Timothy J. Ley - US grants

The long term goal of this project is to define the molecular mechanisms that control the switch from fetal (gamma) to adult (beta) globin synthesis; this switch is controlled at the level of globin gene transcription. Individuals with Hereditary Persistence of Fetal Hemoglobin (HPFH) continue to express gamma-globin genes in adult RBCs, and frequently have mutations just upstream from the gamma-globin genes that probably cause the HPFH phenotype. We intend to use these mutations to define the cis-acting DNA sequences and trans-regulatory factors that control gamma- globin transcription in erythroid cells, as follows: 1) We will define the cis-acting DNA sequences in the human beta-globin gene cluster that silence the gamma-globin gene in mouse erythroleukemia (MEL) cells. gamma-globin genes are minimally expressed in "adult" erythroid MEL cells when introduced on whole chromosomes or cosmids. We will define the sequences that normally silence gamma-globin transcription in MEL cells using stable transfection techniques, and will determine whether these elements interact with HPFH mutations. 2) We will further define an S1 nuclease hypersensitive site (S1-HSS) in the -210 region of the gamma-globin promoter, and define how this site is altered by HPFH mutations. The region from -216 to -208 contains an S1-HSS in supercoiled plasmids; this site is destabilized by some HPFH mutations. We will define the sequences that contribute to the formation of the S1-HSS with site-directed mutations, and will examine the relevance of the site for the binding of potential regulatory factors. 3) We will characterize trans-acting factors that discriminate between wild-type and HPFH-associated gamma-globin alleles. We have identified a MEL cell protein(s) that binds with increased affinity to a gamma-globin allele with the -202 c -> G HPFH substitution. This protein will be characterized, purified, and cloned. A newly region will be further characterized. Functional analysis of this cloned protein will examine its relevance for globin gene switching. 4) We will further characterize the structure and function of the murine beta-globin locus control region (LCR). The region upstream from murine beta-globin LCR 5' HS-2 will be sequenced and examined with a variety of functional assays. Structure/function relationships with the human beta- globin LCR will be established.

The long term goal of this project is to define the molecular mechanisms that control the switch from fetal (gamma) to adult (beta) globin synthesis; this switch is controlled at the level of globin gene transcription. Individuals with Hereditary Persistence of Fetal Hemoglobin (HPFH) continue to express gamma-globin genes in adult RBCs, and frequently have mutations just upstream from the gamma-globin genes that probably cause the HPFH phenotype. We intend to use these mutations to define the cis-acting DNA sequences and trans-regulatory factors that control gamma- globin transcription in erythroid cells, as follows: 1) We will define the cis-acting DNA sequences in the human beta-globin gene cluster that silence the gamma-globin gene in mouse erythroleukemia (MEL) cells. gamma-globin genes are minimally expressed in "adult" erythroid MEL cells when introduced on whole chromosomes or cosmids. We will define the sequences that normally silence gamma-globin transcription in MEL cells using stable transfection techniques, and will determine whether these elements interact with HPFH mutations. 2) We will further define an S1 nuclease hypersensitive site (S1-HSS) in the -210 region of the gamma-globin promoter, and define how this site is altered by HPFH mutations. The region from -216 to -208 contains an S1-HSS in supercoiled plasmids; this site is destabilized by some HPFH mutations. We will define the sequences that contribute to the formation of the S1-HSS with site-directed mutations, and will examine the relevance of the site for the binding of potential regulatory factors. 3) We will characterize trans-acting factors that discriminate between wild-type and HPFH-associated gamma-globin alleles. We have identified a MEL cell protein(s) that binds with increased affinity to a gamma-globin allele with the -202 c -> G HPFH substitution. This protein will be characterized, purified, and cloned. A newly region will be further characterized. Functional analysis of this cloned protein will examine its relevance for globin gene switching. 4) We will further characterize the structure and function of the murine beta-globin locus control region (LCR). The region upstream from murine beta-globin LCR 5' HS-2 will be sequenced and examined with a variety of functional assays. Structure/function relationships with the human beta- globin LCR will be established.

The long-term goals of this proposal are to define the molecular events that control the promyelocyte-specific "program" of normal and leukemic cells, and to define the functions of the promyelocyte-specific enzyme cathepsin G (CG). Our laboratory has determined that the murine and human CG genes are expressed only in promyelocytes; cis-acting sequences within or near the human CG gene contain the promyelocyte-targeting signals. We intend to use this information to further characterize the biology of promyelocytes, via the following Specific Aims: 1. We will define the regulatory DNA sequences that target expression of CG to promyelocytes in transgenic mice. We have shown that a 6.0 kb fragment containing the human CG gene is targeted specifically to myeloid precursors in transgenic mice. We will dissect the sequences within this fragment to define the promyelocyte targeting elements, and identify proteins that interact specifically with these elements. 2. We will study the biology of acute promyelocytic leukemia (APL) by targeting PML-RARalpha and RARalpha-PML cDNAs to murine promyelocytes with CG-derived targeting sequences. We have created a novel targeting vector containing all of the sequences from the 6.0 kb CG transgene. We will use an E2A-PBX-1 cDNA to confirm the accuracy of the targeting vector, an to determine whether promyelocytes can be transformed in vivo. We will then target cDNAs created by the t(15;17) translocation associated with APL. A PML-RARalpha cDNA, and a reciprocal RARalpha-PML cDNA will be individually targeted to promyelocytes in transgenic mice to determine whether either of these cDNAs is capable of causing a block in promyelocyte differentiation or overt APL. Expressing transgenic lines will be bred to one another to determine whether the two transgenes synergize to produce APL. The transgenic animals will be carefully characterized to create models for the study of this disease. 3. We will create a loss of function model for murine CG. We will use standard strategies to create a targeted mutation in the murine CG gene in embryonic stem cells, and crate CG-/-mice. These mice will be examined for defects in neutrophil and macrophage functions in vitro and in vivo. CG may play a role in causing the DIC syndrome associated with human APL; if the "APL" mice defined above develop DIC, we will breed them into the CG-/- background to further define this role.

The Embryonic Stem Cell (ES) core is designed to improve the efficiency of the Program Project Laboratories for all phases of ES cell work. This core will facilitate the production of targeted ES cell lines, will assist laboratories in their production of chimeric animals and mice with germline mutations in targeted alleles, and will explore the use of embryonic stem cells as models of hematopoietic differentiation. The objectives of this core are listed below: 1. The core will serve as a repository for all characterized ES cell lines. 2. The core will produce and distribute mouse embryo fibroblasts (MEFs) from normal and transgenic neo-resistant mice. 3. The ES core is responsible for injecting targeted ES cell lines into blastocysts for the production of chimeric mice. 4. The ES core and transgenic cores are responsible for the breeding programs of chimeric mice to establish germline transmission of target alle s. 5. The ES core will create and characterize new embryonic stem cell lines. These lines are being produced for use in gene targeting experiments, and will improve the efficiency of the core by reducing the number of blastocyst injections required for each targeted ES line.

