Daniel C. Link - US grants

The long term career objectives of this applicant are to obtain an academic position as a basic scientist studying clinically relevant problems. Areas of particular interest include the study of gene regulation as it relates to oncogenesis and hematopoietic differentiation. The faculty of the Washington University Division of Hematology and Oncology have a longstanding record of outstanding and innovative research and a commitment to the development of new investigators. c-fgr is a proto-oncogene belonging to the c-src gene family. It is normally expressed in natural killer cells and terminally differentiated myelomonocytic cells including monocytes, neutrophils and alveolar macrophages. One of the long term objectives of the proposed research is to determine the mechanisms responsible for the tissue- and development- specific regulation of the c-fgr gene. c-fgr is also expressed in Epstein-Barr virus (EBV) infected B-lymphocytes and at least some chronic lymphocytic leukemia (CLL) B-cells, but not in normal or activated B-lymphocytes. Additional long term objectives of the proposed research are to determine the mechanisms and biologic significance of c-fgr gene expression in EBV associated lymphoproliferative disease and CLL. The following specific aims are proposed: 1. We will characterize the cis-acting DNA elements responsible for the tissue- and development- specific expression of the c-fgr gene. 2. We will determine the mechanisms of c-fgr disregulation in EBV infected B-lymphocytes. 3. We will study the regulation and biological significance of c-fgr gene expression in CLL B-cells.

The long range objective of this research is to characterize the molecular mechanisms involved in hematopoietic progenitor cell (HPC) mobilization. The use of HPC to reconstitute hematopoiesis following myeloablative therapy has significantly improved the clinical outcome in patients with a variety of malignancies. Recently, mobilized peripheral blood HPC instead of bone marrow-derived HPC have been used because of their reduce engraftment times, relative ease of collection, and possibly reduced risk of graft-versus-host disease. Currently to mobilize HPC from the bone marrow to blood are well tolerate but not universally effective and are often associated with co-mobilization of neoplastic cells. A better understanding of the mechanisms that regulate HPC mobilization may lead to the design of novel mobilization strategies that overcome these problems. We recently showed that mobilization of HPC in response to cyclophosphamide (CY) or interleukin-8 but not fit-3 ligand is markedly impaired in granulocyte colony-stimulating factor receptor (G-CSFR) deficient mice. These surprising results suggested that G-CSFR signals in hematopoietic- or bone marrow stromal-cells play an important and previously unexpected role in HPC migration. This proposal is designed to characterize these these G- CSFR-dependent mechanisms of HPC mobilization. The following specific aims are proposed. 1. We will characterize in detail the mobilization response in G-CSFR deficient mice to CY, interleukin-12 (IL-12), or stem cell factor (SCF).. HPC mobilization in G-CSFR deficient mice in response to SCF or IL-12 will be analyzed. To explore mechanisms for the mobilization defect in G- CSFR deficient mice, the phenotype of hematopoietic cells, in particular HPC , in the bone marrow of wild-type versus G-CSFR deficient mice after CY treatment will be compared. 2. We will identify the cell type responsible for G-CSFR dependent mobilization. Preliminary studies of HPC mobilization in G-CSFR deficient radiation chimeras suggest that a functional G-CSFR on mature hematopoietic cells but not on HPC or stromal cells is required for CY- induced mobilization. The first objective of this specific aim is to confirm these surprising results and to determine whether primitive HPC are mobilized in a similar fashion. The second objective of this specific aim is to characterize G-CSF induced mobilization in these radiation chimeras. 3. We will define the role of neutrophils in G-CSFR dependent mobilization. A neutropenic mouse line will be generated by driving expression of the attenuated diphtheria toxin A subunit in myeloid cells using murine cathepsin G regulatory sequences. CY- and G-CSF induced HPC mobilization will be characterized in these mice. 4. We will define the regions of the G-CSFR that are required for HPC mobilization. CY- and G-CSF-induced mobilization will be characterized in two recently generated targeted "knock-in" mutations of the G-CSFR. The role of STAT-3 in the generation of the HPC mobilization signal by the G- CSFR will be examined.

