Overexpression of Rheb2 Has Diverse Effects on Hematopoietic Progenitor and Stem Cell Functions.

Abstract Determining which intracellular signaling pathways are important for hematopoietic stem and progenitor cell functions is important for therapeutic uses of these cells. The intracellular serine/threonine kinase mammalian target of rapamycin (mTOR) is important for regulation of the growth, proliferation, survival and differentiation of various cell types in the body, including normal hematopoietic and blood cancer cells. We chose to study the protein Ras homologue enriched in brain-2 (Rheb2), a known activator of mTOR signaling, because there is evidence it is preferentially expressed in highly purified mouse (m) hematopoietic stem cell populations (Ivanova et al. Science , 298(5593):601–04, 2002). Rheb2 is one of two members of the Rheb small GTPase family of proteins known to activate mTOR by mechanisms which are not fully elucidated, but that are regulated by Akt and the tuberous sclerosis complex (TSC1/2). We overexpressed mouse Rheb2 protein in both primary mouse bone marrow (mBM) cells and two mouse hematopoietic cell lines (BaF3 and 32D) and determined effects on progenitor cell colony-formation, delayed IL-3 driven proliferation in cell lines and hematopoietic stem cell function in an in vivo competitive stem cell repopulation assay. Overexpression of Rheb2 in retrovirally transduced mBM cells significantly increased the colony-forming ability of the cells in response to growth factors. The positive effect of Rheb2 was most pronounced on granulocyte-macrophage (CFU-GM) progenitors (greater than two-fold increase over empty vector transduced cells) and was persistent in cells expanded in liquid culture up to one week after FACS sorting of transduced cells. Additionally, Rheb2 overexpression significantly increased the ability of mBM cells to form CFU-GM colonies under conditions of delayed-growth factor addition (at 24 and 48 hours after plating), suggesting a positive role for Rheb2 in the survival of these cells. Interestingly, addition of the pro-survival chemokine SDF-1/CXCL12 to the delayed-growth factor colony assay plates enhanced the survival of the empty vector transduced cells but did not further enhance the survival of the Rheb2 overexpressing cells. Using two IL-3 dependent hematopoietic cell lines, BaF3 and 32D, to further assess Rheb2 function and signaling, we found overexpression of Rheb2 enhanced proliferation after delayed addition of IL-3. These changes correlated with an increase in mTOR signaling in Rheb2 transduced cells. In order to evaluate the effect of Rheb2 overexpression on hematopoietic stem cell function, an in vivo competitive repopulation assay was performed. Transduced donor and non-transduced competitor cells were pooled at two ratios (1:1 and 0.4:1 donor to competitor) and injected into lethally irradiated recipients. Transduced cells were followed in the recipients by measuring GFP+ cell chimerism. In contrast to the data on progenitor cell function, transduced Rheb2 cells had significantly decreased competitive repopulation in the recipient mice (greater that 3-fold decrease compared to empty vector). This suggests that Rheb2 overexpression may negatively affect HSC homing, stable engraftment and/or self-renewal. Overall, these studies demonstrate that overexpression of Rheb2 positively enhances progenitor cell functions such as colony-formation, while negatively affecting stem cell competitive repopulation, thereby providing a possible regulatory mechanistic link between the flow from the stem to progenitor cell compartment.