Michael H. Tomasson - US grants

Acute leukemia arising in the setting of a preexisting myelodysplastic syndrome is especially resistant to treatment. The only curative treatment for these disorders is bone marrow transplantation, a treatment that remains toxic and available only to a subset of patients. Chronic myelomonocytic leukemia (CMML) is a myelodysplastic syndrome characterized by monocytosis, variable bone marrow fibrosis, and progression to acute leukemia. The t(5;12) is associated with CMML and results in the expression of a chimeric protein fusing the amino-terminus of TEL with the intracellular portion of the PDGFbetaR. Our lab cloned the TEL/PDGFbetaR fusion gene and has extensively characterized its in vitro properties. TEL/PDGFbetaR is constitutively tyrosine phosphorylated and forms oligomers mediated by the TEL pointed (PNT) domain in vitro and in COS cells. In Preliminary Results the candidate describes the development of two animal models of hematopoietic malignancy developed using TEL/PDGFbetaR. Mice transplanted with murine bone marrow (BMT) with TEL/PDGFbetaR introduced by retrovirally mediated gene transfer develop a rapidly fatal myeloproliferative syndrome. Furthermore, transgenic mice that have TEL/PDGFbetaR targeted to the lymphoid compartment by the immunoglobulin heavy chain promoter-enhancer (EmuVHP) develop lymphoblastic lymphomas. The focus of this proposal is to increase our understanding of the molecular mechanisms underlying transformation by TEL/PDGFbetaR in these murine models. The specific aims are: 1) To characterize the signaling pathways activated by TEL/PDGFbetaR in vivo, 2) To determine the relevance of activated signaling pathways to the development of hematopoietic malignancy in mice, and 3) To identify the downstream targets of TEL/PDGFbetaR relevant to the transformation of hematopoietic cells. A series of signaling mutants of TEL/PDGFbetaR will be used in repeat BMT experiments to determine residues critical for in vivo transformation. Using coimmunoprecipitation and gel shift assays, the activity of signaling molecules in the Ras and JAK/STAT pathways will be analyzed. Lastly, DNA microarray technology will be used to examine genomic expression of downstream targets of TEL/PDGFbetaR and its mutants in cultured cells. These experiments will provide a detailed understanding of the mechanisms of transformation by TEL/PDGFbetaR, and will provide a basis for the development of rational therapies to cure or halt the progression of CMML.

[unreadable] DESCRIPTION (provided by applicant): The long-term goal of this project is to identify mutations that contribute to myeloma disease progression and to develop treatment strategies based on individual patient genotypes. Multiple myeloma (MM) accounts for 2% of all cancer deaths in the United States and 3% of all cancer deaths in black/African-Americans. In collaboration with the Washington University Genome Sequencing Center (GSC), we propose to extend our current mutational profiling analyses to better our understanding of disease progression in MM. We will define tumor genotypes using bone marrow and skin (germline) DNA from patients with MM using largescale exonic re-sequencing of candidate genes. The R21 phase of this project will develop the translational research infrastructure and solve technical issues associated with mutational profiling. (1) We will capture epidemiological data, disease-related characteristics, prognostic factors, therapeutic information and outcomes for all patients diagnosed with multiple myeloma (MM) seen at the Barnes/Jewish Hospital/Siteman Cancer Center in our comprehensive clinical database. (2A) We will define the threshold level of mutation detection achievable by re-sequencing based on phi29 polymerase-whole genome amplified (WGA) genomic DNA. (2B) We will collect and bank genomic DNA from CD138-enriched myeloma cells and matched skin biopsy samples from 46 patients with newly diagnosed MM and will perform complete exonic re-sequencing often (10) candidate cytokine signaling genes (NRAS, KRAS, FGFR3, ARAF, RAF1, PTPN11, PIK3CA, LTK, INSR, and MERTK). Upon completion of defined milestones, the R33 phase of this project proceed with the following Aims: (3) We will functionally characterize novel gain-of-function mutations, identified in myeloma patient samples, using cell line transformation assays and in a disease model using murine bone marrow transduction/transplantation. (4) We will validate the clinical utility of mutation profiling by high-throughput re-sequencing of cytokine signaling gene mutations in myeloma patient samples collected at distinct stages of disease progression. Accomplishment of these Aims will provide the data necessary to test additional hypotheses requiring larger numbers of patients in a cooperative group setting. Sequencing analysis has, until now, been a relatively underutilized tool, but is a crucial component of a set of genomic technologies (including expression microarrays and proteomics) that will be required to improve outcome in myeloma patients. [unreadable] [unreadable] [unreadable]

