PO-061 BCL-2 family of proteins, BCL-XL and MCL-1, regulate apoptosis and cancer cell survival by different mechanisms

Introduction Abnormal cell survival through resistance to apoptosis is a cardinal feature of most malignancies and plays a key role in chemoresistance. As the major regulators of the intrinsic mitochondrial pathway of apoptosis, the pro-survival BCL-2 family proteins (BCL-2, BCL-XL and MCL-1) are attractive targets for novel cancer therapeutics. The concept of employing BH3 mimetics (small molecule inhibitors of the BCL-2 family) as anticancer agents has been substantiated by the efficacy of selective drugs, such as navitoclax and venetoclax, in treating BCL-2-dependent haematological malignancies. However, most solid tumours depend for survival on both BCL-XL and MCL-1, which are highly amplified in multiple cancers. Several MCL-1 inhibitors that have been generated so far and demonstrate early promise in vitro but most fail to exhibit specificity and potency in a cellular context. Material and methods All cell lines and primary cells, BH3 mimetics and antibodies, unless received as gift from our collaborators, were purchased from commercial vendors. Differential scanning fluorimetry (DSF) was performed using a StepOnePlus Real-Time PCR machine. The extent of cytochrome c release was quantitated using immunofluorescence and apoptosis assessed using an Attune NxT flow cytometer. Gel filtration and immunoprecipitation experiments were performed according to standard protocols. Statistical analysis was performed using a two-way ANOVA with p values: * for p≤0.05, ** for p≤0.005 and *** for p≤0.001. Results and discussions Employing a rapid DSF-based assay, we screened a panel of BH3 mimetics to identify that only S63845 and to a smaller extent, A-1210477, demonstrated enhanced binding to MCL-1 that correlated with a rapid, concentration-dependent apoptosis in relevant cell lines. At higher concentrations, S63845 also appeared to weakly bind BCL-2. Furthermore, S63845 synergized with other BH3 mimetics to induce apoptosis in several cancer cell lines. However, in the colorectal HCT-116 cells, BCL-XL-regulated apoptosis required all known BH3-only members, whereas apoptosis and cellular proliferation regulated by MCL-1 appeared to occur independently of all known BH3-only proteins. Conclusion The anti-apoptotic and cell survival roles of BCL-XL and MCL-1 could be distinct, as antagonising BCL-XL induced BH3-dependent apoptosis, whereas MCL-1 appeared to regulate apoptosis even in the absence of all known BH3-only proteins.