One of the long term goals of this laboratory is to develop genetically-based strategies for the treatment of sickle cell anemia and beta-thalassemia. The goal of this study is to determine whether homologous recombination can be developed as a strategy to repair mutant beta-globin genes in embryonic stem cells and primary hematopoietic progenitors. To accomplish these goals, we propose the following specific aims. Specific Aim 1: We will develop mice with a mutation similar to the betas globin mutation of humans by creating a beta6 mutation in the mouse beta-major globin gene using homologous recombination in embryonic stem cells. We will create embryonic stem cells lines that contain a mutation that causes an alanine yields isoleucine substitution in position 6 of the murine beta-major globin gene (beta6I), and that have a selectable marker cassette (PGK-neo) either retained or excised (via Cre-Lox mediated recombination) downstream from beta-major. These ES cells will be the starting material for Specific Aim 2, and will be used to make mice that bear the mutations. The hematopoietic cells of heterozygous mice with beta6I (and the excised PGK-neo cassette) form the starting material for Specific Aim 3. Specific Aim 2: We will define the efficiency of homologous recombination-mediated repair of the beta6 mutation in embryonic stem cells using targeting vectors of different sizes. To explore the relationship of targeting arm size and homologous recombination efficiency, we will create targeting vectors that contain a total of 8, 16, 60, or 110 kb of wild-type targeting DNA from the mouse beta-globin cluster, and compare the abilities of these vectors to correct the beta6I mutation in ES cells via homologous recombination. Specific Aim 3: We will determine whether hematopoietic progenitors have the ability to correct the beta6I mutation via homologous recombination, using the targeting vectors defined in Specific Aim 2. Functional targeting vectors defined in Specific Aim 2 will be used to determine whether hematopoietic progenitors and/or stem cells have the machinery to perform homologous recombination events within the beta-globin locus. Bone marrow cells purified from mice heterozygous for the beta6I mutation (and PGK-neo deleted) will be transfected with the targeting vectors using physical means of DNA delivery (i.e. electroporation or lipofection). These cells will be selected using either neomycin phosphotransferase or GFP expression (or both), and individual colonies derived from hematopoietic progenitors (LTC-IC and CFU-C) will be analyzed for the frequency of correction of the beta6I mutation using PCR-based techniques. These studies should allow us to determine whether homologous recombination can be rationally developed as a method for correcting mutations in the beta-globin locus within primary hematopoietic progenitor cells.

The long-range goals of the program Project are to elucidate and characterize basic mechanisms that regulate growth related and developmentally related genes as they are normally expressed or inappropriately expressed in neoplastic cells. Substantial progress towards these goals has been achieved with the Program Project in years 01- 04. Cancer may be characterized by the dysregulated growth of cells which phenotypically fail to recapitulate normal differentiation and development; it may also result from a failure of programmed (apoptotic) cell death. This Program Project proposes to analyze the normal and abnormal regulation of growth and development through analyses of different but highly related genetically regulated pathways, through analysis of the sequences and interactive proteins that regulate essential genes within these pathways, and through analysis of loss and gain of function of these genes in mice and embryonic stem (ES) cells. The strategy is common to each of the projects and extends from an in-depth analysis of the regulation of transcription of these genes and the factors that mediate this regulation, the identification and characterization of DNA elements that mediate tissue and developmental stage specific expression, to the use of these genes and regulatory elements to modify lineage and temporal specific expression of growth and development by introduction of them in cells, transgenic mice, and loss of function mice through targeted disruption of these genes. It is hoped to test the hypothesis that understanding these normal and abnormal regulatory mechanisms will lead to logical approaches for the treatment of neoplastic disease in man. We will focus on growth factor genes, genes that appear to direct differentiation functions, genes that function to prevent cell death (apoptosis), genes that encode transcription factors that are activated by growth factors and that direct nuclear signals to program growth factor signals, and highly developmentally regulated genes that are expressed in a lineage specific manner. The tools of molecular biology, cell biology, biochemistry, and immunology will be used. cDNA and genomic cloning, DNA sequencing, identification of cis- acting elements, identification and purification of transacting transcriptional proteins, transcriptional analyses, in situ hybridization and immunoperoxidase analyses, both gain of function and loss of function experiments in the transgenic mouse and in heterozygous and homozygously targeted loss of function mice are planned. Common themes of research interests and goals, technologies, and reagents exist to advance the progress of the Program Project. Core programs with an interactive program of informal and formal meetings will facilitate research and the generation of new ideas and thus to advance the goals of the Program Project. Informal and formal review will ensure orderly and optimum advancement of the research goals. The collective goals, themes, and interactive programs ensure progress to a level that is not achievable without the context of a program project.

(adapted from abstract) The long term goal of this project is to fully characterize the effector mechanisms used by cytotoxic lymphocytes to kill target cells, and to develop strategies to inhibit the activity of the cytotoxic cells in disease states like graft versus host disease, while preserving ability to kill virus infected cells and tumor cells. In this study they will define the roles of granzymes (gzms) A-G for the induction of target cell apoptosis, and define the cytotoxic potential of cells that express granzyme B. To accomplish these goals, they propose the following specific aims: 1) To create new loss-of-function murine models for gzms B-G and to define contributions to cell-mediated cytotoxicity in vitro and in vivo. They will use homologous recombination in embryonic stem cells to create mutant mice that lack gzm B only, that lack the entire gzm B-G cluster, or that lack only gzms C-G. These mice will be intercrossed with granzyme A-/- mice to create mice doubly deficient for the gzm A and B. They will characterize and compare cytotoxic lymphocyte functions in these mice, using in vitro and in vivo models of acute graft versus host disease (GvHD), viral clearance and tumor clearance. 2) They will define the biology and cytotoxic repertoire of lymphocytes that express gzm B. They will utilize homologous recombination in ES cells to make mutant mice that contain an enhanced green fluorescent protein (GFP) cassette in the 5' untranslated (UT) region on the murine gzm B gene. They will analyze and purify GFP+ lymphocytes from these mice using flow cytometric methods, and study the biologic properties of GFP+ cells (i.e., those expressing gzm B) that have been activated in vitro and in vivo. 3) They will create mice that are deficient for gzm B-expressing cells to determine the role that these cells play in GvHD, viral clearance, and tumor clearance. They will utilize homologous recombination in ES cells to make mice that contain an attenuated diphtheria toxin cDNA (or herpes simplex virus thymidine kinase gene) in the 5' UT of the murine gzm B gene. If these mice delete all gzm B-expressing cells, they will be able to fully evaluate the role that this cellular compartment plays in acute GvHD, viral clearance and tumor clearance.

