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BH3 mimetic drugs, which inhibit prosurvival BCL2 family proteins, have limited single-agent activity in solid tumor models. The potential of BH3 mimetics for these cancers may depend on their ability to potentiate the apoptotic response to chemotherapy and targeted therapies. Using a novel class of potent and selective MCL1 inhibitors, we demonstrate that concurrent MEK + MCL1 inhibition induces apoptosis and tumor regression in KRAS-mutant non–small cell lung cancer (NSCLC) models, which respond poorly to MEK inhibition alone. Susceptibility to BH3 mimetics that target either MCL1 or BCL-xL was determined by the differential binding of proapoptotic BCL2 proteins to MCL1 or BCL-xL, respectively. The efficacy of dual MEK + MCL1 blockade was augmented by prior transient exposure to BCL-xL inhibitors, which promotes the binding of proapoptotic BCL2 proteins to MCL1. This suggests a novel strategy for integrating BH3 mimetics that target different BCL2 family proteins for KRAS-mutant NSCLC.Significance:Defining the molecular basis for MCL1 versus BCL-xL dependency will be essential for effective prioritization of BH3 mimetic combination therapies in the clinic. We discover a novel strategy for integrating BCL-xL and MCL1 inhibitors to drive and subsequently exploit apoptotic dependencies of KRAS-mutant NSCLCs treated with MEK inhibitors.See related commentary by Leber et al., p. 1511.This article is highlighted in the In This Issue feature, p. 1494

Supplementary Figure 1-5, Supplementary Tables 1-3

Top 10 correlated genes to BIM (BCL2L11)

Supplemental legend

ZEB1 binding sites found in 1975R2 cells compared to 1975 parental cells

Purpose: Epithelial-to-mesenchymal transition (EMT) confers resistance to a number of targeted therapies and chemotherapies. However, it has been unclear why EMT promotes resistance, thereby impairing progress to overcome it.Experimental Design: We have developed several models of EMT-mediated resistance to EGFR inhibitors (EGFRi) in EGFR-mutant lung cancers to evaluate a novel mechanism of EMT-mediated resistance.Results: We observed that mesenchymal EGFR-mutant lung cancers are resistant to EGFRi-induced apoptosis via insufficient expression of BIM, preventing cell death despite potent suppression of oncogenic signaling following EGFRi treatment. Mechanistically, we observed that the EMT transcription factor ZEB1 inhibits BIM expression by binding directly to the BIM promoter and repressing transcription. Derepression of BIM expression by depletion of ZEB1 or treatment with the BH3 mimetic ABT-263 to enhance “free” cellular BIM levels both led to resensitization of mesenchymal EGFR-mutant cancers to EGFRi. This relationship between EMT and loss of BIM is not restricted to EGFR-mutant lung cancers, as it was also observed in KRAS-mutant lung cancers and large datasets, including different cancer subtypes.Conclusions: Altogether, these data reveal a novel mechanistic link between EMT and resistance to lung cancer targeted therapies. Clin Cancer Res; 24(1); 197–208. ©2017 AACR.

Acquired drug resistance to even the most effective anti-cancer targeted therapies remains an unsolved clinical problem. Although many drivers of acquired drug resistance have been identified1‒6, the underlying molecular mechanisms shaping tumor evolution during treatment are incompletely understood. The extent to which therapy actively drives tumor evolution by promoting mutagenic processes7 or simply provides the selective pressure necessary for the outgrowth of drug-resistant clones8 remains an open question. Here, we report that lung cancer targeted therapies commonly used in the clinic induce the expression of cytidine deaminase APOBEC3A (A3A), leading to sustained mutagenesis in drug-tolerant cancer cells persisting during therapy. Induction of A3A facilitated the formation of double-strand DNA breaks (DSBs) in cycling drug-treated cells, and fully resistant clones that evolved from drug-tolerant intermediates exhibited an elevated burden of chromosomal aberrations such as copy number alterations and structural variations. Preventing therapy-induced A3A mutagenesis either by gene deletion or RNAi-mediated suppression delayed the emergence of drug resistance. Finally, we observed accumulation of A3A mutations in lung cancer patients who developed drug resistance after treatment with sequential targeted therapies. These data suggest that induction of A3A mutagenesis in response to targeted therapy treatment may facilitate the development of acquired resistance in non-small-cell lung cancer. Thus, suppressing expression or enzymatic activity of A3A may represent a potential therapeutic strategy to prevent or delay acquired resistance to lung cancer targeted therapy.

