Abstract 2192A: Combined MERTK and ROCK1/2 inhibition as a potential therapeutic strategy for AML

Abstract

Abstract Acute myeloid leukemia (AML) has a 5-year overall survival rate under 26% in adults and current cytotoxic chemotherapies are toxic, can cause long-term side effects and are often contraindicated for elderly patients. Targeted therapies may have reduced toxicity compared to chemotherapy; however, resistance to single-agents often develops and combination therapies may provide more durable responses. MERTK is aberrantly expressed in >80% of AML patient samples and is a promising therapeutic target. MRX-2843 is a novel small molecule dual MERTK and FMS-like tyrosine kinase 3 (FLT3) inhibitor that is currently in clinical trials. Here, we report synergistic anti-leukemia activity mediated by MRX-2843 and rho-associated, coiled-coil-containing protein kinases 1 and 2 (ROCK1/2) inhibitors. Both MERTK and ROCK1/2 have been implicated in actin/microtubule dynamics and cell cycle progression and previous publications demonstrated induction of apoptosis in AML cells in response to RNAi-mediated MERTK or ROCK1 inhibition. ROCK1/2 inhibitors RKI-1447 or GSK269962A synergized with sub-therapeutic doses of MRX-2843 to reduce cell density as indicated by decreased Presto Blue staining in cultures of 4 of 6 AML cell lines tested and had an additive effect in the remaining 2 cell lines. Similar interactions were observed between RKI-1447 and UNC3997, a MERTK-selective TKI with 15-fold weaker FLT3 activity. Flow cytometric analysis of cells stained with popro-1-iodide and propidium iodide (PI) dyes revealed synergistic induction of cell death in the KG-1, OCI-AML5, and NB4 cell lines and an additive effect in Kasumi-1 cultures. In addition, cells that survived treatment exhibited subsequent defects in expansion, even when they were cultured without inhibitor(s), and these effects were significantly more pronounced in cells treated with the combination therapy compared to single agents. Flow cytometric analysis of PI-stained permeabilized cells revealed an increased fraction of cells in G2/M phase in cultures treated with MRX-2843. This effect was more pronounced in cultures treated with the combination therapy and was accompanied by accumulation of a population with slightly reduced DNA content relative to untreated G2 cells, suggestive of a defect in late S-phase. Thus, the combination therapy mediates anti-leukemia activity by multiple mechanisms, including abrogation of cell cycle progression and induction of cell death. In addition, a preliminary analysis of The Cancer Genome Atlas database revealed significantly poorer overall survival in patients with higher levels of ROCK1 expression (p=0.00147, n=172). Together, these data suggest that combination therapies targeting MERTK and ROCK1/2 may be particularly effective for treatment of AML and support further studies to test the effects of this novel strategy in animal models. Citation Format: Dawn E. Barnes, Xiaodong Wang, Stephen V. Frye, H. Shelton Earp, Deborah DeRyckere, Douglas K. Graham. Combined MERTK and ROCK1/2 inhibition as a potential therapeutic strategy for AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2192A.