The Ryk Receptor Regulates Hematopoietic Stem and Progenitor Cell Proliferation and Their Sensitivity To Myeloablative Agents

The molecular mechanisms that control the balance between quiescence and proliferation of hematopoietic stem and progenitor cells (HSPCs) are critical for maintaining life-long hematopoiesis. In a recent study (Povinelli, et al. Stem Cells, In Press, 2013) we demonstrated that the Wnt5a ligand inhibits HSPC proliferation through a functional interaction with a non-canonical Wnt ligand receptor termed Related to Receptor Tyrosine Kinase (Ryk). Expression of Ryk on HSPCs in vivo was associated with a decreased rate of proliferation. Following treatment with fluorouracil (5-FU), the percentage of Ryk + HSPCs increased at the expense of Ryk -/low HSPCs. Based on these data, we hypothesized that one function of the Ryk receptor is to protect HSPCs from the effects of myeloablative agents. To test this hypothesis, we injected 6-8 week old C57BL/6 mice with 150 mg/kg of 5-FU and analyzed bone marrow 48 hours later for the presence of apoptotic HSPCs, defined as lineage negative (Lin - ), Sca-1 + , CD48 - cells positive for active caspase-3. There was a 2.5-fold decrease in the percentage of apoptotic Ryk + HSPCs (12.9 ± 1.7%) compared to Ryk -/low HSPCs (32.4 ± 5.3%, p - , c-kit + , Sca-1 + , CD150 + , CD48 - immunophenotype or LSK, SLAM). Comparable to the effects of 5-FU, there was a significant 3.0-fold reduction in the percentage of apoptotic Ryk + HSPCs (3.1 ± 0.2%) compared to Ryk -/low HSPCs (9.2 ± 1.5%, p in vivo targeting of the Ryk receptor would increase the sensitivity of HSPCs to myeloablative injury, we utilized a neutralizing rabbit anti-Ryk antibody (α-Ryk). We injected C57BL/6 mice with 5 mg/kg α-Ryk or rabbit IgG isotype for 2 consecutive days. Twenty-four hours after the second dose, we determined the frequency and cell cycle status of LSK SLAM cells. Treatment with α-Ryk significantly increased the percentage of LSK SLAM cells in the S/G 2 /M phases compared to control (α-Ryk: 17.8 ± 2.2%; isotype IgG: 11.6 ± 2.7%, p 1 following treatment with α-Ryk (α-Ryk: 40.5 ± 3.2%, isotype IgG: 51.3 ± 2.2; p 0 LSK SLAM cells was unchanged (α-Ryk: 37.9 ± 2.6, isotype IgG: 35.7 ± 3.1% n = 3) indicating that inhibiting Ryk promoted the exit of LSK SLAM cells from G 1 . Treatment with α-Ryk also increased the percentage of whole bone marrow cells expressing the LSK SLAM phenotype by 1.4-fold compared to controls (p + GFP - cells. There was no difference in engraftment by transplanted bone marrow cells from mice treated with α-Ryk or isotype IgG (α-Ryk: 61.6 ± 6.1% n = 4, isotype IgG: 52.8 ± 13.6%, n = 5), indicating that the neutralizing antibody does not inhibit short-term HSPC function on its own. We then tested whether blocking Ryk function resulted in greater sensitivity of HSPCs to 5-FU. We treated B6.SJL mice with 5 mg/kg α-Ryk or isotype IgG for 2 consecutive days, followed by 150 mg/kg of 5-FU. Forty-eight hours after 5-FU treatment, we transplanted 2x10 6 C57BL/6 whole bone marrow cells into treated B6.SJL mice without additional conditioning. Four weeks after transplant, we determined the percentage of donor-derived CD45.2 + peripheral blood cells. Treatment of recipient mice with α-Ryk prior to 5-FU treatment resulted in increased engraftment of donor bone marrow by 3.6-fold compared to isotype (p Disclosures: No relevant conflicts of interest to declare.