Scaffolding Protein Connector Enhancer of Kinase Suppressor of Ras 1 (CNKSR1) Regulates MAPK Inhibition Responsiveness in Pancreas Cancer via Crosstalk with AKT Signaling.

Combinatorial molecular therapy in pancreatic ductal adenocarcinoma (PDAC) has yielded largely disappointing results in clinical testing to-date as a multitude of adaptive resistance mechanisms is making selection of patients via molecular markers that capture essential, intersecting signaling routes challenging. Here, we report the scaffolding protein connector enhancer of kinase suppressor of Ras 1 (CNKSR1) as mediator of resistance to MAPK (MEK) inhibition. MEK inhibition in CNKSR1high cancer cells induces translocation of CNKSR1 to the plasma membrane where the scaffolding protein interacts with and stabilizes the phosphorylated form of AKT. CNKSR1-mediated AKT activation following MEK inhibition was associated with increased cellular p-PRAS40 levels and reduced nuclear translocation and cellular levels of FoxO1, a negative regulator of AKT signaling. In clinical PDAC specimens, high cytoplasmatic CNKSR1 levels correlated with increased cellular phospho-AKT and mTOR levels. Pharmacological co-blockade of AKT and MEK ranked top in induced synergies with MEK inhibition in CNKSR1high pancreas cancer cells among other inhibitor combinations targeting known CNKSR1 signaling. In vivo, CNKSR1high pancreatic tumors treated with AKT and MEK inhibitors showed improved outcome in the combination arm compared with single-agent treatment, an effect not observed in CNKSR1low models.Our results identify CNKSR1 as regulator of adaptive resistance to MEK inhibition by promoting crosstalk to AKT signaling via a scaffolding function for the phosphorylated form of AKT. CNSKR1 expression might be a possible molecular marker to enrich patients for future AKT-MEK inhibitor precision medicine studies. IMPLICATIONS: The CNKSR1 scaffold, identified within an RNAi screen as a novel mediator of resistance to MEK inhibition in pancreas cancer, connects the MAPK pathway and AKT signaling and may be adopted as a biomarker to select patients for combined MEK AKT blockade.

Distinct resistance mechanisms arise to allosteric vs. ATP-competitive AKT inhibitors.

The AKT kinases have emerged as promising therapeutic targets in oncology and both allosteric and ATP-competitive AKT inhibitors have entered clinical investigation. However, long-term efficacy of such inhibitors will likely be challenged by the development of resistance. We have established prostate cancer models of acquired resistance to the allosteric inhibitor MK-2206 or the ATP-competitive inhibitor ipatasertib following prolonged exposure. While alterations in AKT are associated with acquired resistance to MK-2206, ipatasertib resistance is driven by rewired compensatory activity of parallel signaling pathways. Importantly, MK-2206 resistance can be overcome by treatment with ipatasertib, while ipatasertib resistance can be reversed by co-treatment with inhibitors of pathways including PIM signaling. These findings demonstrate that distinct resistance mechanisms arise to the two classes of AKT inhibitors and that combination approaches may reverse resistance to ATP-competitive inhibition.

Etoposide-induced DNA damage is increased in p53 mutants: identification of ATR and other genes that influence effects of p53 mutations on Top2-induced cytotoxicity.

The functional status of the tumor suppressor p53 is a critical component in determining the sensitivity of cancer cells to many chemotherapeutic agents. DNA topoisomerase II (Top2) plays essential roles in DNA metabolism and is the target of FDA approved chemotherapeutic agents. Topoisomerase targeting drugs convert the enzyme into a DNA damaging agent and p53 influences cellular responses to these agents. We assessed the impact of the loss of p53 function on the formation of DNA damage induced by the Top2 poison etoposide. Using human HCT116 cells, we found resistance to etoposide in cell growth assays upon the functional loss of p53. Nonetheless, cells lacking fully functional p53 were etoposide hypersensitive in clonogenic survival assays. This complex role of p53 led us to directly examine the effects of p53 status on topoisomerase-induced DNA damage. A deficiency in functional p53 resulted in elevated levels of the Top2 covalent complexes (Top2cc) in multiple cell lines. Employing genome-wide siRNA screens, we identified a set of genes for which reduced expression resulted in enhanced synthetic lethality upon etoposide treatment of p53 defective cells. We focused on one hit from this screen, ATR, and showed that decreased expression sensitized the p53-defective cells to etoposide in all assays and generated elevated levels of Top2cc in both p53 proficient and deficient cells. Our findings suggest that a combination of etoposide treatment with functional inactivation of DNA repair in p53 defective cells could be used to enhance the therapeutic efficacy of Top2 targeting agents.

A genome-wide screen uncovers multiple roles for mitochondrial nucleoside diphosphate kinase D in inflammasome activation.

Noncanonical inflammasome activation by cytosolic lipopolysaccharide (LPS) is a critical component of the host response to Gram-negative bacteria. Cytosolic LPS recognition in macrophages is preceded by a Toll-like receptor (TLR) priming signal required to induce transcription of inflammasome components and facilitate the metabolic reprograming that fuels the inflammatory response. Using a genome-scale arrayed siRNA screen to find inflammasome regulators in mouse macrophages, we identified the mitochondrial enzyme nucleoside diphosphate kinase D (NDPK-D) as a regulator of both noncanonical and canonical inflammasomes. NDPK-D was required for both mitochondrial DNA synthesis and cardiolipin exposure on the mitochondrial surface in response to inflammasome priming signals mediated by TLRs, and macrophages deficient in NDPK-D had multiple defects in LPS-induced inflammasome activation. In addition, NDPK-D was required for the recruitment of TNF receptor-associated factor 6 (TRAF6) to mitochondria, which was critical for reactive oxygen species (ROS) production and the metabolic reprogramming that supported the TLR-induced gene program. NDPK-D knockout mice were protected from LPS-induced shock, consistent with decreased ROS production and attenuated glycolytic commitment during priming. Our findings suggest that, in response to microbial challenge, NDPK-D-dependent TRAF6 mitochondrial recruitment triggers an energetic fitness checkpoint required to engage and maintain the transcriptional program necessary for inflammasome activation.

