including pre-BCR+ ALL. Here we utilized shRNA library screening and global transcriptomic analysis to identify several novel genes and pathways that may enhance dasatinib efficacy or mitigate potential resistance in human pre-BCR+ ALL. Depletion of the transcriptional co-activator CBP increased dasatinib sensitivity by activating transcription of the pre-BCR signaling pathway previously associated with dasatinib sensitivity. Acquired resistance was due in part to upregulation of alternative pathways including WNT through a mechanism suggesting transcriptional plasticity. Small molecules that disrupt CBP interactions with the CREB KID domain or beta-catenin showed promising preclinical efficacy in combination with dasatinib. These findings highlight novel modulators of sensitivity to targeted therapies in human pre-BCR+ ALL, which can be reversed by small molecules inhibitors. They also identify promising therapeutic approaches to ameliorate dasatinib sensitivity and prevent resistance in ALL.

View details for PubMedID 30262461

Perspective on Diamond-Blackfan anemia: lessons from a rare congenital bone marrow failure syndrome LEUKEMIA Sakamoto, K. M., Narla, A. 2018; 32 (2): 24951

View details for PubMedID 29182601

View details for PubMedCentralID PMC5808082

Beyond mRNA: The role of non-coding RNAs in normal and aberrant hematopoiesis MOLECULAR GENETICS AND METABOLISM Wilkes, M. C., Repellin, C. E., Sakamoto, K. M. 2017; 122 (3): 28-38
Beyond mRNA: The role of non-coding RNAs in normal and aberrant hematopoiesis. Molecular genetics and metabolism Wilkes, M. C., Repellin, C. E., Sakamoto, K. M. 2017


The role of non-coding Ribonucleic Acids (ncRNAs) in biology is currently an area of intense focus. Hematopoiesis requires rapidly changing regulatory molecules to guide appropriate differentiation and ncRNA are well suited for this. It is not surprising that virtually all aspects of hematopoiesis have roles for ncRNAs assigned to them and doubtlessly much more await characterization. Stem cell maintenance, lymphoid, myeloid and erythroid differentiation are all regulated by various ncRNAs, including microRNAs (miRNAs), long non-coding RNAs (lncRNAs) and various transposable elements within the genome. As our understanding of the many and complex ncRNA roles continues to grow, new discoveries are challenging the existing classification schemes. In this review we briefly overview the broad categories of ncRNAs and discuss a few examples regulating normal and aberrant hematopoiesis.

View details for PubMedID 28757239

Lost in translation: RP and GATA1 mutations in DBA BLOOD Sakamoto, K. M. 2017; 129 (23): 3048-3049

View details for DOI 10.1182/blood-2017-04-778969

View details for Web of Science ID 000402875100003

View details for PubMedID 28596438

Loss of FOXM1 promotes erythropoiesis through increased proliferation of erythroid progenitors. Haematologica Youn, M., Wang, N., LaVasseur, C., Bibikova, E., Kam, S., Glader, B., Sakamoto, K. M., Narla, A. 2017


Forkhead box M1 (FOXM1) belongs to the forkhead/winged-helix family of transcription factors and regulates a network of proliferation-associated genes. Its abnormal upregulation has been shown to be a key driver of cancer progression and an initiating factor in oncogenesis. FOXM1 is also highly expressed in stem/progenitor cells and inhibits their differentiation, suggesting that FOXM1 plays a role in the maintenance of multipotency. However, the exact molecular mechanisms by which FOXM1 regulates human stem/progenitor cells are still uncharacterized. To understand the role of FOXM1 in normal hematopoiesis, human cord blood CD34(+) cells were transduced with FOXM1 short hairpin ribonucleic acid (shRNA) lentivirus. Knockdown of FOXM1 resulted in a 2-fold increase in erythroid cells compared to myeloid cells. Additionally, knockdown of FOXM1 increased bromodeoxyuridine (BrdU) incorporation in erythroid cells, suggesting greater proliferation of erythroid progenitors. We also observed that the defective phosphorylation of FOXM1 by checkpoint kinase 2 (CHK2) or cyclin-dependent kinases 1/2 (CDK1/2) increased the erythroid population in a manner similar to knockdown of FOXM1. Finally, we found that an inhibitor of FOXM1, forkhead domain inhibitor-6 (FDI-6), increased red blood cell numbers through increased proliferation of erythroid precursors. Overall, our data suggest a novel function of FOXM1 in normal human hematopoiesis.

View details for DOI 10.3324/haematol.2016.156257

View details for PubMedID 28154085

A PET/MR Imaging Approach for the Integrated Assessment of Chemotherapy-induced Brain, Heart, and Bone Injuries in Pediatric Cancer Survivors: A Pilot Study. Radiology Theruvath, A. J., Ilivitzki, A. n., Muehe, A. n., Theruvath, J. n., Gulaka, P. n., Kim, C. n., Luna-Fineman, S. n., Sakamoto, K. M., Yeom, K. W., Yang, P. n., Moseley, M. n., Chan, F. n., Daldrup-Link, H. E. 2017: 170073


Purpose To develop a positron emission tomography (PET)/magnetic resonance (MR) imaging protocol for evaluation of the brain, heart, and joints of pediatric cancer survivors for chemotherapy-induced injuries in one session. Materials and Methods Three teams of experts in neuroimaging, cardiac imaging, and bone imaging were tasked to develop a 20-30-minute PET/MR imaging protocol for detection of chemotherapy-induced tissue injuries of the brain, heart, and bone. In an institutional review board-approved, HIPAA-compliant, prospective study from April to July 2016, 10 pediatric cancer survivors who completed chemotherapy underwent imaging of the brain, heart, and bone with a 3-T PET/MR imager. Cumulative chemotherapy doses and clinical symptoms were correlated with the severity of MR imaging abnormalities by using linear regression analyses. MR imaging measures of brain perfusion and metabolism were compared among eight patients who were treated with methotrexate and eight untreated age-matched control subjects by using Wilcoxon rank-sum tests. Results Combined brain, heart, and bone examinations were completed within 90 minutes. Eight of 10 cancer survivors had abnormal findings on brain, heart, and bone images, including six patients with and two patients without clinical symptoms. Cumulative chemotherapy doses correlated significantly with MR imaging measures of left ventricular ejection fraction and end-systolic volume, but not with the severity of brain or bone abnormalities. Methotrexate-treated cancer survivors had significantly lower cerebral blood flow and metabolic activity in key brain areas compared with control subjects. Conclusion The feasibility of a single examination for assessment of chemotherapy-induced injuries of the brain, heart, and joints was shown. Earlier detection of tissue injuries may enable initiation of timely interventions and help to preserve long-term health of pediatric cancer survivors. () RSNA, 2017 Online supplemental material is available for this article.

View details for PubMedID 28777701

RSK Inhibition Suppresses AML Proliferation through Activation of DNA Damage Pathways and S Phase Arrest Dutta, R., Castellanos, M., Tiu, B., Chae, H., Davis, K. L., Sakamoto, K. M. AMER SOC HEMATOLOGY. 2016
The Salicylamide Derivative, Niclosamide, Inhibits CREB Function in Acute Myeloid Leukemia Cells In Vitro and In Vivo Chae, H., Cox, N., Zhang, X., Lee, J., Morgens, D., Bassik, M. C., Smith, M., Sakamoto, K. M. AMER SOC HEMATOLOGY. 2016
Role of Mir-34 Upregulation in Disruption of c-Myc, c-Myb and NOTCH Signaling in Diamond-Blackfan Anemia Wilkes, M., Bibikova, E., Youn, M., Lee, A., Eskin, A., Nelson, S., Glader, B., Narla, A., Sakamoto, K. M. AMER SOC HEMATOLOGY. 2016
Varicella-Zoster Virus Activates CREB, and Inhibition of the pCREB-p300/CBP Interaction Inhibits Viral Replication In Vitro and Skin Pathogenesis In Vivo. Journal of virology Franois, S., Sen, N., Mitton, B., Xiao, X., Sakamoto, K. M., Arvin, A. 2016; 90 (19): 8686-8697


Varicella-zoster virus (VZV) is an alphaherpesvirus that causes varicella upon primary infection and zoster upon reactivation from latency in sensory ganglion neurons. The replication of herpesviruses requires manipulation of cell signaling pathways. Notably, CREB, a factor involved in the regulation of several cellular processes, is activated upon infection of T cells with VZV. Here, we report that VZV infection also induced CREB phosphorylation in fibroblasts and that XX-650-23, a newly identified inhibitor of the phosphorylated-CREB (pCREB) interaction with p300/CBP, restricted cell-cell spread of VZV in vitro CREB phosphorylation did not require the viral open reading frame 47 (ORF47) and ORF66 kinases encoded by VZV. Evaluating the biological relevance of these observations during VZV infection of human skin xenografts in the SCID mouse model of VZV pathogenesis showed both that pCREB was upregulated in infected skin and that treatment with XX-650-23 reduced infectious-virus production and limited lesion formation compared to treatment with a vehicle control. Thus, processes of CREB activation and p300/CBP binding are important for VZV skin infection and may be targeted for antiviral drug development.Varicella-zoster virus (VZV) is a common pathogen that causes chicken pox and shingles. As with all herpesviruses, the infection is acquired for life, and the virus can periodically reactivate from latency. Although VZV infection is usually benign with few or no deleterious consequences, infection can be life threatening in immunocompromised patients. Otherwise healthy elderly individuals who develop zoster as a consequence of viral reactivation are at risk for postherpetic neuralgia (PHN), a painful and long-lasting complication. Current vaccines use a live attenuated virus that is usually safe but cannot be given to many immunodeficient patients and retains the capacity to establish latency and reactivate, causing zoster. Antiviral drugs are effective against severe VZV infections but have little impact on PHN. A better understanding of virus-host cell interactions is relevant for developing improved therapies to safely interfere with cellular processes that are crucial for VZV pathogenesis.

View details for DOI 10.1128/JVI.00920-16

View details for PubMedID 27440893

Navigating your career path in pediatric hematology/oncology: On and off the beaten track. Pediatric blood & cancer Zweidler-McKay, P. A., Hogan, M. S., Jubran, R., Black, V., Casillas, J., Harper, J., Malempati, S., Margolin, J., Felgenhauer, J., Sakamoto, K. M., Franklin, J., Shah, M., Seibel, N., Buchanan, G., Vaiselbuh, S. R., Hastings, C., Hilden, J., Stork, L. C. 2016; 63 (10): 1723-1730

View details for DOI 10.1002/pbc.26094

View details for PubMedID 27295503

CBP/p300 acetyltransferase activity in hematologic malignancies. Molecular genetics and metabolism Dutta, R., Tiu, B., Sakamoto, K. M. 2016; 119 (1-2): 37-43


CREB binding protein (CBP) and p300 are critical regulators of hematopoiesis through both their transcriptional coactivator and acetyltransferase activities. Loss or mutation of CBP/p300 results in hematologic deficiencies in proliferation and differentiation as well as disruption of hematopoietic stem cell renewal and the microenvironment. Aberrant lysine acetylation mediated by CBP/p300 has recently been implicated in the genesis of multiple hematologic cancers. Understanding the effects of disrupting the acetyltransferase activity of CBP/p300 could pave the way for new therapeutic approaches to treat patients with these diseases.

