Rebecca J. Chan, MD/PhD - US grants

DESCRIPTION Understanding the dynamic interaction between kinases and phosphates is a critical biochemical mechanism in the control of cytoplasmic signaling that mediates blood cell development. Much of the information passed from cell surface receptor-ligand interaction to the nucleus is transmitted via phosphorylated proteins. We wish to understand the molecular basis and signal transduction pathways responsible for the commitment and differentiation of hematopoietic stem/progenitor cells. We intend to address this question by investigating the role of Shp-2, a protein tyrosine phosphatase, in hematopoiesis. We will determine the role of Shp-2 in mediating the development of a common precursor for hematopoietic and endothelial cells. We will also determine the significance of the ras-Erk kinase pathway in mediating Shp-2 control of hematopoiesis Our ultimate goal is that the findings from these studies will allow for the development of improved and less toxic therapies for human leukemias.

Myeloproliferative disorder (MPD) is a heterogeneous group of hematologic diseases which share the common characteristic of myeloid cell overproduction. We have been examining the role of activating mutations of PTPN11, which encodes the protein tyrosine phosphatase, Shp-2, and of c-kit, which encodes the receptor protein tyrosine kinase for stem cell factor (SCF), in juvenile myelomonocytic leukemia and systemic mastocytosis, respectively. GM-CSF signaling via Ras hyperactivation is central to the pathogenesis of JMML; however, we have preliminary studies demonstrating correction of myeloid progenitor GM-CSF hypersensitivity induced by activating PTPN11 mutations by the lipid kinase phosphoinsositol-3-kinase (PI3K) inhibitor, LY294002; therefore, we hypothesize hyperactivation of PI3K activity also contributes to the pathogenesis of JMML. Additionally, in a model of systemic mastocytosis, we have evidence demonstrating that genetic disruption of p85a, a regulatory subunit of class IA PI3K, abrogates mast cell proliferation induced by activating c-kit mutations, leading us to hypothesize that the enhanced proliferation, survival, and migration of mast cells expressing activating c-kit mutations is mediated in part via hyperactivation of PI3K. Therefore, the central hypothesis of this application, formulated on the basis of our preliminary data, is that hyperactivation of class IA PI3K induced by activating PTPN11 and c-kit mutations contributes to the etiology of JMML and systemic mastocytosis, respectively. To examine this hypothesis, we will transduce murine hematopoietic cells lacking expression of the regulatory subunit of PI3K, p85alpha, with activating PTPN11 mutants to conduct in vitro and in vivo hematopoietic progenitor, survival, and proliferation assays as well as biochemical analysis in response to GMCSF stimulation and will utilize a genetic and a biochemical approach involving a direct comparison of the mast cells deficient in p85a or engineered to retrovirally express the activating c-Kit (D814V) mutation to look for modulation of growth, survival and activation of downstream signaling pathways in vitro and MPD in vivo. To define additional potential therapeutic targets in JMML and systemic mastocytosis, we will map the proteome and the phosphoproteome of murine mast cells and stem/progenitor cells expressing the activating mutations of c-Kit (c-Kit D814V) and PTPN11, respectively. Collectively, this combined approach of genetic, biochemical, and proteomic experiments will identify a full range of functions that are controlled by Shp-2 and c-kit via p85 subunits of class IA PI3K and will provide novel targets for molecular therapies in the treatment of JMML and systemic mastocytosis, both of which currently have no good treatment options.

