Kathleen M. Sakamoto, Ph.D. - US grants

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a hematopoietic growth factor which promotes the proliferation and maturation of myeloid progenitors and enhances their function. GM-CSF is effective in ameliorating chemotherapy-induced myelosuppression and enhances hematologic recovery following bone marrow transplantation. The biological activity of GM-CSF is mediated by a high-affinity receptor, which consists of an alpha and beta subunit (also shared with the IL-3 and IL-5 receptors). The precise biochemical and molecular events mediating the effects of GM-CSF are presently unknown. Our laboratory has demonstrated the rapid and transient induction of the immediate early gene, Egr-1, in both proliferating and terminally differentiated hematopoietic cells. By using Egr-1 induction as an endpoint, we have begun to identify sequences mediating GM-CSF-induced gene expression. This will allow us to work backwards to identify key steps in the GM-CSF signal transduction pathway. Recombinant constructs containing regions of the human Egr-1 promoter and chloramphenicol acetyltransferase (CAT) reporter gene were transiently transfected into the GM-CSF- or IL-3-dependent cell line, TF-1 . Preliminary results have demonstrated that GM-CSF and IL-3 work through both overlapping and distinct sequences, suggesting that their signaling pathways diverge. The aims of this proposal are to: 1) precisely identify the nucleotide sequences of the human Egr-1 promoter regulating GM-CSF-induced gene expression; 2) fractionate and characterize the nuclear factors interacting with GM-CSF-responsive sequences; and 3) determine the role of these factors in normal and neoplastic target cells. Overall, these investigations will provide new and important information on the precise mechanisms controlling proliferation of myeloid cells and will yield insights into the pathophysiology and treatment of myeloid leukemias. As a Pediatric Hematologist/Oncologist, my ultimate goal is to understand the relationship between the biology and clinical management of patients who have this potentially fatal condition. This project will allow me to pursue my interests in academic pediatric hematology/oncology and to bridge the gap between basic science research and clinical medicine.

This is a Shannon award providing partial support for the research projects that fall short of the assigned Institute's funding range but are in the margin of excellence. The Shannon award is intended to provide support to test the feasibility of the approach; develop further tests and refine research techniques; perform secondary analysis of available data sets; or conduct discrete projects that can demonstrate the PI's research capabilities or lend additional weight to an already meritorious application. The abstract below is taken from the original document submitted by the principal investigator. DESCRIPTION: Myeloid leukemias result from the dysregulation of proliferation and differentiation. Hematopoietic growth factors and their receptors play a critical role in lineage commitment and differentiation of myeloid progenitor cells. Egr-1, or early growth response gene-1, is an immediate early response gene which is required for terminal differentiation of myeloid cells. In response to differentiating agents, the induction of egr-1 is rapid, transient, and not dependent on protein synthesis. Egr-1 is transcriptionally regulated in response Granulocyte-Colony Stimulating Factor (G-CSF) or 12-0- tetradecanoylphorbol-13-acetate (TPA) in myeloid leukemic cell lines. The overall goal of this First Award proposal is to use a molecular and biochemical approach to define the transcriptional regulation of egr-1 by specific DNA-binding proteins activated by myeloid-specific differentiating agents. Myeloid cell lines will be used as a tool to study the transcriptional regulation of egr-1 by growth factors and agents which induce differentiation. The myeloid leukemic cell line, 32Dc13, differentiates but does not proliferate in the presence of G-CSF. TF-1, a human myeloid leukemic cell line, differentiates to macrophages in response to TPA. The specific aims of this proposal are to: 1) define egr-1 promoter sequences critical for signal transduction by myeloid-specific differentiating agents; 2) isolate and purify proteins that interact with egr-1 promoter sequences identified in Aim 1; and finally 3) characterize the role of these proteins in signaling pathways activated by myeloid-specific inducers of differentiation. In this manner, the signaling pathways mediating G-CSF or TPA response will be traced in reverse. First, recombinant constructs containing regions of the egr-1 promoter will be transiently transfected into 32Dc13 or TF-1 cells and stimulated with G-CSF or TPA, respectively. Critical egr-1 promoter sequences will be identified by deletion analysis and site-directed mutagenesis. Second, the proteins binding to these sequences will be characterized by Dnase I footprinting (for novel transcription factor binding sites) and simultaneously, electromobility shift assays with antibodies to proteins binding to known recognition sequences will be performed. The ultimate goal of this proposal is to determine the post-translational modification of transcription factors activated by myeloid-specific differentiating growth factors such as G-CSF.

