Christopher Y Park - US grants

[unreadable] DESCRIPTION (provided by applicant): This 5 year-proposal describes a comprehensive plan for the development of a career in experimental pathology. The principal investigator completed anatomic pathology residency and hematopathology fellowship training at Stanford University and has spent the past two years honing his skills as a diagnostic hematopathologist while spending the majority of his time developing the technical and intellectual skills that will allow him to become an independent investigator. [unreadable] [unreadable] The proposed work will take place in the laboratory of Irving L. Weissman, an internationally recognized leader in the field of stem cell biology, cancer stem cell biology, and hematopoiesis. Given the breadth and depth of intellectual and technological resources, Dr. Weissman's lab represents a unique environment, geared not only to allowing highly creative and innovative projects to come to fruition, but also to train future independent investigators. [unreadable] [unreadable] Given the growing evidence that microRNAs play a role in the development of human cancers, this proposal represents a timely investigation into the pathogenesis of human acute myeloid leukemia (AML). Unlike conventional cancer biology research, the proposed studies will focus on the tumor-initiating, or stem cells, of AML. Because of the unique properties of leukemia stem cells (LSC), including the capacity to initiate tumors, self-renew, and recapitulate disease in xenograft models, it is imperative that we understand the molecular basis of the AML LSC biologic properties. Because of the unique experimental challenges associated with working with primary human samples, a great deal of time has been, and will continue to be, invested in developing improved models to study the role of microRNAs in human AML. [unreadable] [unreadable] The Stanford Institute for Stem Cell Biology and Regenerative Medicine is an ideal setting for these studies. In addition to housing a community of cancer and stem cell biologists, there are numerous opportunities to collaborate with members of other departments through interdisciplinary programs including the Stanford Comprehensive Cancer Center, Immunology Program, and many others. Given the strength of Stanford's clinical divisions (adult and pediatric hematology/oncology, bone marrow transplant, hematopathology), there is significant tissue access as well as opportunities to work with clinical colleagues. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Cellular and molecular basis of microRNA-29a induced acute myeloid leukemia. Acute myeloid leukemia (AML) arises from the accumulation of genetic and/or epigenetic changes in immature hematopoietic cells. While leukemogenesis involves multiple steps including initiation, progression, transformation, and maintenance, studying these processes in human disease presents many practical challenges; however, well-characterized mouse models of AML afford the opportunity to study each of them due to the relative ease of genetically modifying cells and testing cellular function in a variety of in vitroand in vivo assays. While AML arises from immature hematopoietic cells, the exact cell that initiates disease or ultimately transforms into the leukemia stem cell (LSC) is unclear. We previously showed that microRNA-29a (miR-29a) is highly expressed in human HSC and AML LSC, and that ectopic expression of miR-29a in mouse BM cells is sufficient to induce a myeloproliferative neoplasm (MPN)-like disease that progresses to AML. We characterized miR-29a induced disease and showed that self-renewing committed progenitors arise prior to the development of AML and that miR-29a induced AMLs contain LSCs that can be prospectively isolated based on the expression of specific cell surface markers. Although the precise role of the self-renewing progenitors has not been defined, their appearance raises the intriguing possibility that aberrant acquisition of self-renewal by committed progenitors may be an early event in AML pathogenesis. Overall, these data demonstrate that the miR-29a induced AML model is a robust and experimentally tractable model for studying AML pathogenesis. We propose to characterize miR-29a's roles during AML development and to elucidate the molecular basis of miR-29a's regulation of self-renewal in LSCs and aberrantly self-renewing progenitors. We will determine if miR-29a is required for leukemogenesis utilizing our newly developed miR-29a deficient mouse model as well as various mouse models of AML. We will identify the cell population/s that become transformed during miR- 29a driven leukemogenesis, and we will also test the contribution of two miR-29a targets, Dnmt3a and Smpd3 (genes also recurrently mutated in human AML) to miR-29a induced phenotypes. To identify potential genes or mutations that may cooperate with miR-29a to induce leukemia, we will perform RNA-Seq on LSCs and aberrantly self-renewing progenitors. Finally, we will leverage our experience working with the miR-29a induced AML model as well as with primary human AML samples to evaluate the role of miR-29a in LSC function in the serial transplantation setting, the gold-standard assay for self-renewal. Ultimately, we expect these studies to generate new insights into the role of miR-29a in AML, define the molecular mechanisms of miR-29a's actions in pre-leukemic intermediates (including the molecular basis for self-renewal), and determine whether miR-29a or its downstream mediators may serve as potential therapeutic targets in AML.

