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Abstract
While inhibitors of BCL-2 family proteins (BH3 mimetics) have shown promise as anti-cancer agents, the various dependencies or co-dependencies of diverse cancers on BCL-2 genes remain poorly understood. Here we develop a drug screening approach to define the sensitivity of cancer cells from ten tissue types to all possible combinations of selective BCL-2, BCL-XL, and MCL-1 inhibitors and discover that most cell lines depend on at least one combination for survival. We demonstrate that expression levels of BCL-2 genes predict single mimetic sensitivity, whereas EMT status predicts synergistic dependence on BCL-XL+MCL-1. Lastly, we use a CRISPR/Cas9 screen to discover that BFL-1 and BCL-w promote resistance to all tested combinations of BCL-2, BCL-XL, and MCL-1 inhibitors. Together, these results provide a roadmap for rationally targeting BCL-2 family dependencies in diverse human cancers and motivate the development of selective BFL-1 and BCL-w inhibitors to overcome intrinsic resistance to BH3 mimetics.
Introduction
The process of intrinsic apoptosis is tightly regulated by the BCL-2 family of proteins. In human cancers, the anti-apoptotic BCL-2 proteins play a critical role in protecting cells, which are often “primed” for apoptosis, from committing to irreversible cell death1. To date, the most well described of the anti-apoptotic BCL-2 genes are BCL-2, BCL-XL, and MCL-1, and recently, following over a decade of extensive research effort, potent and selective inhibitors of each of these proteins were developed. Much is known about the cancer types that respond well to selective BCL-2 inhibitors, and indeed the BCL-2 inhibitor venetoclax (ABT-199) is now FDA approved to treat certain leukemias such as chronic lymphocytic leukemia (CLL)2,3. In contrast, outside of a small number of studies in select cancer types, little is known regarding which cancers might respond well to single agent BCL-XL or MCL-1 inhibition4,5,6,7. Finally, to the best of our knowledge, no studies have systematically examined the dependencies of cancers on combinations of BCL-2 family proteins.
With these limitations in mind, we set out to address the following questions: What are the dependencies of diverse human cancers with respect to BCL-2, BCL-XL, MCL-1, and their combinations? What are the molecular features of tumors that drive these dependencies? Finally, which cancers fail to respond to BH3 mimetics, and how can this intrinsic resistance be overcome? To answer these questions, we developed a screening strategy to assess the sensitivity of cancer cell lines to all possible combinations of a selective BCL-2 inhibitor (ABT-199), a selective BCL-XL inhibitor (WEHI-539), and a selective MCL-1 inhibitor (A-1210477). Using this approach, we mapped cellular dependencies and co-dependencies on BCL-2, BCL-XL, and MCL-1 across a large number of primary and established cancer cell lines representing 10 distinct cancer types. These data provide new insights into the landscape of sensitivity to BH3 mimetics in human cancers, revealing molecular determinants of sensitivity and a role for a novel endoplasmic reticulum (ER) stress-epithelial-mesenchymal transition (EMT) axis in dictating the frequently observed synergy between BCL-XL and MCL-1 inhibitors in solid tumors. Collectively, these findings may help guide the use of BH3 mimetics as precision therapies in defined cancers.
Results
Mapping of BCL-2 gene dependencies
To begin, we first made several assumptions regarding the BH3 mimetic drugs ABT-199, WEHI-539, and A-1210477 based on prior literature and our own experience. First, we elected to perform screens using a concentration of 1 µM for both ABT-199 and WEHI-539, as complete target inhibition is observed at these concentrations, and concentrations above this level may have off-target effects or may not be achievable in patients. A-1210477 is a first-in-class probe compound, and as such is less potent than ABT-199 or WEHI-539. Therefore, a concentration of 10 µM was selected for this compound, as at this dose MCL-1 is fully inhibited without inhibitory effects on BCL-2 and BCL-XL8. A drug panel consisting of all possible single, double, and triple agent combinations of these drugs, at these concentrations, was then constructed and assayed in cell lines after a 72 h treatment using a conventional viability assay (see Methods) (Fig. 1a). To ensure that this assay accurately reveals BCL-2 family dependencies, we assembled several cell lines previously reported to be dependent on BCL-2, BCL-XL, MCL-1, or combinations of these proteins, then verified the recovery of expected dependencies (Fig. 1b) [6,9,10,11]. In prior studies, we identified Panc 03.27 cells as BCL-XL dependent, and as such this line was included as a control. To further validate this BCL-2 family dependency assay, we compared its results to conventional BH3 profiling assays (Supplementary Fig. 1A–C). Consistent with the reported selective, on-target activities of the BH3 mimetics above, these assays revealed that BCL-XL dependency levels from viability assays correlate strongly on a cell line by cell line basis with the activity of the HRK peptide, which selectively inhibits BCL-XL. Similarly, MCL-1 dependency correlated with the activity of the NOXA peptide, a selective and direct inhibitor of MCL-11. Lastly, we tested structurally independent BCL-XL (A-1331852) and MCL-1 (S63845) inhibitors, as well as a dual BCL-2/BCL-XL inhibitor (ABT-737), in cell lines exhibiting single agent BCL-2, BCL-XL, or MCL-1 inhibitor sensitivity, or a cell line resistant to the inhibition of all three genes (Supplementary Fig. 2). Importantly, the sensitivity of each of these cell lines to these BH3 mimetics recapitulated the sensitivities observed following treatment with ABT-199, WEHI-539, or A-1210477, providing confidence that the latter drugs can be used as reliable probes of BCL-2 family dependencies
SYSTEMATIC MAPPING OF BCL-2 GENE DEPENDENCIES IN CANCER REVEALS MOLECULAR DETERMINANTS OF BH3 MIMETIC SENSITIVITY
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