Vcam1 Is a "Don't-Eat-Me" Signal on Healthy Hematopoietic and Leukemic Stem Cells

Hematopoietic stem cells (HSCs) possess the ability to maintain the entire population of blood cells throughout life and to replenish the hematopoietic system after transplantation into marrow-ablated recipients. In mammalian adults, HSCs predominantly reside in the bone marrow to generate and maintain all blood cell types. HSCs, however, retain their ability to migrate to ectopic niches via the bloodstream, and traffic back to the bone marrow microenvironment via in part the interactions of alpha4 integrins with Vascular Cell Adhesion Molecule-1 (Vcam1, also known as CD106), constitutively expressed by endothelial and stromal cells. In the course of studies to investigate the role of Vcam1 in the macrophage erythroblastic niche, we have found using Vcam1-floxed mice crossed with a Csf1r-iCre (hereafter Vcam1^Δ/Δ) that Vcam1 and Cre, using this transgenic line, were expressed on Lineage⁻ (Lin⁻) c-Kit⁺ Sca1⁺ CD48⁻ CD150⁺ HSCs. Although Vcam1^Δ/Δ mice show no significant steady-state hematopoietic defect, transplantation of Vcam1^Δ/Δ bone marrow, both in competitive and non-competitive settings, failed to engraft irradiated wild-type recipients. Indeed, ~80% of animals transplanted with Vcam1^Δ/Δcells died, and the few that survived failed to successfully engraft secondary recipient mice. Vcam1^Δ/Δ HSC/progenitors homed to bone marrow as well as wild-type counterparts and showed no detectable change in apoptosis in the bone marrow. However, syngeneic Vcam1^Δ/Δ HSC/progenitors were selectively cleared in vivo by bone marrow macrophages over 4 days. The in vivo Vcam1^Δ/Δ HSC/progenitor clearance did not require irradiation-induced damage since in steady-state parabiosis (Vcam1^Δ/Δ and wild-type co-joined pairs), Vcam1^Δ/Δ HSCs did not engraft the partner after G-CSF-induced mobilization whereas wild-type HSCs engrafted the parabiont partner. In addition, in vitro incubation of bone marrow-derived macrophages with Lin⁻ cells revealed rapid phagocytosis by macrophages of Vcam1^Δ/Δ, but not wild-type, cells and phagocytosis was also induced when the integrin alpha4 beta1 (also known as VLA-4) was blocked in wild-type Lin⁻ cells. Thus, these results suggest that Vcam1 expressed on HSCs serves as a novel don't-eat-me signal, controlling the migration in the bone marrow by a macrophage-enabled checkpoint. Since we found that Vcam1 is expressed at higher levels on acute myelogenous leukemia (AML) cells than on healthy HSCs, and high VCAM1 expression correlates with poor prognosis in human AML patients (Ley et. al. NEJM 2013), we examined whether Vcam1 expression by AML cell could affect leukemogenesis in vivo. To test this idea, we generated AML-Vcam1^Δ/Δand AML-Control cells by transduction of bone marrow Lin⁻ c-Kit⁺ Sca1⁺ cells with the pMSCV-MLL-AF9-GFP oncogene. Strikingly, FACS analysis of primary AML recipient bone marrow revealed a marked reduction (>99%) in the number and percentage of phenotypic Lin⁻ IL7Rα⁻ Sca1⁻ MLL-AF9 GFP⁺ c-Kit^high CD34^low FcγRII/III^high leukemic stem cells in AML-Vcam1^Δ/Δmice compared to control. These results were confirmed by imaging analyses, which showed a marked reduction in leukemic infiltration in AML-Vcam1^Δ/Δmice compared to control. Kaplan-Meier survival analysis of secondary recipient mice receiving 20,000 GFP⁺ leukemic cells revealed a significantly greater survival of mice harboring AML-Vcam1^Δ/Δ cells relative to AML-Control (P<0.0001). To assess in a pre-clinical experimental setting whether Vcam1 inhibition can alter the course of AML, we established AML in immunocompetent C57BL/6 recipients and started therapy of moribund leukemic mice (>50% circulating AML-GFP⁺ cells) with a daily injection of saline, IgG control, anti-Vcam1 blocking antibody (100 μg/day), cytarabine (100 mg/kg), or a combination of anti-Vcam1/cytarabine for 5 days. Remarkably, this short treatment with anti-Vcam1 blocking antibody was able to preferentially and significantly reduce the frequency and absolute number of phenotypic bone marrow leukemic stem cells in vivo. In addition, our results suggest that using a blocking anti-Vcam1 antibody synergizes with cytarabine treatment to decrease leukemic burden in vivo. These studies lay the groundwork for the development of a new therapeutic strategy for eliminating leukemia stem cells by modulating the innate immune response.