DESCRIPTION: Myeloid leukemias result from the dysregulation of proliferation and differentiation. Hematopoietic growth factors and their receptors play a critical role in lineage commitment and differentiation of myeloid progenitor cells. Egr-1, or early growth response gene-1 is an immediate early response gene which is required for terminal differentiation of myeloid cells. In response to differentiation agents, the induction of egr-1 is rapid in response to granulocyte-colony stimulating factor (G-CSF) or 12-0 Tetradecanoylphorbol-13-acetate (TPA) and myeloid leukemic cell lines. The overall goal of this FIRST Award proposal is to use a molecular and biochemical approach to define the transcriptional regulation of egr-1 by a specific DNA-binding proteins activated by myeloid-specific differentiating agents. The myeloid cell lines will be used as a tool to study the transcriptional regulation of egr-1 by growth factors and agents which induce differentiation. The myeloid leukemic cell lines, 32Dc13, differentiatesinto granulocyte but does not proliferate in the presence of G-CSF. TF-1, a human myeloid leukemic cell line, differentiates to macrophages in response to TPA.

DESCRIPTION (provided by applicant): Prostate cancer is the most common type of cancer diagnosed in men and the second most common cause of death from cancer. Initial therapy for patients with androgen-dependent prostate cancer includes hormonal therapy to inhibit the biological effects of androgen receptor (AR) signaling. Recent evidence suggests that the AR is critical for tumor growth in androgen-refractory prostate cancer cells. The ubiquitin proteasome pathway is the primary pathway for protein turnover in all eukaryotic cells that involves the assembly of an ubiquitin chain on a substrate, which then targets the multi-ubiquitinated protein for degradation by the 26S proteasome. A protein complex known as, the SCF (Skp1, Cullin, F-box, and Hrtl/Rbx) ubiquitin ligase specifically targets proteins for ubiquitination and subsequent degradation. We previously demonstrated that a bridging molecule or Protac (Proteolysis Targeting Chimeric molecule) promotes in vitro ubiquitination of a stable protein, Methionine Aminopeptidase-2, and an unstable protein, the estrogen receptor, by SCFbeta-TRCP. We have synthesized a Protac (Protac-3) consisting of the ligand of AR, dihydroxytestosterone (DHT), and the kappa(Balpha-phosphopeptide, which binds SCFbeta-TRCP. Our goal is to develop a new technology in which a Protac links the AR to SCFbeta-TRCP, resulting in ubiquitination and degradation of AR. The specific aims of this proposal are to: 1) Test the hypothesis that a phosphopeptide-DHT chimera (Protac-3) will link AR to SCFbeta-TRCP, and direct the ubiquitination and degradation of AR in vitro; 2) Test the hypothesis that a phosphopeptide-DHT chimera (Protac-3) will link AR to SCFbeta-TRCP, and increase the ubiquitination and degradation of AR in cells; and 3) Test the hypothesis that small non-peptidic ligands that bind beta-TRCP can be used to generate testosterone-based Protacs that increase AR ubiquitination and degradation. We hypothesize that AR can be targeted to SCFbeta-TRCP resulting in increased turnover of AR in prostate cancer cells. Development of this technology will lead to novel approaches to treat androgen dependent and -independent disease in hopes of improving the survival of men with prostate cancer.

