P1348: c3g controls megakaryocyte influence on myeloid commitment of hematopoietic stem cells upon chemotherapy-induced myelosuppression.

Topic: 23. Hematopoiesis, stem cells and microenvironment Background: After bone marrow (BM) depletion induced by chemotherapy, hematopoietic stem cell (HSC) self-renewal and differentiation is triggered to restore BM homeostasis through a process tightly regulated by their microenvironment, known as “niche”, where megakaryocytes (MK) and adipocytes (BMA) act in an opposite manner. To avoid either HSC pool depletion or excessive proliferation leading to myeloproliferative diseases, a balanced HSC expansion and differentiation is required. Rap1, a small GTPase highly expressed in BM cells, regulates different crucial cellular processes, such as proliferation, cell adhesion or secretion. Its activation is controlled through a GDP-GTP binding cycle that is regulated by guanine nucleotide-exchange factors (GEFs), which favor the active state, and by GTPase-activating proteins (GAPs), which promote the inactive state. Several hematological diseases arise when Rap1 is dysregulated, both in mouse and humans. C3G, a GEF for Rap1, has been described to affect megakaryopoiesis. However, its role in BM recovery following myelosuppression has not been explored. Aims: Since C3G effect in MK behavior is enhanced in pathological and challenging situations, we wanted to explore its contribution to MK niche function after chemotherapy-induced myelosuppression using in vivo models. Methods: We induced myeloablation in mouse models in which C3G is overexpressed (transgenic) or deleted (knock-out) specifically in MK by a sublethal injection of 5-fluorouracil to analyze its influence in MK niche function. Platelets and hematological parameters were evaluated on an ADVIA 120 hematology analyzer. We assessed MK and BMA content in femur sections by hematoxylin-eosin staining. Hematopoietic progenitors and adipocyte progenitors in BM extracts upon erythrocyte lysis were analyzed by flow cytometry using specific surface markers. Proliferative capability of HSC cells was assessed by an in vivo BrdU assay. Results: C3G levels inversely correlated with the number of MK during BM repopulation due to a promotion of the maturation of MK by C3G, culminating in a higher platelet production 10 days after 5-FU injection. Conversely, the differentiation of CD45-CD31-Sca1+CD24- adipose progenitors into BMA decreased or increased parallel to C3G expression in MK. This modulation of the niche affected the HSC pool. Thus, when MK C3G was ablated, HSC proliferation after 7 days of treatment became slower. Furthermore, HSC commitment was biased to lymphoid population in knock-out mice. In contrast, overexpression of C3G in MK significantly reduced myeloid-skewed progenitor populations 7 days after myelosuppression, which was consistent with the increased platelet production. This would reflect a promotion of myeloid commitment by C3G. Besides, these alterations in the recovery response provoked significant differences in the ability of mice to survive subsequent cycles of chemotherapy. Summary/Conclusion: Although the underlying signaling alterations are yet to be elucidated, these results suggest a putative function of C3G in MK actions on BMAs and HSCs during BM recovery after ablation, by controlling myeloid fate decision. This study opens a potential application of C3G as a prognostic marker in BM niche after chemotherapy-induced myelosuppression. Keywords: Rap1, Chemotherapy, Hematopoietic stem and progenitor cells, Bone marrow niche