The long term goal of this work is to create effective therapies for patients with acute promyelocytic leukemia (APML) who have failed conventional treatment protocols. To accomplish this goal, we will attempt to better understand the biology and immunology of APML by refining and expanding our "first generation" transgenic mouse model of APML as follows: Specific Aim 1. We will develop a "knock-in" system for inserting novel cDNAs into the mouse cathepsin G (CG) locus, and precisely define the myeloid progenitor/precursor compartment in which targeted transgenes first are expressed. We will target an enhanced green fluorescent protein (eGFP) cassette into the murine CG 5' untranslated region (UTR) using homologous recombination, and create transgenic animals bearing this mutation. We will sort GFP positive and negative cells using flow cytometry, and examine these populations for their ability to form hematopoietic colonies in vitro and reconstitute hematopoiesis in vivo. Specific Aim 2. We will create and analyze mice that express both PML- RARalpha and a GFP "tag" in the same early myeloid cells, so that the biology and immunology of these cells can be characterized. We will use homologous recombination to insert a breakpoint 1-derived PML- RARalpha cDNA into the 5-HTR of the cathepsin expressing PML- RARalpha will therefore carry a fluorescent tag that will permit their purification and biological characterization. We will characterize the latency, penetrance, and phenotype of tumors developing in these animals, and characterize them immunologically in Specific Aim 4. Specific Aim 3. We will create and analyze mice that express both RARalpha-PML and a GFP tag in early myeloid cells, and intercross them with the PML-RARalpha mice of Specific Aim 2. We will insert a reciprocal breakpoint 1 RARalpha-PML-IRES-eGFP cDNA into the murine cathepsin G 5'UTR, and define the phenotype of expressing cells. We will intercross these mice with animals containing the cathepsin G targeted PML-RARalpha- cDNA, and compare the latency, penetrance, and phenotype of tumors arising in PML-RARalpha, RARalpha-PML, or double transgenic mice. Specific Aim 4. We will determine whether tumor specific immunity can be developed against APML cells derived from the mouse models. Our preliminary data suggest that immunity can be induced in vivo against APML tumors with a plasmid that expresses PML-RARalpha. In this Aim, we will further explore the type of immunity conferred by DNA vaccines, and will attempt to determine whether immunity can be developed against peptides spanning the PML-RARalpha-PML breakpoint regions.

One of the long term goals of this laboratory is to develop genetically-based strategies for the treatment of sickle cell anemia and beta-thalassemia. The goal of this study is to determine whether homologous recombination can be developed as a strategy to repair mutant beta-globin genes in embryonic stem cells and primary hematopoietic progenitors. To accomplish these goals, we propose the following specific aims. Specific Aim 1: We will develop mice with a mutation similar to the betas globin mutation of humans by creating a beta6 mutation in the mouse beta-major globin gene using homologous recombination in embryonic stem cells. We will create embryonic stem cells lines that contain a mutation that causes an alanine yields isoleucine substitution in position 6 of the murine beta-major globin gene (beta6I), and that have a selectable marker cassette (PGK-neo) either retained or excised (via Cre-Lox mediated recombination) downstream from beta-major. These ES cells will be the starting material for Specific Aim 2, and will be used to make mice that bear the mutations. The hematopoietic cells of heterozygous mice with beta6I (and the excised PGK-neo cassette) form the starting material for Specific Aim 3. Specific Aim 2: We will define the efficiency of homologous recombination-mediated repair of the beta6 mutation in embryonic stem cells using targeting vectors of different sizes. To explore the relationship of targeting arm size and homologous recombination efficiency, we will create targeting vectors that contain a total of 8, 16, 60, or 110 kb of wild-type targeting DNA from the mouse beta-globin cluster, and compare the abilities of these vectors to correct the beta6I mutation in ES cells via homologous recombination. Specific Aim 3: We will determine whether hematopoietic progenitors have the ability to correct the beta6I mutation via homologous recombination, using the targeting vectors defined in Specific Aim 2. Functional targeting vectors defined in Specific Aim 2 will be used to determine whether hematopoietic progenitors and/or stem cells have the machinery to perform homologous recombination events within the beta-globin locus. Bone marrow cells purified from mice heterozygous for the beta6I mutation (and PGK-neo deleted) will be transfected with the targeting vectors using physical means of DNA delivery (i.e. electroporation or lipofection). These cells will be selected using either neomycin phosphotransferase or GFP expression (or both), and individual colonies derived from hematopoietic progenitors (LTC-IC and CFU-C) will be analyzed for the frequency of correction of the beta6I mutation using PCR-based techniques. These studies should allow us to determine whether homologous recombination can be rationally developed as a method for correcting mutations in the beta-globin locus within primary hematopoietic progenitor cells.

DESCRIPTION (provided by applicant): The long term goal of the Genomics of AML (GAML) Program Project is to discover all the genetic alterations that occur in the genomes of AML cells derived from adult patients, and to validate the importance of these alterations for disease susceptibility, initiation, progression, relapse, and resistance. The short-term goal is to define the most frequently occurring genetic changes that affect clinical outcomes, since these are the ones most likely to have an impact on therapy. We will use a variety of experimental platforms to define these genetic changes, including routine cytogenetics/FISH, array-based comparative genomic hybridization (CGH), RNA profiling, and high-throughput DNA resequencing. All of the exons of approximately 450 selected genes will be resequenced in a "discovery set" of 47 patients with FAB subtypes 0-4, and all changes will be evaluated in the germline to see whether they are acquired. A second discovery project will comprehensively analyze the genes of chromosome 7, since loss of part or all of this chromosome occurs frequently in several malignant myeloid diseases. Using a "validation set" of 94 AML samples with mature clinical data from the CALGB, we will attempt to rapidly define the genetic changes that alter clinical outcomes. The importance of genetic changes will also be verified in the Project Laboratories using biochemical, cellular, and murine model systems. Mouse models of AML will be used not only to identify genes for resequencing in humans, but they will be used to verify the importance of mutations discovered by resequencing human samples. Our group is a unique position to perform these studies because of key components already in place at Washington University Medical School, including the Siteman Cancer Center, the Genome Sequencing Center, the Stem Cell Transplant/Leukemia Program, and an extensive AML mouse-modeling program. We will use information gained from these studies to create molecular diagnostic tools for disease stratification, and we will identify candidate genes for targeted therapeutics. The identification of the mutations that occur in the genomes of AML cells will contribute greatly to our understanding of the pathogenesis of this disease, and we hope that it will ultimately improve our ability to cure these patients.

The Embryonic Stem Cell Core (ESC Core) is responsible for: . creating and providing state-of-the-art reagents for the production of targeted mutations in embryonic stem cells. . creating and maintaining quality controlled embryonic stem cell lines from a variety of mouse backgrounds. . teaching the methodology of embryonic stem cell culture and manipulation. The ESC Core also provides consultative services for Siteman Cancer Center (SCC) members when they are designing knock-out or knock-in vectors, and it creates and distributes user-friendly plasmids that facilitate the generation of targeting vectors. The ESC Core also attempts to assist all users with blastocyst injections if injection facilities are not available to that laboratory. By providing a comprehensive service, the ESC Core facilitates the production of gain-of-function and loss-of-function mouse models for SCC faculty.

