Rational Targeting of the Signaling GTPase Cdc42 Establishes a Non-Myeloid Ablative Regimen for Hematopoietic Stem Cell Engraftment In Mouse Models

Abstract 73

Hematopoietic stem cell (HSC) transplantation has become a standard of care for the treatment of many hematological disorders such as Fanconi anemia. However, current myeloid ablative or chemotherapy conditioning regimens are associated with significant morbidity and mortality. In addition, outcomes of transplantation can be limited by low infused stem cell numbers, especially in the setting of umbilical cord blood or autologous transplantation. Thus, availability of agents that could optimize entry of stem cells into bone marrow (BM) niche would be of great value in improving HSC transplant outcome. Recent studies have shown that HSC mobilization before transplantation might vacate BM niche and thus allow improved engraftment of donor HSCs (Blood 113, 4856). Previously we reported that conditional knockout of the Rho GTPase Cdc42 from BM cells led to massive egress of hematopoietic stem/progenitor cells (HSPCs) from BM to peripheral blood (PB) in mice (PNAS 104, 5091). We have also shown that a novel Cdc42 activity-specific inhibitor, CASIN, is able to transiently mimic Cdc42 knockout phenotype in mice in inducing HSPC mobilization from mouse BM by suppressing actin polymerization, adhesion, and directional migration (Blood 112, 68). In the present studies, we hypothesize Cdc42 targeting can open up BM niche to facilitate subsequent HSC engraftment. First, in a conditional Cdc42 knockout mouse transplant model (CD45.2⁺ MxCre;Cdc42^flox/flox BM engrafted in congenic recipients) we showed that poly I:C induction followed by transplant of congenic donor BM cells resulted in significant engraftment of donor blood accompanied by a loss of Cdc42^−/− cells. The donor-derived cells in both BM and PB made up of ∼70% chimerism in multiple blood lineages including Lin⁻Sca⁺c-Kit⁺ primitive progenitors (74.62±5.5%). The multi-lineage donor chimerism was maintained in secondary transplant, indicating effective long-term HSC engraftment. In contrast, no significant engraftment was observed in mice of MxCre;Cdc42^wt/wt BM genotype after similar polyI:C induction. These experiments indicate that genetic deletion of Cdc42 in host HSCs can allow efficient donor engraftment without irradiation or myeloid ablative conditioning. Second, we found CASIN treatment of recipient mice transiently suppressed low density BM cell Cdc42 activity and mimicked the Cdc42 knockout effect in mobilizing long-term HSCs to allow congenic donor HSC engraftment. Three i.p. injections of CASIN to recipient mice prior to infusion of donor BM cells resulted in 8.66±2.3% chimerism of multi-lineage donor derived cells 4 months post-transplant, while the vehicle treated mice showed less than 1% chimerism. Third, similar CASIN preconditioning allowed the engraftment of 5.53±2.68% CD34⁺ human cord blood cells into immunodeficient NSG mice compared with less than 1% by vehicle. Fourth, CASIN preconditioning alone was able to achieve 13.08±2.94% and 11.17±2.86% chimerisms of WT and Fanca gene-corrected Fanca^−/− donor BM cells, respectively, in Fanca^−/− mouse recipients. This effect in engraftment was particularly evident when CASIN was administrated in combination with the immunosuppressant, Fludarabine, as donor chimerism reached 22±3.05% compared with 3.63±0.87% by Fludarabine alone or 11.3±1.08% by CASIN alone. Throughout these studies, no toxicity was detected in CASIN-treated recipient mice based on examinations of CASIN treated blood, BM, spleen, liver, and other organs at one or seven days. Together, our studies demonstrate that Cdc42 is a useful target for enhancing HSC engraftment in transplantation by opening BM niche. The results present a novel preclinical regimen of pharmacological targeting Cdc42 to facilitate the engraftment of murine HSCs and human cord blood HSPCs and to improve Fanconi anemia HSC transplantation therapy, clinical settings in which stem cell numbers critically limit success.