DESCRIPTION (provided by applicant): Leukemia is the most common form of childhood cancer. Children with acute myeloid leukemia (AML) have less than 50% overall survival despite aggressive chemotherapy and bone marrow transplantation. Therefore, it is critical to understand the molecular pathogenesis of AML. We demonstrated that CREB is overexpressed in bone marrow cells from patients with AML but not in normal bone marrow or bone marrow from patients without active leukemia. Furthermore, CREB overexpression was associated with an increased risk of relapse and decreased event-free survival in patients with AML. Our preliminary results suggest that AML is a heterogeneous disease that is not well understood. We hypothesize that there is an uncoupling of differentiation and CREB expression in myeloid leukemia cells. We propose to study the role of CREB in normal and malignant myeloid cells to identify novel mechanisms of leukemogenesis and improve our understanding of the molecular pathways regulating myeloid cell proliferation and differentiation. In Specific Aim 1, we will characterize CREB expression and activation in primary normal myeloid cells and myeloid leukemia cells. Experiments are proposed to determine the expression of CREB in normal mouse embryos at different stages of hematopoietic development. We will also examine CREB expression in normal myeloid progenitor cells at different stages of myeloid differentiation. Finally, we will examine whether CREB is activated in primary leukemia cells. In Specific Aim 2, we will further characterize the biological phenotype of CREB overexpression and down regulation in myeloid leukemia cell lines and primary normal myeloid cells. Our preliminary results demonstrated that CREB overexpression leads to increased proliferation and survival of myeloid leukemia cells. CREB down regulation using RNA interference (RNAi) suppresses the growth and survival of leukemia cells. To study signaling pathways upstream of CREB, we will overexpress activated kinases and use RNAi technology to inhibit expression of kinases. In Specific Aim 3, we will characterize the phenotype of transgenic mice in which CREB overexpression is targeted to myeloid cells. Defects in hematopoiesis and development of leukemia will be determined in both CREB transgenic mice and a mouse bone marrow transplant model. These studies will define the role of CREB in both normal and malignant myelopoiesis.

DESCRIPTION (provided by applicant): Myeloproliferative disease (MPD) represents abnormal proliferation of myeloid cells in the blood and bone marrow, and is considered to be a "stem cell disorder." Although the pathologic classification of MPD has been recently defined, the molecular pathogenesis of disease is not well understood. Most individuals diagnosed with MPD succumb to their disease from either complications of progressive cytopenias (bleeding or infections) or transformation to acute leukemia. Therefore, it is imperative that we identify alternative approaches to treat MPD. We generated CREB transgenic mice in which the cAMP Response Element Binding protein (CREB) is expressed in the myeloid lineage. These mice develop monocytosis, splenomegaly, and MPD. In bone marrow colony assays, we observed increased proliferation, growth factor independence, and blast transformation with tertiary replating of bone marrow from CREB transgenic mice. Bone marrow transplantation with CREB transgenic mouse bone marrow result in enhanced myeloid engraftment and increased numbers of stem cells. CREB is overexpressed in hematopoietic cells from patients with MPD. In this proposal, we hypothesize that the CREB transgenic mouse is a model for human MPD and that CREB plays a role in regulating stem cell self-renewal and possibly transformation to acute leukemia. To test these hypotheses, we will: 1) characterize the MPD phenotype in hMRP8-CREB transgenic mice; 2) characterize the biological and cellular effects of CREB overexpression in hematopoietic stem cells; and 3) characterize the cooperation of CREB with other oncogenes in MPD. In Specific Aim 1, we will study the time course, immunophenotype, colony formation, and development of MPD in CREB transgenic mice over time. We will also correlate our findings with human disease. In Specific Aim 2, we will investigate the phenotype of CREB overexpression in primary stem cells. We will transduce bone marrow progenitor cells with CREB retrovirus or lentivirus at different stages of differentiation and examine the effects of CREB on stem cell proliferation and differentiation in vitro and in vivo. Since 30% of patients with MPD have ras mutations, we will transduce bone marrow from K-rasG12D mice with CREB retrovirus and examine colony formation, immunophenotype, engraftment, and potential transformation to AML. These studies will provide new insights into the molecular pathways leading to MPD.