BH3 mimetic drugs, which inhibit prosurvival BCL2 family proteins, have limited single-agent activity in solid tumor models. The potential of BH3 mimetics for these cancers may depend on their ability to potentiate the apoptotic response to chemotherapy and targeted therapies. Using a novel class of potent and selective MCL1 inhibitors, we demonstrate that concurrent MEK + MCL1 inhibition induces apoptosis and tumor regression in KRAS-mutant non–small cell lung cancer (NSCLC) models, which respond poorly to MEK inhibition alone. Susceptibility to BH3 mimetics that target either MCL1 or BCL-xL was determined by the differential binding of proapoptotic BCL2 proteins to MCL1 or BCL-xL, respectively. The efficacy of dual MEK + MCL1 blockade was augmented by prior transient exposure to BCL-xL inhibitors, which promotes the binding of proapoptotic BCL2 proteins to MCL1. This suggests a novel strategy for integrating BH3 mimetics that target different BCL2 family proteins for KRAS-mutant NSCLC. Significance: Defining the molecular basis for MCL1 versus BCL-xL dependency will be essential for effective prioritization of BH3 mimetic combination therapies in the clinic. We discover a novel strategy for integrating BCL-xL and MCL1 inhibitors to drive and subsequently exploit apoptotic dependencies of KRAS-mutant NSCLCs treated with MEK inhibitors. See related commentary by Leber et al., p. 1511. This article is highlighted in the In This Issue feature, p. 1494

Purpose: Epithelial-to-mesenchymal transition (EMT) confers resistance to a number of targeted therapies and chemotherapies. However, it has been unclear why EMT promotes resistance, thereby impairing progress to overcome it. Experimental Design: We have developed several models of EMT-mediated resistance to EGFR inhibitors (EGFRi) in EGFR-mutant lung cancers to evaluate a novel mechanism of EMT-mediated resistance. Results: We observed that mesenchymal EGFR-mutant lung cancers are resistant to EGFRi-induced apoptosis via insufficient expression of BIM, preventing cell death despite potent suppression of oncogenic signaling following EGFRi treatment. Mechanistically, we observed that the EMT transcription factor ZEB1 inhibits BIM expression by binding directly to the BIM promoter and repressing transcription. Derepression of BIM expression by depletion of ZEB1 or treatment with the BH3 mimetic ABT-263 to enhance “free” cellular BIM levels both led to resensitization of mesenchymal EGFR-mutant cancers to EGFRi. This relationship between EMT and loss of BIM is not restricted to EGFR-mutant lung cancers, as it was also observed in KRAS-mutant lung cancers and large datasets, including different cancer subtypes. Conclusions: Altogether, these data reveal a novel mechanistic link between EMT and resistance to lung cancer targeted therapies. Clin Cancer Res; 24(1); 197–208. ©2017 AACR.

There are currently no effective targeted therapies for KRAS mutant cancers. Therapeutic strategies that combine MEK inhibitors with agents that target apoptotic pathways may be a promising therapeutic approach. We investigated combining MEK and MDM2 inhibitors as a potential treatment strategy for KRAS mutant non-small cell lung cancers (NSCLC) and colorectal carcinomas that harbor wild-type TP53. The combination of pimasertib (MEK inhibitor) and SAR405838 (MDM2 inhibitor) was synergistic and induced the expression of PUMA and BIM, led to apoptosis and growth inhibition in vitro, and tumor regression in vivo. Acquired resistance to the combination commonly resulted from the acquisition of TP53 mutations, conferring complete resistance to MDM2 inhibition. In contrast, resistant clones exhibited marked variability in sensitivity to MEK inhibition, which significantly impacted sensitivity to subsequent treatment with alternative MEK inhibitor-based combination therapies. These results highlight both the potential promise and limitations of combining MEK and MDM2 inhibitors for treatment of KRAS mutant NSCLC and colorectal cancers.

Although mechanisms of acquired resistance of epidermal growth factor receptor (EGFR)-mutant non-small-cell lung cancers to EGFR inhibitors have been identified, little is known about how resistant clones evolve during drug therapy. Here we observe that acquired resistance caused by the EGFR(T790M) gatekeeper mutation can occur either by selection of pre-existing EGFR(T790M)-positive clones or via genetic evolution of initially EGFR(T790M)-negative drug-tolerant cells. The path to resistance impacts the biology of the resistant clone, as those that evolved from drug-tolerant cells had a diminished apoptotic response to third-generation EGFR inhibitors that target EGFR(T790M); treatment with navitoclax, an inhibitor of the anti-apoptotic factors BCL-xL and BCL-2 restored sensitivity. We corroborated these findings using cultures derived directly from EGFR inhibitor-resistant patient tumors. These findings provide evidence that clinically relevant drug-resistant cancer cells can both pre-exist and evolve from drug-tolerant cells, and they point to therapeutic opportunities to prevent or overcome resistance in the clinic.