DESCRIPTION (provided by applicant): The goal of this project is to understand the molecular mechanisms that underlie the pathogenesis of acute promyelocytic leukemia (APL). When PML-RARalpha is expressed in the early myeloid cells of mice, all animals develop myeloproliferative disease, and some develop APL after a long latent period, which suggests that additional mutations are required for APL to progress. Recently, we have learned that APL penetrance is heavily influenced by 1) neutrophil elastase (NE)-induced cleavage of PML-RARalpha and 2) the expression level of PML-RARalpha. To further address the issues raised by these observations, we propose the following Specific Aims: Specific Aim 1: We will create and analyze mice that express an NE-resistant PML-RARalpha cDNA in early myeloid cells. To determine whether the cleavage of PML-RARalpha by NE is relevant for leukemia development, we have created a mutant PML-RARalpha that is minimally cleaved by NE. This cDNA will be targeted to the murine cathepsin G locus using the identical strategy used to make the mCGPR/+ mouse. Mice will be followed for the development of APL, and carefully compared to mCGPR/+ mice expressing wild-type PML-RARalpha. Specific Aim 2: We will create and analyze mice containing a PML-RARalpha cDNA that is conditionally expressed in early myeloid cells under control of the murine PML locus. PML-RARalpha will be targeted to the murine PML 5'UT just downstream from a Lox-stop-Lox cassette. PML-RARalpha will be activated in mice by a cathepsin G-driven Cre transgene. This strategy should limit PML-RARalpha expression to the early myeloid compartment, with expression levels that closely resemble the "physiologic" dose in t(15;17) APL cells. The phenotype of these mice will be carefully compared to our previous models. Specific Aim 3: We will use qenomic approaches to identify genetic events that contribute to APL initiation and progression. High-density BAC arrays will be used in comparative genomic hybridization studies to identify gene copy number changes that may be relevant for APL progression. Affymetrix-based arrays will be used to study the RNA expression profiles of normal and APL promyelocytes. Candidate genes identified by these studies will be resequenced in human AML samples and validated in mouse models.

DESCRIPTION (provided by applicant): The long term goal of this project is to understand the molecular and cellular mechanisms by which cytotoxic lymphocytes (CL) kill their target cells in health (eg. viral and tumor clearance) and disease (eg. Graft vs. Host Disease, and autoimmune states). CL can kill by secreting the contents of their cytotoxic granules (including perforin and granzymes) onto the surface of target cells via the "secretory synapse." Perforin is responsible for delivering and/or trafficking the granzymes, which induce target cell death by cleaving a variety of substrates. Granzymes A and B induce cell death via non-overlapping pathways, but several "orphan" granzymes are expressed in both mice and humans, and may be relevant for specific CL functions. We have made (or are currently making) pure 129/SvJ mice deficient for granzymes A, B, C (and all combinations thereof), and perforin. With this unique set of reagents, we will further examine granzyme activities and substrates via the following specific aims: Specific Aim 1: We will define the roles of individual granzymes for perforin-mediated cytotoxity in vivo. Perforin is required for the clearance of many tumors and viruses, and for CDS8+ mediated GvHD, but the roles of individual granzymes in these processes remains extremely controversial. We will use the precisely strain-matched mice described above to better define the roles of these granzymes for CD8+ and NK mediated killing in vitro and in vivo. Specific Aim 2: We will define the role of the perforin/qranzyme pathway for CD4+ mediated immune regulation in mice. We have shown that human regulatory T cells contain perforin and granzymes, and that they can kill autologous immune targets in a perforin-dependent fashion. We will use precisely strainmatched 129/SvJ mice deficient for perforin or granzymes to assess the importance of these molecules for T regulatory cell function in vitro and in vivo. Specific Aim 3: We will use proteomic approaches to define the substrates of qranzymes. We will use 2-D differential in-gel electrophoresis (2D-DIGE) and mass spectrometry to identify the substrates and cleavage products of granzymes A, B, and C. Novel substrates will be evaluated for their importance in mediating granzyme-induced death in vitro and in vivo.

The long-term goal of this project is to integrate the analysis of all genomic studies of the GAML PPG to identify and validate acquired genetic changes that may contribute to the pathogenesis of AML. Somatic mutations that are predicted to change the function of a gene will be analyzed to assess their consequences on patient outcomes, on patterns of gene expression, and on AML-relevant pathways. To identify these mutations, high-resolution genomic screens and large scale resequencing studies will be required. In the GAML PPG, several cores and projects are generating genome-wide databases that must be carefully coanalyzed to identify candidate genes for resequencing and validation studies. These databases also must be mined to define relationships between the mutations and the biological pathways that they affect. To achieve these goals, we propose these Specific Aims: Specific Aim 1: We will analyze the outputs of multiple genomic screens to prioritize candidate genes for resequencing, and we will define and validate somatic mutations in AML samples. Exonbased resequencing studies (Core D) are difficult and expensive to perform, and candidate genes for these studies must therefore be carefully prioritized using data generated by high-resolution genomic screens. Array-based gene expression profiling, high resolution array-based comparative genomic hybridization, and high resolution array-based SNP genotyping studies will be used to identify genes and/or loci that are deleted, amplified, or duplicated from one parental allele (uniparental disomy), or that have aberrant patterns of expression;whole genome resequencing studies of 10 M1 AML genomes (Project 1) will also be analyzed to define potentially important mutations that will be validated using the 94 matched tumor-germline AML samples from the Discovery Set. When potentially important somatic mutations are identified, we will perform additional studies to verify the mutation and its frequency in the AML sample of interest (i.e. bacterial cloning and resequencing of the mutant exon in 96 clones from the sample), and we will further define the mutation's frequency in 94 fully annotated AML cases obtained from Cancer and Leukemia Group B (CALGB). Specific Aim 2: We will define the clinical and gene expression consequences of validated somatic mutations, and define biologic pathways altered by these mutations. We will use statistical approaches to define the effects of mutations on clinical outcomes. Novel informatics approaches (e.g. promoter analyses, pathway/interaction network construction, etc.) will be used to define the effects of mutations on patterns of gene expression, and to identify potentially relevant biological pathways that are affected by AML mutations. These algorithms will be used to identify additional genes for resequencing studies. Selected mutations and pathways identified by these studies will be biologically validated in the laboratories of PPG members.

