LAUSR

Background: Triple-negative breast cancer (TNBC) is an aggressive disease associated with recurrence, metastasis, and resistance to chemotherapy and radiation; effective targeted therapies are needed. Although the MEK inhibitors (MEKi) selumetinib (AZD6244) and pimasertib (AS703026) showed therapeutic efficacy in preclinical models, they failed as effective single agents in the clinic due to development of intrinsic resistance. To identify potential mediators of this resistance, we performed a synthetic lethal siRNA screen and identified myeloid cell leukemia-1 (MCL1) as a potential mediator of selumetinib resistance. Mcl-1 is an anti-apoptotic protein that is highly amplified in numerous human cancers and associated with cell immortalization and chemoresistance. We hypothesized that Mcl-1 promotes MEKi resistance in TNBC, and sought to confirm this and discern of mechanisms. Results: We established selumetinib- and pimasertib-resistant clones of TNBC cells SUM-149 and MDA-MB-231 to a final concentration of 15µM selumetinib and 11µM pimasertib by continuous exposure to increasing concentrations over a 6-month period. MEKi-resistant cells showed a significantly higher proliferation rate than the parental cells. More colonies were observed among the MEKi-resistant cells than in the parental cells in a colony formation assay (40%, p=0.05) and a similar outcome was observed in a mammosphere assay (65%, p=0.01), suggesting a higher fraction of tumor-initiating cells. To determine if the Mcl-1-specific inhibitor S63845 could sensitize the MEKi-resistant cells, we treated parental and resistant cells with either selumetinib or pimasertib together with S63845, a highly specific Mcl-1 inhibitor. After treatment with the Mcl-1 inhibitor, the resistant SUM-149 and MDA-MB-231 cells had similar cell proliferation rates to those of their parental counterparts. To extrapolate the mechanism of MEKi resistance, we performed an RT2 Profiler PCR Human Mitochondria Array; mitochondrial uncoupling proteins UCP4 and UCP2 were upregulated in the SUM149 and MDA-MB-231 MEKi-resistant cells, respectively. MDA-MB-231 MEKi-resistant cells had higher levels of glycolysis than the parental control cells, suggesting that the MEKi-resistant cells had undergone a metabolic shift to enhance glycolysis. This is consistent with a observation that metabolic alterations play a part in drug resistance. In addition, the Warbug effect, in which tumor cells adopt aerobic glycolysis, is considered the major adaptive mechanism and increased glycolysis is commonly displayed in resistant cells. Furthermore, knockdown of UCP2 in selumetinib-resistant MDA-MB-231 cells made them ~20% more sensitive to selumetinib. Inhibition of glycolysis using 2-deoxy-D-glucose in combination with MEKi increased drug sensitivity by 20-30%. Conclusion: We found preliminary evidence of a link between Mcl-1 and glycolytic metabolism that may be involved in MEK resistance. We will also design combinational studies of MEKi and Mcl-1 inhibitors in vivo in TNBC models. Citation Format: Maria Gagliardi, Moises Tacam, Lakesla Iles, Yuan Qi, Lajos Pusztai, Debu Tripathy, Geoffrey Bartholomeusz, Chandra Bartholomeusz. Mechanism of MEK inhibitor resistance in triple negative breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1897.