DESCRIPTION (provided by applicant): The adult bone marrow is a major reservoir of multipotent stem/progenitor cells including hematopoietic stem cells (HSC), mesenchymal stem cells (MSC), and endothelial progenitor cells (EPC). The ability of HSC to regenerate hematopoietic tissue following myeloablative therapy is well documented and widely used in clinical practice. Emerging data suggest that stem cells in the bone marrow also may be able to regenerate damaged tissue in other organs, including liver and heart. However, repair of damaged tissue by endogenous stem cells may be limited by the rarity of stem cells in the circulation under basal conditions. To circumvent this limitation, the number of HSC, EPC and possibly MSC in the circulation can be dramatically increased or mobilized by treatment with various agents. Cytokine mobilization of bone marrow cells prior to myocardial infarction has been reported to improve survival and cardiac function in mice. Collectively, these data support the hypothesis that rapid mobilization of stem cells from the bone marrow to blood may enhance tissue regeneration following injury. HOWEVER, there are significant gaps in our knowledge of this process that should be addressed prior to the rational design of clinical trials. What are the optimal regimens to mobilize HSC, MSC, and EPC into the blood in a rapid yet sustained fashion? Can the initiation of stem cell mobilization after organ damage lead to significant tissue regeneration? If so, what are the cell types capable of mediating the most robust tissue repair, and what is the optimal time period following tissue injury to deliver these cells? To begin to answer these questions, we recently developed NOD/SCID/MPSVII mice. These mice are deficient in b-glucuronidase (GUSB), a ubiquitously expressed lysosomal enzyme. Importantly, sensitive and specific methods have been developed to detect GUSB-positive cells in these mice following transplantation. In the proposed studies, we will use this novel murine model to examine the stem cell-mediated repair of chemically damaged liver tissue and of cardiac tissue following myocardial infarction. The following specific aims are proposed. 1. Optimize mobilization regimens that lead to rapid and sustained increases in the number of circulating HSC, MSC, and EPC 2. Assess the efficacy of stem cell mobilization to mediate tissue regeneration in a novel murine model of acute liver injury. 3. Examine the efficacy of stem cell mobilization to mediate tissue regeneration in a murine model of acute myocardial infarction.

DESCRIPTION (provided by applicant): The long-range objective of this research is to characterize the mechanisms that regulate the mobilization of hematopoietic progenitor cells (HPC) from the bone marrow to blood. Hematopoietic stem cell transplantation is a potentially curative therapy for patients with advanced hematological malignancies. Recently, mobilized peripheral blood HPC instead of bone marrow-derived HPC have been used because of reduced engraftment times and relative ease of collection. Current mobilization protocols utilizing granulocyte colony stimulating factor (G-CSF) alone are generally well tolerated but not universally effective and are often associated with co-mobilization of neoplastic cells. A better understanding of the mechanisms that regulate HPC mobilization may lead to the design of novel mobilization strategies that overcome these problems. Accumulating evidence suggests that interactions between stromal derived factor-1 (SDF-1, CXCL12) and its cognate receptor, CXCR4, may play a key role in regulating HPC and neutrophil trafficking from the bone marrow. Loss-of-function models for SDF-1 and CXCR4 have established a critical role for these genes in the migration of HPC from the fetal liver to bone marrow. More recently, gain-of-function mutations of the CXCR4 gene have been implicated in the pathogenesis of WHIM syndrome, a syndrome manifested, in part, by impaired neutrophil trafficking from the bone marrow. We recently showed that G-CSF treatment results in a significant decrease in SDF-1alpha protein in the bone marrow of wild type mice. Finally, treatment with AMD3100, a selective antagonist of CXCR4, induces rapid and robust HPC mobilization in mice and humans. Collectively, these data suggest a hypothesis in which disruption of SDF-1/CXCR4 signaling is a key step in HPC mobilization by G-CSF. The objective of this research is to test this hypothesis and define mechanisms by which mobilizing agents regulate SDF-1/CXCR4 signaling. The following specific aims are proposed. 1. We will determine whether gain-of-function mutations of the CXCR4 gene found in patients with WHIM syndrome are sufficient to induce impaired HPC and neutrophil trafficking from the bone marrow. 2. We will identify the mechanisms by which G-CSF regulates SDF-1 expression in the bone marrow. 3. We will determine whether disruption of SDF-1/CXCR4 signaling is a common final pathway by which diverse mobilizing agents mediate HPC mobilization.

DESCRIPTION (provided by applicant): Severe congenital neutropenia (SCN) is a rare inherited syndrome manifested by impaired granulopoiesis and a marked predisposition to develop acute leukemia. G-CSF treatment, while effective in increasing neutrophil counts in most patients, does not prevent progression to leukemia. Allogeneic bone marrow transplantation, while potentially curative, is associated with significant mortality and morbidity. Thus, there is a clear need for more effective therapies for SCN. There is compelling genetic evidence implicating mutations of the ELA2 gene, encoding neutrophil elastase (NE), as the cause of most cases of SCN. Studies of the molecular mechanisms by which ELA2 mutants induce a block in granulocytic differentiation have been limited by the rarity of SCN and the resulting difficulty in obtaining clinical samples. Instead, most recent studies have focused on ectopic NE expression in immortalized cell lines. However, these cell lines have a limited capacity for differentiation and the level of NE expression may not be physiological. Thus, an accurate animal model of SCN would be an invaluable tool to investigators in this field. We propose to generate transgenic mice in which mutant NE is expressed under the regulatory control of the endogenous ELA2 locus. These mice will be used to explore mechanisms of disease pathogenesis and test novel therapies. A second major objective of this application is to explore the feasibility of RNAi-mediated inhibition of ELA2 gene expression to rescue the block in granulocytic differentiation in primary progenitors from patients with SCN. These studies may ultimately lead to a novel molecularly targeted therapy for SCN. The following specific aims are proposed. 1. We will determine whether expression of mutant NE under the regulatory control of the ELA2 gene locus is sufficient to induce an SCN phenotype in mice. Two complementary state-of-the-art transgenic approaches will be used to generate a mouse model of SCN. 2. We will characterize the effect of RNAi-mediated inhibition of ELA2 gene expression on the growth and differentiation of primary myeloid progenitors. Preliminary data show the feasibility of RNAi to attenuate NE protein expression in cell lines. The efficacy of lentiviral RNAi vectors to inhibit NE protein expression and revert the block in granulocytic differentiation in primary progenitors from patients with SCN will be determined. A novel xenotransplantation model of SCN will be developed to study hematopoiesis in vivo.