AML - Acute Myeloid Leukemia; Collaborations; Communication; Genomics; Goals; Grant; Guidelines; Investigators; Leukemia, Myelocytic, Acute; Monitor; Myeloblastic Leukemia, Acute; Myelogenous Leukemia, Acute; P01 Mechanism; P01 Program; Program Project Grant; Program Research Project Grants; Programs (PT); Programs [Publication Type]; Research Personnel; Research Program Projects; Researchers; Visit; acute granulocytic leukemia; acute myeloid leukemia; acute nonlymphocytic leukemia; member; programs

DESCRIPTION (provided by applicant): The long-term goals of the "Genomics of AML" PPG are to define the genetic changes responsible for the development of acute myeloid leukemia in order to create improved molecular tools for diagnosis and disease stratification, and to identify new candidate genes for targeted therapeutic approaches. We intend to identify somatic mutations that are responsible for the initiation and progression of disease (Projects 1, 2, and 4 and Core C), and the genetic changes associated with relapse and chemotherapeutic resistance (Project 3). We also intend to identify mechanisms leading to increased AML susceptibility in patients who have received alkylator therapy (Project 5). To accomplish these aims, pathologic material and clinical data from AML patients is collected in Core A, and patient samples are banked and subjected to array-based genomic screens in Core B. To discover all of the mutations in AML genomes in an unbiased fashion, we proposed to sequence the entire genomes of the AML cells and normal skin cells from 10 individuals with FAB M1 AML at the renewal of the grant (Project 1). We have now accomplished this goal for 1 case (Nature 456:66-72, 2008), and will finish a second and third M1 AML genome during year 1 of the grant cycle. Because of remarkable improvements in the cost and quality of DNA sequence obtained with 'next generation'sequencing platforms, we request $900,000 (300K/year) in supplemental funds in years 2-4 to further accelerate the work in Project 1. If supplemental funds are granted, we will sequence 7 new cytogenetically normal AML M1 genomes in year 2. In year 3, we will be able to sequence 10 total M3 AML genomes bearing t(15;17) as the sole cytogenetic abnormality. Since the PML-RARA fusion protein caused by this translocation is known to initiate M3 AML, we will be able to contrast the kinds of mutations found in these two very distinct, very well defined AML subtypes. The AML genomes selected for year 4 will be directed by the results of years 2 and 3, and could potentially involve dozens of additional carefully selected cases if costs continue to fall. All of the DNA samples required for the study are currently available and consented for whole genome sequencing.

The long-term goal of this project is to use Homology Directed Repair (HDR) induced by meganucleases to correct mutations in the mouse β-glucuronidase gene and the human β-globin gene in induced Pluripotent Stem (iPS) cells. HDR is achieved by creating a double stranded break (DSB) near a mutation of interest with a site-specific meganuclease, followed by repair with a wild-type [unreadable]correcting DNA[unreadable]. Two classes of meganucleases (zinc-finger and homing) are currently being evaluated for their ability to induce site-specific DSBs, but these enzyme classes have not been directly compared for their ability to induce repair. Although meganuclease-initiated HDR is thought to be more efficient than traditional homologous recombination approaches, these enzymes carry a risk of [unreadable]off-target[unreadable] DNA cleavage events and the creation of additional mutations. To address these issues, we propose the following Specific Aims: Specific Aim 1: We will use homology directed repair to correct the point mutation that causes murine Mucopolysaccaridosis Type VII (MPS VII). We have already obtained 6 different homing meganucleases that specifically cleave at or near the point mutation that inactivates the mouse β-glucuronidase (Gusb) gene, causing MPS VII. ES cells, iPS cells, and bone marrow cells derived from Gusb deficient mice will be transfected with cDNAs encoding each of the meganucleases, and quantitatively assessed for restoration of enzyme activity using a highly sensitive flow-based assay for β-glucuronidase activity. Corrected cells will be transferred to Gusb deficient mice to determine whether they can mitigate the disease phenotype. Specific Aim 2: We will use homology directed repair to correct the point mutation that causes sickle cell anemia. We have already obtained 6 different homing meganucleases and 1 zinc-finger meganuclease that cleave within the human β-globin gene. We will transfect the cDNAs encoding these enzymes into iPS cell lines derived from sickle cell patients, and assess HDR efficiency for each. Hematopoietic progenitors obtained from the corrected iPS cells will be tested for their ability to produce functional red blood cells. Specific Aim 3: We will completely sequence the genomes of iPS cell lines that have undergone homology directed repair initiated by meganucleases. We will use [unreadable]next generation[unreadable] DNA sequencing to produce 8x haploid coverage of iPS cells derived from Gusb deficient mice, and gene-corrected subclones from these lines. Similarly, we will generate 25x haploid coverage of the primary fibroblasts, iPS lines, and gene-corrected iPS lines from each of several sickle cell anemia patients. We will define the mutations that were generated during the creation of the iPS lines, and subsequent correction of the Gusb or β-globin mutations. These studies will be essential for understanding the potential risks of using gene-corrected iPS cells for human therapeutic trials.

The Embryonic Stem Cell (ESC) Core is responsible for: ¿ Creating and providing state-of-the-art reagents for the production of targeted mutations in murine embryonic stem cells. ¿ Creating and maintaining quality-controlled embryonic stem cell lines from a variety of mouse backgrounds. ¿ Teaching the methodology of embryonic stem cell culture and manipulation. The ESC Core provides consultative services for Siteman Cancer Center members when they are designing knock-out or knock-in vectors, and it creates and distributes user-friendly plasmids that facilitate the generation of targeting vectors. By providing a comprehensive service, the ESC Core facilitates the production of gain-offunction and loss-of-function mouse models for Siteman Cancer Center faculty.

The long-term goals ofthe "Genomics of AML" PPG are to define the genetic changes responsible forthe developmentof acute myeloid leukemia in order to create improved molecular tools for diagnosis and disease stratification, and to identify new candidate genes for targeted therapeutic approaches. We intend to identify somatic mutations that are responsible forthe initiation and progression of disease (Projects 1, 2, and 4 and Core C), and the genetic changes associated with relapse and chemotherapeutic resistance (Project 3). We also intend to identify mechanisms leading to increased AML susceptibility in patients who have received alkylator therapy (Project 5). To accomplish these aims, pathologic material and clinical data from AML patients is collected in Core A, and patient samples are banked and subjected to array-based genomic screens ih Core B, To discover all ofthe mutations in AML genomes in an unbiased fashion, we proposed to sequence the entire genomes of the AML cells and normal skin cells from 10 individuals with FAB Ml AML at the renewal ofthe grant (Project 1). We have now accomplished this goal for 1 case (Nature 456:66-72, 2008), and will finish a second and third M1 AML genome during year 1 ofthe grant cycle. Because of remarkable improvements in the cost and quality of DNA sequence obtained with 'next generation'sequencing platforms, we request $900,000 (SOOK/year) in supplemental funds in years 2-4 to further accelerate the work in Project 1. If supplemental funds are granted, we will sequence 7,new cytogenetically normal AML Ml genomes in year 2. In year 3, we will be able to sequence 10 total M3 AML genomes bearing t(15;17) as the sole cytogenetic abnormality. Since the PML-RARA fusion protein caused by this translocation is known to initiate M3 AML, we will be able to contrast the kinds of mutations found in these two very distinct, very well defined AML subtypes. The AML genomes selected for year 4 will be directed by the results of years 2 and 3, and could potentially involve dozens of additional carefully selected cases if costs continue to fall. All of the DNA samples required for the study are currently available and consented for whole genome sequencing