View details for DOI 10.1016/j.ymgme.2016.06.013

View details for PubMedID 27380996

The role of Fas-associated phosphatase 1 in leukemia stem cell persistence during tyrosine kinase inhibitor treatment of chronic myeloid leukemia LEUKEMIA Huang, W., Luan, C., Hjort, E. E., Bei, L., Mishra, R., Sakamoto, K. M., Platanias, L. C., Eklund, E. A. 2016; 30 (7): 1502-1509


Chronic myeloid leukemia (CML) is characterized by expression of Bcr-abl, a tyrosine kinase oncogene. Clinical outcomes in CML were revolutionized by development of Bcr-abl-targeted tyrosine kinase inhibitors (TKIs), but CML is not cured by these agents. CML leukemia stem cells (LSCs) are relatively TKI insensitive and persist even in remission. LSC persistence results in relapse upon TKI discontinuation, or drug resistance or blast crisis (BC) during prolonged treatment. We hypothesize that increased expression of Fas-associated phosphatase 1 (Fap1) in CML contributes to LSC persistence and BC. As Fap1 substrates include Fas and glycogen synthase kinase-3 (Gsk3), increased Fap1 activity in CML is anticipated to induce Fas resistance and stabilization of -catenin protein. Resistance to Fas-induced apoptosis may contribute to CML LSC persistence, and -catenin activity increases during BC. In the current study, we directly tested the role of Fap1 in CML LSC persistence using in an in vivo murine model. In TKI-treated mice, we found that inhibiting Fap1, using a tripeptide or small molecule, prevented TKI resistance, BC and relapse after TKI discontinuation; all events observed with TKI alone. In addition, Fap1 inhibition increased Fas sensitivity and decreased -catenin activity in CD34(+) bone marrow cells from human subjects with CML. Therapeutic Fap1 inhibition may permit TKI discontinuation and delay in progression in CML.

View details for DOI 10.1038/leu.2016.66

View details for Web of Science ID 000379504400007

View details for PubMedID 26984787

A Zebrafish Model of 5q-Syndrome Using CRISPR/Cas9 Targeting RPS14 Reveals a p53-Independent and p53-Dependent Mechanism of Erythroid Failure JOURNAL OF GENETICS AND GENOMICS Ear, J., Hsueh, J., Nguyen, M., Zhang, Q., Sung, V., Chopra, R., Sakamoto, K. M., Lin, S. 2016; 43 (5): 307-318


5q-syndrome is a distinct form of myelodysplastic syndrome (MDS) where a deletion on chromosome 5 is the underlying cause. MDS is characterized by bone marrow failures, including macrocytic anemia. Genetic mapping and studies using various models support the notion that ribosomal protein S14 (RPS14) is the candidate gene for the erythroid failure. Targeted disruption of RPS14 causes an increase in p53 activity and p53-mediated apoptosis, similar to what is observed with other ribosomal proteins. However, due to the higher risk for cancer development in patients with ribosome deficiency, targeting the p53 pathway is not a viable treatment option. To better understand the pathology of RPS14 deficiency in 5q-deletion, we generated a zebrafish model harboring a mutation in the RPS14 gene. This model mirrors the anemic phenotype seen in 5q-syndrome. Moreover, the anemia is due to a late-stage erythropoietic defect, where the erythropoietic defect is initially p53-independent and then becomes p53-dependent. Finally, we demonstrate the versatility of this model to test various pharmacological agents, such as RAP-011, L-leucine, and dexamethasone in order to identify molecules that can reverse the anemic phenotype.

View details for DOI 10.1016/j.jgg.2016.03.007

View details for Web of Science ID 000376763300010

View details for PubMedID 27216296

Progressing Toward a Cohesive Pediatric 18F-FDG PET/MR Protocol: Is Administration of Gadolinium Chelates Necessary? Journal of nuclear medicine : official publication, Society of Nuclear Medicine Klenk, C., Gawande, R., Tran, V. T., Leung, J. T., Chi, K., Owen, D., Luna-Fineman, S., Sakamoto, K. M., McMillan, A., Quon, A., Daldrup-Link, H. E. 2016; 57 (1): 70-77


With the increasing availability of integrated PET/MR scanners, the utility and need for MR contrast agents for combined scans is questioned. The purpose of our study was to evaluate whether administration of gadolinium chelates is necessary for evaluation of pediatric tumors on (18)F-FDG PET/MR images.First, in 119 pediatric patients with primary and secondary tumors, we used 14 diagnostic criteria to compare the accuracy of several MR sequences: unenhanced T2-weighted fast spin-echo imaging; unenhanced diffusion-weighted imaging; and-before and after gadolinium chelate contrast enhancement-T1-weighted 3-dimensional spoiled gradient echo LAVA (liver acquisition with volume acquisition) imaging. Next, in a subset of 36 patients who had undergone (18)F-FDG PET within 3 wk of MRI, we fused the PET images with the unenhanced T2-weighted MR images (unenhanced (18)F-FDG PET/MRI) and the enhanced T1-weighted MR images (enhanced (18)F-FDG PET/MRI). Using the McNemar test, we compared the accuracy of the two types of fused images using the 14 diagnostic criteria. We also evaluated the concordance between (18)F-FDG avidity and gadolinium chelate enhancement. The standard of reference was histopathologic results, surgical notes, and follow-up imaging.There was no significant difference in diagnostic accuracy between the unenhanced and enhanced MR images. Accordingly, there was no significant difference in diagnostic accuracy between the unenhanced and enhanced (18)F-FDG PET/MR images. (18)F-FDG avidity and gadolinium chelate enhancement were concordant in 30 of the 36 patients and 106 of their 123 tumors.Gadolinium chelate administration is not necessary for accurate diagnostic characterization of most solid pediatric malignancies on (18)F-FDG PET/MR images, with the possible exception of focal liver lesions.

View details for DOI 10.2967/jnumed.115.161646

View details for PubMedID 26471690

Progressing Toward a Cohesive Pediatric F-18-FDG PET/MR Protocol: Is Administration of Gadolinium Chelates Necessary? JOURNAL OF NUCLEAR MEDICINE Klenk, C., Gawande, R., Vy Thao Tran, V. T., Leung, J. T., Chi, K., Owen, D., Luna-Fineman, S., Sakamoto, K. M., McMillan, A., Quon, A., Daldrup-Link, H. E. 2016; 57 (1): 70-77


With the increasing availability of integrated PET/MR scanners, the utility and need for MR contrast agents for combined scans is questioned. The purpose of our study was to evaluate whether administration of gadolinium chelates is necessary for evaluation of pediatric tumors on (18)F-FDG PET/MR images.First, in 119 pediatric patients with primary and secondary tumors, we used 14 diagnostic criteria to compare the accuracy of several MR sequences: unenhanced T2-weighted fast spin-echo imaging; unenhanced diffusion-weighted imaging; and-before and after gadolinium chelate contrast enhancement-T1-weighted 3-dimensional spoiled gradient echo LAVA (liver acquisition with volume acquisition) imaging. Next, in a subset of 36 patients who had undergone (18)F-FDG PET within 3 wk of MRI, we fused the PET images with the unenhanced T2-weighted MR images (unenhanced (18)F-FDG PET/MRI) and the enhanced T1-weighted MR images (enhanced (18)F-FDG PET/MRI). Using the McNemar test, we compared the accuracy of the two types of fused images using the 14 diagnostic criteria. We also evaluated the concordance between (18)F-FDG avidity and gadolinium chelate enhancement. The standard of reference was histopathologic results, surgical notes, and follow-up imaging.There was no significant difference in diagnostic accuracy between the unenhanced and enhanced MR images. Accordingly, there was no significant difference in diagnostic accuracy between the unenhanced and enhanced (18)F-FDG PET/MR images. (18)F-FDG avidity and gadolinium chelate enhancement were concordant in 30 of the 36 patients and 106 of their 123 tumors.Gadolinium chelate administration is not necessary for accurate diagnostic characterization of most solid pediatric malignancies on (18)F-FDG PET/MR images, with the possible exception of focal liver lesions.

View details for DOI 10.2967/jnumed.115.161646

View details for Web of Science ID 000367862700014

View details for PubMedCentralID PMC4703553

Loss of FoxM1 Promotes Erythroid Differentiation through Increased Proliferation of Erythroid Progenitors Youn, M., Bibikova, E., LaVasseur, C., Glader, B., Sakamoto, K., Narla, A. AMER SOC HEMATOLOGY. 2015
CREB Increases Chemotherapy Resistance through Regulation of the DNA Damage Repair Pathway in AML Cells Dutta, R., Tiu, B., Kaul, A., Mitton, B., Sakamoto, K. M. AMER SOC HEMATOLOGY. 2015
Flow-induced protein kinase A-CREB pathway acts via BMP signaling to promote HSC emergence JOURNAL OF EXPERIMENTAL MEDICINE Kim, P. G., Nakano, H., Das, P. P., Chen, M. J., Rowe, R. G., Chou, S. S., Ross, S. J., Sakamoto, K. M., Zon, L. I., Schlaeger, T. M., Orkin, S. H., Nakano, A., Daley, G. Q. 2015; 212 (5): 633-648


Fluid shear stress promotes the emergence of hematopoietic stem cells (HSCs) in the aorta-gonad-mesonephros (AGM) of the developing mouse embryo. We determined that the AGM is enriched for expression of targets of protein kinase A (PKA)-cAMP response element-binding protein (CREB), a pathway activated by fluid shear stress. By analyzing CREB genomic occupancy from chromatin-immunoprecipitation sequencing (ChIP-seq) data, we identified the bone morphogenetic protein (BMP) pathway as a potential regulator of CREB. By chemical modulation of the PKA-CREB and BMP pathways in isolated AGM VE-cadherin(+) cells from mid-gestation embryos, we demonstrate that PKA-CREB regulates hematopoietic engraftment and clonogenicity of hematopoietic progenitors, and is dependent on secreted BMP ligands through the type I BMP receptor. Finally, we observed blunting of this signaling axis using Ncx1-null embryos, which lack a heartbeat and intravascular flow. Collectively, we have identified a novel PKA-CREB-BMP signaling pathway downstream of shear stress that regulates HSC emergence in the AGM via the endothelial-to-hematopoietic transition.

View details for DOI 10.1084/jem.20141514

View details for PubMedID 25870201

Targeting novel signaling pathways for resistant acute myeloid leukemia. Molecular genetics and metabolism Sakamoto, K. M., Grant, S., Saleiro, D., Crispino, J. D., Hijiya, N., Giles, F., Platanias, L., Eklund, E. A. 2015; 114 (3): 397-402


Acute myeloid leukemia (AML) is a hematologic malignancy that is the most common type of acute leukemia diagnosed in adults and the second most common type in children. The overall survival is poor and treatment is associated with significant complications and even death. In addition, a significant number of patients will not respond to therapy or relapse. In this review, several new signaling proteins aberrantly regulated in AML are described, including CREB, Triad1, Bcl-2 family members, Stat3, and mTOR/MEK. Identifying more effective and less toxic agents will provide novel approaches to treat AML.

View details for DOI 10.1016/j.ymgme.2014.11.017

View details for PubMedID 25533111

View details for PubMedCentralID PMC4355162

TNF-mediated inflammation represses GATA1 and activates p38 MAP kinase in RPS19-deficient hematopoietic progenitors. Blood Bibikova, E., Youn, M., Danilova, N., Ono-Uruga, Y., Konto-Ghiorghi, Y., Ochoa, R., Narla, A., Glader, B., Lin, S., Sakamoto, K. M. 2014; 124 (25): 3791-3798


Diamond-Blackfan anemia (DBA) is an inherited disorder characterized by defects in erythropoiesis, congenital abnormalities, and predisposition to cancer. Approximately 25% of DBA patients have a mutation in RPS19, which encodes a component of the 40S ribosomal subunit. Upregulation of p53 contributes to the pathogenesis of DBA, but the link between ribosomal protein mutations and erythropoietic defects is not well understood. We found that RPS19 deficiency in hematopoietic progenitor cells leads to decreased GATA1 expression in the erythroid progenitor population and p53-dependent upregulation of tumor necrosis factor- (TNF-) in nonerythroid cells. The decrease in GATA1 expression was mediated, at least in part, by activation of p38 MAPK in erythroid cells and rescued by inhibition of TNF- or p53. The anemia phenotype in rps19-deficient zebrafish was reversed by treatment with the TNF- inhibitor etanercept. Our data reveal that RPS19 deficiency leads to inflammation, p53-dependent increase in TNF-, activation of p38 MAPK, and decreased GATA1 expression, suggesting a novel mechanism for the erythroid defects observed in DBA.

