Overexpression of CD200 Is a Stem Cell-Specific Mechanism of Immune Escape in AML

Background: Acute myeloid leukemia (AML) stem cells (LSC), the likely source of relapsed disease, are capable of surviving current standard chemotherapy. Therefore, novel therapeutic approaches specifically engineered to eradicate LSCs are critical for curing AML. We previously introduced a novel bioinformatics approach that harnessed publically available AML gene expression datasets and identified CD200 as significantly over-expressed in LSCs when compared to paired blast cells, as well as when compared to their normal hematopoietic stem cell (HSC) counterparts (Fig 1A; Herbrich et al Blood. 2018; 130:3962). CD200 can identify AML cells with LSC activity in vivo (Ho et al Blood. 2016; 128:1705). Functionally, CD200 has been shown to have an immunosuppressive effect on macrophages (Hoek et al Science. 2000; 290:1768) and NK cells (Coles et al Leukemia. 2012; 26:2148), and correlates with a high prevalence of FOXP3+ regulatory T cells (Coles et al Leukemia. 2012; 26:2146). Additionally, CD200 has been implicated as a poor prognostic marker in AML (Damiani et al Oncotarget. 2015; 6:30212). To date, we have screened 40 primary AML patient samples by flow cytometry, 95% of which are positive for CD200. Methods: To study the functional role of CD200 in AML, we generated a CD200 overexpression model in the human OCI-AML3 cell line (with no basal expression) and characterized changes in proliferation, survival, and gene expression. To examine the immune function of CD200 in AML in vitro, we performed a series of mixed lymphocyte reactions with isolated effector immune cells and target isogenic AML cell lines to assess immune cell-mediated apoptosis, proliferation, and cytokine secretion. To understand the contribution of CD200 immune protection in a physiological setting, we developed a peripheral blood mononuclear cell (PBMC)-humanized mouse in which we tracked the engraftment and overall survival of the CD200+/- OCI-AML3 cells. Lastly, the utility of CD200-blockade using a fully humanized anti-CD200 monoclonal antibody (CD200-IgG1) was evaluated both in vitro and in vivo. Results: In vitro, CD200+ AML significantly inhibited the secretion of inflammatory cytokines and cytotoxic enzymes from healthy PBMCs; a phenomenon that could be largely reversed by blocking the CD200/CD200R interaction with the CD200 antibody (Fig 1B). In vivo, OCI-AML3 CD200+/- cells showed no difference in engraftment, progression, and overall survival in immunodeficient NSG mice (Fig 1C). However, when mice were humanized using healthy PBMCs, CD200+ leukemia progressed rapidly, escaping T cell-mediated elimination, compared to CD200- control leukemic cells (Fig 1D). Cytokine production in PBMC-humanized mice was significantly compromised in those with CD200-expressing leukemia. Transcriptome analysis revealed that T cells from humanized mice exposed to CD200 expressing disease were metabolically quiescent. In humanized mice, CD200-IgG1 therapy eliminated CD200+ AML disease (Fig 1E). The novel CD200-IgG1 antibody also induced potent, specific NK cell-mediated antibody dependent cellular cytotoxicity (ADCC) and macrophage-mediated antibody dependent cellular phagocytosis (ADCP; Fig 1F). Conclusion: We have identified CD200 as a putative stem cell-specific immunomodulatory target that aids in establishing an immunosuppressive microenvironment by significantly suppressing cytokine secretion in response to AML. In a PBMC-humanized mouse model, the presence of cell-surface CD200 was sufficient to protect AML cells from immune-mediated clearance and could be reversed using a blocking anti-CD200 mAb. These findings indicate a utility of CD200 as a novel immune checkpoint target for the development of therapeutic strategies in AML. Download : Download high-res image (761KB) Download : Download full-size image Disclosures Konopleva: Calithera: Research Funding; Kisoji: Consultancy; AbbVie: Consultancy, Research Funding; Reata Pharmaceutical Inc.;: Patents & Royalties: patents and royalties with patent US 7,795,305 B2 on CDDO-compounds and combination therapies, licensed to Reata Pharmaceutical; Ablynx: Research Funding; Genentech: Consultancy, Research Funding; F. Hoffmann La-Roche: Consultancy, Research Funding; Eli Lilly: Research Funding; Cellectis: Research Funding; Amgen: Consultancy; Stemline Therapeutics: Consultancy, Research Funding; AstraZeneca: Research Funding; Sanofi: Research Funding; Agios: Research Funding; Forty-Seven: Consultancy, Research Funding; Rafael Pharmaceutical: Research Funding; Ascentage: Research Funding.