DESCRIPTION (provided by applicant): The long-term goal of this proposal is to define the role of DNMT3A mutations for the pathogenesis of acute myeloid leukemia (AML), so that rational targeted therapies can be designed to counteract these mutations. DNMT3A (which encodes a de novo DNA methyltransferase) is among the most commonly mutated genes in AML (22% of all patients, and 34% of intermediate risk). Most mutations are missense, and the most common of these (60% of all cases) occurs at amino acid R882. However, many AML genomes have loss-of-function mutations in DNMT3A that are reminiscent of inactivating mutations in classical tumor suppressors. All DNMT3A mutations are associated with adverse overall survival. It is critical to understand whether the common DMNT3A mutations (especially at R882) are acting as gain-of-function or loss-of-function alleles, since this information will influence approaches to targeted therapies. We will assess the biochemical consequences of DNMT3A mutations in Aim 1, and the in vivo consequences in Aims 2 and 3, as follows: Specific Aim 1: We will define how AML-associated mutations alter DNMT3A functions in vitro. Recombinant DNMT3A protein with R882 (and other) mutations will be produced in E. coli, and tested for alterations in cytosine methylase activity and specificity, and binding to other cellular proteins. We will determine whether specific mutations cause loss-of-function or gain-of-function properties (and/or have dominant negative activities), and use this information to select additional mutations for study in vivo. Specific Aim 2: We will determine whether the DNMT3A R882H mutation can contribute to AML pathogenesis in vivo. Using retroviral vectors with fluorescent tags, we will express WT and mutant R882H DNMT3A cDNAs in murine bone marrow cells. Transduced cells will be evaluated in colony assays and mice to assess the leukemogenic potential of DNMT3A mutations alone or in combination with other mutations that commonly occur with DNMT3A mutations (e.g. FLT3 ITD, IDH1 R132H, NPMc). Using homologous recombination in ES cells, we will create mice with the R882H mutation at the identical position in the mouse Dnmt3a gene (R878H), and assess development, hematopoiesis, and cancer susceptibility. Specific Aim 3: We will assess the effect of Dnmt3a haploinsufficiency on AML pathogenesis. Mice haploinsufficient for Dnmt3a do not have a measurable hematopoietic phenotype. We will perform tumor watches using mice that are haploinsufficient for Dnmt3a, and we will compare the leukemogenicity of FLT3 ITD, NPMc, and IDH1/2 mutations using WT vs. Dnmt3a haploinsufficient bone marrow cells.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The goal of this project is to measure DNA methylation in patients with acute myeloid leukemia (AML) by LC-MRM-MS using published methods and also to identify other DNA adducts.

The long-term goal of this project is to integrate the analysis of all genomic studies of the GAML PPG to identify and validate acquired genetic changes that may contribute to the pathogenesis of AML. Somatic mutations that are predicted to change the function of a gene will be analyzed to assess their consequences on patient outcomes, on patterns of gene expression, and on AML-relevant pathways. To identify these mutations, high-resolution genomic screens and large scale resequencing studies will be required. In the GAML PPG, several cores and projects are generating genome-wide databases that must be carefully coanalyzed to identify candidate genes for resequencing and validation studies. These databases also must be mined to define relationships between the mutations and the biological pathways that they affect. To achieve these goals, we propose these Specific Aims: Specific Aim 1: We will analyze the outputs of multiple genomic screens to prioritize candidate genes for resequencing, and we will define and validate somatic mutations in AML samples. Exonbased resequencing studies (Core D) are difficult and expensive to perform, and candidate genes for these studies must therefore be carefully prioritized using data generated by high-resolution genomic screens. Array-based gene expression profiling, high resolution array-based comparative genomic hybridization, and high resolution array-based SNP genotyping studies will be used to identify genes and/or loci that are deleted, amplified, or duplicated from one parental allele (uniparental disomy), or that have aberrant patterns of expression; whole genome resequencing studies of 10 M1 AML genomes (Project 1) will also be analyzed to define potentially important mutations that will be validated using the 94 matched tumor-germline AML samples from the Discovery Set. When potentially important somatic mutations are identified, we will perform additional studies to verify the mutation and its frequency in the AML sample of interest (i.e. bacterial cloning and resequencing of the mutant exon in 96 clones from the sample), and we will further define the mutation's frequency in 94 fully annotated AML cases obtained from Cancer and Leukemia Group B (CALGB). Specific Aim 2: We will define the clinical and gene expression consequences of validated somatic mutations, and define biologic pathways altered by these mutations. We will use statistical approaches to define the effects of mutations on clinical outcomes. Novel informatics approaches (e.g. promoter analyses, pathway/interaction network construction, etc.) will be used to define the effects of mutations on patterns of gene expression, and to identify potentially relevant biological pathways that are affected by AML mutations. These algorithms will be used to identify additional genes for resequencing studies. Selected mutations and pathways identified by these studies will be biologically validated in the laboratories of PPG members.

The long term goal of this project is to identify the patients with acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) who are the most likely to respond to decitabine therapy. Decitabine is a hypomethylating agent that has efficacy in both MDS and AML. It can be given as an outpatient, and it is well tolerated in most patients. However, response rates are modest; even with modern aggressive schedules, only 4 5 % of patients achieve a complete response. The molecular basis of decitabine sensitivity and/or resistance is not yet clear. Specific Aims: Aim1. We will define the molecular signature of decitabine responders. We will prospectively bank 125 properly consented patients treated with the current state-of-the-art decitabine protocol. We will comprehensively define patient-specific molecular signatures through exome sequencing and expression profiling, using both mRNA and miRNA based arrays. We will correlate genotyping and expression results with clinical features, including responsiveness to decitabine therapy. These studies will correlate genomic signatures of DNMT3A, I D H I , IDH2, and TET2 with outcomes. In addition, comprehensive, unbiased analysis will determine whether specific molecular signatures are associated with decitabine responses. Aim 2. We will determine whether the rate of AML clearance and persistence of AML-associated subclones corresponds to drug metabolism, molecular, and/or clinical features of AML in each case. We will assess the velocity of patient-specific mutation clearance on day 0, 10, and 28, and the persistence of AML-associated subclones despite blast clearance. We will correlate this with steady-state decitabine drug levels, the reduction of methylcytosine in the total marrow sample (a biomarker of effective dosing), and with clinical response rates and event-free survival.