View details for DOI 10.1182/blood-2014-06-584656

View details for PubMedID 25270909

RPS19 Deficiency Leads to GATA1 Downregulation through TNF-Mediated p38 MAPK Activation Youn, M., Bibikova, E., Danilova, N., Ono-Uruga, Y., Konto-Ghiorghi, Y., Ochoa, R., Narla, A., Glader, B., Lin, S., Sakamoto, K. AMER SOC HEMATOLOGY. 2014
CREB Regulates Cell Cycle Progression through RFC3-PCNA Axis in Acute Myeloid Leukemia Chae, H., Mitton, B., Sakamoto, K. AMER SOC HEMATOLOGY. 2014
The Role of pp90rsk-Mediated CREB Phosphorylation in Acute Myelogenous Leukemia Mitton, B., Dutta, R., Hsu, Y., Sakamoto, K. AMER SOC HEMATOLOGY. 2014
Efficacy and Safety of Eculizumab in Children and Adolescents With Paroxysmal Nocturnal Hemoglobinuria PEDIATRIC BLOOD & CANCER Reiss, U. M., Schwartz, J., Sakamoto, K. M., Puthenveetil, G., Ogawa, M., Bedrosian, C. L., Ware, R. E. 2014; 61 (9): 1544-1550


Paroxysmal nocturnal hemoglobinuria (PNH) is rare in children, but represents a similarly serious and chronic condition as in adults. Children with PNH frequently experience complications of chronic hemolysis, recurrent thrombosis, marrow failure, serious infections, abdominal pain, chronic fatigue, and decreased quality of life with reduced survival. The terminal complement inhibitor eculizumab is proven to be effective and safe in adults and approved by the FDA for treatment of PNH.This 12-week, open-label, multi-center phase I/II study evaluated pharmacokinetics, pharmacodynamics, efficacy, and safety in seven children with PNH 11-17 years of age. Eculizumab was intravenously administered at 600 mg weekly for 4 weeks, 900 mg in week 5, and 900 mg every 2 weeks thereafter ( NCT00867932).Eculizumab therapy resulted in complete and sustained inhibition of hemolysis in all participants with a reduction of lactate dehydrogenase to normal levels. All hematological parameters stabilized. No definitive, study drug-related adverse events were observed. Only one severe SAE of hospitalization due to aplastic anemia occurred, which was not study drug-related.Eculizumab appears to be a safe and effective therapy for children with PNH.

View details for DOI 10.1002/pbc.25068

View details for Web of Science ID 000340540600005

View details for PubMedID 24777716

The role of the DNA damage response in zebrafish and cellular models of Diamond Blackfan anemia. Disease models & mechanisms Danilova, N., Bibikova, E., Covey, T. M., Nathanson, D., Dimitrova, E., Konto, Y., Lindgren, A., Glader, B., Radu, C. G., Sakamoto, K. M., Lin, S. 2014; 7 (7): 895-905


Ribosomal biogenesis involves the processing of pre-ribosomal RNA. A deficiency of some ribosomal proteins (RPs) impairs processing and causes Diamond Blackfan anemia (DBA), which is associated with anemia, congenital malformations and cancer. p53 mediates many features of DBA, but the mechanism of p53 activation remains unclear. Another hallmark of DBA is the upregulation of adenosine deaminase (ADA), indicating changes in nucleotide metabolism. In RP-deficient zebrafish, we found activation of both nucleotide catabolism and biosynthesis, which is consistent with the need to break and replace the faulty ribosomal RNA. We also found upregulation of deoxynucleotide triphosphate (dNTP) synthesis - a typical response to replication stress and DNA damage. Both RP-deficient zebrafish and human hematopoietic cells showed activation of the ATR/ATM-CHK1/CHK2/p53 pathway. Other features of RP deficiency included an imbalanced dNTP pool, ATP depletion and AMPK activation. Replication stress and DNA damage in cultured cells in non-DBA models can be decreased by exogenous nucleosides. Therefore, we treated RP-deficient zebrafish embryos with exogenous nucleosides and observed decreased activation of p53 and AMPK, reduced apoptosis, and rescue of hematopoiesis. Our data suggest that the DNA damage response contributes to p53 activation in cellular and zebrafish models of DBA. Furthermore, the rescue of RP-deficient zebrafish with exogenous nucleosides suggests that nucleoside supplements could be beneficial in the treatment of DBA.

View details for DOI 10.1242/dmm.015495

View details for PubMedID 24812435

Letting microRNAs overcome resistance to chemotherapy in acute myeloid leukemia. Leukemia & lymphoma Sakamoto, K. M. 2014; 55 (7): 1449-1450

View details for DOI 10.3109/10428194.2013.862244

View details for PubMedID 24206095

MicroRNA-34b promoter hypermethylation induces CREB overexpression and contributes to myeloid transformation. Haematologica Pigazzi, M., Manara, E., Bresolin, S., Tregnago, C., Beghin, A., Baron, E., Giarin, E., Cho, E., Masetti, R., Rao, D. S., Sakamoto, K. M., Basso, G. 2013; 98 (4): 602-610


MicroRNA-34b down-regulation in acute myeloid leukemia was previously shown to induce CREB overexpression, thereby causing leukemia proliferation in vitro and in vivo. The role of microRNA-34b and CREB in patients with myeloid malignancies has never been evaluated. We examined microRNA-34b expression and the methylation status of its promoter in cells from patients diagnosed with myeloid malignancies. We used gene expression profiling to identify signatures of myeloid transformation. We established that microRNA-34b has suppressor ability and that CREB has oncogenic potential in primary bone marrow cell cultures and in vivo. MicroRNA-34b was found to be up-regulated in pediatric patients with juvenile myelomonocytic leukemia (n=17) and myelodysplastic syndromes (n=28), but was down-regulated in acute myeloid leukemia patients at diagnosis (n=112). Our results showed that hypermethylation of the microRNA-34b promoter occurred in 66% of cases of acute myeloid leukemia explaining the low microRNA-34b levels and CREB overexpression, whereas preleukemic myelodysplastic syndromes and juvenile myelomonocytic leukemia were not associated with hypermethylation or CREB overexpression. In paired samples taken from the same patients when they had myelodysplastic syndrome and again during the subsequent acute myeloid leukemia, we confirmed microRNA-34b promoter hypermethylation at leukemia onset, with 103 CREB target genes differentially expressed between the two disease stages. This subset of CREB targets was confirmed to associate with high-risk myelodysplastic syndromes in a separate cohort of patients (n=20). Seventy-eight of these 103 CREB targets were also differentially expressed between healthy samples (n=11) and de novo acute myeloid leukemia (n=72). Further, low microRNA-34b and high CREB expression levels induced aberrant myelopoiesis through CREB-dependent pathways in vitro and in vivo. In conclusion, we suggest that microRNA-34b controls CREB expression and contributes to myeloid transformation from both healthy bone marrow and myelodysplastic syndromes. We identified a subset of CREB target genes that represents a novel transcriptional network that may control myeloid transformation.

View details for DOI 10.3324/haematol.2012.070664

View details for PubMedID 23100280

View details for PubMedCentralID PMC3659992

Increased Abscess Formation and Defective Chemokine Regulation in CREB Transgenic Mice PLOS ONE Wen, A. Y., Landaw, E. M., Ochoa, R., Cho, M., Chao, A., Lawson, G., Sakamoto, K. M. 2013; 8 (2)


Cyclic AMP-response element-binding protein (CREB) is a transcription factor implicated in growth factor-dependent cell proliferation and survival, glucose homeostasis, spermatogenesis, circadian rhythms, and synaptic plasticity associated with memory. To study the phenotype of CREB overexpression in vivo, we generated CREB transgenic (TG) mice in which a myeloid specific hMRP8 promoter drives CREB expression. CREB TG mice developed spontaneous skin abscesses more frequently than wild type (WT) mice. To understand the role of CREB in myeloid function and innate immunity, chemokine expression in bone marrow derived macrophages (BMDMs) from CREB TG mice were compared with BMDMs from WT mice. Our results demonstrated decreased Keratinocyte-derived cytokine (KC) in CREB TG BMDMs but not TNF protein production in response to lipid A (LPA). In addition, mRNA expression of KC and IL-1 (Interleukin)-1 was decreased in CREB TG BMDMs; however, there was no difference in the mRNA expression of TNF, MCP-1, IL-6 and IL-12p40. The mRNA expression of IL-1RA and IL-10 was decreased in response to LPA. Nuclear factor kappa B (NFB) expression and a subset of its target genes were upregulated in CREB TG mouse BMDMs. Although neutrophil migration was the same in both CREB TG and WT mice, Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity was significantly increased in neutrophils from CREB TG mice. Taken together, CREB overexpression in myeloid cells results in increased abscess formation in vivo and aberrant cytokine and chemokine response, and neutrophil function in vitro.

View details for DOI 10.1371/journal.pone.0055866

View details for Web of Science ID 000315153400169

View details for PubMedID 23405224

View details for PubMedCentralID PMC3566130

Identification of somatic and germline mutations using whole exome sequencing of congenital acute lymphoblastic leukemia BMC CANCER Chang, V. Y., Basso, G., Sakamoto, K. M., Nelson, S. F. 2013; 13


Acute lymphoblastic leukemia (ALL) diagnosed within the first month of life is classified as congenital ALL and has a significantly worse outcome than ALL diagnosed in older children. This suggests that congenital ALL is a biologically different disease, and thus may be caused by a distinct set of mutations. To understand the somatic and germline mutations contributing to congenital ALL, the protein-coding regions in the genome were captured and whole-exome sequencing was employed for the identification of single-nucleotide variants and small insertion and deletions in the germlines as well as the primary tumors of four patients with congenital ALL.Exome sequencing was performed on Illumina GAIIx or HiSeq 2000 (Illumina, San Diego, California). Reads were aligned to the human reference genome and the Genome Analysis Toolkit was used for variant calling. An in-house developed Ensembl-based variant annotator was used to richly annotate each variant.There were 1-3 somatic, protein-damaging mutations per ALL, including a novel mutation in Sonic Hedgehog. Additionally, there were many germline mutations in genes known to be associated with cancer predisposition, as well as genes involved in DNA repair.This study is the first to comprehensively characterize the germline and somatic mutational profile of all protein-coding genes patients with congenital ALL. These findings identify potentially important therapeutic targets, as well as insight into possible cancer predisposition genes.

View details for DOI 10.1186/1471-2407-13-55

View details for Web of Science ID 000315027900001

View details for PubMedID 23379653

View details for PubMedCentralID PMC3573941

Activity of Egr-1 promoter induced by UV light in human primary dermal fibroblasts transduced with Ad-Egr-1/Luc is repressed by betamethasone. GENE THERAPY AND MOLECULAR BIOLOGY Martinez-Flores, F., Barrera-Lopez, A., Sandoval-Zamora, H. E., Garcia-Cavazos, R., Franco-Bourland, R. E., Madinaveitia-, J. A., Zentella-Dehesa, A., Curiel, D. T., Sakamoto, K. 2013; 15: 92-106
Sox4 cooperates with CREB in myeloid transformation BLOOD Sandoval, S., Kraus, C., Cho, E., Cho, M., Bies, J., Manara, E., Accordi, B., Landaw, E. M., Wolff, L., Pigazzi, M., Sakamoto, K. M. 2012; 120 (1): 155-165


The cAMP response element-binding protein (CREB) is a nuclear transcription factor that is critical for normal and neoplastic hematopoiesis. Previous studies have demonstrated that CREB is a proto-oncogene whose overexpression promotes cellular proliferation in hematopoietic cells. Transgenic mice that overexpress CREB in myeloid cells develop a myeloproliferative disease with splenomegaly and aberrant myelopoiesis. However, CREB overexpressing mice do not spontaneously develop acute myeloid leukemia. In this study, we used retroviral insertional mutagenesis to identify genes that accelerate leukemia in CREB transgenic mice. Our mutagenesis screen identified several integration sites, including oncogenes Gfi1, Myb, and Ras. The Sox4 transcription factor was identified by our screen as a gene that cooperates with CREB in myeloid leukemogenesis. We show that the transduction of CREB transgenic mouse bone marrow cells with a Sox4 retrovirus increases survival and self-renewal of cells in vitro. Furthermore, leukemic blasts from the majority of acute myeloid leukemia patients have higher CREB, phosphorylated CREB, and Sox 4 protein expression. Sox4 transduction of mouse bone marrow cells results in increased expression of CREB target genes. We also demonstrate that CREB is a direct target of Sox4 by chromatin immunoprecipitation assays. These results indicate that Sox4 and CREB cooperate and contribute to increased proliferation of hematopoietic progenitor cells.

