Single-Cell Characterization of Acute Myeloid Leukemia (AML) and Its Microenvironment Identifies Signatures of Resistance to PD-1 Blockade Based Therapy

Background The success of allogenic stem cell transplantation in curing AML suggests that the immune system can be harnessed to eradicate AML. In a phase 2 trial (NCT02397720) in relapsed/refractory (R/R) AML patients, we demonstrated that the azacitidine/nivolumab combination improved response rates and median overall survival compared with similar patients treated on other azacitidine-based studies (Daver et al Cancer Discovery 2019). The heterogenous response profiles and shorter duration of responses than seen in solid tumor patients suggested hitherto undefined tumor intrinsic, tumor microenvironment (TME) and T cell factors may impede PD-1 blockade therapy in AML. Methods We performed single cell RNA sequencing (scRNAseq) of 13,633 healthy bone marrow (BM) donor, and 113,394 BM cells from 22 aspirates (8 pre- and 14 post- treatment) from 8 R/R AML patients (median age 73 years; range 64-88 years) treated with azacitidine/nivolumab (Fig 1A). 3/8 patients were responders (2CR, 1 PR), while 2/8 and 3/8 had stable disease (SD), and no response (NR), respectively, allowing us to evaluate factors involved in response, relapse and resistance to azacitidine/nivolumab. Results A total of 60,753 AML and 52,641 TME cells passed scRNAseq quality check, with the proportion of identified AML cells correlating with clinical flow cytometry (r=0.87, p=1.5x10-7) and immunohistochemistry (r=0.73, p=0.0001). Pre- and post-treatment AML cells clustered by patient and had distinct cell cycle profiles regardless of response type, suggesting significant inter-tumor heterogeneity (Fig 1B). In an aggregate analysis of all cells at the pretreatment timepoint, the 3 responders had lower leukemia stemness (LSC17) scores compared with NR (p