Summary While activating mutations of the serine-threonine kinase BRAF occur in ~8% of solid tumors, they are rare among hematopoietic malignancies except in hairy cell leukemia (HCL) and the systemic histiocytoses (SH) Langerhans Cell Histiocytosis and Erdheim-Chester Disease. The presence of the specific BRAFV600E mutation in nearly 100% of HCL and 40-60% of SH patients has provided major insights into our understanding of the pathophysiology of these poorly understood diseases. The development of targeted inhibitors of BRAF or its downstream mediators to treat solid tumors has led to major therapeutic advances, and more recently this paradigm has been applied in HCL and SH. Our interdisciplinary team has taken advantage of these advances in BRAFV600E mutation biology and therapeutics and recently published its findings tracing the origin of HCL to the hematopoietic stem cell and developed genetically accurate murine models of HCL. More recently, we have confirmed the presence of the BRAFV600E mutation in hematopoietic stem and progenitor cells in SH patients, generated mouse models of SH, identified recurrent mutations co-existing with the BRAFV600E mutation in both HCL and SH, and completed clinical trials of vemurafenib for HCL and SH patients. Although our preliminary data provide substantial evidence that HSPCs contribute to disease pathogenesis in both HCL and SH through their acquisition of BRAFV600E mutations, it is not yet clear how this common mutation drives the development of such phenotypically and clinically distinct disorders. Moreover, although we have noted that HCL and SH patients exhibit remarkable clinical responses to vemurafenib, we have begun to identify genetic mechanisms of vemurafenib resistance in HCL, which provides us with the unique opportunity to develop the next line of therapeutic strategies in the treatment of these disorders. Thus, the overall goal of this proposal is to delineate the cellular and functional requirements for HCL and SH pathogenesis and to utilize this information to identify the origins of resistance mutations that arise in the context of BRAF targeted therapy. We hypothesize that the cell in which the BRAFV600E mutant protein is active and/or the presence of collaborating mutations play a major role in determining disease phenotype and response to BRAF inhibition. We will address this hypothesis in the following Aims: 1) Delineate the functional effects of the BRAFV600E mutation on hematopoiesis based on the cell in which it is active, 2) Identify the constellation of mutations co-existing with the BRAFV600E mutation in HCL and SH, and 3) Identify the mechanisms of BRAF inhibitor resistance. This project will provide a comprehensive characterization of the cellular origins and cooperating mutations that give rise to HCL and SH. Moreover, this work will delineate mechanisms of BRAF inhibitor resistance in hematopoietic malignancies - an effort that may have broader benefits to the larger population of BRAF-mutant cancer patients ineffectively treated with current BRAF inhibitors.