The long-term goal of Project 4 is to validate the biological significance of discovered mutations so that specific molecular targets can be used to guide the treatment of patients with acute myeloid leukemia (AMI). Mutations in genes involved in cytokine signaling (e.g. PL73, KRAS, NRAS, KIT) can be identified in nearly 50% of AML cases. We hypothesized that every AML tumor harbors at least one mutation in a cytokine signaling gene, and that these mutations cooperate to cause disease progression. Focused high-throughput exonic re-sequencing of expression-prioritized receptor tyrosine kinase (RTK), cytoplasmic tyrosine kinase (CTK), and Ras-MAPK pathway genes has been performed on 94 selected primary de novo AML patient samples. Sequence data has been analyzed and novel, non-synonymous mutations have been identified in multiple genes in the Janus kinase (JAK) family, including JAK1, JAK3, and TYK2. We propose the following Specific Aims: Specific Aim 1: We will validate the biological significance of somatic Janus kinase (JAK) family mutations by assessing the growth and differentiation of primary hematopoietic cells expressing mutant JAK1 and TYK2, and we will characterize the mechanisms by which these somatic mutations contribute to leukemic transformation. Our laboratory has validated the functional significance of several myeloid leukemia-associated oncogenes using biochemical, cell culture, and mouse model assays. We will express JAK1, TYK2, and JAK3 mutations in cell lines and primary murine bone marrow assays to determine the effect of these mutations on growth and survival of hematopoietic cells. The effects of these mutations on the subcellular localization of the mutant proteins themselves, and associated proteins will be examined using confocal microscopy, biochemical analysis, and localization-tagged mutants. Specific Aim 2: We will validate the biological significance of high-priority germline single nucleotide polymorphisms (SNPs) in JAK family genes TYK2 and JAK3 by characterizing their functions using cell culture and animal model systems. We have found known and previously unidentified nonsynonymous SNPs in our AML discovery set. To prioritize these SNPs for further study, we will collaborate with Project 5 to determine the frequency of these SNPs in control populations. We will express high-priority non-synonymous SNPs in cell lines and in murine bone marrow transduction-transplantation assays to characterize their role in transformation. We will also perform biological validation experiments with mutations and SNPs in cooperation as they are found in our patients to rigorously assess the contribution of discovered mutations to disease pathogenesis.

DESCRIPTION (provided by applicant): Multiple myeloma (MM) is the second most common hematologic malignancy in the United States, is a malignancy of morphologically differentiated plasma B-cells and is largely incurable. Chromosome 13 deletions (del[13]) are found in approximately one half (50%) of all patient samples and is associated with worse prognosis, but previous efforts have failed to conclusively identify the MM tumor suppressor gene on chromosome 13. We used a unique ultra high-resolution array comparative genomic hybridization (aCGH) platform to analyze MM patient samples, and identified neurobeachin (NBEA) as a novel target of recurrent interstitial deletions. NBEA encodes a protein kinase A anchoring protein (AKAP) that is localized to the trans-Golgi, transport vesicles and post-synaptic membranes of neurons where it plays a functional role in neuronal cell synaptic transmission. Our Preliminary Data demonstrated not only that NBEA is a target of chromosomal deletions at 13q13, but that NBEA expression at the RNA and protein level is significantly dysregulated in MM patient samples. Our hypothesis is that del[13] contributes to dysregulation of the NBEA gene, and that NBEA over- expression contributes to MM disease progression. To determine the potential role of NBEA as a tumor marker in MM, we propose the following aims: Aim 1: Characterize the relationship between the NBEA gene and MM disease stage. We have assembled a unique MM tissue bank with both tumor and germline patient samples, and we will characterize the NBEA locus in depth in our MM patient samples. We will correlate NBEA genotypes and expression data with clinical patient data, and anticipate that NBEA genotypes will be associated with poor outcomes in MM. Aim 2: Develop novel assays for NBEA