DESCRIPTION (provided by applicant): A fundamental property of leukemic stem cells (LSCs) is clonal dominance. Clonal dominance refers to the clonal expansion of LSCs within the bone marrow microenvironment at the expense of normal hematopoietic cells. The mechanisms by which mutations that accumulate during leukemogenesis confer clonal dominance are largely unknown. Severe congenital neutropenia (SCN) is a bone marrow failure syndrome characterized by a marked propensity to develop acute myeloid leukemia. Truncation mutations of CSF3R, encoding the G-CSF receptor (G-CSFR), are common early mutations associated with leukemic progression in patients with SCN. We previously described transgenic mice (termed d715 G-CSFR) carrying a knock-in mutation of Csf3r that reproduces a mutation found in SCN. Our preliminary data show that the d715 G-CSFR confers a strong clonal advantage at the hematopoietic stem cell (HSC) level. Importantly, the clonal HSC advantage is dependent upon G-CSF administration, providing a novel, physiological, and "inducible" model to study clonal dominance. We propose to use this model to characterize the molecular mechanisms by which HSCs expressing mutant CSF3R gain clonal dominance. A better understanding of these mechanisms may provide novel strategies to develop molecular therapies that specifically target LSCs. This is highly relevant, since there is evidence that LSCs are inherently resistant to most current chemotherapy. Preliminary data suggest that accentuated STAT5 activation by the d715 G-CSFR is a proximal signal mediating the clonal HSC advantage. This hypothesis will be tested by conditionally deleting Stat5 and assessing the effect on HSC function. In specific aim 2, we will identify genes that are dysregulated by G-CSF in d715 G-CSFR HSCs and prioritize them for biological validation. Preliminary mRNA expression profiling studies have identified two candidate genes that are differentially induced by G-CSF in d715 G-CSFR HSC: Cdkn1a (p21cip1/waf1) and Enah. We hypothesize that induction of Cdkn1a expression by the d715 G-CSFR allows for HSC proliferation without loss of self-renewal. We also hypothesize that increased Enah expression favorably alters the interaction of HSCs with bone marrow stromal. The following specific aims are proposed. Aim 1. We will determine whether Stat5 mediates the clonal advantage of HSCs expressing the d715 G-CSFR. Aim 2. We will identify genes dysregulated by G-CSF in d715 G-CSFR HSCs that contribute to clonal dominance. Aim 3. We will define the role of Cdkn1a and Enah in the clonal advantage of HSCs expressing the d715 G-CSFR. PUBLIC HEALTH RELEVANCE: Clonal dominance is a fundamental but poorly understood property of all leukemias that allows the leukemic stem cell to expand at the expense of normal blood cells. The goal of this research is to improve our understanding of the pathways that mediate clonal dominance. We believe this research will lead to novel strategies to specifically target the leukemic stem cell and ultimately lead to improved cure rates in individuals with leukemia.

