HSC/P transplants are used to replace the endogenous hematopoiesis of patients in the treatment of cancer and some genetic disorders ^1–7. The success of these transplants depends on the number and quality of HSCs infused, a receptive host marrow and, in the case of allogeneic transplants, on the immunotolerance of the recipient for the progeny engrafted HSCs.

In adults, HSC/Ps reside in the marrow and are largely absent from the peripheral blood (PB) ⁸. The endosteal space of the adult marrow is enriched in HSC/Ps which are, located in putative “niches” where a specialized microenvironment supports and nurtures them, and allows the maintenance of an equilibrium with a minority of HSC/Ps circulating in PB. While marrow harvests were previously utilized to collect transplantable HSCs, current clinical practice mostly applies the biological phenomenon of HSC/P mobilization from the marrow into the PB to allow leukoapheresis harvest⁹ as this does not require general anesthesia and is typically associated with shorter periods of post-pancytopenia ^7,10.

Administration of cytokines, such as granulocyte-colony stimulating factor (G-CSF) ² or stem cell factor (SCF) ¹¹, chemokine-receptor inhibitors (e.g. AMD3100), ¹² and cytotoxic drugs (e.g. cyclophosphamide), ^2–6 have been used clinically to increase the number of circulating HSC/Ps. A goal of PB stem cell transplant for hemato-oncological diseases is to optimize the number of HSCs to ensure low levels of the morbidity and mortality that is associated with rescue in the myeloablative setting ^5,10. In addition, PBSCs are ideal targets for cell and gene therapies ¹³. The administration of G-CSF is currently the major method for mobilization of HSC/Ps for clinical usage. However, over 40% of patients who have undergone intensive chemotherapy, and between 10% and 20% of all patients and normal individuals, fail to mobilize sufficient numbers of HSC/Ps for successful PB stem cell transplant ⁹.

Cytoskeleton rearrangements are controlled by members of the Rho GTPase family. One of the best studied subfamilies of Rho GTPases in the context of HSC/Ps functions is Rac. Signaling associated with all homing and migration-related activities is driven through Rac GTPases. Rac integrates signals from β₁-integrins and c-kit in HSC/P and mast cells ^28,29. CXCL 12 (SDF-1)-induced chemoattraction is mediated by Rac and is at least partly mediated by the GEF Tiam-1³⁰. Signaling of c-kit to Rac/Cdc42 is mediated through the GEF Vav^31,32. However, other GEFs may be crucial in Rac activation in the control of HSC/P functions. Three highly related Rac proteins are expressed in mammalian cells, Rac1, Rac2 and Rac3. Rac 1 and 3 are widely expressed^33,34, while Rac2 expression is hematopoietic specific ^35,36. The three Rac proteins share a high degree of protein homology. However, each appears to have overlapping as well as unique roles in HSC/P activity. Studies using dominant negative mutants lacked specificity, and their conclusions were significantly modified in in vivo studies. A summary of the phenotypes of HSC/Ps described using these dominant negative mutants and in vivo gene targeting of Rac proteins is included in Table 1. Initial studies in Rac2^−/− mice demonstrated that Rac2 is important for HSC/P adhesion and migration, and Rac2^−/− mice showed increased numbers of circulating HSC/Ps and hypermigration³⁷, suggesting an important role for Rac2 in retaining cells in the marrow hematopoietic microenvironment (HM). Rac2 activity is also important in maintaining normal Rac1 and Cdc42 function, confirming in primary cells ‘the existence of a cross talk’ between Rac2 and other Rho GTPases involved in regulating HSC/P migration^37,38. Although Rac2 is highly homologous to Rac1, Rac2 and Rac1 regulate distinct HSC/P functions. Rac1-deficient HSC/P demonstrated defective proliferative signaling via the receptor tyrosine kinase, c-kit, while loss of Rac2 activity leads to a pro-apoptotic phenotype²⁸. Double-deficient Rac1^Δ/Δ/ Rac2^−/− HSC/P showed a combination of reduced proliferation and increased apoptosis²⁸ and at the same time, Rac1^Δ/Δ;Rac2^−/− HSC/Ps show decreased adhesion to fibronectin, despite the normal expression of CD49d (α₄β₁)- and CD49e (α₅β₁)-integrin adhesion molecules on these cells⁶. Since Rac1^Δ/Δ HSC/Ps have normal adhesion to fibronectin, and Rac2^−/− HSC/Ps display an intermediate level of adhesion, it appears that Rac2 has a predominant but overlapping role with Rac1 in integrin-mediated HSC/P adhesion. In addition, Rac1^Δ/Δ;Rac2^−/− HSC/Ps have decreased migration in response to CXCL12 compared with wild-type cells while having a significantly increased expression of CXCR4, the receptor for the CXCL12.⁶ Since Rac GTPases are key components of the signaling pathways downstream of the SCF-receptor, c-kit,⁷ the chemokine receptor CXCR4,⁸ and the β₁-integrin-receptors for fibronectin,^9,10 all critical mediators of HSC/P interaction with the marrow HM, it is not surprising that a combined deficiency of Rac1 and Rac2 results in massive mobilization of progenitor colony-forming unit cells (CFU-C) into the peripheral circulation and results in increased homing of CFU-C in the spleen.³⁹ CXCL12 gradients controlling homing and mobilization of HSCs control Rac activation through the incorporation of CXCR4 into membrane lipid rafts⁴⁰ and through the downstream activation of the Rac effector Wave2⁴¹.