protein detection in MM patient samples. Quantitative detection of NBEA protein in MM clinical samples will provide a useful adjunct to nucleic acid based detection methods. We will correlate NBEA protein levels with clinical patient data, and anticipate that high NBEA protein expression will be associated with poor outcomes in MM. Aim 3: Establish the role of NBEA in MM disease progression. Genetically defined murine models will be used to definitively address the role of NBEA in myeloma development. Our long-term goal is to develop novel disease prevention strategies based on understanding the genetic contribution, both germline and somatic, to MM disease development. PUBLIC HEALTH RELEVANCE: Multiple Myeloma (MM) is the second most common hematologic malignancy in the United States accounting for approximately 10% of all hematologic neoplasms. We have i) established a unique MM tissue bank to provide critical tools for genetic studies and ii) have developed a database to prospectively collect clinical data on MM patients seen at the Siteman Comprehensive Cancer Center, so that we can trace molecular abnormalities back to clinical outcomes. We have identified NBEA as a novel candidate gene on chromosome 13 that is targeted by interstitial deletions and whose expression is dysregulated. We will validate the role of NBEA in MM and will develop assays to characterize NBEA in MM clinical specimens.

DESCRIPTION (provided by applicant): Monoclonal gammopathy of uncertain significance (MGUS) is a common, pre-malignant plasma cell disorder found in 3.2 and 5.3 percent of individuals over the ages of 50 and 70 years respectively (Kyle RA, NEJM, 2006). MGUS is characterized by monoclonal serum immunoglobulin, an increased risk of thrombosis, an increased risk of osteoporosis and bone fractures and a risk of developing malignancy (predominantly multiple myeloma at 1% per year (REFS Kyle RA, NEJM, 2002). MGUS has a significant component of inherited risk, and is found at 2-3 fold higher rates in African Americans, and 2-fold higher rates in family members of MGUS patients. Neither the genetic basis nor the environmental factors contributing to MGUS/MM risk have been defined. Our long-term goal is to develop screening and prevention strategies based on a detailed understanding of MGUS/MM genetics. The C57BL/KaLwRij (KaLwRij) mouse strain, described decades ago (Radl J, Clin Exp Immunol, 1984), develops MGUS at high frequency with many of the same features of the human disease including an increased risk of developing MM. Since the most common somatic mutations to occur in MGUS/MM are chromosomal translocations involving immunoglobulin heavy chain switch regions, our hypothesis is that germline susceptibility to MGUS is the consequence of abnormal immunoglobulin isotype switching. We found highly significant differences in antibody isotype responses by ELISA between KaLwRij and 11 separate mouse strains. We also found that ionizing radiation and vitamin D deprivation, two environmental factors associated with MM, induced significant and strain-specific changes in antibody responses in mice. To advance our understanding of MGUS/MM risks, we propose: Specific Aim 1: Characterize effects of ionizing radiation, vitamin D deprivation and strain background on immunoglobulin isotype responses and monoclonal gammopathy (MGUS) development in mice;Specific Aim 2: Map quantitative trait loci (QTL) for MGUS/MM risk and identify somatic mutations associated with disease progression. The experiments in SA1 will provide valuable insights into the effects of strain and relevant environmental factors to MGUS development. The experiments in SA2 will provide an MGUS-specific QT data set and matched DNA samples that will allow us to identify QTL's in mice associated with MGUS risk. These mice will be used as a platform to explore the relationship between inherited MGUS risk and environmental factors and the data we generate will inform ongoing genome-wide association (GWA) studies in humans (a collaborative effort between Washington University and the Mayo Clinic). This project will be part of a coordinated effort to identify the genetic factors that drive MGUS and MM in humans. PUBLIC HEALTH RELEVANCE: Monoclonal gammopathy of uncertain significance (MGUS) occurs in 3.2% of persons over the age of 50, and in 5.3% of individuals 70 years or older. MGUS patients are at increased risk for thrombosis, bone fractures and progression to multiple myeloma (MM), an invariably fatal cancer. Environmental factors such as vitamin D deficiency and ionizing radiation are associated with increased risk of MM. Identification of gene tests for MM risk and predisposition will facilitate the long-term goal of this project which is to develop screening and prevention strategies for MGUS/MM.