DESCRIPTION (provided by applicant): Prostate cancer is the most common type of cancer diagnosed in men and the second most common cause of death from cancer. Initial therapy for patients with androgen-dependent prostate cancer includes hormonal therapy to inhibit the biological effects of androgen receptor (AR) signaling. Recent evidence suggests that the AR is critical for tumor growth in androgen-refractory prostate cancer cells. The ubiquitin proteasome pathway is the primary pathway for protein turnover in all eukaryotic cells that involves the assembly of an ubiquitin chain on a substrate, which then targets the multi-ubiquitinated protein for degradation by the 26S proteasome. A protein complex known as, the SCF (Skp1, Cullin, F-box, and Hrtl/Rbx) ubiquitin ligase specifically targets proteins for ubiquitination and subsequent degradation. We previously demonstrated that a bridging molecule or Protac (Proteolysis Targeting Chimeric molecule) promotes in vitro ubiquitination of a stable protein, Methionine Aminopeptidase-2, and an unstable protein, the estrogen receptor, by SCFbeta-TRCP. We have synthesized a Protac (Protac-3) consisting of the ligand of AR, dihydroxytestosterone (DHT), and the kappa(Balpha-phosphopeptide, which binds SCFbeta-TRCP. Our goal is to develop a new technology in which a Protac links the AR to SCFbeta-TRCP, resulting in ubiquitination and degradation of AR. The specific aims of this proposal are to: 1) Test the hypothesis that a phosphopeptide-DHT chimera (Protac-3) will link AR to SCFbeta-TRCP, and direct the ubiquitination and degradation of AR in vitro; 2) Test the hypothesis that a phosphopeptide-DHT chimera (Protac-3) will link AR to SCFbeta-TRCP, and increase the ubiquitination and degradation of AR in cells; and 3) Test the hypothesis that small non-peptidic ligands that bind beta-TRCP can be used to generate testosterone-based Protacs that increase AR ubiquitination and degradation. We hypothesize that AR can be targeted to SCFbeta-TRCP resulting in increased turnover of AR in prostate cancer cells. Development of this technology will lead to novel approaches to treat androgen dependent and -independent disease in hopes of improving the survival of men with prostate cancer.

Myeloproliferative disease (MPD) represents abnormal proliferation of myeloid cells in the blood and bone marrow, and is considered to be a "stem cell disorder." Although the pathologic classification of MPD has been recently defined, the molecular pathogenesis of disease is not well understood. Most individuals diagnosed with MPD succumb to their disease from either complications of progressive cytopenias (bleeding or infections) or transformation to acute leukemia. Therefore , it is imperative that we identify alternative approaches to treat MPD. We generated CREB transgenic mice in which the cAMP Response Element Binding protein (CREB) is expressed in the myeloid lineage. These mice develop monocytosis, splenomegaly, and MPD. In bone marrow colony assays, we observed increased proliferation, growth factor independence, and blast transformation with tertiary replating of bone marrow from CREB transgenic mice. Bone marrow transplantation with CREB transgenic mouse bone marrow result in enhanced myeloid engraftment and increased numbers of stem cells. CREB is overexpressed in hematopoietic cells from patients with MPD. In this proposal, we hypothesize that the CREB transgenic mouse is a model for human MPD and that CREB plays a role in regulating stem cell self-renewal and possibly transformation to acute leukemia. To test these hypotheses, we will: 1) characterize the MPD phenotype in hMRP8-CREB transgenic mice; 2) characterize the biological and cellular effects of CREB overexpression in hematopoietic stem cells; and 3) characterize the cooperation of CREB with other oncogenes in MPD. In Specific Aim 1, we will study the time course, immunophenotype, colony formation, and development of MPD in CREB transgenic mice over time. We will also correlate our findings with human disease. In Specific Aim 2, we will investigate the phenotype of CREB overexpression in primary stem cells. We will transduce bone marrow progenitor cells with CREB retrovirus or lentivirus at different stages of differentiation and examine the effects of CREB on stem cell proliferation and differentiation in vitro and in vivo. Since 30% of patients with MPD have ras mutations, we will transduce bone marrow from K-rasG12D mice with CREB retrovirus and examine colony formation, immunophenotype, engraftment, and potential transformation to AML. These studies will provide new insights into the molecular pathways leading to MPD.