The long-term goal of this project is to determine whether the clonal architecture of AML samples is relevant for clinical outcomes, and whether that information can be translated into clinical testing to predict prognosis. Our group has recently discovered that the clonal architecture of AML samples can be deduced by whole genome sequencing (which defines ali mutations in every case), followed by deep digital sequencing of ail mutations to define their variant allele frequencies (VAFs). Groups of mutations with similar VAFs represent subclones, Ali AML samples have a founding clone, and most contain 1-3 additional subclones that are derived from the founding clone. The dominant clone at relapse, however, is often a subclone that has acquired addition mutations. These data suggest that specific subclones contain mutations that are relevant for altered grov/th properties and/or altered drug sensitivity, which may contribute to refractory disease or relapse. In this project, we will study the clonal architecture of AML genomes, and determine their relevance for ciinical outcomes, via the following specific Aims: Specific Aim 1: We will use custom capture reagents and deep digital sequencing to determine the rate of clearance of individual AML subclones after induction chemotherapy. We will utilize DNA derived from bone marrow biopsies of 89 AML samples that have already undergone whole genome sequencing. gDNA from bone marrow biopsies obtained 2-4 weeks after initiation of therapy will be subjected to custom capture for all known mutations in each genome, and deep digital sequencing will be performed to assess the rate of clearance of the founding clone and all subclones. Clearance patterns will be correlated with clinical parameters to define impact on outcomes. Specific Aim 2: We will use a stromal-based culture system and xenotransplantation to evaluate the clonal architecture of AML samples grown in vitro and in vivo. Our stromal-based culture system allows for the expansion of most primary AML samples for at least 7 days without significantly altered physical properties or clonal drift. We will treat cultured AML cells with a variety of drugs, and responses will be measured using cell cycle assays and deep digital sequencing to define the drug sensitivity of each sample and each subclone. We will also analyze AML cells that expand in immunodeficient mice to determine whether specific subclones have a growth advantage in mice. These data will be used to identify mutations in subclones that may negatively influence prognosis.

? DESCRIPTION (provided by applicant): In this proposal, we will attempt to determine the precise molecular mechanisms by which acute myeloid leukemia (AML)-initiating mutations act, and to exploit these mechanisms therapeutically. The vast majority of patients who develop AML still die from their disease. New therapies that are more efficacious and less toxic are urgently needed. Recent AML genome sequencing studies have taught us that virtually all AML tumors are clonally heterogeneous. Each tumor originates from a founding clone that was created by an initiating mutation that allowed a single hematopoietic stem/progenitor cell (HSPC) to achieve a clonal advantage. This `preleukemic' clone acquires additional, cooperating mutations that lead to the development of a founding clone, and clinically apparent AML. Subclones arise from the founding clone, or can evolve from other subclones. Regardless, all subclones contain the founding clone mutations. Although cooperating mutations are often attractive for targeted therapies (e.g. FLT3 and/or IDH1/2 inhibitors), they are sometimes found in subclones (i.e. they are only in a fraction of the total leukemic cell population); therapeutic targeting of subclones cannot be expected to be curative. The central hypothesis of this work is that a complete understanding of the consequences of initiating mutations is required to fully understand AML pathogenesis. We also hypothesize that therapeutic approaches directed against initiating mutations are the most likely to provide long-term benefit for AML patients. We will fully characterize two common, well-validated AML-initiating mutations (PML-RARA and DNMT3A R882H) that are both associated with profound epigenetic alterations in hematopoietic cells. We will utilize state-of-the-art techniques (including comprehensive, strand-specific RNA-seq of large and small RNAs, whole genome bisulfite sequencing, chromatin accessibility studies, and ChIP-seq studies for oncogene binding and histone modifications) to pinpoint the key genomic targets of these initiating mutations, and unbiased proteomic techniques to comprehensively identify proteins that interact specifically with the mutant proteins. We will integrate these data to identify genes, RNAs, loci, and pathways that are altered by the initiating mutations, and develop new hypotheses regarding mechanisms that may be relevant for AML pathogenesis. We will model AML-initiating mutations and downstream pathways both in human embryonic stem cells, and in transgenic mice expressing PML-RARA or DNMT3A R882H, to fully explore the contributions of pathways (e.g. DNA methylation and/or histone modifiers) and/or cooperating mutations that may be critical for their actions. As a translational goal of thi work, we will attempt to develop a novel drug that will inhibit the action of the mutant DNMT3A R882H protein, which acts as a dominant negative inhibitor of WT DNMT3A, thereby suppressing de novo DNA methylation in HSPCs. This mutation causes in focal, canonical, DNA hypomethylation, an event that may be reversed by an effective inhibitor, which may restore normal HSPC function.

The long term goal of this project is to identify the patients with acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) who are the most likely to respond to decitabine therapy. Decitabine is a hypomethylating agent that has efficacy in both MDS and AML. It can be given as an outpatient, and it is well tolerated in most patients. However, response rates are modest; even with modern aggressive schedules, only 4 5 % of patients achieve a complete response. The molecular basis of decitabine sensitivity and/or resistance is not yet clear. Specific Aims: Aim1. We will define the molecular signature of decitabine responders. We will prospectively bank 125 properly consented patients treated with the current state-of-the-art decitabine protocol. We will comprehensively define patient-specific molecular signatures through exome sequencing and expression profiling, using both mRNA and miRNA based arrays. We will correlate genotyping and expression results with clinical features, including responsiveness to decitabine therapy. These studies will correlate genomic signatures of DNMT3A, I D H I , IDH2, and TET2 with outcomes. In addition, comprehensive, unbiased analysis will determine whether specific molecular signatures are associated with decitabine responses. Aim 2. We will determine whether the rate of AML clearance and persistence of AML-associated subclones corresponds to drug metabolism, molecular, and/or clinical features of AML in each case. We will assess the velocity of patient-specific mutation clearance on day 0, 10, and 28, and the persistence of AML-associated subclones despite blast clearance. We will correlate this with steady-state decitabine drug levels, the reduction of methylcytosine in the total marrow sample (a biomarker of effective dosing), and with clinical response rates and event-free survival.