DESCRIPTION (provided by applicant): Monoclonal gammopathy of uncertain significance (MGUS) is a common, pre-malignant plasma cell disorder found in 3.2 and 5.3 percent of individuals over the ages of 50 and 70 years respectively (Kyle RA, NEJM, 2006). MGUS is characterized by monoclonal serum immunoglobulin, an increased risk of thrombosis, an increased risk of osteoporosis and bone fractures and a risk of developing malignancy (predominantly multiple myeloma at 1% per year (REFS Kyle RA, NEJM, 2002). MGUS has a significant component of inherited risk, and is found at 2-3 fold higher rates in African Americans, and 2-fold higher rates in family members of MGUS patients. Neither the genetic basis nor the environmental factors contributing to MGUS/MM risk have been defined. Our long-term goal is to develop screening and prevention strategies based on a detailed understanding of MGUS/MM genetics. The C57BL/KaLwRij (KaLwRij) mouse strain, described decades ago (Radl J, Clin Exp Immunol, 1984), develops MGUS at high frequency with many of the same features of the human disease including an increased risk of developing MM. Since the most common somatic mutations to occur in MGUS/MM are chromosomal translocations involving immunoglobulin heavy chain switch regions, our hypothesis is that germline susceptibility to MGUS is the consequence of abnormal immunoglobulin isotype switching. We found highly significant differences in antibody isotype responses by ELISA between KaLwRij and 11 separate mouse strains. We also found that ionizing radiation and vitamin D deprivation, two environmental factors associated with MM, induced significant and strain-specific changes in antibody responses in mice. To advance our understanding of MGUS/MM risks, we propose: Specific Aim 1: Characterize effects of ionizing radiation, vitamin D deprivation and strain background on immunoglobulin isotype responses and monoclonal gammopathy (MGUS) development in mice; Specific Aim 2: Map quantitative trait loci (QTL) for MGUS/MM risk and identify somatic mutations associated with disease progression. The experiments in SA1 will provide valuable insights into the effects of strain and relevant environmental factors to MGUS development. The experiments in SA2 will provide an MGUS-specific QT data set and matched DNA samples that will allow us to identify QTL's in mice associated with MGUS risk. These mice will be used as a platform to explore the relationship between inherited MGUS risk and environmental factors and the data we generate will inform ongoing genome-wide association (GWA) studies in humans (a collaborative effort between Washington University and the Mayo Clinic). This project will be part of a coordinated effort to identify the genetic factors that drive MGUS and MM in humans.