Myeloproliferative disorder (MPD) is a heterogeneous group of hematologic diseases which share the common characteristic of myeloid cell overproduction. We have been examining the role of activating mutations of PTPN11, which encodes the protein tyrosine phosphatase, Shp-2, and of c-kit, which encodes the receptor protein tyrosine kinase for stem cell factor (SCF), in juvenile myelomonocytic leukemia and systemic mastocytosis, respectively. GM-CSF signaling via Ras hyperactivation is central to the pathogenesis of JMML; however, we have preliminary studies demonstrating correction of myeloid progenitor GM-CSF hypersensitivity induced by activating PTPN11 mutations by the lipid kinase phosphoinsositol-3-kinase (PI3K) inhibitor, LY294002; therefore, we hypothesize hyperactivation of PI3K activity also contributes to the pathogenesis of JMML. Additionally, in a model of systemic mastocytosis, we have evidence demonstrating that genetic disruption of p85a, a regulatory subunit of class IA PI3K, abrogates mast cell proliferation induced by activating c-kit mutations, leading us to hypothesize that the enhanced proliferation, survival, and migration of mast cells expressing activating c-kit mutations is mediated in part via hyperactivation of PI3K. Therefore, the central hypothesis of this application, formulated on the basis of our preliminary data, is that hyperactivation of class IA PI3K induced by activating PTPN11 and c-kit mutations contributes to the etiology of JMML and systemic mastocytosis, respectively. To examine this hypothesis, we will transduce murine hematopoietic cells lacking expression of the regulatory subunit of PI3K, p85alpha, with activating PTPN11 mutants to conduct in vitro and in vivo hematopoietic progenitor, survival, and proliferation assays as well as biochemical analysis in response to GMCSF stimulation and will utilize a genetic and a biochemical approach involving a direct comparison of the mast cells deficient in p85a or engineered to retrovirally express the activating c-Kit (D814V) mutation to look for modulation of growth, survival and activation of downstream signaling pathways in vitro and MPD in vivo. To define additional potential therapeutic targets in JMML and systemic mastocytosis, we will map the proteome and the phosphoproteome of murine mast cells and stem/progenitor cells expressing the activating mutations of c-Kit (c-Kit D814V) and PTPN11, respectively. Collectively, this combined approach of genetic, biochemical, and proteomic experiments will identify a full range of functions that are controlled by Shp-2 and c-kit via p85 subunits of class IA PI3K and will provide novel targets for molecular therapies in the treatment of JMML and systemic mastocytosis, both of which currently have no good treatment options.

DESCRIPTION (provided by applicant): Noonan syndrome (NS) is a common (1 in 1500 to 2500 live births) autosomal-dominant disorder caused by mutations in PTPN11 (50%), SOS1 (20%), or KRAS (<5%) and is characterized by dysmorphic facial features, congenital heart malformations, skeletal anomalies, and a variety of hematologic abnormalities including a predisposition to juvenile myelomonocytic leukemia (JMML). Although the anomalies observed in NS are diverse, several of the complications can be attributed to the increased function or number of macrophages or monocyte-derived cells. The most extreme and lethal example of macrophage overproduction in NS patients is the childhood leukemia, JMML. JMML is characterized clinically by overproduction of myelomonocytic cells and by the in vitro phenotype of hematopoietic progenitor hypersensitivity to granulocyte-macrophage colony- stimulating factor (GM-CSF). In addition to GM-CSF-stimulated hyperproliferation and elevated levels of phospho-Erk, we found that activating Shp2 mutations (Shp2E76K and Shp2D61Y) promote a shift toward monocytic differentiation at the expense of other myeloid lineages, consistent with the disease phenotype observed in JMML patients. This skewed differentiation toward the monocytic lineage is associated with increased c-Jun and decreased GATA2 expression. Based on these findings, we hypothesize that activating Shp2-induced Ras hyperactivation alters the transcriptional profile leading to enhanced monocytic differentiation at the expense of alternative myeloid lineages. Mechanistically, we propose that Ras hyperactivation produces constitutive c-Jun expression permitting, in collaboration with PU.1, excessive monocytic differentiation and reduced GATA2 expression. The objectives of this application are to 1) delineate if JMML associated Shp2 gain-of-function mutations induce c-Jun expression and promote aberrant monocytic differentiation in a JNK-dependent or a JNK-independent manner;2) investigate the potential of trichostatin A, a histone deacetylase inhibitor, to reduce c-Jun and PU.1 and to re-activate GATA2 and C/EBP? expression in mutant Shp2-expressing hematopoietic progenitors;and 3) examine hematopoietic- specific transcription factor levels in human samples of JMML compared to normal controls. We anticipate that results of these studies will lead to novel therapeutic strategies in JMML, a lethal childhood leukemia and complication of Noonan syndrome. PUBLIC HEALTH RELEVANCE: This proposal, "Aberrant Monocytic Differentiation Induced by Gain-of-Function Shp2 Mutants" focuses on a particularly lethal form of childhood myeloid leukemia called juvenile myelomonocytic leukemia (JMML). JMML is a lethal leukemia of children less than 5 years of age characterized by massive overproduction of monocytic cells. Unfortunately, JMML is resistant to chemotherapy and most afflicted children succumb to disease due to organ infiltration with malignant monocytes and macrophages, ending terminally in bleeding and infection. Experiments outlined in this proposal are directed at identifying the molecular mechanisms that enhance monocyte overproduction in order to delineate novel targets for therapeutic intervention in JMML.