Summary While activating mutations of the serine-threonine kinase BRAF occur in ~8% of solid tumors, they are rare among hematopoietic malignancies except in hairy cell leukemia (HCL) and the systemic histiocytoses (SH) Langerhans Cell Histiocytosis and Erdheim-Chester Disease. The presence of the specific BRAFV600E mutation in nearly 100% of HCL and 40-60% of SH patients has provided major insights into our understanding of the pathophysiology of these poorly understood diseases. The development of targeted inhibitors of BRAF or its downstream mediators to treat solid tumors has led to major therapeutic advances, and more recently this paradigm has been applied in HCL and SH. Our interdisciplinary team has taken advantage of these advances in BRAFV600E mutation biology and therapeutics and recently published its findings tracing the origin of HCL to the hematopoietic stem cell and developed genetically accurate murine models of HCL. More recently, we have confirmed the presence of the BRAFV600E mutation in hematopoietic stem and progenitor cells in SH patients, generated mouse models of SH, identified recurrent mutations co-existing with the BRAFV600E mutation in both HCL and SH, and completed clinical trials of vemurafenib for HCL and SH patients. Although our preliminary data provide substantial evidence that HSPCs contribute to disease pathogenesis in both HCL and SH through their acquisition of BRAFV600E mutations, it is not yet clear how this common mutation drives the development of such phenotypically and clinically distinct disorders. Moreover, although we have noted that HCL and SH patients exhibit remarkable clinical responses to vemurafenib, we have begun to identify genetic mechanisms of vemurafenib resistance in HCL, which provides us with the unique opportunity to develop the next line of therapeutic strategies in the treatment of these disorders. Thus, the overall goal of this proposal is to delineate the cellular and functional requirements for HCL and SH pathogenesis and to utilize this information to identify the origins of resistance mutations that arise in the context of BRAF targeted therapy. We hypothesize that the cell in which the BRAFV600E mutant protein is active and/or the presence of collaborating mutations play a major role in determining disease phenotype and response to BRAF inhibition. We will address this hypothesis in the following Aims: 1) Delineate the functional effects of the BRAFV600E mutation on hematopoiesis based on the cell in which it is active, 2) Identify the constellation of mutations co-existing with the BRAFV600E mutation in HCL and SH, and 3) Identify the mechanisms of BRAF inhibitor resistance. This project will provide a comprehensive characterization of the cellular origins and cooperating mutations that give rise to HCL and SH. Moreover, this work will delineate mechanisms of BRAF inhibitor resistance in hematopoietic malignancies - an effort that may have broader benefits to the larger population of BRAF-mutant cancer patients ineffectively treated with current BRAF inhibitors.

SUMMARY MDS represents a group of acquired bone marrow failure syndromes arising from hematopoietic stem cells (HSCs). While most MDS patients experience progressive cytopenias, a significant minority (30%) will progress to acute myeloid leukemia (AML); however, the mechanisms that determine whether patients experience progressive cytopenias or leukemia transformation are poorly understood. While the ineffectiveness of MDS therapies is due to their inability to effectively eliminate MDS clones and/or restore normal differentiation, it remains unclear whether hypomethylating agents (HMAs) act on HSCs or committed progenitors to induce hematologic improvement and/or reductions in blast count, or if HMAs act through similar or unique mechanisms in these distinct cell populations. We and others have shown that MDS HSCs exhibit markedly different gene expression profiles than CD34+ hematopoietic stem/progenitor cells (HSPCs) and that they are also genetically and transcriptionally heterogeneous6-10; however, these prior studies were not designed to specifically capture committed progenitor contributions to disease progression or therapeutic responses to HMAs. We hypothesize that HSCs and committed progenitors from MDS patients who experience progressive cytopenias or leukemic transformation exhibit unique transcriptional signatures prior to, and in response to, HMA therapy. Secondarily, we hypothesize that HMAs induce unique transcriptional and functional changes in MDS HSCs and committed progenitors, and that their ability to induce specific transcriptional programs determines whether or not patients respond with hematologic improvements and/or blast reductions. We propose to elucidate the transcriptional basis of HSC and committed progenitor responses from MDS patients at the single cell level using novel full-length cDNA scRNA-seq technologies that will allow simultaneous characterization of the transcriptome and mutational data within individual cells from paired pre- and post-therapy samples from MDS patients with different types of disease progression and responses to HMA therapy. We also will evaluate the clinical relevance of our findings by evaluating MDS-associated RNA features in larger cohorts of MDS patients for whom transcriptome data and clinical outcome data are available. Assessments of the contribution of dysregulated transcripts to MDS progression will be evaluated using mouse models of MDS as well as primary MDS patient cells. Overall, we expect our studies to: 1) Identify the genes and biological pathways that determine whether MDS patients will experience progressive cytopenia versus leukemic transformation; 2) Elucidate the roles of different HSPC populations in determining clinical responses to HMA therapy; 3) Identify novel biomarkers for prognostication of clinical outcomes and therapy responses in MDS; 4) Provide the biological rationale for future clinical studies of novel pathway- or genetically-targeted agents in combination with HMAs.

