Abstract
The BH3-only apoptosis agonists BAD and NOXA target BCL-2 and MCL-1 respectively and co-operate to induce apoptosis. On this basis, therapeutic drugs targeting BCL-2 and MCL-1 might have enhanced activity if used in combination. We identified anti-leukaemic drugs sensitising to BCL-2 antagonism and drugs sensitising to MCL-1 antagonism using the technique of dynamic BH3 profiling, whereby cells were primed with drugs to discover whether this would elicit mitochondrial outer membrane permeabilisation in response to BCL-2-targeting BAD-BH3 peptide or MCL-1-targeting MS1-BH3 peptide. We found that a broad range of anti-leukaemic agents–notably MCL-1 inhibitors, DNA damaging agents and FLT3 inhibitors–sensitise leukaemia cells to BAD-BH3. We further analysed the BCL-2 inhibitors ABT-199 and JQ1, the MCL-1 inhibitors pladienolide B and torin1, the FLT3 inhibitor AC220 and the DNA double-strand break inducer etoposide to correlate priming responses with co-operative induction of apoptosis. ABT-199 in combination with pladienolide B, torin1, etoposide or AC220 strongly induced apoptosis within 4 hours, but the MCL-1 inhibitors did not co-operate with etoposide or AC220. In keeping with the long half-life of BCL-2, the BET domain inhibitor JQ1 was found to downregulate BCL-2 and to prime cells to respond to MS1-BH3 at 48, but not at 4 hours: prolonged priming with JQ1 was then shown to induce rapid cytochrome C release when pladienolide B, torin1, etoposide or AC220 were added. In conclusion, dynamic BH3 profiling is a useful mechanism-based tool for understanding and predicting co-operative lethality between drugs sensitising to BCL-2 antagonism and drugs sensitising to MCL-1 antagonism. A plethora of agents sensitised cells to BAD-BH3-mediated mitochondrial outer membrane permeabilisation in the dynamic BH3 profiling assay and this was associated with effective co-operation with the BCL-2 inhibitory compounds ABT-199 or JQ1.
Highlights
The modes of action of diverse cytotoxic agents generally converge on mitochondrial apoptotic pathways [1]
We dichotomise drugs as either agents sensitising to BCL-2 antagonism or agents sensitising to MCL-1 antagonism, and we demonstrate the efficacy of combining an agent from each category in apoptosis assays
MV4-11 cells were used for screening drugs, as these cells were highly sensitive to apoptosis induced by either BCL-2 or MCL-1 targeting (S1 Fig)
Summary
The modes of action of diverse cytotoxic agents generally converge on mitochondrial apoptotic pathways [1]. BAX and BAK activation can be triggered by BH3-only proapoptotic BCL-2 family members such as BID and BIM, PUMA, BAD and NOXA. These are opposed by BCL-2 family prosurvival members, such as MCL-1 and BCL-2 itself, that sequester pro-apoptotic family members to hold apoptosis in check. With many new drugs on the market or in the pipeline [3, 4], there is a need to establish rational principles for predicting suitable drug combinations. One such principle is cooperation between agents that activate complementary components of pro-apoptotic pathways. When BCL-2 is inhibited, e.g. by the binding agents ABT-737 or ABT-199, the apoptosis activator BIM is released [7, 10,11,12,13], but the released BIM can be taken up by MCL-1, so protection from apoptosis is maintained unless MCL-1 is antagonised [10, 14,15,16]
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