Despite the success of targeted cancer therapies, acquired drug resistance remains a significant clinical challenge, as in the case of EGFR mutant non-small cell lung cancers (NSCLC) treated with EGFR inhibitor therapy. Although molecular mechanisms of acquired resistance to EGFR inhibitors have been identified, little is known about how resistant clones evolve during drug therapy. In some cases, clones with clinically validated genetic resistance mechanisms may exist prior to drug exposure and are simply selected by treatment. Alternatively, it has been hypothesized that drug tolerant (or “persister”) cells without bona fide resistance mechanisms may survive initial drug treatment by epigenetic adaptations, and undergo further evolution over time to acquire validated genetic resistance mechanisms. Although this would have immediate implications for new therapeutic strategies to prevent resistance, there has not been any direct evidence that drug tolerant cells can undergo such evolution. Using EGFR mutant NSCLC models, we observe that acquired resistance caused by the T790M gatekeeper mutation, which is observed in 50% of patients with acquired resistance to EGFR inhibitor therapy, can occur by either selection of pre-existing T790M clones or via evolution of T790M-negative drug tolerant cells that develop the mutation during months of drug treatment. Additionally, the path to resistance impacts the biology of the resistant clone, as those that evolved from drug tolerant cells have a diminished apoptotic response to third generation EGFR inhibitors that target T790M EGFR and can be sensitized by BCL-XL inhibitors. In total, these findings provide evidence that clinically relevant drug resistant cancer cells can both pre-exist and evolve from drug tolerant cells, and point to new therapeutic opportunities to prevent or overcome resistance in the clinic. Citation Format: Aaron N. Hata, Matthew Niederst, Maria Gomez-Caraballo, Hannah Archibald, Faria Siddiqui, Hillary Mulvey, Haichuan Hu, Lecia Sequist, Jeffrey Engelman. Dynamic evolution of resistance to EGFR blockade from drug tolerant cancer cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B53.

The development of acquired drug resistance limits the effectiveness of targeted therapies for lung cancer. In 50% of patients with EGFR mutant non-small cell lung cancer (NSCLC), acquired resistance to EGFR inhibitors can be attributed to the development of a secondary T790M mutation that prevents drug binding. Third generation irreversible EGFR inhibitors that effectively inhibit EGFR with T790M are currently under clinical development. Using patient-derived EGFR mutant NSCLC models of T790M+ acquired resistance, we observed that the presence of a mesenchymal phenotype was associated with a decreased response to subsequent irreversible EGFR inhibitor therapy. Epithelial-to-mesenchymal transformation (EMT) led to suppression of pro-apoptotic signaling and a blunted apoptotic response to EGFR inhibition. Combination BCL-XL and irreversible EGFR inhibitor therapy restored the apoptotic response and resulted in regressions of T790M+ resistant tumors that exhibited poor response to single agent irreversible EGFR inhibitors. This suggests that combining BCL-XL and irreversible EGFR inhibitors may be an effective treatment strategy for T790M+ resistant EGFR mutant lung cancers with suppressed apoptotic signaling due co-acquisition of a mesenchymal phenotype. Citation Format: Aaron N. Hata, Matthew J. Niederst, Hannah L. Archibald, Hillary Mulvey, Jungoh Ham, Maria Gomez-Caraballo, Anuj Kalsy, Anthony C. Faber, Jeffrey Engelman. Co-acquisition of T790M and EMT in resistant EGFR mutant non-small cell lung cancer can be overcome by combined irreversible EGFR and BCL-XL inhibition. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2845. doi:10.1158/1538-7445.AM2015-2845

There are currently no effective targeted therapies for KRAS mutant NSCLC. Combination drug strategies that target downstream pathways activated by mutant KRAS are currently being investigated, however efficacy may be limited by inability to induce a sufficient apoptotic response. MDM-2 (murine double minute 2) is a ubiquitin ligase that negatively regulates p53 function by promoting its degradation. Inhibitors of the MDM2-p53 interaction lead to p53 activation and transcription of genes promoting cell cycle arrest and apoptosis. We investigated the efficacy of combined MEK and MDM2 inhibition for KRAS mutant NSCLC. In KRAS mutant NSCLC cell lines with wild-type p53, pimasertib (MEKi) and SAR405838 (MDM-2i) led to upregulation of BIM and PUMA respectively. Induction of apoptosis and cell cycle arrest correlated with in vitro efficacy, and the combination exhibited marked in vivo activity against KRAS mutant p53 wild-type NSCLC xenograft tumors. These studies provide rationale for future clinical investigation of combined MEK and MDM-2 inhibition for KRAS mutant NSCLC. Citation Format: Aaron N. Hata, Hannah L. Archibald, Maria D. Gomez-Caraballo, Laurent R. DeBussche, Sukhvinder Sidhu, James Watters, Jeffrey A. Engelman. Combined inhibition of MDM2 and MEK for KRAS mutant non-small cell lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 741. doi:10.1158/1538-7445.AM2014-741