Approximately 13,000 new cases of adult AML are diagnosed each year in the U.S. Unfortunately for these patients, treatment options have remained essentially unchanged for 30 years, and clinical outcomes remain poor. Moreover, little is known about the genes that regulate leukemia stem cells (LSCs), which represent the population of blasts that is resistant to chemotherapy and are critical for maintaining disease and re-initiating disease after therapy. Thus, eradication of the LSC is a prerequisite for cure. We recently identified a novel LSC antigen, CD99, that is expressed in the vast majority (~85%) of human AMLs [3]. We have shown that CD99 is expressed on leukemic blasts and can be used to prospectively separate leukemic blasts from residual hematopoiesis, similar to LSC antigens such as TIM3 and CD47, but CD99 exhibits several unique features: 1) CD99 is the most commonly expressed LSC antigen; 2) CD99 does not just mark LSCs, but regulates blast growth and survival; 3) CD99 allows isolation of LSCs from blasts with CD99hi blasts enriched ~10-100 fold for leukemia initiating-cell activity over CD99low cells; 4) CD99 is the only LSC antigen that can identify LSCs in both CD34+ and CD34- AMLs; and, 5) Novel monoclonal antibodies (mAbs) against CD99 induce cell death directly by activating Src-family kinases (SFKs). We have evaluated the consequences of CD99 loss in both LSCs and hematopoietic stem cells (HSCs). Our studies indicate that decreased expression of CD99 in both LSCs and HSCs results in global upregulation of protein synthesis and loss of self-renewal. Moreover, cytotoxic mAbs against CD99 mimic the effects of CD99 loss, activating protein synthesis and inducing similar gene expression changes to those observed in CD99 null HSCs or CD99low blasts in most genetic subtypes of AML tested. Collectively, these data support a model in which LSCs require highly regulated levels of protein synthesis, similar to HSCs, and based on our work in normal HSCs, we expect these alterations in translation to result in selective recruitment of mRNA?s to active translating ribosomes (polysomes). Overall, we hypothesize that CD99 constrains the translation of specific mRNA?s, thereby promoting a translational program required for LSC self-renewal. Given our ability to enrich for LSCs, our group is in a unique position to investigate the role of mRNA translation in LSC function. We have painstakingly optimized methods to perform polysome profiling and RNA- sequencing from polysome fractions from small numbers of cells, and we have developed a computational pipeline to identify mRNA?s that are preferentially translated in LSCs. These tools, in combination with unique reagents such as our CD99 KO mice and cytotoxic CD99 mAbs that mimic CD99 loss, place us in an excellent position to investigate how CD99 regulates translation in LSCs. Understanding the molecular pathways that regulate protein synthesis has the potential to help better characterize a poorly understood process in AML biology and to credential targeting translation using mAbs as a potential therapeutic strategy in AML.

