Qing Li - US grants

ABSTRACT Mutations of oncogenes and tumor suppressor genes transform normal hematopoietic stem cells (HSC) into leukemic stem cells (LSC) in a multi-step process. As the first step of leukemogenesis, the ?pre-leukemia? mutations dys-regulate HSC functions to promote clonal expansion by increasing proliferation, competitiveness and self-renewal. Pre-LSCs are further transformed into LSCs by additional mutations. Targeting pre-LSCs and LSCs has the potential to eliminate leukemia. Although many oncogenes and tumor suppressor genes have been proposed to transform normal HSCs to LSCs, very little is known about what signaling mechanism underlies this transformation to allow development of therapies to target pre-LSCs and LSCs. The long term goal of our research is to identify signaling mechanisms through which oncogenes and tumor suppressor genes transform normal HSCs into LSCs. Clonal expansion has been difficult to recapitulate experimentally because mutations that drive HSCs into cycling often lead to reduced HSC self-renewal and HSC depletion. We recently found that an oncogenic Nras mutation commonly found in human leukemias, G12D, dys-regulates HSCs and transform them into pre-LSC with increased proliferation, competitiveness and self-renewal. The objective of this proposal is to identify the signaling mechanism underlying this Nras induced transformation. Based on our preliminary results, our central hypothesis is that JAK2/STAT5 signaling is critical in the Nras induced transformation to pre-LSCs and further mediates transformation to LSCs. We will test this hypothesis by 1) Identify the mechanism by which NrasG12D activates STAT5 to dys-regulate HSC functions; 2) Define the role of JAK2 in NrasG12D induced HSC dys-regulation and 3) determine the role of JAK2/STAT5 signaling in fully transformed LSCs. The approach is innovative because 1) it takes advantage of our newly developed clonal expansion model; 2) it combines mouse genetic analysis and cellular and biochemical analysis on rare populations to investigate signaling mechanisms in pre-LSCs and LSCs, and 3) it evaluates the role of non-canonical Ras effectors that may be responsible for the effects of oncogenic Ras in a population highly relevant to leukemogenesis. The proposed research is significant because it is expected to advance knowledge of the signaling mechanisms underlying the transformation from normal HSCs to LSCs, and therefore to inform novel therapeutic intervention that will target pre-LSCs and LSCs to eradicate leukemia.

Abstract: It is well established that quiescence or dormancy preserves the self-renewal and long-term reconstituting potential of long-term HSCs (LT-HSC). HSCs that are quiescent give rise to much higher reconstitution than proliferating HSCs in transplant recipients, and signals that drive HSCs into proliferation cycle often lead to HSC differentiation and exhaustion. However, the mechanisms that coordinate HSC quiescence, proliferation and differentiation remains to be investigated. Recently, we and others reported that protein homeostasis at endoplasmic reticulum (ER) plays important role in preserving HSC functions under stressed condition. However, it remains to be investigated whether protein quality control is important for HSCs under steady state, when the majority of HSCs remain in a deeply dormant state with profoundly reduced protein synthesis rate and metabolic activity. ER associated degradation (ERAD) is a critical component of protein homeostasis, and ensures protein quality control by degrading inappropriately folded or assembled proteins in ER. ERAD complexes recognize misfolded proteins in ER and translocate them to cytosol for proteasomal degradation. Our preliminary studies indicate that protein quality control via ERAD governs HSC quiescence and self-renewal. The Sel1L/Hrd1 ERAD genes are enriched in the quiescent and inactive HSCs, and conditional knockout of Sel1L in hematopoietic tissues drives HSCs to hyper-proliferation, which leads to complete loss of HSC self-renewal and HSC depletion. ERAD deficiency via Sel1L knockout induces a non-apoptotic ER stress and activates all three main pathways of unfolded protein response (UPR). Furthermore, we found that mTOR signaling is activated in Sel1L knockout HSCs and inhibition of mTOR via rapamycin rescues Sel1L knockout-induced HSC defects. We therefore hypothesize that Sel1L maintains HSC quiescence and self-renewal by restricting mTORC activity. Here, we propose three aims to determine the mechanism by which ERAD modulates mTOR signaling to preserve HSC quiescence and self-renewal: 1) Determine the role of Akt/mTOR signaling branches in Sel1L-mediated HSC regulation; 2) Dissect the interaction of ERAD and UPR signaling; and 3) Determine the role of Rheb in ERAD deficiency-induced HSC dysregulation. These studies will establish Sel1L/Hrd1 ERAD as the master regulator of HSC quiescence, and provide novel insights into how protein quality control systems coordinate with proliferation signaling pathways to determine HSC fate.