DESCRIPTION (provided by applicant): Acute myeloid leukemia (AML) is a lethal disease which dramatically increases in incidence in individuals >65 years of age, the fastest growing population in the United States. Regrettably, the 5 year survival rate drops dramatically in patients over 65 years (4.3%) compared to patients less than 65 years (34.45%). These dismal statistics have led scientists to investigate the molecular mechanism(s) underlying the transforming process leading to AML in an effort to develop novel, molecularly-targeted, effective, and less toxic therapies for use in this lethal disease. Internal tandem duplications (ITD), an in-frame mutation leading to insertion or duplication of several amino acids near the juxtamembrane domain, in fms-like tyrosine kinase receptor (FLT3), are seen in nearly 25% of all AML patients and confer a poor prognosis. Expression of the Shp2 protein tyrosine phosphatase is consistently elevated in primary leukemia cell specimens from multiple adult acute leukemias compared to Shp2 levels in bone marrow mononuclear cells from healthy controls. However, how Shp2 contributes to myeloid leukemogenesis is unknown. We present preliminary studies demonstrating that Shp2 is constitutively associated with FLT3 in mutant N51-FLT3- and N73-FLT3-bearing cells and that genetic disruption of Shp2 and pharmacologic inhibition of Shp2 preferentially reduces N51-FLT3-induced hyperproliferation compared to that observed in WT FLT3-bearing cells. As a corollary, since Shp2 has been shown to regulate the activation of the Rac subfamily of Rho-GTPases in hematopoietic cells, we predicted that Rac1 and/or Rac2 may also be relevant effectors of FLT3-ITDs. Consistently, we present preliminary findings demonstrating that pharmacologic Rac inhibition using NSC23766 or genetic disruption of Rac2 results in significantly reduced N51-FLT3-induced hyperproliferation. Based on our preliminary data, the central role of STAT5 hyperactivation in FLT3-ITD-induced leukemia, and the reported positive roles of Shp2 and Rac1/Rac2 promoting activated STAT5 nuclear accumulation, the central hypothesis of this application is that, mechanistically, Shp2 and Rac1/Rac2 contribute to FLT3-ITD-induced leukemia by facilitating STAT5 nuclear localization and the expression of STAT5-responsive pro-leukemogenic genes. The objectives of this application are to define the consequences of genetic disruption of Shp2 and Rac1/Rac2 on the activation and nuclear localization of STAT5 and on the development of FLT3-ITD-induced myeloproliferative disorder (MPD) in vivo, to examine the efficacy of a Shp2 inhibitor and a Rac1 inhibitor in an AML xenograft model in vivo, and to define the intracellular tyrosines within the juxtamembrane and the duplicated juxtamembrane of FLT3-ITD that contribute to ligand-independent proliferation. PUBLIC HEALTH RELEVANCE: Acute myeloid leukemia (AML) is a lethal disease which dramatically increases in incidence in individuals >65 years of age, the fastest growing population in the United States. We present studies that will examine the role of the protein tyrosine phosphatase, Shp2, and the Rac subfamily of Rho GTPases in the pathogenesis of AMLs bearing internal tandem duplications (ITDs) of the fms-like tyrosine kinase receptor (FLT3). Results from these studies are expected to identify new therapeutic targets for treating AML.

DESCRIPTION (provided by applicant): The blood is a specialized tissue that performs several crucial physiologic functions including carrying oxygen, fighting infections, and clotting wounds. The etiology of blood cell dysfunction is diverse and results in a myriad of blood disorders including anemia, myeloproliferative disorder, leukemia, infections, bleeding disorders, and hypercoagulable states. Despite considerable advances in the diagnosis and treatment of blood disorders, these diseases continue to cause significant patient morbidity and mortality. Additionally, autologous and allogeneic stem cell transplantation continues to be a mainstay of therapy for several congenital disorders and acquired malignancies. Thus, collectively, improved understanding of normal hematopoiesis, stem cell biology, and hematopoietic disease states is relevant to our national interest in the United States in terms of preventive measures and improved therapies for blood disorders, congenital disorders, and cancers. The Midwest Blood Club Symposium was initiated in 2003 as a forum to facilitate collaborative ties between investigators at Midwestern universities with a mutual interest in hematopoiesis and hematopoietic stem cell biology. The objectives of this meeting have been to foster regional collaborations in the area of hematopoiesis and stem cell biology within the Midwest and to provide a collegial atmosphere for the training of graduate students and post-doctoral fellows in platform and poster presentations. As we move forward, we are seeking to attract more physician scientists involved in both patient care and basic research, in an effort to underscore the clinical significance and potential application of the basic science studies traditionally presented at this meeting. By meeting on an annual basis, investigators can harness strengths of collaborating colleagues and institutions, resulting in a synergistic increase in productivity. Because of these inter-institutional interactions and collaborations, institutional and extramural research dollars are more efficiently utilized and leveraged for high quality research outcomes. We aim to 1) provide a venue for dialogue of ideas and exchange of expertise and services among investigators located in the Midwest focused on hematopoiesis and hematopoietic stem cell biology; 2) engender an amiable, yet challenging, environment to educate and train graduate students and post-doctoral fellows in the presentation, explanation, and justification of scientific data and interpretation; and 3) broaden the scope of the meeting to include more physician scientists to provide context and perspective on the significance of the ongoing basic science studies and to present clinically-based studies.

