At an accelerating pace, the genomic characterization of human cancer is elucidating the molecular basis of the disease. Recent large-scale analyses of gene copy number in cancer demonstrated that the genes encoding the BCL2-family proteins MCL1 and BCL-xL are frequent targets of amplification. Low-level MCL1
amplification is particularly notable, representing one of the most common copy number abnormalities in all of human cancer (Beroukhim et al., 2010
). In support of a functionally important role of MCL1, numerous studies have elucidated the critical role of MCL1 in preventing tumor cell death (Warr and Shore, 2008
Using a multiplexed Luminex bead-based assay, we screened for compounds that reduced MCL1 expression while preserving the expression of proapoptotic genes. Although the compounds that emerged from this screen were general transcriptional repressor (TR) compounds (as opposed to specifically targeting the MCL1 locus), they preferentially repressed MCL1 because of the short half-life of MCL1 mRNA and protein. Multiple lines of evidence suggest that TR compounds induce apoptosis in cancer cells primarily through repression of MCL1 expression including 1) upon treatment with TR compounds, MCL1 protein levels decreased rapidly and preceded caspase activation; 2) ectopic expression of physiological levels of MCL1 rescued cancer cells from TR compounds, despite the expression of other genes still being repressed; 3) the pattern of TR compound sensitivity across a panel of cancer cell lines closely mirrored the pattern of sensitivity of those cell lines to MCL1 knock-down by RNAi; 4) of over 40,000 genomic features measured, the top feature that predicted sensitivity to TR compounds was the low expression of BCL-xL, which shares redundant function with MCL1; 5) Ectopic expression of BCL-xL rescued cancer cells from TR compounds; 6) MCL1 repression by TR compounds resulted in the release of, pro-apoptotic protein BAK protein from MCL1; and 7) Bak deficiency protected cells from TR compounds. These results suggest that the mechanism of cell death induced by TR compounds is best explained by MCL1 inhibition.
This indicated that some of the widely used chemotherapeutic drugs such as anthracyclines may preferentially repress MCL1
to induce apoptosis in tumor cells. Although the anti-tumor effect of anthracyclines has long been speculated to be related to the drug’s inhibition of DNA topoisomerase II (Desmedt et al., 2011
; Moretti et al., 2009
), and an association between low TOP2A expression and anthracycline response in ER-negative breast cancer patients has been reported (Martin, 2011
), our data suggest that their activity may be largely explained by inhibition of transcription, leading most dramatically to the repression of short-lived MCL1
transcripts. While it is possible that multiple mechanisms of action explain the anti-tumor effects of anthracyclines, at least in the experimental cancer models studied here, anthracycline gene expression consequences most reflected transcriptional inhibition rather than DNA topoisomerase II inhibition. Furthermore, the similar pattern of sensitivity of cell lines to MCL1
knockdown compared to anthracycline treatment is also consistent with an MCL1
-mediated transcriptional inhibitory effect. Lastly, our observation that BCL-xL
expression is predictive of resistance to MCL1
repression both in model systems and in patients with breast cancer further strengthens the anthracycline-MCL1
connection. We note that the concentration of doxorubicin used in our experiments approximates that observed in human tumor tissues (1.9–24.4 µM) (Rossi et al., 1987
). Doxorubicin stimulates topoisomerase II-mediated DNA cleavage only at low concentrations, whereas at doses greater than ~ 0.4 µM, topoisomerase II-mediated DNA cleavage is lost (Tewey et al., 1984
). These data therefore suggest that at clinically relevant concentrations, anthracyclines act as transcriptional repressors, as opposed to DNA damaging agents.
The transcriptional inhibitory role of anthracyclines is also of importance when considering anthracycline-based combination therapies. The transcriptional induction of pro-apoptotic proteins has been reported to be crucial for the efficacy of many classes of anti-neoplastic agents including radiation (Jeffers et al., 2003
; Villunger et al., 2003
), the proteasome inhibitor bortezomib (Gomez-Bougie et al., 2007
; Voortman et al., 2007
), the HDAC inhibitor vorinostat (Wiegmans et al., 2011
), and the kinase inhibitors imatinib (Kuroda et al., 2006
) and erlotinib (Gong et al., 2007
). Anthracyclines may block the induction of such pro-apoptotic proteins and counteract, rather than synergize, with those therapies. For example, we found that doxorubicin treatment actually rescues cancer cells from bortezomib- and vorinostat-induced killing (Figure S2
). Such antagonistic actions may be preventable by adjusting the dosing schedule of combination therapies, but the results serve as a reminder that knowledge of mechanisms of action should ideally be considered in developing combination strategies.
Taken together, the results reported here elucidate a strategy for the development of MCL1 inhibitors as cancer therapeutics. The multiplexed, gene-expression-based high-throughput screening approach described here holds promise for the future discovery of specific inhibitors of MCL1 expression and for the use of chemical genomic approaches to elucidate small-molecule mechanisms of action. The study also highlights the power of genomically-characterized cell lines for the discovery of predictive biomarkers of drug response. Most immediately, the work suggests an approach to the clinical development of any MCL1 inhibitor in breast and NSCLC tumors, focusing on tumors expressing low levels of BCL-xL as a patient-selection strategy.