View details for DOI 10.1182/blood-2011-05-357418

View details for Web of Science ID 000307411100023

View details for PubMedID 22627767

View details for PubMedCentralID PMC3390953

The neutropenic diet... still ageless? Oncology (Williston Park, N.Y.) Aftandilian, C. C., Milotich, C., Sakamoto, K. M. 2012; 26 (6): 586-?

View details for PubMedID 22870544

The Neutropenic Diet ... Still Ageless? C. Aftandilian Article Reviewed ONCOLOGY-NEW YORK Aftandilian, C. C., Milotich, C., Sakamoto, K. M. 2012; 26 (6): 586-589
Editorial: Granulopoiesis versus monopoiesis: a consequence of transcription factors dancing with the right partners JOURNAL OF LEUKOCYTE BIOLOGY Sakamoto, K. M. 2011; 90 (4): 637-638

View details for DOI 10.1189/jlb.0411187

View details for Web of Science ID 000295372100001

View details for PubMedID 21965310

View details for PubMedCentralID PMC3177698

Tubacin suppresses proliferation and induces apoptosis of acute lymphoblastic leukemia cells LEUKEMIA & LYMPHOMA Aldana-Masangkay, G. I., Rodriguez-Gonzalez, A., Lin, T., Ikeda, A. K., Hsieh, Y., Kim, Y., Lomenick, B., Okemoto, K., Landaw, E. M., Wang, D., Mazitschek, R., Bradner, J. E., Sakamoto, K. M. 2011; 52 (8): 1544-1555


Over the past decade, histone deacetylase inhibitors have increasingly been used to treat various malignancies. Tubacin (tubulin acetylation inducer) is a small molecule that inhibits histone deacetylase 6 (HDAC6) and induces acetylation of -tubulin. We observed a higher antiproliferative effect of tubacin in acute lymphoblastic leukemia (ALL) cells than in normal hematopoietic cells. Treatment with tubacin led to the induction of apoptotic pathways in both pre-B and T cell ALL cells at a 50% inhibitory concentration (IC(50)) of low micromolar concentrations. Acetylation of -tubulin increases within the first 30min following treatment of ALL cells with tubacin. We also observed an accumulation of polyubiquitinated proteins and poly(ADP-ribose) polymerase (PARP) cleavage. Furthermore, the signaling pathways activated by tubacin appear to be distinct from those observed in multiple myeloma. In this article, we demonstrate that tubacin enhances the effects of chemotherapy to treat primary ALL cells in vitro and in vivo. These results suggest that targeting HDAC6 alone or in combination with chemotherapy could provide a novel approach to treat ALL.

View details for DOI 10.3109/10428194.2011.570821

View details for Web of Science ID 000292747300021

View details for PubMedID 21699378

Increasing Diversity in Pediatric Hematology/Oncology PEDIATRIC BLOOD & CANCER Fruge, E., Lakoski, J. M., Luban, N., Lipton, J. M., Poplack, D. G., Hagey, A., Felgenhauer, J., Hilden, J., Margolin, J., Vaiselbuh, S. R., Sakamoto, K. M. 2011; 57 (1): 147-152


Diversity is necessary for the survival and success of both biological and social systems including societies. There is a lack of diversity, particularly the proportion of women and minorities in leadership positions, within medicine [Leadley. AAMC 2009. Steinecke and Terrell. Acad Med 2010;85:236-245]. In 2009 a group of ASPHO members recognized the need to support the career advancement of women and minority members. This article reports the results of a survey designed to characterize the comparative career pathway experience of women and minority ASPHO members.A group of ASPHO members modified a published Faculty Worklife survey [Pribbenow et al. High Educ Policy 2010;23:17-38] for use by Pediatric Hematologist-Oncologists (PHOs). A link to an online version of the survey was sent to all ASPHO members.Of 1,228 ASPHO members polled, 213 responded (17%). Women and minority PHOs reported less satisfaction than their counterparts on 70 of the 90 issues addressed in the survey including the hiring process, access to resources as well as integration and satisfaction with their organizations. Women also expressed greater dissatisfaction with issues of work-life balance, support for family obligations and personal health.The current literature suggests that there are significant disparities in career opportunities, compensation and satisfaction for women compared to men and minority compared to majority faculty in academic medicine [Nivet. J Vasc Surg 2010;51:53S-58S; Peterson et al. J Gen Intern Med 2004;19:259-265; DesRoches et al. Acad Med 2010;85:631-639; Castillo-Page. AAMC 2008]. Our data, derived from a survey of ASPHO members, suggests that this holds true for PHOs as well.

View details for DOI 10.1002/pbc.22977

View details for Web of Science ID 000290452400024

View details for PubMedID 21284078

View details for PubMedCentralID PMC3092025

The Role of HDAC6 in Cancer JOURNAL OF BIOMEDICINE AND BIOTECHNOLOGY Aldana-Masangkay, G. I., Sakamoto, K. M. 2011


Histone deacetylase 6 (HDAC6), a member of the HDAC family whose major substrate is -tubulin, has become a target for drug development to treat cancer due to its major contribution in oncogenic cell transformation. Overexpression of HDAC6 correlates with tumorigenesis and improved survival; therefore, HDAC6 may be used as a marker for prognosis. Previous work demonstrated that in multiple myeloma cells, inhibition of HDAC6 results in apoptosis. Furthermore, HDAC6 is required for the activation of heat-shock factor 1 (HSF1), an activator of heat-shock protein encoding genes (HSPs) and CYLD, a cylindromatosis tumor suppressor gene. HDAC6 contributes to cancer metastasis since its upregulation increases cell motility in breast cancer MCF-7 cells and its interaction with cortactin regulates motility. HDAC6 also affects transcription and translation by regulating the heat-shock protein 90 (Hsp90) and stress granules (SGs), respectively. This review will discuss the role of HDAC6 in the pathogenesis and treatment of cancer.

View details for DOI 10.1155/2011/875824

View details for Web of Science ID 000285611100001

View details for PubMedID 21076528

View details for PubMedCentralID PMC2975074

Self-Renewal of Acute Lymphocytic Leukemia Cells Is Limited by the Hedgehog Pathway Inhibitors Cyclopamine and IPI-926 PLOS ONE Lin, T. L., Wang, Q. H., Brown, P., Peacock, C., Merchant, A. A., Brennan, S., Jones, E., McGovern, K., Watkins, D. N., Sakamoto, K. M., Matsui, W. 2010; 5 (12)


Conserved embryonic signaling pathways such as Hedgehog (Hh), Wingless and Notch have been implicated in the pathogenesis of several malignancies. Recent data suggests that Hh signaling plays a role in normal B-cell development, and we hypothesized that Hh signaling may be important in precursor B-cell acute lymphocytic leukemia (B-ALL). We found that the expression of Hh pathway components was common in human B-ALL cell lines and clinical samples. Moreover, pathway activity could be modulated by Hh ligand or several pathway inhibitors including cyclopamine and the novel SMOOTHENED (SMO) inhibitor IPI-926. The inhibition of pathway activity primarily impacted highly clonogenic B-ALL cells expressing aldehyde dehydrogenase (ALDH) by limiting their self-renewal potential both in vitro and in vivo. These data demonstrate that Hh pathway activation is common in B-ALL and represents a novel therapeutic target regulating self-renewal and persistence of the malignant clone.

View details for DOI 10.1371/journal.pone.0015262

View details for Web of Science ID 000285792500027

View details for PubMedID 21203400

View details for PubMedCentralID PMC3011010

The Role of the Transcription Factor CREB in Immune Function JOURNAL OF IMMUNOLOGY Wen, A. Y., Sakamoto, K. M., Miller, L. S. 2010; 185 (11): 6413-6419


CREB is a transcription factor that regulates diverse cellular responses, including proliferation, survival, and differentiation. CREB is induced by a variety of growth factors and inflammatory signals and subsequently mediates the transcription of genes containing a cAMP-responsive element. Several immune-related genes possess this cAMP-responsive element, including IL-2, IL-6, IL-10, and TNF-. In addition, phosphorylated CREB has been proposed to directly inhibit NF-B activation by blocking the binding of CREB binding protein to the NF-B complex, thereby limiting proinflammatory responses. CREB also induces an antiapoptotic survival signal in monocytes and macrophages. In T and B cells, CREB activation promotes proliferation and survival and differentially regulates Th1, Th2, and Th17 responses. Finally, CREB activation is required for the generation and maintenance of regulatory T cells. This review summarizes current advances involving CREB in immune function--a role that is continually being defined.

View details for DOI 10.4049/jimmunol.1001829

View details for Web of Science ID 000284311500004

View details for PubMedID 21084670

Germline CBL mutations cause developmental abnormalities and predispose to juvenile myelomonocytic leukemia NATURE GENETICS Niemeyer, C. M., Kang, M. W., Shin, D. H., Furlan, I., Erlacher, M., Bunin, N. J., Bunda, S., Finklestein, J. Z., Sakamoto, K. M., Gorr, T. A., Mehta, P., Schmid, I., Kropshofer, G., Corbacioglu, S., Lang, P. J., Klein, C., Schlegel, P., Heinzmann, A., Schneider, M., Stary, J., van den Heuvel-Eibrink, M. M., Hasle, H., Locatelli, F., Sakai, D., Archambeault, S., Chen, L., Russell, R. C., Sybingco, S. S., Ohh, M., Braun, B. S., Flotho, C., Loh, M. L. 2010; 42 (9): 794-U93


CBL encodes a member of the Cbl family of proteins, which functions as an E3 ubiquitin ligase. We describe a dominant developmental disorder resulting from germline missense CBL mutations, which is characterized by impaired growth, developmental delay, cryptorchidism and a predisposition to juvenile myelomonocytic leukemia (JMML). Some individuals experienced spontaneous regression of their JMML but developed vasculitis later in life. Importantly, JMML specimens from affected children show loss of the normal CBL allele through acquired isodisomy. Consistent with these genetic data, the common p.371Y>H altered Cbl protein induces cytokine-independent growth and constitutive phosphorylation of ERK, AKT and S6 only in hematopoietic cells in which normal Cbl expression is reduced by RNA interference. We conclude that germline CBL mutations have developmental, tumorigenic and functional consequences that resemble disorders that are caused by hyperactive Ras/Raf/MEK/ERK signaling and include neurofibromatosis type 1, Noonan syndrome, Costello syndrome, cardiofaciocutaneous syndrome and Legius syndrome.

View details for DOI 10.1038/ng.641

View details for Web of Science ID 000281388400018

View details for PubMedID 20694012

Targeting CREB for Cancer Therapy: Friend or Foe CURRENT CANCER DRUG TARGETS Xiao, X., Li, B. X., Mitton, B., Ikeda, A., Sakamoto, K. M. 2010; 10 (4): 384-391


The cyclic-AMP response element-binding protein (CREB) is a nuclear transcription factor activated by phosphorylation at Ser133 by multiple serine/threonine (Ser/Thr) kinases. Upon phosphorylation, CREB binds the transcriptional co-activator, CBP (CREB-binding protein), to initiate CREB-dependent gene transcription. CREB is a critical regulator of cell differentiation, proliferation and survival in the nervous system. Recent studies have shown that CREB is involved tumor initiation, progression and metastasis, supporting its role as a proto-oncogene. Overexpression and over-activation of CREB were observed in cancer tissues from patients with prostate cancer, breast cancer, non-small-cell lung cancer and acute leukemia while down-regulation of CREB in several distinct cancer cell lines resulted in inhibition of cell proliferation and induction of apoptosis, suggesting that CREB may be a promising target for cancer therapy. Although CREB, as a transcription factor, is a challenging target for small molecules, various small molecules have been discovered to inhibit CREB phosphorylation, CREB-DNA, or CREB-CBP interaction. These results suggest that CREB is a suitable transcription factor for drug targeting and therefore targeting CREB could represent a novel strategy for cancer therapy.