Project Summary The long-term goal of this project is to identify the patients with acute myeloid leukemia (AML) who are the most likely to respond to decitabine therapy, and to determine the molecular mechanisms of decitabine responses. We recently reported that TP53 mutated AML and MDS patients, which have a high risk of relapse, and very poor outcomes, respond consistently to decitabine, a hypomethylating agent that can be given as an outpatient, and which is well tolerated in most patients. However, most responding patients did not have TP53 mutations, suggesting that other pathways can also influence decitabine sensitivity. The molecular mechanisms associated with decitabine responses and subsequent relapse are currently unclear. To refine and extend these findings, we propose the following specific aims: Aim 1. We will determine the efficacy of decitabine salvage therapy in AML patients with TP53 mutations. Patients with relapsed/refractory AML and with TP53 mutations represent an ultra-high-risk population with extremely poor outcomes, representing an unmet therapeutic need. We will therefore treat 60 relapsed/refractory AML patients known to have TP53 mutations with decitabine on days 1-10 of 28-day cycles at 3 centers (Washington University, Fred Hutchinson Cancer Research Center, and the University of Iowa). Responding patients will undergo allogeneic transplantation for consolidation therapy, if possible. We will determine the overall survival at 1 year, as well as response rates, time to transplant, time to leukemia relapse, and the average number of hospital days during cycles 1 and 2. Aim 2. We will define the genomic and epigenomic signatures associated with decitabine responses. We will use enhanced whole genome sequencing to determine whether TP53 wild-type patients have recurrent, non-genic mutations, and whether recurrent mutations are acquired at relapse, regardless of TP53 status. We will integrate whole genome bisulfite sequencing with RNA-Seq to determine whether decitabine causes specific and canonical patterns of DNA hypomethylation, whether these changes result in consistent transcriptional signatures, and whether any of these patterns correlate with clinical outcomes.

Project Summary/Abstract Project 1: Genomics of intermediate-risk AML progression and relapse. The long-term goal of this project is to better understand the genetic and epigenetic events that contribute to the progression and relapse of patients with intermediate-risk AML, and exploit them therapeutically. Intermediate-risk AML (which is most commonly initiated by DNMT3A mutations) can have vastly different outcomes, for reasons that are still not well understood. In this proposal, we will explore the genetic and epigenetic factors that influence how a pre-leukemic, ancestral clone progresses to AML (i.e. ?progression?), and then evolves to recur after a remission (i.e. ?relapse?). Virtually all AML samples are clonally heterogeneous at presentation, generally containing one or more subclones derived from a founding clone (or other subclones). Subclones often display different susceptibilities to therapies, and therapy-resistant subclones often evolve with new mutations that are not recognized as drivers at relapse. To determine whether epigenetic factors may also be relevant for subclonal evolution, we have performed pilot studies using single-cell RNA-sequencing (scRNA- seq), and defined transcriptional evolution at relapse, and detected relapse-specific, differentially expressed genes that were not detectable by bulk RNA-sequencing. In this proposal, we will exploit single cell methods to better understand subclonal evolution at relapse, and evaluate the role of DNMT3A mutations for patterns of gene expression at presentation and relapse. We will also determine whether initiating mutations in DNMT3A are required only for creating the preleukemic ?state?, or whether they are also relevant for maintaining fully transformed AML cells. The studies of both aims may improve risk assessment and therapeutics for AML: Specific Aim 1: We will define the events that contribute to clonal evolution and relapse in intermediate-risk AML patients. We will perform enhanced whole genome sequencing (eWGS) and scRNA- seq on matched presentation and relapse samples from intermediate-risk AML samples, which will allow us to impute the expression signatures of subclones, how they progress at relapse, and identify genes and/or pathways that are commonly dysregulated in dominant relapse subclones. Specific Aim 2: We will define the role of DNMT3A mutations for AML initiation and maintenance. We have generated Dnmt3a deficient mice with an inducible WT DNMT3A transgene (?Dnmt3a null-3A addback? mice) that can accurately remethylate the genomes of transplanted bone marrow cells. We will generate similar addback mice with a conditional Dnmt3aR878H mutation, define their DNA methylation phenotype, and characterize their remethylation kinetics and accuracy with DNMT3A restoration. We will create a variety of cooperating mutations in both models to cause AML, and then determine whether these AMLs can have their growth and/or differentiation altered by restoring DNMT3A expression. These studies will inform preclinical trials that will study the effects of drugs designed to target the DNMT3AR882H mutation in human AML cells.

PROJECT SUMMARY/ABSTRACT The Administrative Core will provide executive oversight and administrative support for all of the projects and cores that comprise the Genomics of Acute Myeloid Leukemia (GAML) Program Project Grant. The goal of the Administrative 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 the External Advisory Board. Accordingly, the specific aims of the Administrative Core are as follows: Specific Aim/Core Service 1: We will facilitate communication and collaboration among investigators. Specific Aim/Core Service 2: We will provide administrative oversight and support for all projects and cores. Specific Aim/Core Service 3: We will provide budgetary management for all projects and cores. Specific Aim/Core Service 4: We will coordinate all GAML PPG-related meetings, including those with External Advisory Board members.

Overview: Project Summary/Abstract The long-term goal of the Genomics of Acute Myeloid Leukemia Program Project Grant (GAML PPG) is to define the genetic and epigenetic events that drive AML progression, relapse, and response to therapy, and to use this information to improve risk assessment and treatment. In the past five years, we have defined the roles of subclonal heterogeneity and evolution for AML progression and relapse, but the essential molecular events that are responsible have not been fully defined by exome and/or bulk RNA- sequencing. We will therefore exploit single cell transcriptomics to more fully define the epigenetic features of clonal evolution (Projects 1 and 3). We have also learned that hematopoietic stem/progenitor cells (HSPCs) that contain functional TP53 mutations are often selected for by chemotherapy and other stressors; the cell- intrinsic and extrinsic processes that contribute to the progression of clonal hematopoiesis to AML will be defined in Project 4. Project 2 will use genomic approaches to define minor histocompatibility antigens that are expressed specifically in AML cells to improve the immunologic therapy of AML. With these studies, we hope to define new targets and novel therapies for high-risk AML patients, via the following projects and cores: Project 1 (Ley): Genomics of Intermediate-Risk AML Progression and Relapse. This project will define the genetic and epigenetic features of intermediate-risk AML with post-chemo relapse, and determine whether the initiating DNMT3AR882H mutation represents a therapeutic target for fully transformed AML cells. Project 2 (DiPersio): Genomics of Allogeneic Transplantation. Genomic approaches will be used to define minor histocompatibility antigens that contribute to the Graft vs. Leukemia effect of allotransplantation; a mouse model of human AML (Dnmt3aR878H + FLT3-ITD) will be used to better define allo-resistance. Project 3 (Walter): Genomics of Secondary AML Progression. This project will define the genetic and epigenetic characteristics of subclones in MDS patients that contribute to progression to secondary AML, and model MDS disease progression in mice using well-defined MDS-initiating mutations (e.g. U2AF1). Project 4 (Link): Genomics of TP53-mutant AML. This project will use genomic approaches to define genetic and epigenetic events that contribute to the initiation and progression of AML initiated by TP53 mutations, and use mouse models to define extrinsic stressors that may contribute to AML progression. Core A (Westervelt). Clinical Database: provides patient enrollment, sample collection, clinical annotation, database management, and statistical support for all projects. Core B (Payton). Specimen Database: provides sample annotation, storage, and preparation for all projects. Core C (Miller). Sequencing and Analysis: provides all data production (scRNA-seq, eWGS, error-corrected sequencing, and custom studies) and comprehensive integrative analyses for all projects. Core D (Ley). Administration: provides administrative support for projects, cores, and the EAB.