Improved Engraftment of Bone Marrow and Mobilized Peripheral Blood Stem Cells in a Fanconi Anemia Murine Model.
Authors: Lee Andrew W. , Milsom Michael D. , Zheng Yi , Cancelas Jose A. , Williams David A. 
Published: October 2019

Fanconi anemia (FA) is a genetic syndrome characterized by development of progressive bone marrow failure and cancer predisposition. The difficulty in harvesting FA HSC and their fragility during subsequent in vitro manipulations has proven a confounding factor in attempted gene therapy of this disease. Mobilization of HSC/P in FA patients is poor (Croop et al. Blood, 2001) probably due to HSC deficiency. We have previously shown that the genetic deletion of the Rac GTPases 1 and 2 results in an increase in circulating hematopoietic stem cells and progenitors (HSC/P) (Gu et al. Science 2003). In addition, administration of a single dose of a small molecule inhibitor of Rac GTPases, NSC23766, results in a transient mobilization of engraftable stem cells (Cancelas et al. Nat. Med., 2005). Here, we analyzed the role of NSC23766 in mobilizing HSC/P in FA A (Fanca −/ −) mice (Cheng et al., Hum. Mol. Genet., 2000; kindly provided by M. Grompe, OHSU). First, we validated that this murine model of FA demonstrated a stem cell phenotype by a competitive repopulation assay of BM HSC. We found that Fanca −/ − HSC contribute decreased chimerism in short-term engraftment (52.6 ± 2.6% donor engraftment) compared to wild-type (WT) controls (63.8 ± 1.0%, respectively, p < 0.005). BM and spleen homing of Fanca −/ − HSC/P at sixteen-hours post infusion was not impaired (7.0% in BM and 6.1% in spleen) compared to WT mice (7.8% in BM and 5.4% in spleen) and there was no difference in expression of CXCR4, a4-integrin, a5-integrin and L-selectin between Lin−/c-kit+/Sca-1+ BMC and mobilized PBC derived from Fanca −/ − and WT mice, also supporting an intrinsic HSC defect. We then analyzed the ability of NSC23766, alone or in combination with G-CSF, to mobilize HSC. We observed that Fanca −/ − mice also show an impaired mobilization response to G-CSF administration (200 mcg/Kg/day for five days), which can be partially rescued by administering a single dose of NSC23766, 6 hours before peripheral blood harvest (Table1). We additionally demonstrated the impaired engraftment of in vitro manipulated Fanca −/ − BMC in a competitive transplant assay. This engraftment defect could be completely ameliorated by treatment with Diprotin A (5.9±2.0% donor engraftment untreated vs. 12.0±4.4% treated; p value = 0.01). Diprotin A is an inhibitor of CD26 peptidase which has been shown to cleave SDF1alpha/CXCL12. The combined use of G-CSF and NSC23766 may constitute a future novel approach to induce mobilization of Fanconi anemia HSC and, when coupled with Diprotin A treatment, could act to enhance the engraftment of cells undergoing genetic correction.

Table 1. Competitive Repopulating Units (normalized to WT GCSF) Short Term Engraftment (+1 month) Long Term Engraftment (+4 months) * p value < 0.05, compared to WT GCSF; ** p value < 0.001, compared to WT GCSF, *** p value < 0.05 compared to Fanca −/ − GCSF WT GCSF (%) 100 ± 34.08 100 ± 52.34 WT GCSF + NSC 23766 (%) 77.43 ± 37.11 53.34 ± 54.61 Fanca −/− GCSF (%) 11.25 ± 5.92 * 2.54 ± 2.59 ** Fanca −/− GCSF + NSC 23766 (%) 24.45 ± 1.89 39.01 ± 29.47 ***