Abstract
Hematopoietic stem cell transplantation has become a standard of care for the treatment of many hematological diseases. Transplantation of mobilized peripheral blood stem cells has replaced bone marrow (BM) transplantation as the preferred method for hematopoietic recovery. To date, G-CSF mobilized hematopoietic stem/progenitor cell (HSPC) harvest is the main FDA-approved preparative regiment for transplantation protocols, but this application has several limitations in utilities including diverse individual variability and potential side effects in several patient populations. Although AMD3100, a chemical CXCR4-blocker, has been found effective for HSPC mobilization, the development of additional HSPC mobilization agents that work through well defined molecular mechanisms remains in need. Previously our laboratory has shown in a conditional knockout mouse model that deficiency of the Rho GTPase Cdc42 in the BM causes impaired adhesion, homing, lodging and retention of HSPCs, leading to massive egress of HSPCs from BM to the peripheral blood without compromising their proliferative potential. From an array of small molecule inhibitors of PIP2-induced actin-polymerization discovered in a high throughput screening, we identified CASIN, a novel Cdc42 Activity-Specific Inhibitor, that is effective in suppressing Cdc42 activity in a dose-dependent manner in murine fibroblasts and low density bone marrow (LDBM) cells and human CD34+ umbilical cord blood (HCB) cells in vitro, and in murine LDBM cells in vivo. The inhibitory effect by CASIN appears to be specific to Cdc42 and is reversible. We subsequently tested the hypothesis that pharmacological targeting Cdc42 by CASIN may transiently mimic the Cdc42 knockout phenotype leading to HSPC mobilization. In the dose range of 5–10 uM, CASIN does not show detectable toxicity in wild type or Cdc42 knockout HSPCs in cell survival and colony-forming unit activity assays. CASIN treatment of 32D murine myeloid progenitor cells or freshly isolated progenitor cells results in a reversible inhibition of F-actin polymerization induced by SDF-1α and blockade of α5β1 integrin mediated adhesion to fibronectin fragment CH296. Its effects on actin organization and adhesion are associated with an inhibition of directional migration of the colony-forming cells toward SDF-1α. In contrast, CASIN does not show a detectable effect on the adhesion and migration activities of Cdc42 knockout HSPCs, suggesting that it works specifically through Cdc42 to affect cell actin structure and adhesion. Upon injection into mice (5mg/Kg, intraperitoneally), CASIN is effective in stimulating mobilization of progenitor activity into the peripheral blood (~ 6-fold increase compared to control at 40 hrs post injection). Subsequent serial transplantation experiments show that the PB harvested from CASIN treated mice could reconstitute various lineages of blood cells in primary, secondary, and tertiary recipients, indicating that long-term hematopoietic stem cells were mobilized from the BM of CASIN-treated donor mice. Consistent with the mobilization phenotype, FACS analysis shows that intravenous injection of CASIN can cause transient reduction of long-term hematopoietic stem cells (IL7Ra−Lin−Sca-1+c-Kit+CD34−) and short-term hematopoietic stem cells (IL7Ra−Lin−Sca-1+c-Kit+CD34+) from BM. Similar to the effects on murine HSPCs, CASIN is active on CD34+ HCB cells in transiently suppressing F-actin assembly, adhesion to fibronectin, and SDF-1α induced migration without detectable toxicity in vitro. Whether CASIN is effective in mobilizing HCB-engrafted NOD/SCID mice is currently under investigation. Our studies suggest that the novel concept of pharmacological targeting of Cdc42, that transiently and reversibly mimics the effect of Cdc42 knockout, may be developed into a mobilization regiment with a well defined molecular and cellular mechanism.
An in vitro limiting-dilution assay of long-term repopulating hematopoietic stem cells in the mouse