Regulation of Rho GTPases by the Hematopoietic-Specific Guanine Nucleotide Exchange Factor Vav1 Is Critical for Hematopoietic Stem Cell Retention in the Endosteal Niche and Engraftment.

Abstract 80 Background: Rho GTPases are molecular switches that regulate actin cytoskeleton dynamics, cell proliferation and survival. In hematopoietic stem cells and progenitors (HSC/P), several Rho GTPases (including Rac1, Rac2 and Cdc42) function as critical regulators of engraftment through the integration of diverse extracellular signals, such as those transmitted by growth factor, chemokine and adhesion receptors. In addition, Rac-deficient mice show significantly increased numbers of mobilized HSC/P. GTPase activation downstream of these and other receptors is mediated by a large family of guanine nucleotide exchange factors (GEF). Functional interactions between receptors, GEF and Rho GTPases are potentially complex and the crucial biochemical pathways regulating HSC activity have not been defined. Among the Rho/Rac GEFs, Vav1 shows hematopoietic-specific expression and has been previously implicated in immune cell processes, such as immunoreceptor signaling in lymphocytes and neutrophil migration. To further explore the mechanism of Rho GTPase regulation of HSC engraftment, we investigated the role of Vav1 GEF in Rho GTPase activation after ligation of multiple HSC receptors and the effect of genetic deletion of Vav1 on HSC homing, retention and engraftment in the hematopoietic microenvironment. Methods: GTPase activation (Rac, Cdc42, RhoA) was analyzed by in vitro pulldown assays. The HSC/P compartment of Vav1−/− mice was studied by flow cytometry, colony forming cell (CFC) assays, progenitor (CFC) homing, competitive and non-competitive repopulation assays. HSC localization in the endosteal niche was determined by intravital microscopy 1 h and 48 h after transplant. Results: At the biochemical level, Vav1−/− hematopoietic progenitors showed a dysfunctional Rho GTPase activation pattern, with increased baseline levels of GTP-bound Rac, Cdc42 and RhoA; however, in the absence of Vav1, these GTPases were unresponsive to stimulation by stem cell factor and SDF1α, critical proteins in HSC engraftment. In spite of this biochemical abnormality, Vav1−/− mice at baseline had nearly normal numbers of immunophenotypically defined HSC, myeloid and lymphoid progenitors in the bone marrow (BM), and normal hematopoietic progenitor content as defined by CFC, although reduced rather than increased circulating HSC/P. Vav1−/− HSC/P transplanted into irradiated recipients exhibited normal BM CFC homing efficiency (∼5%) and normal early endosteal localization of HSC in vivo (1 h after injection) as determined by intravital microscopy. Surprisingly-but in concordance with the normal BM homing of HSC/P in vivo - the loss of Vav1 did not affect hematopoietic progenitor chemotaxis or short-term adhesion to fibronectin in vitro . However, there was a significant decrease in the retention of HSC in the endosteal space at 48 h after transplant ( Vav1−/− HSC numbers were reduced to 46%, relative to WT HSC) and this defect was associated with a profound loss of short- and long-term engraftment. In competitive repopulation assays, Vav1−/− cells virtually did not contribute to the graft ([Table 1][1]), whereas in a non-competitive setting, they either failed to rescue the recipient (60% survival vs 100% at 1 month, Vav1−/− vs WT) or showed significantly delayed hematopoietic reconstitution ([Table 2][2]). Conclusions: The hematopoietic-specific GEF Vav1 is essential for the appropriate microenvironment-induced Rho GTPase activation in HSC/P after transplant and is required for the retention of HSC/P in the BM endosteal niche and subsequent engraftment. | | time after transplant (months) | WT | Vav1−/− | |:---------------- | ------------------------------ | ----------- | -------------- | | Peripheral blood | 1 | 53.4 ± 6.0 | 1.1 ± 2.4 (**) | | | 2 | 63.2 ± 5.1 | 0.9 ± 1.2 (**) | | | 3 | 60.1 ± 14.0 | 1.4 ± 1.7 (**) | | | 4 | 57.4 ± 18.5 | 0.1 ± 0.1 (**) | | Bone marrow | 4 | 66.3 ± 33.0 | 0.2 ± 0.4 (*) | | Spleen | 4 | 55.0 ± 13.3 | 0.4 ± 0.5 (**) | Table 1. Percentage of donor chimerism in lethally irradiated recipients transplanted with either WT or Vav1−/− CD45.2 BM cells (3 × 106 cells/recipient) and an equal number of WT CD45.1 competitor BM cells. Values represent mean ± SD. (*) p < 0.05, (**) p < 0.005. | | time after transplant (months) | WT | Vav1−/− | |:---------------- | ------------------------------ | ---------- | -------------- | | Peripheral blood | 0.5 | 4.1 ± 1.4 | 1.1 ± 0.6 (**) | | | 1 | 14.5 ± 3.8 | 5.6 ± 4.7 (**) | | | 2 | 14.2 ± 6.4 | 10.7 ± 6.4 | Table 2. Peripheral blood leukocyte count (× 103/μl) of lethally irradiated recipients transplanted with either WT or Vav1−/− BM cells (3 × 106 cells/recipient). Values represent mean ± SD. (**) p < 0.005. Disclosures: No relevant conflicts of interest to declare. [1]: #T1 [2]: #T2