MCB-613 exploits a collateral sensitivity in drug resistant EGFR-mutant non-small cell lung cancer through covalent inhibition of KEAP1.

Targeted therapies have revolutionized cancer chemotherapy. Unfortunately, most patients develop multifocal resistance to these drugs within a matter of months. Here, we used a high-throughput phenotypic small molecule screen to identify MCB-613 as a compound that selectively targets EGFR-mutant, EGFR inhibitor-resistant non-small cell lung cancer (NSCLC) cells harboring diverse resistance mechanisms. Subsequent proteomic and functional genomic screens involving MCB-613 identified its target in this context to be KEAP1, revealing that this gene is selectively essential in the setting of EGFR inhibitor resistance. In-depth molecular characterization demonstrated that (1) MCB-613 binds KEAP1 covalently; (2) a single molecule of MCB-613 is capable of bridging two KEAP1 monomers together; and, (3) this modification interferes with the degradation of canonical KEAP1 substrates such as NRF2. Surprisingly, NRF2 knockout sensitizes cells to MCB-613, suggesting that the drug functions through modulation of an alternative KEAP1 substrate. Together, these findings advance MCB-613 as a new tool for exploiting the selective essentiality of KEAP1 in drug-resistant, EGFR-mutant NSCLC cells.

Cellphone enabled point-of-care assessment of breast tumor cytology and molecular HER2 expression from fine-needle aspirates.

Management of breast cancer in limited-resource settings is hindered by a lack of low-cost, logistically sustainable approaches toward molecular and cellular diagnostic pathology services that are needed to guide therapy. To address these limitations, we have developed a multimodal cellphone-based platform-the EpiView-D4-that can evaluate both cellular morphology and molecular expression of clinically relevant biomarkers directly from fine-needle aspiration (FNA) of breast tissue specimens within 1 h. The EpiView-D4 is comprised of two components: (1) an immunodiagnostic chip built upon a "non-fouling" polymer brush-coating (the "D4") which quantifies expression of protein biomarkers directly from crude cell lysates, and (2) a custom cellphone-based optical microscope ("EpiView") designed for imaging cytology preparations and D4 assay readout. As a proof-of-concept, we used the EpiView-D4 for assessment of human epidermal growth factor receptor-2 (HER2) expression and validated the performance using cancer cell lines, animal models, and human tissue specimens. We found that FNA cytology specimens (prepared in less than 5 min with rapid staining kits) imaged by the EpiView-D4 were adequate for assessment of lesional cellularity and tumor content. We also found our device could reliably distinguish between HER2 expression levels across multiple different cell lines and animal xenografts. In a pilot study with human tissue (n = 19), we were able to accurately categorize HER2-negative and HER2-positve tumors from FNA specimens. Taken together, the EpiView-D4 offers a promising alternative to invasive-and often unavailable-pathology services and may enable the democratization of effective breast cancer management in limited-resource settings.

CRISPR screens in cancer spheroids identify 3D growth-specific vulnerabilities.

Cancer genomics studies have identified thousands of putative cancer driver genes1. Development of high-throughput and accurate models to define the functions of these genes is a major challenge. Here we devised a scalable cancer-spheroid model and performed genome-wide CRISPR screens in 2D monolayers and 3D lung-cancer spheroids. CRISPR phenotypes in 3D more accurately recapitulated those of in vivo tumours, and genes with differential sensitivities between 2D and 3D conditions were highly enriched for genes that are mutated in lung cancers. These analyses also revealed drivers that are essential for cancer growth in 3D and in vivo, but not in 2D. Notably, we found that carboxypeptidase D is responsible for removal of a C-terminal RKRR motif2 from the α-chain of the insulin-like growth factor 1 receptor that is critical for receptor activity. Carboxypeptidase D expression correlates with patient outcomes in patients with lung cancer, and loss of carboxypeptidase D reduced tumour growth. Our results reveal key differences between 2D and 3D cancer models, and establish a generalizable strategy for performing CRISPR screens in spheroids to reveal cancer vulnerabilities.

Using antagonistic pleiotropy to design a chemotherapy-induced evolutionary trap to target drug resistance in cancer.

Local adaptation directs populations towards environment-specific fitness maxima through acquisition of positively selected traits. However, rapid environmental changes can identify hidden fitness trade-offs that turn adaptation into maladaptation, resulting in evolutionary traps. Cancer, a disease that is prone to drug resistance, is in principle susceptible to such traps. We therefore performed pooled CRISPR-Cas9 knockout screens in acute myeloid leukemia (AML) cells treated with various chemotherapies to map the drug-dependent genetic basis of fitness trade-offs, a concept known as antagonistic pleiotropy (AP). We identified a PRC2-NSD2/3-mediated MYC regulatory axis as a drug-induced AP pathway whose ability to confer resistance to bromodomain inhibition and sensitivity to BCL-2 inhibition templates an evolutionary trap. Across diverse AML cell-line and patient-derived xenograft models, we find that acquisition of resistance to bromodomain inhibition through this pathway exposes coincident hypersensitivity to BCL-2 inhibition. Thus, drug-induced AP can be leveraged to design evolutionary traps that selectively target drug resistance in cancer.