DESCRIPTION (provided by applicant): Myeloproliferative disease (MPD) represents abnormal proliferation of myeloid cells in the blood and bone marrow, and is considered to be a "stem cell disorder." Although the pathologic classification of MPD has been recently defined, the molecular pathogenesis of disease is not well understood. Most individuals diagnosed with MPD succumb to their disease from either complications of progressive cytopenias (bleeding or infections) or transformation to acute leukemia. Therefore, it is imperative that we identify alternative approaches to treat MPD. We generated CREB transgenic mice in which the cAMP Response Element Binding protein (CREB) is expressed in the myeloid lineage. These mice develop monocytosis, splenomegaly, and MPD. In bone marrow colony assays, we observed increased proliferation, growth factor independence, and blast transformation with tertiary replating of bone marrow from CREB transgenic mice. Bone marrow transplantation with CREB transgenic mouse bone marrow result in enhanced myeloid engraftment and increased numbers of stem cells. CREB is overexpressed in hematopoietic cells from patients with MPD. In this proposal, we hypothesize that the CREB transgenic mouse is a model for human MPD and that CREB plays a role in regulating stem cell self-renewal and possibly transformation to acute leukemia. To test these hypotheses, we will: 1) characterize the MPD phenotype in hMRP8-CREB transgenic mice; 2) characterize the biological and cellular effects of CREB overexpression in hematopoietic stem cells; and 3) characterize the cooperation of CREB with other oncogenes in MPD. In Specific Aim 1, we will study the time course, immunophenotype, colony formation, and development of MPD in CREB transgenic mice over time. We will also correlate our findings with human disease. In Specific Aim 2, we will investigate the phenotype of CREB overexpression in primary stem cells. We will transduce bone marrow progenitor cells with CREB retrovirus or lentivirus at different stages of differentiation and examine the effects of CREB on stem cell proliferation and differentiation in vitro and in vivo. Since 30% of patients with MPD have ras mutations, we will transduce bone marrow from K-rasG12D mice with CREB retrovirus and examine colony formation, immunophenotype, engraftment, and potential transformation to AML. These studies will provide new insights into the molecular pathways leading to MPD.

DESCRIPTION (provided by applicant): Studies in pediatric blood disorders provide an important paradigm for understanding the fundamental mechanisms regulating hematopoiesis. Unlike adult hematologic disorders, many pediatric diseases result from intrinsic genetic defects that control blood cell development or function. Very little is understood about the molecular pathogenesis of most of the hematologic diseases in children. At UCLA, we have a tremendous resource of interdisciplinary investigators whose research focuses on a wide spectrum of topics related to developmental hematology. Therefore, we propose a unique training program that focuses specifically on pediatric hematologic diseases by integrating a variety of disciplines, including molecular and cellular hematopoiesis, alternative organism models, stem cell transplantation, hemostasis and thrombosis, transfusion medicine, novel technologies (RNA interference, proteomics, genomics), hematologic malignancies, therapeutics, and mathematical modeling. Given the declining number of physician-scientists in Pediatric Hematology, a training program in Developmental Hematology would be critical to advance the field. In this application, we seek funding for 4 postdoctoral fellows per year for 5 years. The fellows will be either MDs, MD/PhDs, or PhDs. Trainees will have the opportunity to develop research in one of the 22 faculty member's laboratories. Trainees will be selected from a large pool of postdoctoral fellows (approximately 80 per year) based on their academic and research achievements. Appointments will be for two years, the second year appointment being dependent on a progress report. Trainees will participate in journal clubs, lectures related to pediatric hematology, and seminars. They will also take a required course on Developmental Hematology through the Department of Pathology. All trainees will be expected to meet with a Scholarship Oversight Committee every 6 months to monitor progress and productivity. They will present their work at national meetings. Many investigators have pre-existing collaborations and publications. We hope to utilize the strengths of UCLA, including the breadth and depth of investigators across many disciplines, to train future researchers and leaders in the field of Pediatric Hematology. (End of Abstract)

DESCRIPTION (provided by applicant): Diamond Blackfan Anemia (DBA) is a heterogeneous disease in which patients present with pure red cell aplasia, congenital abnormalities, and an increased risk of cancer. Mutations in ribosomal protein subunits (RPS) 19 and 11 have been described. We propose to characterize the molecular pathways resulting from deficiencies of RPS19 and RPL11 in the zebrafish and in mouse and human hematopoietic model systems to understand the pathogenesis of DBA. Based on our preliminary data, we will study how decreased levels of RPS19 and RPL11 alter the p53 network of proteins and the regulation of p53 through Mdm2 and other regulators. We hypothesize that p53 is differentially regulated during development. Our results and published work by others suggest that p53 is primarily regulated transcriptionally during early development, but post-translationally in more mature or adult cells. In Specific Aim 1, we will develop different zebrafish models with RPS19 or RPL11 insufficiency to characterize the role of p53 related proteins in DBA and malignant transformation. In Specific Aim 2, we will study p53 signaling pathways in human and mouse primary hematopoietic cells with RPS19 and RPL11 deficiency in vitro and in vivo. The role of inhibin as a downstream target of RPS19 insufficiency will also be investigated. In Specific Aim 3, we will evaluate known compounds regulating p53 activity in RPS19 and RPL11 knockdown hematopoietic cells as possible therapeutic approaches to treat DBA. We also propose to use zebrafish embryos to identify FDA approved drugs that rescue the RPL11 phenotype. In this manner, the signaling pathways will be linked to developmental anomalies in zebrafish and defects in hematopoiesis. Our studies will provide novel insights into the molecular pathogenesis of DBA and lead to new avenues for treatment of DBA patients.

