Mechanisms of necrosis regulation of hematopoietic stem cell function

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Normal hematopoiesis requires stringent control of production of new cells (proliferation) and removal of aging or damaged cells (programmed cell death). Patients with bone marrow failure disorders such as Myelodysplastic syndrome (MDS) have increased bone marrow programmed cell death, and increased levels of death-inducing cytokines such as TNFa. Discoveries in the last several years have demonstrated that in addition to apoptosis, TNFa also activates a novel form of cell death, programmed necrosis. Apoptotic cells implode in caspase-driven and immune silent process, whereas necrotic cells explode in a Rip kinase-driven process, releasing cellular contents (DAMPS) and eliciting an immune response. We find increased Rip1 kinase expression in 70% of MDS patient samples tested suggesting that necroptosis is activated in MDS. We also find that bone marrow from mouse models harboring known genetic mutations found in MDS (Asxl1-/-, Asxl1-/-Tet2-/-) display increased Rip1 kinase, suggesting that MDS genetic/epigenetic alterations result in increased necroptosis signaling that contributes to bone marrow cell death. Substantial data demonstrate that MDS is a clonal stem cell disorder(Graubert et al., 2012; Walter et al., 2011; Walter et al., 2012; Walter et al., 2013). A paradox inherent in MDS is that although the MDS-propagating clone has increased competitive ability, its expansion ultimately results in bone marrow failure. The presence of MDS stem cells and dying progenitor cells confers decreased function to the coexisting normal stem cells, suggesting that MDS stem and progenitor cells create a bone marrow environment that is killing normal hematopoietic stem cells. In our mouse model with unrestrained hematopoietic necrosis, mice die of bone marrow failure with the majority of the features of human MDS. Furthermore, bone marrow from these mice displayed increased competitive repopulating ability against wild type bone marrow, but transplanted mice die of bone marrow failure at four months despite the persistence of wild type bone marrow, suggesting that these necrotic HSC and progenitor cells can kill wild type HSCs to cause bone marrow failure. Our mice thus shed light on how an MDS clone can cause bone marrow failure: Our overarching hypothesis is that programmed necrosis in MDS cells triggers an inflammatory response that kills normal hematopoietic stem cells. This in term enables mutant stem and progenitor cells to expand and take over the bone marrow, thus driving bone marrow failure. Interrupting the cell death signaling pathway or altering the inflammatory signaling pathway has the potential to prevent cell death and re establish bone marrow homeostasis for therapeutic benefit. Aim 1: Will evaluate cell death and cytokine signaling in genetic mouse models of unrestrained necroptosis, as well as MDS mutations, to identify the molecular decision drivers, and how these drivers alter bone marrow cell death Aim 2: Will determine whether inhibiting necrosis or inflammatory signaling in HSC and progenitor cells in the above mouse models by inhibiting necrosis signaling (Rip1/Rip3 inhibitors) or inhibiting inflammatory signaling (Jak1/2 inhibitors) can reset hematopoietic homeostasis and prevent MDS bone marrow failure. Impact: The goal is to identify how HSCs and progenitor cells harboring MDS mutations execute cell death, and how they kill normal HSCs, and determine whether interrupting this cell death can rescue bone marrow function.