Deciphering JunB Function in Regulating Hematopoietic Stem Cell Functions.

The AP-1 transcription factor JunB plays a key role in controlling normal homeostasis of the hematopoietic stem cell (HSC) compartment and acts as a tumor suppressor in mice. We have previously shown that inactivating JunB expression in HSC causes an aberrant stem cell expansion leading to myeloproliferative disorder (MPD) development and leukemia progression. JunB-deficient HSC are the leukemia-initiating (leukemic) stem cells (LSC) responsible for the initiation and maintenance of this disease. We have now investigated the mechanisms by which loss of JunB alters HSC properties. We found that loss of JunB severely impaired HSC transplantability and cell cycle regulation. Limit dilution transplantation experiments with purified HSC (Lin−/c-Kit+/Sca-1+/Flk2- cells) revealed that junB-deficient HSC are extremely poor at providing engraftment on a cell-by-cell basis. Competitive bone marrow transplantation experiments confirmed that junB-deficient cells are on average 50% less efficient than normal cells in providing engraftment, although in every case where they did engraft, they mediated multilineage reconstitution followed by myeloid expansion and MPD development. JunB-deficient HSC also displayed rapid exhaustion of their self-renewal activity following serial transplantation. At the cellular level, the absence of JunB profoundly deregulated HSC proliferation and cell cycle distribution. Direct analysis of junB-deficient HSC revealed a striking decrease in the number of quiescent G0 cells (30% vs. 70% in normal HSC) and a correlative increase in the number of cycling cells. Quantitative RT-PCR analysis of junB-deficient HSC indicated a global decrease in the expression level of early G1 cyclins and almost all cyclin-dependant kinase inhibitors, associated with an increase in the expression level of late G1 and S-G2/M cyclins. These results provide a molecular understanding of junB-deficient HSC proliferation and strongly suggests that faster trafficking through the cell cycle is a central feature of their leukemic behavior. We also found that loss of JunB severely impaired HSC migratory response, with junB-deficient HSC displaying increased random diffusion and severely blunted response to the chemoattractant SDF1α. In fact, flow cytometry, qRT-PCR and microarray analyses revealed that several molecules involved in cell adhesion/migration (including CXCR4, LFA-1 and VLA-4) are deregulated in junB-deficient HSC. Finally, using short-term in vivo homing assays and intrafemoral injections we established that the engraftment defect exhibited by junB-deficient HSC is not due to a defect in their ability to home to the bone marrow (BM) cavity but to their defective ability to respond and to be maintained in this microenvironment. We are now studying how junB-deficient HSC localize/interact with cellular and molecular components of the BM niches contributes to their loss of quiescence and leukemic expansion. Taken together, these results provide a cellular and molecular understanding of how JunB controls the homeostasis of the stem cell compartment by coordinating stem cell maintenance with stem cell migration. They raise the exciting possibility that interfering with the deregulated cell cycle and altered maintenance of LSC in the BM microenvironment could lead to their specific eradication without impacting on normal HSC function.