DESCRIPTION (provided by applicant): This application addresses broad Challenge Area (08) Genomics and specific Challenge Topic, 08-CA-09-003: Micro-RNAs in Cancer. The objective of this challenge grant is to define the contribution of microRNAs (miRNAs) to transformation in acute myelogenous leukemia (AML). There is emerging data suggesting that non-coding RNAs, in particular miRNAs, play an important role in the regulation of gene expression. Moreover, dysregulation of miRNAs, through deletion, point mutation, or promoter methylation has been implicated in the pathogenesis of human cancer. We hypothesize that altered miRNA expression and/or function play a significant role in the pathogenesis of AML. Most current studies utilize array-based or quantitative reverse-transcription-polymerase chain reaction (RT-PCR) approaches to measure miRNA expression. However, these approaches do not interrogate all known (or predicted) miRNAs and are unable to detect mutations in miRNAs. Our preliminary data show that next-generation sequencing technologies allow for the efficient detection of miRNA genetic variants, the identification of novel expressed miRNAs, and the characterization of miRNA isomer (isomiRs) expression patterns. Specifically, in our analysis of a patient with AML, more than 70 novel miRNAs were detected. In addition, 3 novel single nucleotide variants and 3 novel small insertions/deletions within the precursor region of miRNA genes were detected. In this challenge grant, we propose to use these technological advances to identify commonly mutated or dysregulated miRNAs in AML. Using the information gained from these studies, we intend to create molecular diagnostic tools for risk stratification, and we will identify candidate genes for targeted therapeutic approaches. The following specific aims are proposed. Aim 1. We will characterize miRNA expression using massive parallel sequencing in the leukemic blasts of at least 40 patients with de novo AML. Aim 2. We will identify genetic variants of miRNA genes and define the frequencies of somatic mutations in 400 cases of de novo AML PUBLIC HEALTH RELEVANCE: Acute myelogenous leukemia (AML) is a blood cancer with a poor prognosis. Our research is directed at understanding the genetic mutations that contribute to AML. This research will improve our understanding of the pathogenesis of AML and may lead to the development of new therapeutics.

The long-term goals ofthe Hematopoietic Development & Malignancy Program (HDMP) are to elucidate basic mechanisms regulating normal and malignant hematopoiesis and to use this information to develop strategies for the prevention, diagnosis, prognostic stratification, and treatment of hematopoietic malignancies. We have identified areas of institutional strength and developed the following specific translational goals of the HDMP: 1) to leverage local expertise in cancer genomics to identify novel recurrent mutations in hematopoietic malignancies and develop their translational potential; 2) to expand translational research in multiple myeloma and lymphoma; 3) to take advantage of local expertise in fundamental hematopoiesis research to expand translational research in bone marrow failure syndromes; 4) to utilize local expertise in fundamental immunology and stem cell biology research to expand translational research in stem cell transplantation. Working groups in leukemia, lymphoma & myeloma, bone marrow failure and transplantation biology have been established to develop, review, prioritize and conduct translational research. The HDMP will foster collaborative translational research and provide training of junior investigators through research seminars, journal clubs, work-in-progress meetings and the annual HDMP retreat. The HDMP has 29 members from four Departments and one School. The HDMP is supported by $11,684,316 in funding, of which $4,798,898 is NCI funding and $6,264,597 is other peer reviewed funding. In the last grant period, members ofthe HDMP published 260 manuscripts, of which 34.6% represent interprogrammatic and 13.1% resulted from intra-programmatic collaborations.

The mechanisms regulating hematopoietic stem cell (HSC) proliferation, self-renewal, and differentiation are fundamental to our understanding and treatment of a number of hematopoietic disorders, including bone marrow failure syndromes and hematopoietic malignancies. Accumulating evidences suggests that stromal cells in the bone marrow provide key signals regulating HSC, yet the nature of these signals and even the stromal cell types that comprise the stem cell niche are poorly understood. Recent studies from our laboratory show that treatment with granulocyte colony-stimulating factor (G-CSF) results in marked changes in the bone marrow microenvironment that culminate in the mobilization of HSC into the blood. In this proposal, we plan to continue this research to answer the two following questions: 1) what are the cellular components of the stem cell niche; 2) what are the signals that regulate the number and function of stem cell niche cells. This research has direct clinical relevance for stem cell transplantation, since it may lead to novel strategies to increase HSC mobilization yields and enhance HSC homing and engraftment following transplantation. Preliminary data support the hypothesis that factor(s) produced by cells of the monocyte lineage in the bone marrow provide key signals that regulate osteoblast function/survival and that expression of these factor(s) is regulated by G-CSF. In Aim 1, we will identify candidate factors produced by monocytic cells and characterize their contribution to G-CSF-induced HSC mobilization and osteoblast suppression. The identification of factors that regulate osteoblasts may lead to the development of small molecule mimetics (or antagonists) that are able to stimulate (or inhibit) osteoblast (and possibly HSC) function. The chemokine Cxcl12 provides a key signal regulating HSC quiescence, survival, and trafficking. Within the bone marrow, Cxcl12 is expressed in osteoblasts, osteoblast precursors, endothelial cells, and Cxcl12- abundant reticular (CAR) cells. In Aim 2, we will selectively delete Cxcl12 in selected stromal cel types and characterize their effect on HSC function and trafficking. To this end, we have generated transgenic mice carrying a floxed null allele of Cxcl12. These studies should provide new insight into the cellular composition of the stem cell niche and may lead to better strategies to augment HSC function in bone marrow failure syndromes or after stem cell transplantation. The following specific aims are proposed. Aim 1. We will define the contribution of bone marrow monocytes/macrophages to HSC mobilization by G- CSF. Aim 2. We will define the contribution of Cxcl12 expression by osteoblasts and endothelial cells to HSC function and trafficking.