Inhibition of BCL2 Family Members Increases the Efficacy of Copper Chelation in BRAFV600E-Driven Melanoma.

The principal unmet need in BRAFV600E-positive melanoma is lack of an adequate therapeutic strategy capable of overcoming resistance to clinically approved targeted therapies against oncogenic BRAF and/or the downstream MEK1/2 kinases. We previously discovered that copper (Cu) is required for MEK1 and MEK2 activity through a direct Cu-MEK1/2 interaction. Repurposing the clinical Cu chelator tetrathiomolybdate (TTM) is supported by efficacy in BRAFV600E-driven melanoma models, due in part to inhibition of MEK1/2 kinase activity. However, the antineoplastic activity of Cu chelators is cytostatic. Here, we performed high-throughput small-molecule screens to identify bioactive compounds that synergize with TTM in BRAFV600E-driven melanoma cells. Genetic perturbation or pharmacologic inhibition of specific members of the BCL2 family of antiapoptotic proteins (BCL-W, BCL-XL, and MCL1) selectively reduced cell viability when combined with a Cu chelator and induced CASPASE-dependent cell death. Further, in BRAFV600E-positive melanoma cells evolved to be resistant to BRAF and/or MEK1/2 inhibitors, combined treatment with TTM and the clinically evaluated BCL2 inhibitor, ABT-263, restored tumor growth suppression and induced apoptosis. These findings further support Cu chelation as a therapeutic strategy to target oncogene-dependent tumor cell growth and survival by enhancing Cu chelator efficacy with chemical inducers of apoptosis, especially in the context of refractory or relapsed BRAFV600E-driven melanoma. SIGNIFICANCE: This study unveils a novel collateral drug sensitivity elicited by combining copper chelators and BH3 mimetics for treatment of BRAFV600E mutation-positive melanoma.

Genomically informed small-molecule drugs overcome resistance to a sustained-release formulation of an engineered death receptor agonist in patient-derived tumor models.

Extrinsic pathway agonists have failed repeatedly in the clinic for three core reasons: Inefficient ligand-induced receptor multimerization, poor pharmacokinetic properties, and tumor intrinsic resistance. Here, we address these factors by (i) using a highly potent death receptor agonist (DRA), (ii) developing an injectable depot for sustained DRA delivery, and (iii) leveraging a CRISPR-Cas9 knockout screen in DRA-resistant colorectal cancer (CRC) cells to identify functional drivers of resistance. Pharmacological blockade of XIAP and BCL-XL by targeted small-molecule drugs strongly enhanced the antitumor activity of DRA in CRC cell lines. Recombinant fusion of the DRA to a thermally responsive elastin-like polypeptide (ELP) creates a gel-like depot upon subcutaneous injection that abolishes tumors in DRA-sensitive Colo205 mouse xenografts. Combination of ELPdepot-DRA with BCL-XL and/or XIAP inhibitors led to tumor growth inhibition and extended survival in DRA-resistant patient-derived xenografts. This strategy provides a precision medicine approach to overcome similar challenges with other protein-based cancer therapies.

Systematic mapping of BCL-2 gene dependencies in cancer reveals molecular determinants of BH3 mimetic sensitivity.

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.

Dysregulation of mitochondrial dynamics proteins are a targetable feature of human tumors.

Altered mitochondrial dynamics can broadly impact tumor cell physiology. Using genetic and pharmacological profiling of cancer cell lines and human tumors, we here establish that perturbations to the mitochondrial dynamics network also result in specific therapeutic vulnerabilities. In particular, through distinct mechanisms, tumors with increased mitochondrial fragmentation or connectivity are hypersensitive to SMAC mimetics, a class of compounds that induce apoptosis through inhibition of IAPs and for which robust sensitivity biomarkers remain to be identified. Further, because driver oncogenes exert dominant control over mitochondrial dynamics, oncogene-targeted therapies can be used to sensitize tumors to SMAC mimetics via their effects on fission/fusion dynamics. Collectively, these data demonstrate that perturbations to the mitochondrial dynamics network induce targetable vulnerabilities across diverse human tumors and, more broadly, suggest that the altered structures, activities, and trafficking of cellular organelles may facilitate additional cancer therapeutic opportunities.

Codon bias imposes a targetable limitation on KRAS-driven therapeutic resistance.

KRAS mutations drive resistance to targeted therapies, including EGFR inhibitors in colorectal cancer (CRC). Through genetic screens, we unexpectedly find that mutant HRAS, which is rarely found in CRC, is a stronger driver of resistance than mutant KRAS. This difference is ascribed to common codon bias in HRAS, which leads to much higher protein expression, and implies that the inherent poor expression of KRAS due to rare codons must be surmounted during drug resistance. In agreement, we demonstrate that primary resistance to cetuximab is dependent upon both KRAS mutational status and protein expression level, and acquired resistance is often associated with KRASQ61 mutations that function even when protein expression is low. Finally, cancer cells upregulate translation to facilitate KRASG12-driven acquired resistance, resulting in hypersensitivity to translational inhibitors. These findings demonstrate that codon bias plays a critical role in KRAS-driven resistance and provide a rationale for targeting translation to overcome resistance.