Despite advances in our understanding of the genetic origins of AML, treatment options have remained essentially unchanged for 30 years, and clinical outcomes remain poor. Leukemia stem cells (LSCs) represent the population of blasts that are resistant to chemotherapy and re-initiate AML after therapy; thus, this subset of blasts must be eradicated to cure disease. Unfortunately, therapies developed specifically to target LSCs have yet to be validated in the clinic. We and others recently identified a novel AML antigen, CD97, that is expressed in the vast majority of human AMLs. Our recently published studies have revealed several features of CD97 that suggest that it may be an excellent therapeutic target in AML: 1) CD97 is one of the most commonly expressed AML antigens; 2) CD97 regulates blast growth, survival, and differentiation; 3) CD97 regulates LSC function, as demonstrated in serial transplantation experiments of primary AML; and, 4) CD97 is not required for HSC function, suggesting low toxicity of CD97-targeting therapeutics. Highlighting its clinical importance, CD97 mRNA expression is an independent predictor of disease-free and overall survival in AML. CD97 is an adhesion class G-protein coupled receptor (aGPCR) characterized by a long, extracellular ligand- binding domain and a GPCR-Autoproteolysis-INducing (GAIN) domain that can induce signals that may or may not require extracellular domain shedding. Isoforms of CD97 produced by alternative splicing differ in the composition of the ligand-binding domain, but at present, it is unclear if the various CD97 isoforms mediate unique or overlapping roles in AML. Our overall hypothesis is that the various CD97 isoforms play distinct roles in leukemogenesis and LSC self-renewal by virtue of their unique ligand binding and/or signaling properties. Our specific goals are to determine the role of CD97 isoforms in leukemic transformation and LSC function, to identify the molecular and structural requirements for CD97 activity, and to utilize novel human synthetic antibodies (sAbs) against CD97 with different epitope specificities to evaluate the function of CD97 as well as test their anti-leukemic activity. We will determine the roles of the various structural subdomains of CD97 required for LSC function utilizing our novel CD97 Abs, CD97 constructs expressing multiple naturally occurring and engineered structural variants of CD97, and complementary in vitro and in vivo models of mouse and human AML. Given our team's complementary expertise in LSC biology, antibody engineering, and aGPCR biology, we are uniquely positioned to investigate the mechanisms of CD97 signaling and function in LSCs. Collectively, these studies will dramatically increase our understanding of the molecular mechanisms that regulate LSC self- renewal and help expedite translation of CD97 antibody therapies to the clinic. Finally, these studies may have broader consequences since CD97 plays disease-modifying roles in other human cancers.

PROJECT SUMMARY The mTOR pathway regulates longevity as well as stem cell function, and inhibition of mTOR dramatically extends lifespan in model organisms and laboratory animals. While the role of mTOR has been investigated in many tissues, studies on the role of mTOR in regulating lifespan have taken place almost exclusively in the context of pan-tissue knockout strains, and possible tissue-specific roles of mTOR in regulating longevity have not been explored. The aging hematopoietic system undergoes numerous changes including increased myeloid cell production, decreased lymphocyte production, reduced red cell output, and decreased hematopoietic stem cell (HSC) self-renewal. It has been proposed that aging HSCs promote age-related diseases by establishing and/or perpetuating the systemic inflammation observed in aging due to their altered ability to give rise to cells of the innate and cellular immune responses. Consistent with this hypothesis, recent studies from our group and others indicate that HSCs may directly regulate longevity. While most studies investigating hematopoietic aging have focused on cell-intrinsic mechanisms that regulate HSC aging, a growing body of work has firmly established the role of the bone marrow microenvironment in promoting HSC aging. Indeed, our work has demonstrated that bone marrow endothelial cells (BMECs) are major contributors to this process. Intriguingly, in contrast to HSCs where reductions in mTOR activity are thought to promote HSC activity, low mTOR activity in BMECs leads to an enhancement of HSC aging phenotypes. Thus, while many have suggested that systemic mTOR inhibition may be a potential strategy to reverse aging, this view is based on an incomplete picture regarding the role of mTOR in cell-extrinsic regulation of HSC aging. Given recent published and unpublished data confirming that the hematopoietic system is an important contributor to longevity, it is critical to not only clarify the role of mTOR in HSC aging, but determine if hematopoietic aging contributes to other important age- related phenotypes. Moreover, it will be important to determine if hematopoietic regulation of longevity is due to alterations in HSC function and/or their downstream progeny. Given the importance of developing rationally designed strategies to prevent/reverse organismal aging and the potential of inhibiting/reversing HSC aging to promote both healthspan and lifespan, we propose to determine the mTOR-dependent cell-intrinsic and cell- extrinsic mechanisms that regulate HSC aging. Finally, given the uncertainty surrounding direct inhibition of mTOR as a unitary strategy to ameliorate hematopoietic aging, it is important to identify novel regulators of HSC aging. We have identified such a candidate in Thrombospondin-1 (TSP1), and thus will test whether TSP1 inhibition can not only inhibit/reverse HSC aging phenotypes, but also improve health- and lifespan.