The goal of this project is to identify genetic alterations that contribute to the pathogenesis of therapyrelated acute myeloid leukemia and myelodysplastic syndrome (t-AML/t-MDS). These disorders are emerging as a major clinical problem, now accounting for 10-20% of all new cases of AML, with a rising incidence. The available treatments for these diseases have limited efficacy, and the prognosis is very poor, highlighting the need for new therapies. However, the genetic alterations that contribute to t-AML/t- MDS pathogenesis are largely unknown, limiting the development of novel targeted therapies. Major improvements in the clinical management of patients with t-AML/t-MDS are unlikely without a better understanding of the genetic alterations contributing to t-AML/t-MDS susceptibility, transformation, and resistance to chemotherapy. To identify these genetic alterations, the following specific aims are proposed. Aim 1. We will identify novel recurring somatic mutations in t-AML/t-MDS. We propose to sequence at least 150 t-AML/t-MDS genomes over the 5-year grant period. These data will be compared with cfe novo AML/MDS to determine whether prior exposure to chemotherapy or radiotherapy affects the spectrum of somatic mutations found in the tumors. Biological studies will be performed on selected novel recurring mutations to define their contribution to leukemic transformation. Aim 2. We will characterize clonal progression in t-AML/t-MDS. We will take advantage of hematopoietic tissue banked prior to the development of t-AML/t-MDS to address the following questions: 1) does clonal hematopoiesis develop after exposure to intensive chemotherapy; 2) are certain mutations preferentially acquired early during; 3) are mutations that confer resistance to chemotherapy present in hematopoietic stem cell (HSC) clones prior to exposure to chemotherapy; and 4) in cases of t-AML/t-MDS arising after lymphoma, do these tumors share mutations, suggesting a common founder HSC clone? Aim 3. We will determine whether specific small non-coding RNAs (SncRNAs) are consistently dysregulated or mutated in t-AML/t-MDS. We propose to sequence the small RNA transcriptome in t- AML/t-MDS. This data will be use to: 1) identify sncRNAs that are dysregulated in t-AML/t-MDS relative to CD34+ progenitors from healthy donors and leukemic blasts from de novo AML, and 2) to identify novel sncRNAs and determine whether they are targets for somatic mutation in AML. Biological studies will be performed to define the contribution of selected sncRNAs to leukemic transformation.

Advocate; leukemia; Patients; programs; Research;

DESCRIPTION (provided by applicant): The Washington University SPORE in Leukemia is a highly dynamic translational cancer research program that focuses specifically on leukemias and myelodysplastic syndromes (MDS). We have assembled an outstanding group of investigators with complementary expertise in basic and clinical leukemia research. In this SPORE, we leverage expertise in cancer genomics, immunology, and hematopoiesis to develop innovative translational research in leukemia. Our long term goal is to develop novel biomarkers and treatments for leukemias and MDS and to recruit and promote innovative translational leukemia research. To achieve these goals, the following specific aims are proposed. Aim 1. To exploit recent advances in cancer genomics to develop novel biomarkers and treatments for leukemias and MDS. Washington University has been at the forefront of genomic studies in AML and MDS. Through sequencing of primary acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) genomes, we identified several novel recurring mutations of genes involved in DNA methylation (DNMT3A and IDH1) and RNA splicing (U2AF1). This basic research has led to the development of the following two translational SPORE projects. Project 1. Molecular determinants of decitabine responsiveness Project 3. Development of RNA splicing modulators for myelodysplastic syndromes and AML. Aim 2. To leverage local expertise in immunology and hematopoiesis to develop novel treatments for leukemias and myelodvsplastic syndromes. Institutional expertise in basic immunology and hematopoiesis research has led to the development of two innovative translational projects in leukemia. Project 2. Targeting the bone marrow microenvironment in acute lymphocytic leukemia. Project 4. Epigenetic modulation of graft versus host disease and graft versus leukemia. Aim 3. To enhance the infrastructure that supports translational leukemia research. This SPORE will support the following Shared Research Resources: 1) Core A. Biospecimen Processing; 2) Core B. Biostatistics; and 3) Core C. Administration. Aim 4. To recruit and train new investigators in translational research. Aim 5. To facilitate inter-SPORE collaboration.

