Asxl1 gene is mutated and/or deleted with high frequencies in multiple forms of myeloid malignancies including CMML, MDS, MPN and AML. The majority of the Asxl1 mutations in these patients are heterozygous, leading to nonsense/frameshift, suggesting loss of function. Mutations in ASXL1 are associated with poor prognosis. Therefore, ASXL1 has been speculated to be a putative tumor suppressor gene that is strongly implicated in the pathogenesis of myeloid malignancies. The objective of this project is to define the physiological function of ASXL1 and its role in the pathogenesis of myeloid malignancies. We generated several Asxl1-targeted murine models. Haploinsufficiency of Asxl1 (+/-) leads to the development of MDS in mice, which can progress to MDS/MPN and leukemia as they age. The Asxl1-targeted mouse model, therefore, recapitulates the pathologic situation of patients with myeloid malignancy, thus allows us to gain the needed information about Asxl1 biology in a timely fashion. Deleting Asxl1 leads to increased apoptosis and mitosis of bone marrow cells, characteristic cellular feature of MDS. A competitive reconstitution assay showed that Asxl1-/- hematopoietic stem/progenitor cells (HSC/HPCs) had a decreased hematopoietic repopulating capacity. We, therefore, hypothesize that Asxl1 acts as a tumor suppressor in myelopoiesis by altering the behavior of HSC/HPCs. We will test this hypothesis in 3 specific aims:
Aim 1 : To determine if Asxl1 acts as a tumor suppressor in myelopoiesis by characterizing the phenotype of various Asxl1-deficient mice, including MxCre or Vav1Cre mediated Asxl1 conditional knock-out mice. In addition, we will evaluate the role of Asxl1 haploinsufficiency in cooperation with other genetic alterations, such as Nf1 haploinsufficiency, for triggering myeloid malignancies.
Aim 2 : To define the cellular mechanisms by which loss of Asxl1 function in mice leads to myeloid malignancies. Specifically, we will examine the effects of Asxl1 deletion/haploinsufficiency on the proliferation, differentiation, apoptosis and cell cycle o HSC/HPCs. In addition, we will determine the effect of Asxl1 deletion/haploinsufficiency on self-renewal and differentiation potential of HSC/HPCs by serial transplantation.
Aim 3 : To define the molecular mechanisms by which Asxl1 regulates normal hematopoiesis and exerts its tumor suppressor function in myelopoiesis. We will identify Asxl1-target genes by mapping the genomic distribution of Asxl1 and its interacting histone modifying enzymes by ChIP-Seq in Asxl1:Tag and WT or Asxl1-/- HSC/HPCs, respectively. Furthermore, we will dissect Asxl1- deletion induced misregulation of H3 methylation and H2A monoubiquitination in HSC/HPCs with ChIP-Seq and correlate with the gene expression profiling. Accomplishment of these studies allows us to uncover the role of Asxl1 in normal hematopoiesis and in the multiple-step pathogenesis of myeloid malignancies, which may lead to the identification of novel molecular targets for the treatment of patients with myeloid malignancies.
Asxl1 gene is mutated and/or deleted with high frequencies in multiple forms of myeloid malignancies including CMML, MDS, MPN and AML. However, the role of Asxl1 in the development of myeloid malignancies and in normal hematopoiesis remains largely unknown. The major goal of this proposal is to solve these important scientific questions using our newly generated Asxl1-targeted murine models.
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