View details for PubMedID 20370681

Macroautophagy modulates cellular response to proteasome inhibitors in cancer therapy DRUG RESISTANCE UPDATES Wu, W. K., Sakamoto, K. M., Milani, M., Aldana-Masankgay, G., Fan, D., Wu, K., Lee, C. W., Cho, C. H., Yu, J., Sung, J. J. 2010; 13 (3): 87-92


Macroautophagy and the ubiquitin-proteasome system are two complementary pathways for protein degradation. The former degrades long-lived proteins and damaged organelles while the later degrades short-lived proteins. Recent findings indicate that suppression of the ubiquitin-proteasome system by proteasome inhibitors induces macroautophagy through multiple pathways, including (1) accumulation of ubiquitinated proteins and activation of HDAC6; (2) activation of the IRE1-JNK pathway; (3) proteasomal stabilization of ATF4; (4) inhibition of mTOR complex 1 signaling; (5) reduced proteasomal degradation of LC3. Induction of macroautophagy attenuates the antitumor effect of proteasome inhibitors in various types of cancer. These findings suggest that inhibition of macroautophagy may represent a novel strategy to enhance cellular sensitivity to proteasome inhibition.

View details for DOI 10.1016/j.drup.2010.04.003

View details for Web of Science ID 000279649500004

View details for PubMedID 20462785

ABT-869 Inhibits the Proliferation of Ewing Sarcoma Cells and Suppresses Platelet-Derived Growth Factor Receptor beta and c-KIT Signaling Pathways MOLECULAR CANCER THERAPEUTICS Ikeda, A. K., Judelson, D. R., Federman, N., Glaser, K. B., Landaw, E. M., Denny, C. T., Sakamoto, K. M. 2010; 9 (3): 653-660


The Ewing Sarcoma (EWS) family of tumors is one of the most common tumors diagnosed in children and adolescents and is characterized by a translocation involving the EWS gene. Despite advances in chemotherapy, the prognosis of metastatic EWS is poor with an overall survival of <30% after 5 years. EWS tumor cells express the receptor tyrosine kinases, platelet-derived growth factor receptor (PDGFR) and c-KIT. ABT-869 is a multitargeted small-molecule inhibitor that targets Fms-like tyrosine kinase-3, c-KIT, vascular endothelial growth receptors, and PDGFRs. To determine the potential therapeutic benefit of ABT-869 in EWS cells, we examined the effects of ABT-869 on EWS cell lines and xenograft mouse models. ABT-869 inhibited the proliferation of two EWS cell lines, A4573 and TC71, at an IC(50) of 1.25 and 2 mumol/L after 72 h of treatment, respectively. The phosphorylation of PDGFRbeta, c-KIT, and extracellular signal-regulated kinases was also inhibited. To examine the effects of ABT-869 in vivo, the drug was given to mice injected with EWS cells. We observed inhibition of growth of EWS tumor cells in a xenograft mouse model and prolonged survival in a metastatic mouse model of EWS. Therefore, our in vitro and in vivo studies show that ABT-869 inhibits proliferation of EWS cells through inhibition of PDGFRbeta and c-KIT pathways.

View details for DOI 10.1158/1535-7163.MCT-09-0812

View details for Web of Science ID 000278487300012

View details for PubMedID 20197394

View details for PubMedCentralID PMC2837519

Targeting steroid hormone receptors for ubiquitination and degradation in breast and prostate cancer ONCOGENE Rodriguez-Gonzalez, A., Cyrus, K., Salcius, M., Kim, K., Crews, C. M., Deshaies, R. J., Sakamoto, K. M. 2008; 27 (57): 7201-7211


Proteolysis targeting chimeric molecules (Protacs) target proteins for destruction by exploiting the ubiquitin-dependent proteolytic system of eukaryotic cells. We designed two Protacs that contain the peptide 'degron' from hypoxia-inducible factor-1alpha, which binds to the Von-Hippel-Lindau (VHL) E3 ubiquitin ligase complex, linked to either dihydroxytestosterone that targets the androgen receptor (AR; Protac-A), or linked to estradiol (E2) that targets the estrogen receptor-alpha (ERalpha; Protac-B). We hypothesized that these Protacs would recruit hormone receptors to the VHL E3 ligase complex, resulting in the degradation of receptors, and decreased proliferation of hormone-dependent cell lines. Treatment of estrogen-dependent breast cancer cells with Protac-B induced the degradation of ERalpha in a proteasome-dependent manner. Protac-B inhibited the proliferation of ERalpha-dependent breast cancer cells by inducing G(1) arrest, inhibition of retinoblastoma phosphorylation and decreasing expression of cyclin D1, progesterone receptors A and B. Protac-B treatment did not affect the proliferation of estrogen-independent breast cancer cells that lacked ERalpha expression. Similarly, Protac-A treatment of androgen-dependent prostate cancer cells induced G(1) arrest but did not affect cells that do not express AR. Our results suggest that Protacs specifically inhibit the proliferation of hormone-dependent breast and prostate cancer cells through degradation of the ERalpha and AR, respectively.

View details for DOI 10.1038/onc.2008.320

View details for Web of Science ID 000261384100007

View details for PubMedID 18794799

Role of the aggresorne pathway in cancer: Targeting histone deacetylase 6-dependent protein degradation CANCER RESEARCH Rodriguez-Gonzalez, A., Lin, T., Ikeda, A. K., Sinnns-Waldrip, T., Fu, C., Sakamoto, K. M. 2008; 68 (8): 2557-2560


Misfolded or aggregated proteins have two fates: they are either refolded with the help of chaperones or degraded by the proteasome. Cells also have an alternative pathway that involves intracellular "storage bins" for misfolded intracellular proteins known as aggresomes. Aggresomes recruit motor proteins that transport misfolded or aggregated proteins to chaperones and proteasomes for subsequent destruction. There is emerging evidence that inhibiting the aggresome pathway leads to accumulation of misfolded proteins and apoptosis in tumor cells through autophagy. We discuss the role of aggresomes in cancer and the potential to target this pathway for therapy.

View details for DOI 10.1158/0008-5472.CAN-07-5989

View details for Web of Science ID 000255100500002

View details for PubMedID 18413721

CREB is a critical regulator of normal hematopoiesis and leukemogenesis BLOOD Cheng, J. C., Kinjo, K., Judelson, D. R., Chang, J., Wu, W. S., Schmid, I., Shankar, D. B., Kasahara, N., Stripecke, R., Bhatia, R., Landaw, E. M., Sakamoto, K. M. 2008; 111 (3): 1182-1192


The cAMP-responsive element binding protein (CREB) is a 43-kDa nuclear transcription factor that regulates cell growth, memory, and glucose homeostasis. We showed previously that CREB is amplified in myeloid leukemia blasts and expressed at higher levels in leukemia stem cells from patients with myeloid leukemia. CREB transgenic mice develop myeloproliferative disease after 1 year, but not leukemia, suggesting that CREB contributes to but is not sufficient for leukemogenesis. Here, we show that CREB is most highly expressed in lineage negative hematopoietic stem cells (HSCs). To understand the role of CREB in hematopoietic progenitors and leukemia cells, we examined the effects of RNA interference (RNAi) to knock down CREB expression in vitro and in vivo. Transduction of primary HSCs or myeloid leukemia cells with lentiviral CREB shRNAs resulted in decreased proliferation of stem cells, cell- cycle abnormalities, and inhibition of CREB transcription. Mice that received transplants of bone marrow transduced with CREB shRNA had decreased committed progenitors compared with control mice. Mice injected with Ba/F3 cells expressing either Bcr-Abl wild-type or T315I mutation with CREB shRNA had delayed leukemic infiltration by bioluminescence imaging and prolonged median survival. Our results suggest that CREB is critical for normal myelopoiesis and leukemia cell proliferation.

View details for DOI 10.1182/blood-blood-2007-04-083600

View details for Web of Science ID 000252792900040

View details for PubMedID 17975014

View details for PubMedCentralID PMC2214769

Expression profile of CREB knockdown in myeloid leukemia cells. BMC cancer Pellegrini, M., Cheng, J. C., Voutila, J., Judelson, D., Taylor, J., Nelson, S. F., Sakamoto, K. M. 2008; 8: 264-?


The cAMP Response Element Binding Protein, CREB, is a transcription factor that regulates cell proliferation, differentiation, and survival in several model systems, including neuronal and hematopoietic cells. We demonstrated that CREB is overexpressed in acute myeloid and leukemia cells compared to normal hematopoietic stem cells. CREB knockdown inhibits leukemic cell proliferation in vitro and in vivo, but does not affect long-term hematopoietic reconstitution.To understand downstream pathways regulating CREB, we performed expression profiling with RNA from the K562 myeloid leukemia cell line transduced with CREB shRNA.By combining our expression data from CREB knockdown cells with prior ChIP data on CREB binding we were able to identify a list of putative CREB regulated genes. We performed extensive analyses on the top genes in this list as high confidence CREB targets. We found that this list is enriched for genes involved in cancer, and unexpectedly, highly enriched for histone genes. Furthermore, histone genes regulated by CREB were more likely to be specifically expressed in hematopoietic lineages. Decreased expression of specific histone genes was validated in K562, TF-1, and primary AML cells transduced with CREB shRNA.We have identified a high confidence list of CREB targets in K562 cells. These genes allow us to begin to understand the mechanisms by which CREB contributes to acute leukemia. We speculate that regulation of histone genes may play an important role by possibly altering the regulation of DNA replication during the cell cycle.

View details for DOI 10.1186/1471-2407-8-264

View details for PubMedID 18801183

View details for PubMedCentralID PMC2647550

Cancer stem cells: The root of the problem PEDIATRIC RESEARCH Lin, T. L., Fu, C., Sakamoto, K. M. 2007; 62 (3): 239-239

View details for Web of Science ID 000248973200001

View details for PubMedID 17712286

Potential role of CREB as a prognostic marker in acute myeloid leukemia. Future oncology Cheng, J. C., Esparza, S., Sandoval, S., Shankar, D., Fu, C., Sakamoto, K. M. 2007; 3 (4): 475-480


The cAMP response element binding protein (CREB) is a leucine zipper transcription factor that regulates genes responsible for cell proliferation, differentiation and survival. CREB is overexpressed in the bone marrow from most patients with acute leukemia. Overexpression of CREB occurs both at the protein and at the transcript levels and is associated with gene amplification in leukemic blast cells. Higher levels of CREB correlate with a less favorable prognosis in a small cohort of adult patients with acute myeloid leukemia. In one study, patients whose bone marrow over-expresses CREB had an increased risk of relapse and decreased event-free survival. Mice that overexpress CREB in myeloid cells develop a myeloproliferative/myelodysplastic syndrome. These findings suggest that CREB plays an important role in the pathogenesis of acute leukemia and is a potential biomarker of disease.

View details for PubMedID 17661722

PEG-asparaginase EXPERT OPINION ON PHARMACOTHERAPY Fu, C. H., Sakamoto, K. M. 2007; 8 (12): 1977-1984


L-asparaginases have been established components in the treatment of acute leukemias for nearly 40 years. Their antitumor effect results from the depletion of asparagine, an amino acid essential to leukemic cells, and subsequent inhibition of protein synthesis leading to considerable cytotoxicity. The efficacy of L-asparaginases has been limited by a high rate of hypersensitivity reactions and development of anti-asparaginase antibodies, which neutralize their activity. PEG-asparaginase, a form of Escherichia coli L-asparaginase covalently linked to polyethylene glycol, was rationally synthesized to decrease immunogenicity of the enzyme and prolong its half-life. In recent years, clinical trials have established the importance of intramuscular PEG-asparaginase in frontline pediatric and adult acute lymphoblastic leukemia therapy. Present studies are evaluating the feasibility of intravenous PEG-asparaginase administration.