The Administration Core will provide executive oversight and administrative support for all of the projects and cores that comprise the Leukemia SPORE. The goal of the Administration Core is to monitor the activities of all of the program components, to comply with all local and federal guideline for grant administration, and to facilitate communication and collaboration among the program members. Accordingly, the specific aims of the Administration Core are as follows: ? Aim 1. To facilitate intra- and inter-SPORE communication and collaboration. We will organize monthly SPORE meetings and the development and maintenance of a Leukemia SPORE web site. ? Aim 2. To provide administrative and fiscal oversight and support for all SPORE components. This core will be responsible for interacting with the Grants and Contracts Office at Washington University and with staff at the NCI, including preparation and submission of annual progress reports. ? Aim 3. To coordinate all SPORE-related meetings. This core will coordinate the meetings of the External Advisory Board, attendance at the annual SPORE workshop, and monthly meetings of the Leukemia SPORE Steering Committee. ? Aim 4. To coordinate administrative activities of the SPORE Developmental Research Program. This core will solicit applications for pilot projects and coordinate the review of these applications. ? Aim 5. To coordinate the SPORE Career Development Program. This core will assist in the recruitment and monitoring of candidates in this program. ? Aim 6. To assist investigators with the preparation of scholarly presentations, publications, regulatory documents, and all other SPORE-related paperwork. ? Aim 7. To enhance participation of minorities in SPORE activities. ? Aim 8. To ensure advocacy issues are addressed and included in all aspects fo research with patient participants.

? DESCRIPTION (provided by applicant): Hematopoietic stem cells (HSCs) reside in specialized microenvironments (niches) in the bone marrow. There is emerging evidence that distinct populations of mesenchymal stromal cells in the bone marrow regulate specific subsets of hematopoietic stem/progenitor cells (HSPC), including HSCs themselves. The transforming growth factor (TGF) superfamily includes TGF-?s, bone morphogenetic proteins (BMPs), growth differentiation factors (GDFs), and activins. The contribution of TGF family member signaling to the establishment and maintenance of hematopoietic niches is largely unknown. Our preliminary data show that loss of TGF-? signaling in BM stromal cells results in the disruption of the B-lymphoid niche but preserves the stem cell niche. In contrast, prior studies suggest that BMP or activin/GDF signaling in BM stromal cells contributes to the maintenance of the stem cell and erythroid niches, respectively. These observations suggest the hypothesis that different TGF family members may regulate distinct types of hematopoietic niches. There is evidence that expression of TGF family members in the bone marrow is regulated by environmental signals. Indeed, our preliminary data show that TGF-? signaling is activated in stromal cells following treatment with G-CSF. These data suggest the hypothesis that TGF family signaling in mesenchymal stromal cells is an important mechanism to modify hematopoietic niches in response to environmental stresses. Since drugs that modulate the activity of several TGF family members are in development, this research may suggest novel approaches to modulate hematopoietic niches for therapeutic benefit. Aim 1. To characterize the contribution of TGF family member signaling in the maintenance and function of mesenchymal stromal cells implicated in hematopoietic niches. We will generate mice carrying deletions of the three type II TGF receptors expressed in mesenchymal stromal cells: Tgfbr2, which mediates TFG-? signaling; Acvr2a, which mediates activin, GDF, and BMP signaling; and Bmpr2, which mediates BMP signaling. We also will postnatally delete Smad4, which is required for all canonical TGF family member signaling. In each case, we will quantify and assess the function of stromal cells implicated in hematopoietic niche maintenance, including mesenchymal stem cells, pericytes, CXCL12-abundant reticular (CAR) cells, and mature osteoblasts in the bone marrow of adult mice. Aim 2. To characterize the role of TGF family member signaling in mesenchymal stromal cells in the regulation of hematopoiesis. Here, we will characterize basal and stress hematopoiesis in the transgenic mice carrying targeted deletions of TGF receptors described in Aim 1. In particular, we will assess the contribution of TGF family member signaling in G-CSF induced HSPC mobilization and suppression of B lymphopoiesis, the stress erythropoiesis response, and hematopoietic recovery from myeloablative chemotherapy.

PROJECT SUMMARY/ABSTRACT The Administration Core will provide executive oversight and administrative support for all of the projects and cores that comprise the Leukemia SPORE. The goal of the Administration Core is to monitor the activities of all of the program components, to comply with all local and federal guideline for grant administration, and to facilitate communication and collaboration among the program members and with other Leukemia SPOREs. Accordingly, the specific aims of the Administration Core are as follows: Aim 1. To facilitate intra- and inter-SPORE communication and collaboration. Aim 2. To provide administrative and fiscal oversight and support for all SPORE components. Aim 3. To coordinate administrative activities of the SPORE Developmental Research Program. Aim 4. To coordinate the SPORE Career Enhancement Program. Aim 5. To assist investigators with the preparation of scholarly presentations, publications, regulatory documents, and all other SPORE-related paperwork. Aim 6. To enhance participation of minorities in SPORE activities. Aim 7. To ensure advocacy issues are addressed and included in all aspects of research with patient participants.