PROJECT SUMMARY/ABSTRACT Juvenile myelomonocytic leukemia (JMML) is the most common myeloproliferative neoplasm (MPN) in childhood, and tends to occur in very young children less than 4 years of age. JMML is traditionally characterized as being Ras-driven due to mutations in NF1, CBL, KRAS, NRAS, or PTPN11. Traditional cytotoxic chemotherapeutic agents are ineffective in JMML, and the only curative modality is allogeneic hematopoietic stem cell transplantation. Unlike other MPNs, JMML rarely progresses to blast crisis; rather, mortality is due to extramedullary tumor cell expansion leading to organ failure, respiratory failure, bleeding, or infection. Notably, following allogeneic stem cell transplant, 50% of children succumb to leukemia relapse. This relapse rate in JMML is substantially higher than that of individuals who receive allogeneic stem cell transplant for chronic myelogenous leukemia (CML) in chronic phase (approximately 7% leukemia relapse), implicating a strong hematopoietic stem cell (HSC)-independent component of JMML development and progression. We envision two distinct mechanisms that potentially account for a HSC-independent means of JMML relapse after allogeneic HSC transplant. First, the JMML-initiating malignant cells may emerge during embryonic development prior to and independently from HSCs, and persist postnatally as self-replenishing malignant tissue macrophages. Alternatively, regardless of the origin of the JMML cells, the hyperinflammatory nature of JMML may damage the bone marrow microenvironment, prohibiting the expansion of normal donor cells following transplant, permitting residual leukemia cells to outcompete the normal graft, and leading to leukemia relapse. To address these possibilities, we will use the tamoxifen-inducible Cre recombinase system, which will permit yolk sac-restricted expression of the common JMML mutation, Shp2D61Y, to determine if yolk sac-restricted oncogene expression is sufficient for the post-natal development of MPN. Further, we will examine if inhibition of the pro-inflammatory protein, PI3K p110?, improves homing, engraftment, expansion, and myeloid differentiation of WT donor cells into diseased, Shp2D61Y-expressing recipients.

DESCRIPTION (provided by applicant): This is a new application for an institutional NRSA sponsored training program to provide hypothesis-driven biomedical research experience to students during the early stages of their medical education at Indiana University School of Medicine (IUSM). Our purpose is to provide a structured research environment that engages medical students' interest in biomedical research, creating opportunities for basic and translational research experience, and education in research ethics. The main objective of the program is to serve as a portal to train and recruit physician-scientists. The proposed program is based on a highly successful mentorship and training program at IUSM, the Student Research Program in Academic Medicine (SRPAM). Medical students with strong academic credentials are selected for SRPAM, which pairs students with highly qualified faculty mentors for 12 week summer research internships with an accompanying lecture series focused on research communication and writing, ethics, and translational investigations. The current application seeks support to enroll 24 students/year in the program to conduct basic and translational research within the basic and clinical departments and centers at IUSM. The program is designed: a) to increase student awareness of the value of doing biomedical research by challenging students to take on independent projects during the internship; and b) to strongly support students interested in a career in academic medicine by providing access to opportunities in the MD/PhD program and assistance with research fellowship applications to HHMI and NIH. Trainees will engage in mentored research experience in areas that reflect the school's strength and international reputation in hematopoiesis, host defense and pulmonary biology, cancer biology, cardiovascular, diabetes and medical informatics. The strengths of our program are: i) a cadre of highly successful mentors including physician-scientists with extramural funding, outstanding training records and solid experience with short-term trainees; ii) an outstanding scientific environment characterized by an intense interdisciplinary spirit and access to cutting-edge technologies and excellent resources; iii) a supportive community and a solid network providing students with opportunities for leadership and mentoring; iv) integration with the medical school curriculum with student credit for level III competency in biomedical knowledge; and v) an unequivocal commitment from the leadership at the Indiana University School of Medicine. The long term goal of the program is to increase the number of physician scientists nationally by exposing students in their early years of medical education to hypothesis-driven research focused on the molecular and cellular basis of disease and the potential for clinical translation.