Bone Marrow Stromal Cells Hamper Leukemia Cell Induced NK Cell Activation and Cytotoxicity

Natural killer (NK) cells are emerging as a promising therapeutic option in hematological malignancies including acute myeloid leukemia (AML). However, the complete eradication of AML cells from the bone marrow (BM) represents a major clinical challenge. The 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 that occur in the BM stromal niche inducing AML cell resistance to NK cell attack, we performed functional co-cultures of NK, AML and stromal cells.

We designed a co-culture system mimicking the BM stromal compartment using HS-5 stromal cells and 3 AML (MOLM-14, OCI-M1 and THP-1) and 1 CML (K562) cell lines. After culturing leukemia cells with HS-5 cells for 24h, primary expanded NK cells were added, and the viability of the tumor cells was measured by flow cytometry after 24h. To understand how NK and leukemia cells respond to their interaction within the stromal microenvironment, we performed single-cell RNA sequencing (scRNA-seq), genome-scale loss-of-function CRISPR screen and analysis of secreted soluble factors.

After 24h of pre-stimulation of leukemia cells with HS-5 cells and 24h of culture with NK cells, we observed reduced killing of target leukemia cells in the presence of stromal cells compared to NK cell and target cell alone conditions (viability of K562 cells: 46 vs 38%, p=0.0130; MOLM-14: 63 vs 48%, p=0.0165; OCI-M1: 56 vs 35%, p=0.0163, and THP-1: 58 vs 33% p=0.0394). The analysis of soluble factors revealed an increase of CCL2, G-CSF, GM-CSF, IL-1β, IL-4, IL-6, IL-8 and IL-10 when tumor and NK cells interacted within the stromal niche suggesting a role of these soluble mediators in the resistance of leukemic cells. Interestingly, genome-scale CRISPR screen also identified the loss of IL-10 gene as a sensitizer of THP-1 cells to NK cell mediated-cytotoxicity in the stromal microenvironment.

To understand the dynamic changes that happen in various cell types during the co-culture, we pooled all different scRNA-seq conditions together and performed an unsupervised clustering separating expanded NK, leukemia and HS-5 cells. The co-culture with K562 CML cells induced an activated state (73%) of NK cells characterized by the expression of TNFRSF9 (4-1BB), TNFRSF18 (GITR), HAVCR2 (TIM-3) and TIGIT, whereas co-culture with MOLM-14 cells did not induce strong NK cell activation (58% of cells were in the resting state). The co-culture with OCI-M1 (47%) and THP1 (68%) cells led to an activated type I interferon (IFN) state (OAS1, OAS2, MX1, MX2, ISG15) in NK cells. Stromal cells induced a distinct NK cell phenotype characterized by the activation of the IL2/STAT5 and apoptosis signaling and a decrease of the NK cell activation receptor NKp30 expression and an increase of exhaustion markers TIM-3 and TIGIT. Furthermore, during NK, leukemia (K562, MOLM-14 or THP-1) and stromal cells co-culture, an increase of the TGFβ, IL-6/JAK/STAT3, TNFα/NFKB and hypoxia signaling was observed in NK cells. In addition, a decrease of the activated (73 vs 31% in NK with K562 cells) and type I IFN state of NK cells (68 vs 6% in NK with THP-1 cells) was highlighted when stromal cells were present.

In K562, THP-1 and MOLM-14 tumor cells, the interaction with the stromal cells decreased NK cell-induced IFNα and IFNγ response resulting in the downregulation of MHC class I (HLA-B, HLA-F) and class II (HLA-DMB, HLA-DQA1, HLA-DRA) genes. A decrease of TNFα pathway and a downregulation of IL-32 was also observed in the tumor cells. As a common mechanism in all tumor cells, including OCI-M1 cells, we noted an induction of TGFβ and downregulation of the IL-6/JAK/STAT3 signaling resulting in the resistance to NK 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. Based on our findings, 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.

Myllymäki:Gilead: Research Funding. Mustjoki:Dren Bio: Honoraria; Pfizer: Research Funding; Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding.