Deletion of Fak in Hematopoietic Stem Cells Leads to Enhanced Engraftment.

Abstract

Hematopoietic stem and progenitor cells (HSPC) can be induced to leave the bone marrow (BM) and such mobilized hematopoietic stem cell (HSC) enriched cell populations when infused intravenously rapidly home back to the marrow and transmigrate into the extravascular compartment where they lodge in specialized niches. Niche induced signals coupled to intrinsic factors, promote HSC maintenance and regulate HSC localization, migration and/or differentiation. A better understanding of molecular pathways governing these HSC fate decisions could identify new targets for ex vivo HSC expansion and improvement of transplantation. Focal adhesion kinase (Fak) is a non-receptor protein tyrosine kinase, whose function has been well studied in fibroblasts, where it plays an important role in cell adhesion, survival and motility. Fak is also differentially expressed in HSPC, with highest expression in long-term HSCs (LT-HSCs, Lin-Sca-1+c-Kit +CD34-). Its functional role in hematopoiesis has not been well studied due to early embryonic lethality (E.8) of Fak−/− mice. We have generated conditional Fak deleted mice (Fakfl/flMx1-Cre+) to define how Fak signaling contributes to different components of HSC transplantation. We observed that the total cellularity in BM was comparable between Fak knockout (Fak−/−Mx1-Cre+) and control (Fakfl/fl) mice. The frequencies of both Lin-Sca-1+c-Kit + cells (LSKs) and short-term HSCs (ST-HSCs, Lin-Sca-1+c-Kit +CD34+) increased 2-fold in BM of Fak knockout mice compared to WT controls (n=5; p<0.05), while LT-HSCs appeared unaffected. Moreover, the number of LSKs in the spleens of Fak knockout mice increased by 3-fold relative to controls (n≥7, P<0.01). In vitro colony-forming assay also showed that the functionally defined progenitor cell numbers significantly increased in Fak knockout mice spleen. However, Fak deletion did not affect HPC populations including common myeloid progenitors (CMP), granulocyte macrophage progenitors (GMP), megakaryocyte erythrocyte progenitors (MEP) and common lymphoid progenitors (CLP). Importantly, in the competitive repopulation assay Fak−/− HSCs exhibited 2-fold enhanced engraftment for 20 weeks post-transplant, while lineage reconstitution in peripheral blood (PB) was similar between wild-type and knockout donor-derived cells suggesting Fak deletion did not affect PB mature B, T and myeloid cell survival under non-stress conditions. These findings indicate that FAK may play an important role in HSC homeostasis and that targeting the FAK pathway ex vivo may lead to innovative approaches in human HSPC transplantation.