P-080: Single-cell transcriptomic analysis of bone marrow NK cells reveals loss of activated cytotoxic NK cells in Multiple Myeloma

Abstract

<h3>Background</h3> Multiple Myeloma (MM) disease progression and therapy response are influenced by cues of the microenvironment including tumor control by a cytotoxic immune response. Natural killer (NK) cells are notable mediators of the cytotoxic immune response to MM, and important effector cells in recent immune-mediated therapies. NK cells are drivers of antibody-dependent-cellular cytotoxicity in therapies based on anti-CD38 monoclonal antibodies such as Daratumumab. Classically, NK cells are divided based upon CD56 expression into a cytokine-producing CD56bright subset, releasing cytokines such as IFN-γ, TNF-a and GM-CSF, and a cytotoxic CD56dim subset. However, accumulating evidence suggests much larger heterogeneity in the NK cell compartment and modulation of these NK cell subsets could impact response to NK cell-driven immunotherapies. Here, we used single-cell RNA sequencing to investigate the heterogeneity and MM-driven alterations of the NK cell compartment in the bone marrow of newly diagnosed MM patients undergoing first-line Daratumumab-containing therapy. <h3>Methods</h3> We performed single-cell RNA sequencing of the CD38+ and CD38- fractions of viably frozen bone marrow aspirates from 19 newly diagnosed MM patients and 5 non-cancer control patients. NK cells were identified in silico by transcription of KLRF1, KLRD1, GNLY and NKG7 resulting in a single-cell transcriptomic dataset of 30,373 NK cells from MM patients and 8,865 NK cells from control patients. <h3>Results</h3> After integration of the datasets, bone marrow NK cells formed eight distinct transcriptomic clusters. Conventional CD56bright and CD56dim NK-cells were identified by increased transcription of GZMK or GZMB, respectively. The GZMK+CD56bright NK cells contained both a cluster of naive and a cluster of activated NK cells. The GZMB+CD56dim NK cells consisted of 5 subclusters. To identify MM-induced alterations in NK cell subsets we compared GZMK+CD56bright vs GZMB+CD56dim cluster composition and distribution between control and MM patients. Control bone marrow was dominated by GZMB-transcribing cytotoxic CD56dim NK cells, represented by a low cytokine-producing GZMK+CD56bright vs cytotoxic GZMB+CD56dim ratio. In contrast, MM bone marrow was characterized by heterogeneity in the ratio of cytokine-producing GZMK+CD56bright vs cytotoxic GZMB+CD56dim NK cells. A subset of patients presented with a complete reversal of this ratio. This altered composition was due to a loss of cytotoxic GZMB+CD56dim NK cells, and more specifically a loss of NK cells with a transcriptome suggesting recent activation. <h3>Conclusion</h3> Here we present a transcriptomic overview of NK cells in MM bone marrow at the single-cell level. A subset of MM patients has a loss of activated cytotoxic GZMB+CD56dim NK cells, suggestive of reduced cytotoxic anti-tumor responses. Current analyses are focused on clinical implications of NK cell alterations in response to Daratumumab-based therapies.