DESCRIPTION (provided by applicant): The t(4;14) chromosomal translocation found in 20% of multiple myeloma (MM) cases leads to a shorter survival rate in an already incurable disease. The candidate oncogene overexpressed from the t(4;14) translocation, WHSC1 (MMSET), is unable to transform cells in culture or drive tumorigenesis in mice on its own. Examination of a 2 MB region around the t(4;14) breakpoint revealed the overexpression of a 125 bp orphan H/ACA class non-coding RNA, ACA11, which was confirmed to be up-regulated in t(4;14) MM cell lines and patient samples. Further preliminary studies demonstrate ACA11 localizes to the nucleolus as part of an small nuclear ribonucleoprotein (snRNP) complex. In addition, ACA11 overexpression reduces the levels of reactive oxygen species (ROS) in response to oxidative stress and increases cell survival in the presence of chemotherapeutic agents. We hypothesize that the overexpression of ACA11 in t(4;14) MM reduces ROS-induced cell death leading to the development and/or progression of MM. The ultimate goal of this proposal is to elucidate the mechanism by which ACA11 exerts its inhibitory effects on ROS-induced cell cycle inhibition and cell death in t(4;14) MM cell lines. We hypothesize that the overexpression of the ACA11 snoRNA in t(4;14) MM results in the altered splicing of specific RNA transcripts involved in the cellular response to ROS. These selected transcripts will encode either RNA or proteins critical for B-cell survival in MM. Therefore, ACA11 and its associated snRNP's will be potential novel targets for MM treatment or diagnosis. Three specific aims of the grant are proposed: Aim 1: To test the hypothesis that ACA11 must interact with snRNPs to exert its inhibition of oxidative stress induced cell death Aim 2: To test the hypothesis that ACA11 regulates ROS levels through modulating splicing of snoRNAs from ribosomal protein transcripts Aim 3: To test the hypothesis that overexpression of ACA11 leads to an inhibition of nucleolar stress-induced, p53-regulated ROS production

Multiple Myeloma (MM) is the second most common hematologic malignancy in the United States, diagnosed in approximately 14,500 Americans each year, and is responsible for 2% of all cancer deaths (SEER.cancer.gov). MM is an incurable malignancy of antibody-secreting plasma B-cells whose etiology is poorly understood. Chromosomal translocation t(4;14)(p16.3;q32.33) is associated with shorter overall survival and is found in 15% of MM patients. The candidate oncogene WHSC1 (MMSET) at 4p16.3 encodes a histone methyltransferase over-expressed as a result of the t(4;14) in MM cells. Surprisingly, over-expression of WHSC1 cDNA's failed to transform cell lines or to induce any phenotype when expressed in mice. We identified ACA11, an orphan box H/ACA small nucleolar RNA (snoRNA) encoded hosted within WHSC1 at 4p16.3, to be up-regulated in cell lines and MM patient samples harboring t(4;14). ACA11 modulates oxidative stress, contributes to MM cell growth and confers resistance to chemotherapy. ACA11 is a novel oncogenic non-coding RNA and is also expressed in other cancer types. Mutations that cooperate with t(4;14) in myeloma initiation are unknown, but chromosome 13 deletions (Del(13)) are highly correlated with the t(4;14) in MM. Del(13) is found in 85-94% of patients with t(4;14) and associated with poor prognosis. We mapped a minimally deleted region in MM patient samples to the retinoblastoma (RB1) gene at 13q14. Our model is that ACA11 expression cooperates with RB1 loss to cause poor-prognosis MM. We propose to characterize the role of ACA11 in multiple myeloma. Specific Aim 1: Cooperation between ACA11 and the histone methyltransferase WHSC1. We hypothesize that ACA11 cooperates with MMSET protein expression in myeloma pathogenesis and that WHSC1 histone methyltransferase activity is required for cooperation. We will co-express wild-type WHSC1 or HMT-dead WHSC1 alone or with ACA11 in cell lines and mice. Specific Aim 2: Aim 2: How does ACA11 affect levels of ROS and sensitivity to chemotherapy? What domains and partners of ACA11 are required for these effects? Our data suggest that ACA11 modulates reactive oxygen species and resistance to chemotherapy in myeloma. We will determine which residues of ACA11 are required for this function. We hypothesize that ACA11 RNA binding residues will be dispensable for ACA11's oxidative stress function, but that ACA11's protein-binding domains will be critical. Specific Aim 3: ACA11-mediated malignant transformation in the context of Rb1 deletion. The long arm of chromosome 13 is deleted in 50% of all MM patients, and in 90% of patients with the t(4;14). We hypothesize that RB1 loss cooperates with the t(4;14) in myeloma pathogenesis. We will test the effects of ACA11 and WHSC1 expression on B-cell development, oxidative stress and transformation in the context of Rb1 deficiency. Lastly, we will [identify target genes of t(4;14) by RNA- sequencing patient samples.] A betting understanding of how the t(4;14) contributes to myelomagenesis has broad implications to cancer biology and will facilitate the development of novel therapeutic approaches to patients with poor-prognosis MM.