View details for DOI 10.1517/14656566.8.12.1977

View details for Web of Science ID 000248937600015

View details for PubMedID 17696798

ABT-869, a multitargeted receptor tyrosine kinase inhibitor: inhibition of FLT3 phosphorylation and signaling in acute myeloid leukemia BLOOD Shankar, D. B., Li, J., Tapang, P., McCall, J. O., Pease, L. J., Dai, Y., Wei, R., Albert, D. H., Bouska, J. J., Osterling, D. J., Guo, J., Marcotte, P. A., Johnson, E. F., Soni, N., Hartandi, K., Michaelides, M. R., Davidsen, S. K., Priceman, S. J., Chang, J. C., Rhodes, K., Shah, N., Moore, T. B., Sakamoto, K. M., Glaser, K. B. 2007; 109 (8): 3400-3408


In 15% to 30% of patients with acute myeloid leukemia (AML), aberrant proliferation is a consequence of a juxtamembrane mutation in the FLT3 gene (FMS-like tyrosine kinase 3-internal tandem duplication [FLT3-ITD]), causing constitutive kinase activity. ABT-869 (a multitargeted receptor tyrosine kinase inhibitor) inhibited the phosphorylation of FLT3, STAT5, and ERK, as well as Pim-1 expression in MV-4-11 and MOLM-13 cells (IC(50) approximately 1-10 nM) harboring the FLT3-ITD. ABT-869 inhibited the proliferation of these cells (IC(50) = 4 and 6 nM, respectively) through the induction of apoptosis (increased sub-G(0)/G(1) phase, caspase activation, and PARP cleavage), whereas cells harboring wild-type (wt)-FLT3 were less sensitive. In normal human blood spiked with AML cells, ABT-869 inhibited phosphorylation of FLT3 (IC(50) approximately 100 nM), STAT5, and ERK, and decreased Pim-1 expression. In methylcellulose-based colony-forming assays, ABT-869 had no significant effect up to 1000 nM on normal hematopoietic progenitor cells, whereas in AML patient samples harboring both FLT3-ITD and wt-FLT3, ABT-869 inhibited colony formation (IC(50) = 100 and 1000 nM, respectively). ABT-869 dose-dependently inhibited MV-4-11 and MOLM-13 flank tumor growth, prevented tumor formation, regressed established MV-4-11 xenografts, and increased survival by 20 weeks in an MV-4-11 engraftment model. In tumors, ABT-869 inhibited FLT3 phosphorylation, induced apoptosis (transferase-mediated dUTP nick-end labeling [TUNEL]) and decreased proliferation (Ki67). ABT-869 is under clinical development for AML.

View details for DOI 10.1182/blood-2006-06-029579

View details for Web of Science ID 000245658500048

View details for PubMedID 17209055

View details for PubMedCentralID PMC1852258

Report on the workshop "New Technologies in Stem Cell Research," Society for Pediatric Research, San Francisco, California, April 29, 2006. Stem cells Cheng, J. C., Horwitz, E. M., Karsten, S. L., Shoemaker, L., Kornblum, H. I., Malik, P., Sakamoto, K. M. 2007; 25 (4): 1070-1088

View details for PubMedID 17255523

Molecular targets and the treatment of myeloid leukemia MOLECULAR GENETICS AND METABOLISM Ikeda, A., Shankar, D. B., Watanabe, M., Tamanoi, F., Moore, T. B., Sakamoto, K. M. 2006; 88 (3): 216-224


Leukemia is a multistep process involving accumulation of genetic alterations over time. These genetic mutations destroy the delicate balance between cell proliferation, differentiation, and apoptosis. Traditional approaches to treatment of leukemia involve chemotherapy, radiation, and bone marrow transplantation. In recent years, specific targeted therapies have been developed for the treatment of leukemia. The success of treatment of acute promyelocytic leukemia with All Trans Retinoic Acid (ATRA) and CML with imatinib have lead to increased efforts to identify targets that can be inhibited by small molecules for treatment of hematological malignancies. In this review, we describe the current advances in the development of targeted therapy in acute myeloid leukemia.

View details for DOI 10.1016/j.ymgme.2006.03.011

View details for Web of Science ID 000239615900003

View details for PubMedID 16678459

Calcium-dependent upregulation of E4BP4 expression correlates with glucocorticoid-evoked apoptosis of human leukemic CEM cells BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Priceman, S. J., Kirzner, J. D., Nary, L. J., Morris, D., Shankar, D. B., Sakamoto, K. M., Medh, R. D. 2006; 344 (2): 491-499


Glucocorticoid (GC)-evoked apoptosis of T-lymphoid cells is preceded by increases in the intracellular Ca2+ concentration ([Ca2+]i), which may contribute to apoptosis. This report demonstrates that GC-mediated upregulation of the bZIP transcriptional repressor gene, E4BP4, is dependent on [Ca2+]i levels, and correlates with GC-evoked apoptosis of GC-sensitive CEM-C7-14 cells. Calcium chelators EGTA and BAPTA reduced [Ca2+]i levels and protected CEM-C7-14 cells from Dex-evoked E4BP4 upregulation as well as apoptosis. In the GC-resistant sister clone, CEM-C1-15, Dex treatment did not induce [Ca2+]i levels, E4BP4 expression or apoptosis, however, the calcium ionophore A23187 restored Dex-evoked E4BP4 upregulation and apoptosis. CEM-C7-14 cells were more sensitive to GC-independent increases in [Ca2+]i levels by thapsigargin, and a corresponding increase in E4BP4 expression and cell death, compared to CEM-C1-15 cells, suggesting a direct correlation between [Ca2+]i levels, E4BP4 expression, and apoptosis.

View details for DOI 10.1016/j.bbrc.2006.03.169

View details for Web of Science ID 000237410000006

View details for PubMedID 16630563

View details for PubMedCentralID PMC2763529

FMS-like tyrosine kinase 3 in normal hematopoiesis and acute myeloid leukemia STEM CELLS Parcells, B. W., Ikeda, A. K., Simms-Waldrip, T., Moore, T. B., Sakamoto, K. M. 2006; 24 (5): 1174-1184


Ligand-mediated activation of the FMS-like tyrosine kinase 3 (FLT3) receptor is important for normal proliferation of primitive hematopoietic cells. However, activating mutations in FLT3 induce ligand-independent downstream signaling that promotes oncogenesis through pathways involved in proliferation, differentiation, and survival. FLT3 mutations are identified as the most frequent genetic abnormality in acute myeloid leukemia and are also observed in other leukemias. Multiple small-molecule inhibitors are under development to target aberrant FLT3 activity that confers a poor prognosis in patients.

View details for DOI 10.1634/stemcells.2005-0519

View details for Web of Science ID 000240639200005

View details for PubMedID 16410383

Role of cyclic AMP response element binding protein in human leukemias CANCER Shankar, D. B., Cheng, J. C., Sakamoto, K. M. 2005; 104 (9): 1819-1824


Acute myeloid leukemia (AML) in adults has a 20% 5-year disease-free survival despite treatment with aggressive cytotoxic chemotherapy. Previous work from our laboratory demonstrated that the majority of patients with acute lymphoid and myeloid leukemia overexpress CREB in the bone marrow. CREB overexpression is associated with poor initial outcome of clinical disease in AML patients. CREB is a transcription factor that functions in glucose homeostasis, growth-factor-dependent cell survival, and memory. Signaling by hematopoietic growth factors, such as GM-CSF, results in activation of CREB and up-regulation of CREB target genes. To study its role in hematopoiesis, we overexpressed CREB in leukemia cell lines and in mice. CREB overexpression resulted in increased survival and proliferation of myeloid cells and blast-transformation of bone marrow progenitor cells from transgenic mice expressing CREB in the myeloid lineage. CREB transgenic mice also develop myeloproliferative disease after 1 year. Thus, CREB acts as a protooncogene to regulate hematopoiesis and contributes to the leukemia phenotype. Our results suggest that CREB-dependent pathways may serve as targets for directed therapies in leukemia in the future.

View details for DOI 10.1002/cncr.21401

View details for Web of Science ID 000232755500005

View details for PubMedID 16196046

The role of CREB as a proto-oncogene in hematopoiesis CELL CYCLE Kinjo, K., Sandoval, S., Sakamoto, K. M., Shankar, D. B. 2005; 4 (9): 1134-1135


Cyclic-AMP response element binding protein (CREB) is a transcription factor that functions in glucose homeostasis, growth-factor- dependent cell survival, proliferation and memory. Signaling by hematopoietic growth factors, such as GM-CSF, results in activation of CREB and upregulation of CREB target genes. Data from our laboratory shows that a majority of patients with acute lymphoid and myeloid leukemia overexpress CREB in the bone marrow. CREB overexpression is associated with poor initial outcome of clinical disease in AML patients. To study its role in hematopoiesis, we overexpressed CREB in leukemia cell lines and in mice. CREB overexpression resulted in increased survival and proliferation of myeloid cells and blast-transformation of bone marrow progenitor cells from transgenic mice expressing CREB in the myeloid lineage. CREB transgenic mice also develop myeloproliferative disease after one year. Thus, CREB acts as a proto-oncogene to regulate hematopoiesis and contributes to the leukemia phenotype. Our results suggest that CREB-dependent pathways may serve as targets for directed therapies in leukemia in the future.

View details for Web of Science ID 000231512900002

View details for PubMedID 16096372

Chimeric molecules to target proteins for ubiquitination and degradation UBIQUITIN AND PROTEIN DEGRADATION, PT B Sakamoto, K. M. 2005; 399: 833-?


Protein degradation is one of the tactics used by the cell for irreversibly inactivating proteins. In eukaryotes, ATP-dependent protein degradation in the cytoplasm and nucleus is carried out by the 26S proteasome. Most proteins are targeted to the 26S proteasome by covalent attachment of a multiubiquitin chain. A key component of the enzyme cascade that results in attachment of the multiubiquitin chain to the target or labile protein is the ubiquitin ligase that controls the specificity of the ubiquitination reaction. Defects in ubiquitin-dependent proteolysis have been shown to result in a variety of human diseases, including cancer, neurodegenerative diseases, and metabolic disorders. The SCF (Skp1-Cullin-F-box-Hrt1) complex is a heteromeric ubiquitin ligase that multiubiquitinates proteins important for signal transduction and cell cycle progression. A technology was developed known as Protac (Proteolysis Targeting Chimeric Molecule) that acts as a bridge, bringing together the SCF ubiquitin ligase with a protein target, resulting in its ubiquitination and degradation. The Protac contains an SCF-binding peptide moiety at one end that is recognized by SCF that is chemically linked to the binding partner or ligand of the target protein. The first demonstration of the efficacy of Protac technology was the successful recruitment, ubiquitination, and degradation of the protein methionine aminopeptidase-2 (MetAP-2) through a covalent interaction between MetAP-2 and Protac. Subsequently, we demonstrated that Protacs could effectively ubiquitinate and degrade cancer-promoting proteins (estrogen and androgen receptors) through noncovalent interactions in vitro and in cells. Finally, cell-permeable Protacs can also promote the degradation of proteins in cells. This chapter includes experiments to test the ability of Protacs to target proteins in vitro and in cells.

View details for DOI 10.1016/S0076-6879(05)99054-X

View details for Web of Science ID 000233597300054

View details for PubMedID 16338398

Topics in pediatric leukemia--acute lymphoblastic leukemia. MedGenMed : Medscape general medicine Esparza, S. D., Sakamoto, K. M. 2005; 7 (1): 23-?

View details for PubMedID 16369328

View details for PubMedCentralID PMC1681386

Topics in pediatric leukemia--acute lymphoblastic leukemia and late effects in long-term survivors. MedGenMed : Medscape general medicine Casillas, J., Sakamoto, K. M. 2005; 7 (1): 22-?

View details for PubMedID 16369327

View details for PubMedCentralID PMC1681412

Topics in pediatric leukemia--myelodysplastic and myeloproliferative disorders of childhood. MedGenMed : Medscape general medicine Lasky, J., Sakamoto, K. M. 2005; 7 (1): 21-?

View details for PubMedID 16369326

View details for PubMedCentralID PMC1681411

Topics in pediatric leukemia--Fanconi's anemia: new insights. MedGenMed : Medscape general medicine Federman, N., Sakamoto, K. M. 2005; 7 (2): 23-?