Despite the role of Rac1 in HSC proliferation²⁸, induced deletion of Rac1 in mixed chimeric mice does not induce hematopoietic failure, indicating that Rac1 is dispensable for steady-state hematopoiesis³⁹.

The funding from the National Blood Foundation (NBF) helped me to develop an experimental strategy to analyze the specific roles of Rac proteins in vivo in two major instances. First, it helped me to analyze the mechanisms controlled by Rac in HSC activity. Through a series of innovative approaches that include the use of inducible triple transgenic animals, competitive repopulation assays, anatomical microlocalization of HSC/Ps within the marrow cavity, and limiting-dilution analysis of long-term marrow cultures, we found that Rac1 deficiency induces a defective localization in the endosteal space of the marrow cavity after transplantation³⁹. In contrast, Rac2 deficiency induces loss of retention of HSC/Ps in the marrow^37,39 while the deletion of both Rac1 and Rac2 leads to the massive mobilization of HSC/Ps from the marrow to PB due to decreased adhesion and retention in the marrow^28,39. These data strongly suggest that at the molecular level HSC/P homing and retention in the marrow are not mirror image processes and that Rac1 and Rac2 have overlapping and distinct roles in HSC/P trafficking. Secondly, the NBF grant helped me to explore the translational possibilities of Rac inhibition on HSC mobilization. We were fortunate enough to have on the extraordinary collaboration of Dr. Yi Zheng (Division of Experimental Hematology and Cancer Biology, Cincinnati Children’s Hospital Medical Center) who had discovered a first-generation, rationally-designed small molecule, NSC23766, which has with inhibitory activity on Rac activation, and Dr. David Williams (Boston Children’s Hospital Medical Center, Harvard Medical School), my mentor, who has extensive experience in analyzing the role of Rho family GTPases in HSC activity. Together, we discovered that in vivo Rac inhibition induced mobilization of HSCs which could then be harvested and, after drug removal, were engraftable in myeloablated syngenic animals. These data opened up the possibility of Rac targeting to improve HSC mobilization in patients resistant to mobilization-inducing conventional therapies.

Rho GTPases appear to be key regulators of the localization of HSCs within the HM and the interaction of HSCs with cellular niches. Further research into the roles of Rho GTPases in the microenvironment and in relation to the osteoblastic and vascular HSC niches is needed. Finally, the development of small molecule inhibitors, while difficult due to the affinity of the interactions of GEFs with Rho GTPases, offers new opportunities for therapeutic intervention at the HSC level in cell, gene, and cancer therapies.