ABSTRACT The Washington University SPORE in Leukemia is a highly dynamic translational cancer research program that focuses specifically on leukemias and myelodysplastic syndromes (MDS). We have assembled an outstanding group of investigators with complementary expertise in basic and clinical leukemia research. In this SPORE, we leverage expertise in cancer genomics, immunology, and hematopoiesis to develop innovative translational research in leukemia. Our long-term goal is to develop novel biomarkers and treatments for leukemias and myelodysplastic syndromes and to develop and promote innovative translational leukemia research. To achieve these goals, the following specific aims are proposed. Aim 1. We will exploit institutional expertise in cancer genomics, immunology, and hematopoiesis to develop novel biomarkers and treatments for leukemias and myelodysplastic syndromes. Basic research at WUSM has led to the development of the following five translational research projects, all featuring innovative investigator-initiated therapeutic trials for leukemias or MDS. · Project 1. Molecular determinants of decitabine responsiveness · Project 2. Targeted therapies for T cell acute lymphoblastic leukemia (T-ALL) · Project 3. Novel therapies for splicesome-mutant MDS · Project 4. Bi-specific antibody-based therapies for AML · Project 5. Memory-like NK cell augmented hematopoietic cell transplantation for AML Aim 2. We will enhance the infrastructure that supports translational leukemia research. This SPORE will support the following Shared Research Resources: 1) Core A. Biospecimen Processing; 2) Core B. Biostatistics; and 3) Core C. Administration. Aim 3. We will recruit and train new investigators in translational research. This SPORE will support a Career Enhancement Program (CEP) to recruit and mentor new investigators in translational leukemia research. The SPORE has established a successful minority post-baccalaureate training program. The SPORE also will support a Developmental Research Program (DRP) to support innovative translational concepts. Aim 4. We will facilitate inter-SPORE collaboration. Four of the SPORE projects include multi-institutional clinical trials, including three at other Leukemia SPORE institutions. We have established CEP educational exchange and grant review programs with peer Leukemia SPORE institutions. We will continue to organize and participate in joint meetings of Leukemia SPOREs at MD Anderson and Harvard.

ABSTRACT The overall goal of the Leukemia SPORE Developmental Research Program (DRP) is to recruit and support developmental research projects in leukemia for future peer-reviewed funding and/or future independent SPORE projects. The types of studies to be supported include projects in basic research, clinical research, epidemiologic studies, and cancer prevention and control research in leukemia. Projects supported under the DRP will expand the scope of translational research and increase the number of investigators committed to leukemia research. The DRP will work in tandem with the Career Enhancement Program (CEP) to assist in the development of junior investigators and in the recruitment and mentoring of minority investigators. To accomplish these goals, the following specific aims are proposed Aim 1. To support developmental research projects in leukemia for future incorporation as full SPORE projects and for applications for other major peer-reviewed funding. New research projects will be solicited annually. A total of $300,000 has been committed per year to support the combined CEP and DRP (including $50,000 per year each for the CEP and DRP from SPORE funds and $200,000 per year of matching institutional funds). DRP awards will be for up to $70,000 for 1 year, with a competitive renewal allowed for a second year of funding allowed. Funds may be used for salary support or laboratory supplies. Aim 2. To foster collaborations between basic and clinical researchers. The DRP chairs will facilitate interaction between basic and clinical researchers through shared weekly meetings, the annual SPORE retreat, and small group meetings. Aim 3. To provide mentoring to junior faculty. All investigators submitting developmental research projects will receive a written scientific and statistical review, and the DRP chairs will be available to discuss the projects in detail. Where appropriate, mentors will be identified to work with junior faculty. All DRP awardees will present their research progress to the SPORE steering committee twice per year. Aim 4. To promote participation of women, minorities, and disabled investigators in clinical leukemia research and to promote recruitment of minorities to clinical leukemia trials.

