Abstract 1793: Combination treatment with a MCL1 Inhibitor AZD5991 overcomes resistance to MEK inhibitor in TNBC cells
Authors: Mughees Mohd , Tacam Moises , Tan Alex , Iles Lakesla , White Michael , Tripathy Debu , Bartholomeusz Geoffrey , Bartholomeusz Chandra 
Published: June 2022

Abstract
Background: Triple-negative breast cancer (TNBC), which is associated with poor prognosis, accounts for 15-20% of breast cancers (BCs) but up to 30% of BC deaths, likely because of its high recurrence rate and limited targeted therapy options. Basal type-BC cells are sensitive to MEKi (MEK Inhibitor), and 77% of basal-type BCs are TNBCs. We previously showed that MEKi selumetinib (AZD6244) effectively inhibits tumor growth and metastasis in a TNBC xenograft model. However, clinical studies have shown that MEKi as single agents have only modest activity against solid tumors. Therefore, combinatorial therapeutic strategies represent an urgent unmet clinical need for TNBC patients. Using a high-throughput whole-genome siRNA screen, we identified and validated the antiapoptotic protein MCL1, which is overexpressed in many human cancers, including TNBC, as a potential candidate that could be therapeutically targeted alongside MEK. We hypothesized that the combination of AZD6244 plus AZD5991 overcomes MEKi resistance in TNBC.
Methods and Results: We previously established two cell lines resistant to AZD6244 to a final concentration of 15 µM (MDA-MB-231-R and SUM-149-R). Both these MDA MB-231-R and SUM-149-R cells showed increased proliferation with IC50 of 24.01 and 22.76μM as compared to the parental IC50 2.7 and 3.80µM respectively. The colony-forming rates of the MDA-MB-231-R and SUM-149-R cells were more than 50% higher than their parental cells (p ≤ 0.05), and the resistant cells had lower proportions of the cells in G0/G1 phase (49.03% and 22.89%, respectively) than their parental cells did (73.01% and 60.03%, respectively). Compared with their parental cells, SUM 149-R (86.26% vs 66.04%) and MDA MB-231-R (95.27% vs. 73.47%) had a higher proportion of CD44+CD24-/low cells and higher average mammosphere count (330 vs. 55) and (892 vs. 273). The IC50 values for SUM149-R (1.68 µM) and MDA-MB-231-R (7.2 µM) cells treated with AZD6244 in combination with the MCL inhibitor AZD5991 was significantly lower than that for cells treated with AZD6244 alone. To validate this finding, we used siRNA to knock down MCL1 and confirmed 80-90% silencing of both resistant and parental cells via western blotting. Furthermore, MCL1 silencing sensitized both MDA-MB-231-R and SUM-149-R cells to AZD6244, as MCL1-knockout cells treated with AZD6244 had significant reduction in cell proliferation and colony formation as compared to the respective controls.
Conclusions and Future Directions: Our findings support our hypothesis that the combination of AZD6244 plus AZD5991 overcomes MEKi resistance in TNBC. To further translate these interesting findings, we plan to evaluate the effect of this drug combination in vivo in TNBC models. We have also performed a whole-proteome analysis of MDA-MB-231 parental and MDA-MB-231-R cell lines using LC-MS/MS and are now validating the genes/pathways involved in MEK resistance
Citation Format: Mohd Mughees, Moises Tacam, Alex Tan, Lakesla Iles, Michael White, Debu Tripathy, Geoffrey Bartholomeusz, Chandra Bartholomeusz. Combination treatment with a MCL1 Inhibitor AZD5991 overcomes resistance to MEK inhibitor in TNBC cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1793.