PIK3CA mutations enable targeting of a breast tumor dependency through mTOR-mediated MCL-1 translation.

Therapies that efficiently induce apoptosis are likely to be required for durable clinical responses in patients with solid tumors. Using a pharmacological screening approach, we discovered that combined inhibition of B cell lymphoma-extra large (BCL-XL) and the mammalian target of rapamycin (mTOR)/4E-BP axis results in selective and synergistic induction of apoptosis in cellular and animal models of PIK3CA mutant breast cancers, including triple-negative tumors. Mechanistically, inhibition of mTOR/4E-BP suppresses myeloid cell leukemia-1 (MCL-1) protein translation only in PIK3CA mutant tumors, creating a synthetic dependence on BCL-XL This dual dependence on BCL-XL and MCL-1, but not on BCL-2, appears to be a fundamental property of diverse breast cancer cell lines, xenografts, and patient-derived tumors that is independent of the molecular subtype or PIK3CA mutational status. Furthermore, this dependence distinguishes breast cancers from normal breast epithelial cells, which are neither primed for apoptosis nor dependent on BCL-XL/MCL-1, suggesting a potential therapeutic window. By tilting the balance of pro- to antiapoptotic signals in the mitochondria, dual inhibition of MCL-1 and BCL-XL also sensitizes breast cancer cells to standard-of-care cytotoxic and targeted chemotherapies. Together, these results suggest that patients with PIK3CA mutant breast cancers may benefit from combined treatment with inhibitors of BCL-XL and the mTOR/4E-BP axis, whereas alternative methods of inhibiting MCL-1 and BCL-XL may be effective in tumors lacking PIK3CA mutations.

Targeting MCL-1/BCL-XL Forestalls the Acquisition of Resistance to ABT-199 in Acute Myeloid Leukemia.

ABT-199, a potent and selective small-molecule antagonist of BCL-2, is being clinically vetted as pharmacotherapy for the treatment of acute myeloid leukemia (AML). However, given that prolonged monotherapy tends to beget resistance, we sought to investigate the means by which resistance to ABT-199 might arise in AML and the extent to which those mechanisms might be preempted. Here we used a pathway-activating genetic screen to nominate MCL-1 and BCL-XL as potential nodes of resistance. We then characterized a panel of ABT-199-resistant myeloid leukemia cell lines derived through chronic exposure to ABT-199 and found that acquired drug resistance is indeed driven by the upregulation of MCL-1 and BCL-XL. By targeting MCL-1 and BCL-XL, resistant AML cell lines could be resensitized to ABT-199. Further, preemptively targeting MCL-1 and/or BCL-XL alongside administration of ABT-199 was capable of delaying or forestalling the acquisition of drug resistance. Collectively, these data suggest that in AML, (1) the selection of initial therapy dynamically templates the landscape of acquired resistance via modulation of MCL-1/BCL-XL and (2) appropriate selection of initial therapy may delay or altogether forestall the acquisition of resistance to ABT-199.

Systematic identification of signaling pathways with potential to confer anticancer drug resistance.

Cancer cells can activate diverse signaling pathways to evade the cytotoxic action of drugs. We created and screened a library of barcoded pathway-activating mutant complementary DNAs to identify those that enhanced the survival of cancer cells in the presence of 13 clinically relevant, targeted therapies. We found that activation of the RAS-MAPK (mitogen-activated protein kinase), Notch1, PI3K (phosphoinositide 3-kinase)-mTOR (mechanistic target of rapamycin), and ER (estrogen receptor) signaling pathways often conferred resistance to this selection of drugs. Activation of the Notch1 pathway promoted acquired resistance to tamoxifen (an ER-targeted therapy) in serially passaged breast cancer xenografts in mice, and treating mice with a γ-secretase inhibitor to inhibit Notch signaling restored tamoxifen sensitivity. Markers of Notch1 activity in tumor tissue correlated with resistance to tamoxifen in breast cancer patients. Similarly, activation of Notch1 signaling promoted acquired resistance to MAPK inhibitors in BRAF(V600E) melanoma cells in culture, and the abundance of Notch1 pathway markers was increased in tumors from a subset of melanoma patients. Thus, Notch1 signaling may be a therapeutic target in some drug-resistant breast cancers and melanomas. Additionally, multiple resistance pathways were activated in melanoma cell lines with intrinsic resistance to MAPK inhibitors, and simultaneous inhibition of these pathways synergistically induced drug sensitivity. These data illustrate the potential for systematic identification of the signaling pathways controlling drug resistance that could inform clinical strategies and drug development for multiple types of cancer. This approach may also be used to advance clinical options in other disease contexts.