DESCRIPTION (provided by applicant): Women and minorities are underrepresented among the leadership in pediatrics and pediatric subspecialties, including Hematology/Oncology. The American Society of Pediatric Hematology/Oncology (ASPHO) is a multidisciplinary organization of members who study and treat childhood cancer and blood disorders. A recent survey of ASPHO members demonstrated that although 44% of the members were women, fewer than 35% of committee and board members are female. Furthermore, according to the survey, 26% of members were classified as non-White/Caucasian. However, less than 10% of the members of the ASPHO leadership are non-White/Caucasian. These data suggest that minorities are underrepresented in Pediatric Hematology/Oncology, but even more, in leadership positions within the society. In 2009, we performed a needs assessment with a second survey of the society's members. The results of the survey identified significant differences in job satisfaction stress levels, and career advancement among women and minorities compared to male and non-minority members. In this R13 application, we are requesting one year of funding to support a workshop that will address specific barriers to Networking identified in the survey so that women and underrepresented minorities will become successful leaders in the field. We hypothesize that greater awareness of issues related to networking and practical solutions suggested by experts and ASPHO members will increase diversity among the leadership of ASPHO. We also hypothesize that if we can increase women and underrepresented minorities in positions of leadership, this will promote diversity within the workforce for the next generatio of Pediatric Hematologist/Oncologists. The overall objectives of this workshop are to: 1) Provide a national forum for trainees and faculty to address issues related to Networking for women and minorities in the field of Pediatric Hematology- Oncology. 2) Identify barriers and practical solutions to enhance Networking for women and minorities in the field of Pediatric Hematology-Oncology. 3) Invite outside speakers and experts to provide an opportunity to share their Networking experiences in their respective positions of leadership in Pediatrics or Pediatric Hematology-Oncology. 4) Provide opportunities for mentorship to facilitate Networking between trainees or junior faculty and experienced faculty and leaders in the field. 5) Disseminate information about Networking obtained from the workshop to other Pediatric subspecialties both nationally and internationally through manuscripts, newsletters, and websites.

DESCRIPTION (provided by applicant): Acute Myeloid Leukemia (AML) is a heterogeneous malignancy that arises from the bone marrow, resulting in less than 50% of patients with AML being cured. The molecular mechanisms of AML are not well understood. The specific aims of my previous R01 grant addressed the overall hypothesis that the cAMP Response Element Binding Protein (CREB) contributes to leukemogenesis. In the initial funding period, we described the overexpression of CREB in AML. We successfully completed the three specific aims and demonstrated that 1) CREB is expressed in less differentiated normal hematopoietic stem and progenitor cells; 2) CREB overexpression is associated with an increased risk of relapse and decreased event-free survival; 3) overexpression of CREB in myeloid cells results in increased proliferation and survival in vitro; 4) hMRP8-CREB transgenic mice develop myeloproliferative disease; and 5) CREB knockdown in vitro and in vivo inhibits leukemic progression but not long-term hematopoietic stem cell engraftment. In this revised competitive renewal application, we propose to examine the molecular pathways to understand the mechanism of CREB function in normal and aberrant hematopoiesis. We propose four specific aims. In Aim 1, we will study the role of CREB on hematopoietic dynamics by transplanting bone marrow from CREB transgenic mice into lethally irradiated mice and assessing molecular changes in the myeloid compartment. Our collaborators and I found that the microRNA, miR-34b, recognizes a sequence in the 3' UTR of the CREB gene. In Aim 2, we will characterize the regulation of CREB by the microRNA, miR-34b. Our preliminary data showed that Meis1 and Pbx1 are upregulated 33- and 28-fold by expression profiling. Meis1 and Pbx1 are direct targets of CREB. Thus, Aim 3 describes experiments to study the requirement of Meis1 and Pbx1 downstream of CREB. Finally, we identified sox4 and gfi1 in a retroviral insertional mutagenesis screen in which CREB transgenic mice were infected with the MOL4070LTR retrovirus. CREB transgenic mice infected with this retrovirus developed AML with a shortened latency compared to control mice. In Aim 4, we will examine the role of sox4 and gfi1 as genes that cooperate with CREB and contribute to the development of AML. These studies will provide new insights into how CREB regulates normal hematopoiesis and contributes to leukemogenesis.

