A fundamental property of leukemic stem cells (LSCs) is clonal dominance. Clonal dominance refers to the clonal expansion of LSCs within the bone marrow microenvironment at the expense of normal hematopoietic cells. The mechanisms by which mutations that accumulate during leukemogenesis confer clonal dominance are largely unknown. Severe congenital neutropenia (SCN) is a bone marrow failure syndrome characterized by a marked propensity to develop acute myeloid leukemia. Truncation mutations of CSF3R, encoding the G-CSF receptor (G-CSFR), are common early mutations associated with leukemic progression in patients with SCN. We previously described transgenic mice (termed d715 G-CSFR) carrying a knock-in mutation of Csf3r that reproduces a mutation found in SCN. Our preliminary data show that the d715 G-CSFR confers a strong clonal advantage at the hematopoietic stem cell (HSC) level. Importantly, the clonal HSC advantage is dependent upon G-CSF administration, providing a novel, physiological, and "inducible" model to study clonal dominance. We propose to use this model to characterize the molecular mechanisms by which HSCs expressing mutant CSF3R gain clonal dominance. A better understanding of these mechanisms may provide novel strategies to develop molecular therapies that specifically target LSCs. This is highly relevant, since there is evidence that LSCs are inherently resistant to most current chemotherapy. Preliminary data suggest that accentuated STAT5 activation by the d715 G-CSFR is a proximal signal mediating the clonal HSC advantage. This hypothesis will be tested by conditionally deleting Stat5 and assessing the effect on HSC function.
In specific aim 2, we will identify genes that are dysregulated by G-CSF in d715 G-CSFR HSCs and prioritize them for biological validation. Preliminary mRNA expression profiling studies have identified two candidate genes that are differentially induced by G-CSF in d715 G-CSFR HSC: Cdkn1a (p21cip1/waf1) and Enah. We hypothesize that induction of Cdkn1a expression by the d715 G-CSFR allows for HSC proliferation without loss of self-renewal. We also hypothesize that increased Enah expression favorably alters the interaction of HSCs with bone marrow stromal. The following specific aims are proposed.
Aim 1. We will determine whether Stat5 mediates the clonal advantage of HSCs expressing the d715 G-CSFR.
Aim 2. We will identify genes dysregulated by G-CSF in d715 G-CSFR HSCs that contribute to clonal dominance.
Aim 3. We will define the role of Cdkn1a and Enah in the clonal advantage of HSCs expressing the d715 G-CSFR.
Clonal dominance is a fundamental but poorly understood property of all leukemias that allows the leukemic stem cell to expand at the expense of normal blood cells. The goal of this research is to improve our understanding of the pathways that mediate clonal dominance. We believe this research will lead to novel strategies to specifically target the leukemic stem cell and ultimately lead to improved cure rates in individuals with leukemia.
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