Hematopoietic stem cells (HSCs) in the bone marrow are maintained in a quiescent state (i.e. without cell division), yet must be able to exit this stae and proliferate rapidly in response to injury or stress. In cancers, such Acute Leukemia (AML) or in Myeloproliferative diseases, a subpopulation of cells persistently enters cell cycle, disrupting the normal mechanisms of quiescence. Similarly, after injury, HSCs lose quiescence and enter cell cycle inappropriately, causing a gradual "exhaustion" of stem cell function. Understanding the genes that regulate normal HSC quiescence can therefore define new targets for leukemia as well as help us to understand the age-related dysfunction of stem cells. In prior work (Raaijmakers and Mukherjee, jointly authored, Nature 2010), we noted that HSC quiescence is maintained by response to signals from mesenchymal stromal cells (MSCs) in the bone marrow microenvironment ("niche"). We were thus interested in defining downstream genes in HSCs that regulate quiescence. Previous studies have implicated a few genes in the maintenance of quiescence, such as p21 and Gfi-1, but other pathways remain unknown. We performed a novel RNA-interference screen to identify quiescence-regulatory genes in HSCs. Building on prior work, we discovered that our search required the context of a physiological microenvironment defined by marrow mesenchymal stem cells (MSCs). We term this method "niche-based" screening. Niche-based screening revealed a novel gene - Yes-1 - as a candidate regulator of quiescence in HSCs. Notably we found that Yes-1 was highly expressed in long-term HSCs, and strikingly downregulated in cycling progenitors and in mouse and human AMLs caused by the MLL-AF9 translocation. Knockdown of Yes-1 in mouse HSCs caused entry into cell-cycle, and increase in proliferation. Yes-1 -/- mice were viable and fertile, but strikingly, HSCs from Yes-/- mice had increased progenitor activity, evident in their increased ability to repopulate mice in primary transplants (compared to Yes WT cells). However, in secondary transplants, Yes-/- HSCs gradually lost their self-renewal capacity, suggesting that increased progenitor activity was accompanied by a concomitant loss of long- term self-renewal. Overexpression of Yes-1 in human HSCs increased retention in G0, and functional transplantability. Yes-1 was also downregulated in cycling AML-SCs created by misexpression of human MLL-AF9, and forced expression of Yes-1 inhibited proliferation of leukemia cells. Here, we wish to define the roles of Yes-1 in HSC and AML-SC quiescence. We will use mouse knockout models to ablate the genes conditionally and observe the effects on HSC self-renewal and leukemogenesis. We will also define the mechanisms by which Yes-1 modulates HSC quiescence using cell cycle, transplant assays and complementation of the phenotype using targeted overexpression of candidate pathways (p21 and Gfi-1).
Blood stem cells switch between quiescence (non-dividing) and rapidly proliferating (dividing) states in response to injury or stress while in cancers, such s leukemia, this controlled switching is disrupted, and cancer cells constantly proliferate. We have devised a novel technology to identify a new gene - Yes-1- that plays a role in this switch. The goal of this grant is define the roles of this gene in blood generation, and to assess if they can be therapeutically targeted in Acute Myelogenous Leukemia.
|Worthley, Daniel L; Churchill, Michael; Compton, Jocelyn T et al. (2015) Gremlin 1 identifies a skeletal stem cell with bone, cartilage, and reticular stromal potential. Cell 160:269-84|