Data for Flt3? LSK and LKS? cells are summarized in the bar graph on the right (mean SD, n=5, one-way ANOVA, all values were significantly different from the comparators (#) by Dunnetts multiple comparison test)

Data for Flt3? LSK and LKS? cells are summarized in the bar graph on the right (mean SD, n=5, one-way ANOVA, all values were significantly different from the comparators (#) by Dunnetts multiple comparison test). E. corresponding stress resistance provides a selective advantage to Runx1 deficient HSPCs, allowing them to expand in the bone marrow and outcompete normal HSPCs. Introduction Myelodysplastic syndrome (MDS) and acute myelogenous leukemia (AML) begin with the acquisition of a driver TAK-981 mutation that generates a pre-leukemic stem cell (pre-LSC) (Pandolfi et al., 2013). The pre-LSC is self-renewing and capable of competing with normal hematopoietic stem cells (HSCs) to ensure its survival and expansion in the bone marrow. Additional mutations gradually accumulate in the pre-LSC and its downstream progeny, giving rise to MDS or AML (Welch et al., 2012). Early mutations in the leukemogenic process often occur in genes encoding chromatin regulators such as and (Welch et al., 2012; Xie et al., 2014). These genes mediate processes such as DNA methylation, histone modification, or chromatin looping, altering the epigenetic landscape of the pre-LSC (Corces-Zimmerman et al., 2014; Jan et al., 2012; Shlush et al., 2014). Mutations that activate signal transduction pathways, such as internal duplication of are also common in AML, but most often occur as later TAK-981 events in downstream progenitor populations (Corces-Zimmerman et al., 2014). is a DNA binding transcription factor that is mutated in and therapy-related AML, MDS, chronic myelomonocytic leukemia (CMML), acute lymphocytic leukemia (ALL), and in the autosomal dominant pre-leukemia syndrome familial platelet disorder with predisposition to acute myeloid leukemia (FPD/AML) (Mangan and Speck, 2011). In mice, loss-of-function (LOF) mutations cause defects in lymphocyte and megakaryocytic development, and alterations in hematopoietic stem and progenitor cells (HSPCs) that include an increase in the number of committed erythroid/myeloid TAK-981 progenitors and expansion of the lineage negative (L) Sca1+ Kit+ (LSK) population in the bone marrow (Cai et al., 2011; Growney et al., 2005; Ichikawa et al., 2004). Runx1 deficiency has only a modest adverse effect on the number of functional long term repopulating hematopoietic stem cells (LT-HSCs), reducing their frequency in the bone marrow by 3 fold at most, without affecting their self-renewal properties (Cai et al., 2011; Jacob et al., 2009). LOF mutations may also confer increased resistance to genotoxic stress, as several small-scale studies of MDS/AML patients who were previously exposed to radiation, or treated with alkylating agents, revealed a high incidence (~40%) of somatic single nucleotide variants or insertion/deletion mutations in as compared to the overall 6-10% of MDS patients with LOF mutations (Bejar et al., 2011; Haferlach et al., 2014; Harada et al., 2003; Walter et al., 2013; Zharlyganova et al., 2008). The higher association of mutations with exposure to genotoxic agents suggests two possibilities: either mutations are preferentially induced by these agents, or more likely, that pre-existing mutations conferred a selective advantage to pre-LSCs exposed to these providers. mutations can be early or later on events in the progression of MDS and AML (Jan et al., 2012; Welch et al., 2012). That they can become early events is definitely demonstrated unequivocally from the observation that FPD/AML individuals who harbor germline mutations in have a ~35% lifetime risk developing MDS/AML (Ganly et al., 2004; Michaud et al., 2002; Music et al., 1999). Although it has been shown that mutations that happen in pre-LSCs cause them to selectively increase in the bone marrow (Busque et al., 2012; Xie et al., 2014), the mechanisms underlying this trend are not well understood. Here we targeted to elucidate the molecular mechanisms by which LOF mutations generate an expanded human population of HSPCs. Counter-intuitively, we find that Runx1 deficiency in HSPCs results in a slow growth, low biosynthetic, small cell phenotype, accompanied by markedly decreased ribosome biogenesis (Ribi). Furthermore, Runx1 deficient HSPCs have lower levels of p53 and an attenuated unfolded protein response, and are less apoptotic following exposure to genotoxic stress. These observations lead to a model whereby LOF mutations generate stress resistant HSPCs that are able to perdure and increase TEF2 by virtue of their sluggish growth properties and decreased rates of apoptosis as compared to normal HSPCs. Results We previously shown that Runx1 deficient murine HSPCs have a decreased percentage of apoptotic cells (Cai et al., 2011). To determine if Runx1 deficiency also shields against radiation-induced apoptosis, we generated hematopoietic-specific LOF alleles with Vav1-Cre (Cai et al., 2011). We irradiated control ((/) mice and measured the percentage of apoptotic HSPCs 24 hours later. HSPCs were analyzed using CD34 and Flt3 markers, since we previously showed that CD48 and CD150 are dysregulated.