Synergistic Induction of Senescence-Driven Immune Responses and Myeloma Cell Cytotoxicity By CDK4/6 Inhibitors and Daratumumab Combination Therapy

Background: Progressive immunosuppression is associated with the development of multiple myeloma (MM) and strategies aimed at enhancing immune function, including antibody-based therapy, important therapeutic significance. While CD38-directed monoclonal antibody Daratumumab (DARA) is highly effective as single agents and in combination regimens by leveraging natural killer (NK) cells as key effectors, disease relapse persists. Therefore, strategies to further enhance efficacy and overcome drug resistance are needed. In this study we show that CDK4/6 inhibitors (Abemaciclib and Palbociclib) induce cell cycle arrest and a senescent phenotype in MM cells, and act in combination with DARA to provoke an NK cell surveillance program leading to myeloma cell death. Method: We evaluated the response of MM cell lines and primary bone marrow samples from MM patients to CDK4/6 inhibition using both Abemaciclib (Abe) and Palbociclib (Pal). Transcriptional changes were investigated with bulk RNA sequencing and qPCR; while flow cytometry analysis was used to investigate the changes in NK cell receptors and ligands. Senescence and senescence-associated-secretory phenotype (SASP) induction were evaluated by C12FDG (β-galactosidase staining) and cytokine array respectively. The synergistic effect of Abe/Pal and DARA was measured in vivo in a NSG mouse model reconstituted with human-derived NK cells and engrafted with RPMI-8226-Luc MM cells. Results: We observed significant senescence-associated beta-galactosidase (SA-β-gal) activity and potent SASP induction. RNA-seq analysis of MM cell lines after drug treatment revealed a reduction in proliferation genes and increase in SASP factor expression, including type I interferon (IFN I) signaling and enhanced expression of IFN-stimulated genes, compared with control cells. Genes and cytokines related to innate immune responses were significantly increased. Moreover, the NK cell ligands required for activation of NK cell cytotoxicity and tumor cell targeting -NKG2D and -DNAM-1 were increased preferentially on βGal^high compared to βGal^low cells. Overall, these data suggest that, in addition to more stable cell cycle arrest conferred by RB-mediated senescence, CDK4/6 inhibition may promote NK cell immune surveillance through induction of the SASP program. We indeed observed an increase in NK cell degranulation and IFN-γ production using an in vitro NK-MM coculture assay. Importantly, combination with DARA significantly increased DARA -induced MM cell lysis by NK cells both in vitro and in vivo in a humanized model of MM. Conclusion: Strategies to exploit and enhance NK cell immune surveillance may complement existing efforts to harness adaptive immune surveillance in MM. Our results suggest that cytostatic agents inducing senescence such as CDK4/6 inhibitors can be combined in MM with CD38 antibody-based therapy to enhance NK cell activity and the clearance of senescent cells, providing the basis for clinical evaluation of this combination therapy to further improve patient outcome in MM.