View details for PubMedID 16369402

View details for PubMedCentralID PMC1681561

Knocking down human disease: Potential uses of RNA interference in research and gene therapy PEDIATRIC RESEARCH Sakamoto, K. A. 2004; 55 (6): 912-913
Comparison of bone marrow aspirates and biopsies in pediatric patients with ALL at days 7 and 14 of induction therapy LEUKEMIA & LYMPHOMA Countouriotis, A., Landaw, E. M., Naeim, F., Moore, T. B., Sakamoto, K. M. 2004; 45 (4): 745-747


The percentage of blasts in the bone marrow aspirates at day 7 or 14 of induction therapy in pediatric ALL patients is an indicator of rapid early response and an independent prognostic factor for long term outcome. Discrepancies between the percentages of blasts in bone marrow aspirates compared to biopsies have been reported. In a retrospective study on 44 consecutive patients diagnosed with ALL between 1998 and 2001, important differences were observed in the percentage of blasts between bone marrow aspirates and biopsies at days 7 and 14 of induction therapy.

View details for DOI 10.1080/10428190310001625863

View details for Web of Science ID 000187327900017

View details for PubMedID 15160950

Chemical genetic control of protein levels: Selective in vivo targeted degradation JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Schneekloth, J. S., Fonseca, F. N., Koldobskiy, M., Mandal, A., Deshaies, R., Sakamoto, K., Crews, C. M. 2004; 126 (12): 3748-3754


Genetic loss of function analysis is a powerful method for the study of protein function. However, some cell biological questions are difficult to address using traditional genetic strategies often due to the lack of appropriate genetic model systems. Here, we present a general strategy for the design and syntheses of molecules capable of inducing the degradation of selected proteins in vivo via the ubiquitin-proteasome pathway. Western blot and fluorometric analyses indicated the loss of two different targets: green fluorescent protein (GFP) fused with FK506 binding protein (FKBP12) and GFP fused with the androgen receptor (AR), after treatment with PROteolysis TArgeting Chimeric moleculeS (PROTACS) incorporating a FKBP12 ligand and dihydrotestosterone, respectively. These are the first in vivo examples of direct small molecule-induced recruitment of target proteins to the proteasome for degradation upon addition to cultured cells. Moreover, PROTAC-mediated protein degradation offers a general strategy to create "chemical knockouts," thus opening new possibilities for the control of protein function.

View details for DOI 10.1021/ja039025z

View details for Web of Science ID 000220440400034

View details for PubMedID 15038727

BUBR1 deficiency results in abnormal megakaryopoiesis BLOOD Wang, Q., Liu, T. Y., Fang, Y. Q., Xie, S. Q., Huang, X., Mahmood, R., Ramaswamy, G., Sakamoto, K. M., Darzynkiewicz, Z., Xu, M., Dai, W. 2004; 103 (4): 1278-1285


The physiologic function of BUBR1, a key component of the spindle checkpoint, was examined by generating BUBR1-mutant mice. BUBR1(-/-) embryos failed to survive beyond day 8.5 in utero as a result of extensive apoptosis. Whereas BUBR1(+/-) blastocysts grew relatively normally in vitro, BUBR1(-/-) blastocysts exhibited impaired proliferation and atrophied. Adult BUBR1(+/-) mice manifested splenomegaly and abnormal megakaryopoiesis. BUBR1 haploinsufficiency resulted in an increase in the number of splenic megakaryocytes, which was correlated with an increase in megakaryocytic, but a decrease in erythroid, progenitors in bone marrow cells. RNA interference-mediated down-regulation of BUBR1 also caused an increase in polyploidy formation in murine embryonic fibroblast cells and enhanced megakaryopoiesis in bone marrow progenitor cells. However, enhanced megakaryopoiesis in BUBR1(+/-) mice was not correlated with a significant increase in platelets in peripheral blood, which was at least partly due to a defect in the formation of proplatelet-producing megakaryocytes. Together, these results indicate that BUBR1 is essential for early embryonic development and normal hematopoiesis.

View details for DOI 10.1182/blood-2003-06-2158

View details for Web of Science ID 000188828200023

View details for PubMedID 14576056

The role of cyclic-AMP binding protein (CREB) in leukemia cell proliferation and acute leukemias LEUKEMIA & LYMPHOMA Shankar, D. B., Sakamoto, K. M. 2004; 45 (2): 265-270


Leukemia is a result of accumulating genetic alterations. The collaboration of mutations that offer survival and proliferative signals, together with mutations that result in lack of differentiation, is thought to cause a leukemic phenotype. The cyclic-AMP Response Element Binding Protein (CREB) is a transcription factor that is known to be a downstream component of the GM-CSF and IL-3 signaling pathways. We previously showed that CREB is overexpressed in blast cells from patients with acute leukemias. In this paper, we review the role of CREB in hematopoiesis, cell proliferation and acute leukemias.

View details for DOI 10.1080/1042819031000151095

View details for Web of Science ID 000186393200007

View details for PubMedID 15101710

Combined radiation and cytochrome CYP4B1/4-ipomeanol gene therapy using the EGR1 promoter ANTICANCER RESEARCH Hsu, H., Rainov, N. G., QUINONES, A., Eling, D. J., Sakamoto, K. M., Spear, M. A. 2003; 23 (3B): 2723-2728


Cytochrome p450 isozyme CYP4B1 converts the inert prodrug 4-ipomeanol (4-IM) into toxic alkylating metabolites. Induction of cytotoxicity by 4-IM combined with ionizing radiation (IR) in cells transfected with a fusion protein of rabbit cytochrome CYP4B1 under control of the radiation inducible EGR1 promoter was investigated. The capability of activated 4-IM to sensitize cells to IR was also assessed.Survival fractions of cells, determined by MTT assays, stably transfected with EGR1-CYP4B1 were compared with that of cells transfected with a control plasmid after IR followed by 4-IM. Radiosensitization was tested by comparing clonogenic survival curves of cells transfected with the CYP4B1 cassette under a CMV promoter instead of EGR-1, irradiated with or without 4-IM.MTT assays for cytotoxicity indicated a decrease in relative survival fractions (survival with 4-IM/survival without 4-IM) of the EGR1-CYP4B1 transfected cells with increasing radiation dosage, but not of control cells. Clonogenic assays revealed decreased survival fractions with increasing radiation doses (CYP4B1 transfected and control cells) and 4-IM concentrations (CYP4B1 transfected cells), but showed no significant differences in slope of survival curves with 4-IM.The results indicate IR potentiates the cytotoxic activity of the EGR1-CYP4B1/4-IM transgene system, but activated 4-IM does not sensitize cells to IR. Thus, the EGR1-CYP4B1/4-IM system is a viable radiation-gene therapy system that may allow for improved spatial and temporal control of cytotoxicity by therapeutic radiation fields.

View details for Web of Science ID 000184359200072

View details for PubMedID 12894565

The role of p55CDC in cell cycle control and mammalian cell proliferation, differentiation, and apoptosis EXPERIMENTAL AND MOLECULAR PATHOLOGY Lin, M., Chang, J. K., Shankar, D., Sakamoto, K. M. 2003; 74 (2): 123-128


The p55CDC (cell division cycle) protein is a key regulator of the cell cycle. p55CDC is related to both the CDC20 and the CDH1 proteins in yeast. p55CDC has been shown to activate the ubiquitin ligase anaphase promoting complex (APC), which is involved in degradation of proteins that control mitosis. To define the role of p55CDC during the mammalian cell cycle, we overexpressed this protein in the murine myeloid cell line 32Dcl3. 32Dcl3 cells are an ideal model system because these cells can be induced to proliferate, differentiate, or activate cellular programs leading to apoptosis. Our work suggests that p55CDC participates in cell growth, maturation, and death. Thus, p55CDC may play a more diverse role in modulating cellular functions in addition to controlling the cell cycle.

View details for DOI 10.1016/S0014-4800(02)00021-7

View details for Web of Science ID 000182695100006

View details for PubMedID 12710943

Fizzy-related RNA expression patterns in mammalian development and cell lines MOLECULAR GENETICS AND METABOLISM Mendoza, M. J., Wang, C. X., Lin, M., Braun, J., Sakamoto, K. M. 2002; 76 (4): 363-366

View details for Web of Science ID 000178018200016

View details for PubMedID 12208143

Expression of cyclic adenosine monophosphate response-element binding protein in acute leukemia BLOOD Crans-Vargas, H. N., Landaw, E. M., Bhatia, S., Sandusky, G., Moore, T. B., Sakamoto, K. M. 2002; 99 (7): 2617-2619


Cyclic adenosine monophosphate response-element binding protein (CREB) is a nuclear protein that regulates expression of genes that control cell proliferation, differentiation, and survival. To analyze CREB expression in leukemia cells, we conducted Western blot analysis of bone marrow cells obtained from patients with acute lymphoblastic leukemia, patients with acute myeloid leukemia, and patients without active leukemia. CREB was expressed at a higher frequency in bone marrow cells from patients with acute lymphoid or myeloid leukemia than in patients with leukemia remission or without leukemia. Our results indicate that CREB expression could be a useful marker for leukemia in patients with acute disease and suggest a role for CREB in leukemogenesis.

View details for Web of Science ID 000174559300051

View details for PubMedID 11895805

Bacterial peptidoglycan-induced tnf-alpha transcription is mediated through the transcription factors Egr-1, Elk-1, and NF-kappa B JOURNAL OF IMMUNOLOGY Xu, Z. J., Dziarski, R., Wang, Q. L., Swartz, K., Sakamoto, K. M., Gupta, D. 2001; 167 (12): 6975-6982


Bacteria and their ubiquitous cell wall component peptidoglycan (PGN) activate the innate immune system of the host and induce the release of inflammatory molecules. TNF-alpha is one of the highest induced cytokines in macrophages stimulated with PGN; however, the regulation of tnf-alpha expression in PGN-activated cells is poorly understood. This study was done to identify some of the transcription factors that regulate the expression of the tnf-alpha gene in macrophages stimulated with PGN. Our results demonstrated that PGN-induced expression of human tnf-alpha gene is regulated by sequences proximal to -182 bp of the promoter. Mutations within the binding sites for cAMP response element, early growth response (Egr)-1, and kappaB3 significantly reduced this induction. The transcription factor c-Jun bound the cAMP response element site, Egr-1 bound the Egr-1 motif, and NF-kappaB p50 and p65 bound to the kappaB3 site on the tnf-alpha promoter. PGN rapidly induced transcription of egr-1 gene and this induction was significantly reduced by specific mutations within the serum response element-1 domain of the egr-1 promoter. PGN also induced phosphorylation and activation of Elk-1, a member of the Ets family of transcription factors. Elk-1 and serum response factor proteins bound the serum response element-1 domain on the egr-1 promoter, and PGN-induced expression of the egr-1 was inhibited by dominant-negative Elk-1. These results indicate that PGN induces activation of the transcription factors Egr-1 and Elk-1, and that PGN-induced expression of tnf-alpha is directly mediated through the transcription factors c-Jun, Egr-1, and NF-kappaB, and indirectly through the transcription factor Elk-1.

View details for Web of Science ID 000172613400036

View details for PubMedID 11739517

p55CDC/hCDC20 is associated with BUBR1 and may be a downstream target of the spindle checkpoint kinase ONCOGENE Wu, H. Y., Lan, Z. D., Li, W. Q., WU, S. C., WEINSTEIN, J., Sakamoto, K. M., Dai, W. 2000; 19 (40): 4557-4562


Eukaryotic cells have evolved a mechanism that delays the progression of mitosis until condensed chromosomes are properly positioned on the mitotic spindle. We have been studying genes that regulated the spindle checkpoint in human cells. Enforced expression of human BUBR1, but not a BUBR1 mutant allele, enhances accumulation of mitotic cells. Yeast two-hybrid system and GST-pulldown analyses show that p55CDC/hCdc20, a protein known to link spindle checkpoint components such as MAD2 to anaphase promoting complex (APC), interacts with BUBR1. In addition, p55CDC is capable of pulling down BUBR1 in sf-9 cells infected with both p55CDC and His6-BUBR1 recombinant baculoviruses but not in the cells infected with p55CDC baculoviruses or with the baculoviral vector alone. Moreover, immunoprecipitation followed by Western blot analyses confirmed that native p55CDC is associated with BUBR1 in HeLa cells. Spindle checkpoint activation by nocodazole treatment enhances the association between p55CDC and His6-BUBR1. In nocodazole-arrested mitotic cells, both CDC16 and hyperphosphorylated CDC27, two APC components, preferentially associate with His6-BUBR1 resins, but not the control resins. Furthermore, BUBR1 phosphorylates p55CDC in vitro, and the phosphorylation of p55CDC by BUBR1 appears to be correlated with spindle checkpoint activation. Together, our studies strongly suggest that BUBR1 may target APC via p55CDC.

