Role of ThPOK in HSC Maintenance and Leukemogenesis

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 DESCRIPTION (provided by applicant): There is substantial overlap between genes involved in hematopoietic stem cell (HSC) function and in hematological cancers, demonstrating how disregulation of HSC function may predispose to transformation. Indeed, resistance of some forms of leukemia to chemotherapy has been attributed to a small fraction of leukemic-stem cells (LSCs), which are capable of self-renewal and differentiation like HSCs, but lack proper control mechanisms. We have compelling data to suggest important novel functions for the transcription factor ThPOK both as a normal regulator of HSC maintenance/differentiation, and as a mediator of leukemogenesis. We propose to define these functions according to two specific aims: Aim 1: Defining the role of ThPOK in HSC differentiation/maintenance. HSC function depends on a careful balance between HSC self-renewal and differentiation. This is disrupted in ThPOK-deficient mice, since long-term self-renewing HSCs are diminished while myeloid progenitors are expanded. To assess the role of ThPOK in control of HSC self renewal and differentiation, we will test the effect of ThPOK ablation on: a) HSC frequency by limited dilution bone marrow transplantation (BMT) assay, b) Long-term HSC reconstitution by non-competitive serial BMT assays, c) Ability to competitively reconstitute hematopoiesis versus wt HSCs by competitive BMT assay, d) Proliferative capacity of HSCs and progenitor populations in vivo and in vitro, and also e) Identify target genes of ThPOK in HSCs, to begin to define the underlying mechanism/s by which ThPOK regulates HSC balance. Aim 2: Elucidating the role of ThPOK in leukemogenesis. Leukemia-initiating cells (LICs) or leukemic- stem-cells (LSCs) are postulated to have simultaneous capacity for self-renewal and differentiation and to represent critical mediators of drug-resistant leukemia. Our preliminary results indicate that ThPOK-deficient Lin- BM cells are partly protected from leukemogenesis in an in vitro Hoxa9/Meis1-dependent acute myeloid leukemia (AML) model, potentially due to diminished generation of LICs. Conversely, we show that overexpression of ThPOK in the T cell lineage promotes appearance of a long-lived lymphoma progenitor cell population. Therefore, in the current aim, we propose to address the following key questions: a) Is ThPOK required for initiation of AML in the well-established in vivo MLL-AF9 and AML-ETO models of AML? b) Is ThPOK necessary for the maintenance of AML in vivo? c) Does ThPOK promote the leukemia-initiating activity of L-HSCs and L-GMPs? d) Does inhibition of ThPOK in human AML cell lines reduce leukemia cell growth and induce differentiation?