DESCRIPTION (provided by applicant): As faculty at academic institutions are living longer and remaining productive, many are facing challenges of transitioning in the later stages of their career. Professors are being forced to retire and have little guidance or mentorship as to their options. Challenges for senior faculty are unique since preparation requires consideration of both personal and academic aspects of the transition to new roles that can include but are not limited to retirement. The American Society of Pediatric Hematology/Oncology (ASPHO) is a multidisciplinary organization of members who study and treat childhood cancer and blood disorders. In 2008, a workshop for mid- and late career members was conducted at the annual meeting for the American Society of Pediatric Hematology/Oncology that functioned as a systematic needs assessment for these stages of professional development. The results of this workshop, which included a survey component, identified a set of important topics, including work-life balance, transition and succession, management and finances, and keeping up to date with the field. In this R13 application, we are requesting funds to support a workshop that will be open to all ASPHO members and addresses concerns about late career transition that were raised in the previous 2008 workshop on mid- to late-career transitions. The funds will be used to pay for travel expenses, meeting registration, and hotel costs of the invited speakers who would not otherwise attend the ASPHO meeting. We hypothesize that greater awareness of these issues and practical solutions suggested by experts will provide important mentoring and advice to the other members of ASPHO on 1) how to best prepare for late career transitions and 2) what factors to take into consideration when making these critical life and career decisions. We also hypothesize that Pediatric Hematologist/Oncologists can lead the way and pioneer novel approaches to help late career stage professionals in other fields of academic medicine.

DESCRIPTION (provided by applicant): Studies in pediatric blood disorders provide an important paradigm for understanding the fundamental mechanisms regulating hematopoiesis and stem cell biology. Unlike adult hematologic disorders, many pediatric diseases result from intrinsic genetic defects that control blood cell development or function. Very little is understod about the molecular pathogenesis of many of the hematologic diseases in children. We plan to develop an innovative, multidisciplinary training program in pediatric nonmalignant hematology and stem cell biology with investigators who have unique expertise and can foster team science. Therefore, we propose a training program that integrates a variety of disciplines and expertise, including molecular and cellular hematopoiesis, alternative organism models, stem cell transplantation, novel technologies in proteomics/genomics, and bioinformatics. Currently, there is no program at Stanford University that supports the training of MD, MD/PhD, or PhD fellows to study Pediatric Nonmalignant Hematology and Stem Cell Biology. Given the declining number of translational and basic researchers in this area, a training program in nonmalignant hematology and stem cell biology will be critical to advance the field. In this application, we seek funding for two MD, MD/PhD, or PhD postdoctoral fellows per year for two years. Trainees will have the opportunity to develop research in one of the 23-faculty member's laboratories. Co-mentors and team science will be highly encouraged. Trainees will be selected from a pool of approximately 80 internal postdoctoral fellow candidates in addition to external candidates based on their academic potential, career goals, and research achievements. The second year of funding will depend on progress. Trainees will be required to participate in multidisciplinary journal clubs, hematology and stem cell biology lectures, clinic conferences, and joint research seminars. Trainees will also take a required course on Pediatric Nonmalignant Hematology and Stem Cell Biology (Pathology 290) with lectures given by the faculty mentors in the training program. Pediatric Hematology/Oncology MD or MD/PhD fellows will meet with their Scholarship Oversight Committees every 6 months to monitor progress and productivity. Trainees will present their work at national meetings, such as ASH, Keystone, and FASEB meetings. We hope to utilize the strengths of Stanford University to develop research collaborations in pediatric nonmalignant hematology and stem cell biology, including the breadth and depth of investigators across many disciplines, to train future leaders in the field.

