Novel Small-Scale Phosphoproteomic Discovery Of Therapeutic Targets For Hematopoietic Stem and Progenitor Cell Mobilization

Hematopoietic stem and progenitor cell (HSPC) mobilization from the bone marrow is critical for successful maintenance of hematologic homeostasis, and is important clinically in hematopoietic stem cell transplant and for recovery after chemotherapy. Pharmacologic mobilization of HSPCs induces proliferation and migration of progenitor cells into the peripheral circulation, hastening hematologic recovery. However, there are very few agents that can achieve robust mobilization of HSPCs in vivo, partly because the mechanisms by which HSPCs emigrate from the bone marrow are diverse and complex. Understanding the central pathways responsible for HSPC mobilization promises to facilitate the development of highly effective mobilizing agents.

We compared resting bone marrow HSPCs (bmHSPCs) with mobilized HSPCs (mobHSPCs) to identify unique pathways responsible for blood cell mobilization. This type of comparison has been performed at a transcriptional level, but cellular functions are ultimately effected by proteins. Additionally, mRNA abundance correlates poorly with levels of corresponding proteins and is insensitive to changes in functional activity of those proteins. Therefore, we developed a mass spectrometry-based platform to allow for the detection of comprehensive phosphoproteomes from small numbers of cells, enabling for the first time analysis of rare cell populations like HSPCs, which constitute only 0.01-0.1% of all bone marrow hematopoietic cells. Using flow cytometry, stable-isotope labeling, and a novel multidimensional nanoscale phosphoproteomic platform, we have successfully compared the phosphoproteomes of rigorously defined bmHSPCs and mobHSPCs. Analysis of as few as 2x10⁵ flow-sorted cells by 3D RP-SAX-RP-MS/MS coupled to an Orbitrap Velos mass spectrometer resulted in detection of more than 3,600 unique phosphopeptide sequences, corresponding to about 1,000 proteins. Hierarchical clustering and pathway analysis generated priority lists of candidate proteins that are more phosphorylated either in the resting state or in the mobilized state. Of these, we focused on the novel Rac-GAP Arhgap25 as a protein potentially central to the process of HSPC mobilization. Arhgap25 is highly phosphorylated in mobilized HSPCs, but not in resting HSPCs. Although the function of Arhgap25 phosphorylation is unknown, Rac is well-described as necessary for HSPC engraftment and retention in the bone marrow. Thus, absence of Arhgap25 would be anticipated to increase Rac activation and promote bone marrow retention of HSPCs; this suggests that the phosphorylation of Arhgap25 may be inhibitory. Arhgap25 knockout mice were generated; as predicted, Arhgap25^-/- mice have significantly higher numbers of Lin^-Sca-1⁺c-Kit⁺(LSK) cells in the bone marrow, and lower numbers of LSKs in the spleen and peripheral blood, as compared to control mice. The hematopoietic compartment of these mice was further analyzed in both the mobilized state and in a transplant setting. These studies have delineated the role of Arhgap25 in HSC and HPC mobilization and engraftment.

We have developed a nanoscale phosphoproteomics platform able to analyze at high resolution small numbers of rare but biologically important cells. This technology has identified many proteins that are activated in HSPC mobilization, and has identified Arhgap25 as a key moderator of HSPC mobility. Additional candidates are undergoing validation. Further studies are underway to take therapeutic advantage of Arhgap25’s role in mobilization.