View details for Web of Science ID 000089438200001

View details for PubMedID 11030144

Ubiquitin-mediated proteolysis and human disease MOLECULAR GENETICS AND METABOLISM Vu, P. K., Sakamoto, K. M. 2000; 71 (1-2): 261-266

View details for Web of Science ID 000089693000031

View details for PubMedID 11001820

Granulocyte colony-stimulating factor induces egr-1 up-regulation through interaction of serum response element-binding proteins JOURNAL OF BIOLOGICAL CHEMISTRY Mora-Garcia, P., Sakamoto, K. M. 2000; 275 (29): 22418-22426


Granulocyte colony-stimulating factor (G-CSF) stimulates the proliferation and maturation of myeloid progenitor cells both in vitro and in vivo. We showed that G-CSF rapidly and transiently induces expression of egr-1 in the NFS60 myeloid cell line. Transient transfections of NFS60 cells with recombinant constructs containing various deletions of the human egr-1 promoter identified the serum response element (SRE) between nucleotides (nt) -418 and -391 as a critical G-CSF-responsive sequence. The SRE (SRE-1) contains a CArG box, the binding site for the serum response factor (SRF), which is flanked at either side by an ETS protein binding site. We demonstrated that a single copy of the wild-type SRE-1 in the minimal promoter plasmid, pTE2, is sufficient to induce transcriptional activation in response to G-CSF and that both the ETS protein binding site and the CArG box are required for maximal transcriptional activation of the pTE2-SRE-1 construct. In electromobility shift assays using NFS60 nuclear extracts, we identified SRF and the ETS protein Fli-1 as proteins that bind the SRE-1. We also demonstrated through electrophoretic mobility shift assays, using an SRE-1 probe containing a CArG mutation, that Fli-1 binds the SRE-1 independently of SRF. Our data suggest that SRE-binding proteins potentially play a role in G-CSF-induced egr-1 expression in myeloid cells.

View details for Web of Science ID 000088363800090

View details for PubMedID 10806199

Stress-induced stimulation of early growth response gene-1 by p38/stress-activated protein kinase 2 is mediated by a cAMP-responsive promoter element in a MAPKAP kinase 2-independent manner JOURNAL OF BIOLOGICAL CHEMISTRY Rolli, M., Kotlyarov, A., Sakamoto, K. M., Gaestel, M., Neininger, A. 1999; 274 (28): 19559-19564


The p38/stress-activated protein kinase2 (p38/SAPK2) is activated by cellular stress and proinflammatory cytokines. Several transcription factors have been reported to be regulated by p38/SAPK2, and this kinase is involved in the control of expression of various genes. In human Jurkat T-cells, induction of the early growth response gene-1 (egr-1) by anisomycin is completely inhibited by SB203580, a specific inhibitor of p38/SAPK2a and -b. Northern blot and reporter gene experiments indicate that this block is at the level of mRNA biosynthesis. Using mutants of the egr-1 promoter, we demonstrate that a distal cAMP-responsive element (CRE; nucleotides -134 to -126) is necessary to control egr-1 induction by p38/SAPK2. Pull-down assays indicate that phospho-CRE binding protein (CREB) and phospho-activating transcription factor-1 (ATF1) bind to this element in a p38/SAPK2-dependent manner. In response to anisomycin, two known CREB kinases downstream to p38/SAPK2, MAPKAP kinase 2 (MK2) and mitogen- and stress-activated kinase 1 (MSK1), show increased activity. However, in MK2 -/- fibroblasts derived from mice carrying a disruption of the MK2 gene, the phosphorylation of CREB and ATF1 and the expression of egr-1 reach levels comparable with wild type cells. This finding excludes MK2 as an involved enzyme. We conclude that egr-1 induction by anisomycin is mediated by p38/SAPK2 and probably by MSK1. Phosphorylated CREB and ATF1 then bind to the CRE of the egr-1 promoter and cause a stress-dependent transcriptional activation of this gene.

View details for Web of Science ID 000081377300009

View details for PubMedID 10391889

Analysis of functional elements in the human Egr-1 gene promoter RHEUMATOLOGY INTERNATIONAL Aicher, W. K., Sakamoto, K. M., Hack, A., Eibel, H. 1999; 18 (5-6): 207-214


The early growth response (Egr)-1 gene encoding a zinc-finger transcription factor is transiently induced in many different cell types upon various differentiation signals. However, in synovial fibroblasts of rheumatoid arthritis patients, Egr-1 is constitutively expressed at high levels, and several genes with Egr-1 binding sites in their promoter regions have been associated with disease progression of RA. We analyzed the control of Egr-1 transcription by characterizing those regulatory elements in the Egr-1 promoter that induce Egr-1 expression in fibroblasts. Using reporter gene assays and deletion mutants of the Egr-1 promoter we could demonstrate that Egr-1 transcription is mainly activated by a single serum response element, whereas other transcription factor binding sites, including binding sites for AP-1 or Egr-1, were found to play a minor role. Furthermore, we identified a novel regulatory element in the human Egr-1 promoter similar to a NF kappa-B binding site. Deletion of this element enhanced Egr-1 promoter activity in 3T3 but not in L929 fibroblasts. Stimulation by phorbolester induced only transient Egr-1 expression in 3T3 fibroblasts but a extended expression of Egr-1 in L929 cells. These data suggest that in fibroblasts the most proximal serum response element in the Egr-1 promoter represents the major activation site, whereas binding of the NFkB-like factor may serve as negative regulatory signal for Egr-1 transcription in fibroblasts.

View details for Web of Science ID 000081087100008

View details for PubMedID 10399797

Analysis of cell death in myeloid cells inducibly expressing the cell cycle protein p55Cdc EXPERIMENTAL HEMATOLOGY Lin, M., Mendoza, M., Kane, L., WEINSTEIN, J., Sakamoto, K. M. 1998; 26 (10): 1000-1006


p55Cdc, a cell cycle protein is expressed in cycling mammalian cells and is required for normal cell division. Expression of this protein is regulated during the cell cycle, peaking in late G1 and S. We have previously shown that constitutive expression of p55Cdc results in inhibition of granulocyte differentiation. Degradation of p55Cdc is also required for apoptosis in growth factor and serum starved cells. In the present study we prepared stably transfected cells conditionally expressing p55Cdc in response to zinc stimulation to investigate the role of inducible p55Cdc expression in apoptosis of myeloid cells. We report that inducible expression of p55Cdc in the myeloid leukemic cell line 32Dc13 resulted in increased cell death. p55Cdc overexpression led to a statistically significant decrease in the viability of 32Dc13 cells compared with that of control cells. Furthermore, cell staining and flow cytometry analysis revealed that p55Cdc-overexpressing 32Dc13 cells progressed to apoptosis much earlier than uninduced cells. These results suggest that inducible expression of p55Cdc leads to earlier increases in cell death in the absence of growth factor and serum in myeloid leukemic cells.

View details for Web of Science ID 000075653600011

View details for PubMedID 9728936

Genomic organization, 5 ' flanking enhancer region, and chromosomal assignment of the cell cycle gene, p55Cdc MOLECULAR GENETICS AND METABOLISM WEINSTEIN, J., Karim, J., Geschwind, D. H., Nelson, S. F., Krumm, J., Sakamoto, K. M. 1998; 64 (1): 52-57


p55Cdc is a mammalian homologue of a family of cell cycle proteins from widely divergent species, which contains WD repeats and has been implicated in cell cycle-regulated ubiquitin-mediated proteolysis. p55Cdc is highly expressed in proliferating but not in differentiated or growth-arrested cells. The expression, phosphorylation, and degradation of this protein have been shown to be cell cycle-regulated. We analyzed a 5.3-kb genomic region that contains the entire rat p55Cdc gene. The gene contains 10 exons ranging in size from 97 to 373 bp. The promoter region has a TAT box, four E-box consensus sequences, and potential binding sites for cell cycle-specific transcription factors. In transient transfection assays, a construct containing a 1000-nucleotide p55Cdc promoter region upstream of the chloramphenicol acetyltransferase reporter gene demonstrated a 12-fold increase in transcriptional activity. Finally, using radiation hybrid mapping techniques, we localized this gene to the human chromosome, 9q13-21.

View details for Web of Science ID 000074925700008

View details for PubMedID 9682218

Characterization of cis-acting sequences and trans-acting signals regulating early growth response 1 and c-fos promoters through the granulocyte-macrophage colony-stimulating factor receptor in BA/F3 cells BLOOD Watanabe, S., Kubota, H., Sakamoto, K. M., Arai, K. 1997; 89 (4): 1197-1206


Human granulocyte-macrophage colony-stimulating factor (hGM-CSF) activates a set of genes such as c-fos, jun, myc, and early growth response gene 1 (egr-1). Studies on BA/F3 cells that express hGM-CSF receptor (hGMR) showed that two different signaling pathways controlled by distinct regions within the beta subunit are involved in activation of c-fos/c-jun genes and in c-myc, respectively. However, the region(s) of the beta subunit responsible for activation of the egr-1 gene and other regulatory genes has not been identified. We describe here how egr-1 promoter is activated by hGMR through two regions of the beta subunit, with these regions being required for activation of the c-fos promoter. Coexpression of dominant negative (dn) Ras (N17ras) or dn JAK2 almost completely suppressed the activation of egr-1 and c-fos promoters. Deletion analysis of egr-1 promoter showed two cis-acting regions responsible for activation by hGM-CSF or mouse interleukin-3 (mIL-3), one between nucleotide positions (nt) -56 and -116, and the other between nt -235 and -480, which contains tandem repeats of the serum response element (SRE) sites. Similar experiments with the c-fos promoter showed that cis-acting regions containing the SRE/AP-1 sites is sufficient for activation by hGM-CSF. Based on these observations, we propose that signaling pathways activating egr-1 and c-fos promoters are controlled by SRE elements, either through the same or overlapping pathways that involve JAK2 and Ras.

View details for Web of Science ID A1997WG79700010

View details for PubMedID 9028942

The molecular mechanism of action of granulocyte-macrophage colony-stimulating factor JOURNAL OF INVESTIGATIVE MEDICINE Kwon, E. M., Sakamoto, K. M. 1996; 44 (8): 442-446

View details for Web of Science ID A1996VR07100005

View details for PubMedID 8952224

Regulation of egr-1 gene expression by retinoic acid in a human growth factor-dependent cell line INTERNATIONAL JOURNAL OF HEMATOLOGY Horie, M., Sakamoto, K. M., Broxmeyer, H. E. 1996; 63 (4): 303-309


Retinoic acid (RA) has profound suppressive effects on growth and survival of human growth factor-dependent cell line, M07e. Treatment of M07e cells by RA reduced expression of egr-1 gene, while the levels of c-myc gene expression remained similar. Suppression of egr-1 gene expression by RA was dosage-dependent and reached maximum at 4 h after RA addition. The decay of egr-1 mRNA was similar in M07e cells treated with or without RA. The transcriptional activity of the promoter region up to -600 or -480 bp upstream of the egr-1 gene was greatly reduced by RA treatment. These data suggest that biological effects of RA on hematopoietic cells may, in part, be mediated by transcriptional suppression of egr-1 gene through its promoter region within -480 bp.

View details for Web of Science ID A1996UQ71600005

View details for PubMedID 8762813



GM-CSF is a hematopoietic growth factor that regulates myeloid cell proliferation and maturation and enhances the function of terminally differentiated effector cells. Results of clinical trials with GM-CSF in a number of disease states suggest a potential role of this growth factor to stimulate hematopoiesis. Future use of GM-CSF will depend on further studies to optimize its therapeutic efficacy.

View details for Web of Science ID A1991FB29200043

View details for PubMedID 1999769