Project Summary/Abstract The role of Ribosomal Protein Subunit 14 (RPS14) deficiency in the pathogenesis of del(5q) Myelodysplastic Syndromes (MDS) is not well understood. Despite treatment of MDS patients with lenalidomide, 50% of patients will not respond and these patients have an increased risk of acute myeloid leukemia (AML). Patients with anemia may require chronic red cell transfusions resulting in impaired quality of life. Haploinsufficiency of RPS14 is responsible for the anemia phenotype in del(5q) MDS. Therefore, it is critical to understand the mechanisms underlying the defects in erythropoiesis associated with RPS14 deficiency in del(5q) MDS and develop new therapies to treat this disease. To study the molecular pathways downstream of ribosomal protein insufficiency and bone marrow failure, we performed RNA-seq with RPS19 deficient human CD34+ hematopoietic stem and progenitor cells to model Diamond Blackfan Anemia, and found genes that were aberrantly regulated in both RPS19 and RPS14-deficient hematopoietic progenitor cells compared to normal cells. Several of these genes were cytokines and chemokines that regulate inflammatory pathways. The goal of this research is to further define the signaling pathways that contribute to the pathogenesis of RPS14 deficiency in del(5q) MDS and test immune modulatory and anti-inflammatory drugs to rescue the anemia using both human and zebrafish models. We propose three specific aims. In Aim 1, we will characterize signaling pathways regulating erythropoiesis in RPS14- deficient human MDS models. In Aim 2, we will characterize signaling pathways regulating erythropoiesis in RPS14-deficient zebrafish. In Aim 3, we will identify and test known compounds to develop potentially novel therapies to treat erythroid defects in del(5q) MDS. Our studies will increase our understanding of MDS and lead to potentially new approaches to treat del(5q) MDS.

Project Summary/Abstract Very little is understood about the molecular pathogenesis of many hematologic diseases in children. We have developed an innovative, multidisciplinary training program in pediatric nonmalignant hematology and stem cell biology with investigators who have unique expertise and can foster multidisciplinary research. Currently, there is no other program at Stanford University that supports the training of MD, MD/PhD, or PhD fellows to study Pediatric Nonmalignant Hematology and Stem Cell Biology. Given the declining number of translational and basic scientists in this area of research, our training program is critical to continue advancements in the field and maintain a pipeline of researchers focusing on pediatric nonmalignant hematology and stem cell biology. We propose a training program that integrates a variety of disciplines and expertise, including molecular and cellular hematopoiesis, alternative organism models, stem cell transplantation, proteomics/genomics, translational research, and bioinformatics. In this revised renewal application, we have added new faculty with expertise in novel, cutting edge technologies; new aspects of the program in translational research in collaboration with experts in Silicon Valley including those in pharmaceutical and biotechnology industries; an expert in bioinformatics and a new bioinformatic course for trainees, and new junior faculty who will be mentored to become T32 faculty mentors in the future. In this application, we seek funding for two MD, MD/PhD, or PhD postdoctoral fellows per year for a maximum of two years. Trainees will have the opportunity to develop research in one of the 22 established faculty mentor?s laboratories. Trainees will be selected from a pool of approximately 54 eligible internal postdoctoral fellow candidates in addition to external candidates based on their academic potential, career goals, and research achievements. The second year of funding will depend on their progress reports. Trainees will participate in journal clubs, hematology and stem cell biology lectures, clinic conferences, and research seminars. Career development workshops for trainees include Grant Writing, Professionalism, and Leadership. Trainees will also be required to take a course on Pediatric Nonmalignant Hematology and Stem Cell Biology and Ethics and Scientific Integrity. Trainees will meet with the Program Directors and Scholarship Oversight Committees every 6 months to monitor progress and will present their work at national and international meetings. The External Advisory Committee will review the program and meet with trainees every year. We will utilize the strengths of Stanford University to develop research collaborations in Pediatric Nonmalignant Hematology and Stem Cell Biology, including the breadth and depth of investigators across many disciplines, to train future leaders in the field.