Project 4: Genomics of TP53-mutated AML. The long-term goal of this project is to characterize mechanisms by which TP53 mutations contribute to AML progression and to exploit this knowledge to develop new therapies for this high-risk subset of AML. Mutations of TP53 are present in approximately 10% of de novo AML and 35% of therapy-related AML (tAML). Only ~25% of patients with TP53-mutated AML respond to standard induction therapy, and median survival is only approximately 4.2 months. Thus, there is a pressing unmet clinical need for new therapies for TP53-mutated AML. Due to the cytogenetic complexity and clonal heterogeneity of TP53-mutated AML, genetic and transcriptional alterations that contribute to its transformation and/or resistance to chemotherapy are largely unknown. In Aim 1, we will use single cell RNA sequencing along with phased whole genome sequencing to address this knowledge gap and to identify early transcriptional changes associated with the clonal expansion of TP53-mutated hematopoietic stem/progenitor cells (HSPCs). Our recent studies show that certain hematopoietic stressors, such as genotoxic stress or ribosome biogenesis stress, result in the selective expansion of hematopoietic stem progenitor cells (HSPCs) carrying heterozygous TP53 mutations. In Aim 2, we will test the following two hypotheses: 1) both cell-intrinsic factors and cell-extrinsic stressors contribute to the development of clonal hematopoiesis; and 2) repeated genotoxic or hematopoietic stress is important for the evolution from clonal hematopoiesis to AML. Specific Aim 1: We will identify genetic and transcriptional alterations associated with TP53-mutated AML. We will perform phased whole genome sequencing and comprehensive transcriptome sequencing, including single cell RNA sequencing, on a minimum of 50 fully annotated cases of aneuploid AML, 25 of which will carry TP53 mutations in the founding clone. These data will be analyzed to address the following questions: 1) is there a consistent pattern of gene dysregulation in TP53-mutated AML; 2) is there a consistent pattern of gene dysregulation in aneuploid AML compared to other AMLs; and 3) are there distinct structural alterations associated with TP53-mutated AML? We will perform single cell RNA sequencing on leukapheresis samples from patients with TP53-mutated clonal hematopoiesis to characterize gene expression in TP53- mutated HSPCs that do not carry other AML-relevant genetic alterations. Specific Aim 2: We will characterize the contribution of hematopoietic stressors to the development and progression of tAML/tMDS. We will use a mouse model of TP53-mutated clonal hematopoiesis to address three main questions: 1) what are the hematopoietic stressors that contribute to the expansion of TP53-mutated HSPCs or Dnmt3a-mutated HSPCs; and 2) what are the initial cooperating mutations that contribute to the clonal evolution of TP53-mutated HSPCs to tAML; and 3) what role does persistent or recurrent hematopoietic stress play in leukemic transformation?

Project Summary/Abstract The objective of this project is to utilize high dimensional single cell approaches to delineate the spatial architecture of human bone marrow. Functions of hematopoietic stems cells are known to be modulated by signals generated by stromal cells in the bone marrow. Geographic relationships between hematopoietic cells and specific stromal cell populations are thought to be a critical component of this interaction. While studies in animal models have elucidated many of these key relationships, similar studies in human bone marrow have been limited. We therefore propose to utilize imaging mass cytometry (IMC) to directly interrogate the cellular architecture of the human bone marrow microenvironment. We will couple this approach with single cell RNA sequencing to enable the identification of novel markers of specific stromal cell populations. These findings will be leveraged to further inform the IMC approach. Within this context, we will develop a road map of normal human bone marrow, which can serve as the framework for subsequent studies of both normal and diseased hematopoietic states.

The long-term goal of this of this research is to identify new genetic causes of congenital neutropenia and characterize their molecular mechanisms of disease pathogenesis. Severe congenital neutropenia (SCN) is an inborn disorder of granulopoiesis characterized by severe chronic neutropenia from birth, premature death secondary to infectious complications, and transformation to myeloid malignancy. Approximately one-third of cases do not have a known genetic cause. We performed whole exome sequencing of 85 cases of congenital neutropenia. Heterozygous missense mutations of CLPB, encoding caseinolytic peptidase B, were identified in 6 of 45 (13%) ELANE-negative SCN cases; we subsequently identified heterozygous CLPB mutations in an additional 3 cases of SCN that were not part of our original cohort. CLPB encodes for caseinolytic peptidase B, an ATPase implicated in protein folding and mitochondrial function. Prior studies showed that biallelic mutations of CLPB are associated with a syndrome of 3-methylglutaconic aciduria, cataracts, neurologic disease, and variable neutropenia. However, these mutations are distinct from those seen in our series, which are heterozygous and cluster near the ATP binding pocket. Preliminary data show that CRISPR-Cas9 gene editing to inactivate CLPB or lentiviral-mediated overexpression of mutant CLPB in human CD34+ cells results in impaired granulocytic differentiation. Collectively, these data suggest that heterozygous mutations of CLPB are a new and relatively common cause of SCN. In this proposal, we will examine molecular mechanisms by which mutant CLPB disrupts granulopoiesis. We also will identify and biologically validate, using a similar approach, other potential neutropenia-causing gene mutations identified by current and ongoing sequencing of congenital neutropenia cases. The following specific aims are proposed. Aim 1: To define the spectrum of CLPB mutations that disrupt granulopoiesis. We will use lentiviral- mediated overexpression in human CD34+ cells to systematically assess the impact on granulopoiesis of CLPB mutations identified in patients with congenital neutropenia, including the biallelic mutations found in syndromic cases. Aim 2. To characterize mechanisms by which mutations of CLPB disrupt granulopoiesis. We will test the hypothesis that mutant CLPB acts in a dominant fashion to disrupt the chaperone function of CLPB, resulting in impaired activation of the mitochondrial unfolded protein response (UPRMT) and induction of apoptosis in granulocytic precursors. Aim 3. To identify and validate new genetic causes of congenital neutropenia. We will perform exome sequencing of at least 100 additional cases of congenital neutropenia with no known cause. These data will be interrogated to identify candidate genes for functional validation.