Natural killer (NK) cells are emerging as a promising therapeutic option in acute myeloid leukemia (AML). Bone marrow (BM) stromal microenvironment plays an important role in AML cells’ survival and resistance to chemotherapy, whereas the impact of stromal cells on NK cell function has not yet been fully discovered. To decipher the molecular modulations within the BM stromal niche inducing AML cell resistance to NK cell attack, we performed functional co-cultures of NK, AML and stromal cells.

AML (MOLM-14, OCI-M1, THP-1) and CML (K562) cell lines were pre-stimulated with HS-5 stromal cells for 24h before the addition of primary expanded NK cells. To understand how NK and leukemia cells respond to their interaction within the stromal microenvironment, we performed flow cytometry, single-cell RNA sequencing (scRNA-seq), genome-scale loss-of-function CRISPR screen and analysis of secreted soluble factors.

The analysis of soluble factors revealed an increase of IL-10 secretion when tumor and NK cells interact within the stromal niche and the genome-scale CRISPR screen identified the loss of IL-10 gene as a sensitizer of THP-1 cells to NK cell mediated-cytotoxicity in the stromal niche. scRNA-seq revealed a distinct NK cell phenotype induced by the stromal cells characterized by a decrease of the NK cell activation receptor NKp30 expression and an increase of exhaustion markers TIM-3 and TIGIT. Interaction with stromal cells induced a downregulation of MHC class I (HLA-B, HLA-F) and class II (HLA-DMB, HLA-DQA1, HLA-DRA) genes in leukemia cells.

Collectively, at single-cell resolution, our findings reveal the dynamic transcriptomic changes and soluble dialogue of tumor and NK cells interacting within the stromal BM niche. Stromal cells impact both NK cells hampering their activation potential and tumor cells making them more resistant to NK mediated-cytotoxicity. This highlights the need for potential combination therapies to enhance the efficacy of NK cell-based treatments in AML.