ARAF mutations confer resistance to the RAF inhibitor belvarafenib in melanoma.

Although RAF monomer inhibitors (type I.5, BRAF(V600)) are clinically approved for the treatment of BRAFV600-mutant melanoma, they are ineffective in non-BRAFV600 mutant cells1-3. Belvarafenib is a potent and selective RAF dimer (type II) inhibitor that exhibits clinical activity in patients with BRAFV600E- and NRAS-mutant melanomas. Here we report the first-in-human phase I study investigating the maximum tolerated dose, and assessing the safety and preliminary efficacy of belvarafenib in BRAFV600E- and RAS-mutated advanced solid tumours (NCT02405065, NCT03118817). By generating belvarafenib-resistant NRAS-mutant melanoma cells and analysing circulating tumour DNA from patients treated with belvarafenib, we identified new recurrent mutations in ARAF within the kinase domain. ARAF mutants conferred resistance to belvarafenib in both a dimer- and a kinase activity-dependent manner. Belvarafenib induced ARAF mutant dimers, and dimers containing mutant ARAF were active in the presence of inhibitor. ARAF mutations may serve as a general resistance mechanism for RAF dimer inhibitors as the mutants exhibit reduced sensitivity to a panel of type II RAF inhibitors. The combination of RAF plus MEK inhibition may be used to delay ARAF-driven resistance and suggests a rational combination for clinical use. Together, our findings reveal specific and compensatory functions for the ARAF isoform and implicate ARAF mutations as a driver of resistance to RAF dimer inhibitors.

A high-throughput genome-wide RNAi screen identifies modifiers of survival motor neuron protein.

Spinal muscular atrophy (SMA) is a debilitating neurological disorder marked by degeneration of spinal motor neurons and muscle atrophy. SMA results from mutations in survival motor neuron 1 (SMN1), leading to deficiency of survival motor neuron (SMN) protein. Current therapies increase SMN protein and improve patient survival but have variable improvements in motor function, making it necessary to identify complementary strategies to further improve disease outcomes. Here, we perform a genome-wide RNAi screen using a luciferase-based activity reporter and identify genes involved in regulating SMN gene expression, RNA processing, and protein stability. We show that reduced expression of Transcription Export complex components increases SMN levels through the regulation of nuclear/cytoplasmic RNA transport. We also show that the E3 ligase, Neurl2, works cooperatively with Mib1 to ubiquitinate and promote SMN degradation. Together, our screen uncovers pathways through which SMN expression is regulated, potentially revealing additional strategies to treat SMA.

Machine-Learning and Chemicogenomics Approach Defines and Predicts Cross-Talk of Hippo and MAPK Pathways.

Hippo pathway dysregulation occurs in multiple cancers through genetic and nongenetic alterations, resulting in translocation of YAP to the nucleus and activation of the TEAD family of transcription factors. Unlike other oncogenic pathways such as RAS, defining tumors that are Hippo pathway-dependent is far more complex due to the lack of hotspot genetic alterations. Here, we developed a machine-learning framework to identify a robust, cancer type-agnostic gene expression signature to quantitate Hippo pathway activity and cross-talk as well as predict YAP/TEAD dependency across cancers. Further, through chemical genetic interaction screens and multiomics analyses, we discover a direct interaction between MAPK signaling and TEAD stability such that knockdown of YAP combined with MEK inhibition results in robust inhibition of tumor cell growth in Hippo dysregulated tumors. This multifaceted approach underscores how computational models combined with experimental studies can inform precision medicine approaches including predictive diagnostics and combination strategies. SIGNIFICANCE: An integrated chemicogenomics strategy was developed to identify a lineage-independent signature for the Hippo pathway in cancers. Evaluating transcriptional profiles using a machine-learning method led to identification of a relationship between YAP/TAZ dependency and MAPK pathway activity. The results help to nominate potential combination therapies with Hippo pathway inhibition.This article is highlighted in the In This Issue feature, p. 521.

Transcriptional Subtypes Resolve Tumor Heterogeneity and Identify Vulnerabilities to MEK Inhibition in Lung Adenocarcinoma.

PURPOSE: Lung adenocarcinomas comprise the largest fraction of non-small cell lung cancer, which is the leading cause of cancer-related deaths. Seventy-five percent of adenocarcinomas lack targeted therapies because of scarcity of druggable drivers. Here, we classified tumors on the basis of signaling similarities and discovered subgroups within this unmet patient population. EXPERIMENTAL DESIGN: We leveraged transcriptional data from >800 early- and advanced-stage patients. RESULTS: We identified three robust subtypes dubbed mucinous, proliferative, and mesenchymal with respective pathway phenotypes. These transcriptional states lack discrete and causative mutational etiology as evidenced by similarly distributed oncogenic drivers, including KRAS and EGFR. The subtypes capture heterogeneity even among tumors lacking known oncogenic drivers. Paired multi-regional intratumoral biopsies demonstrated unified subtypes despite divergently evolved prooncogenic mutations, indicating subtype stability during selective pressure. Heterogeneity among in vitro and in vivo preclinical models is expounded by the human lung adenocarcinoma subtypes and can be leveraged to discover subtype-specific vulnerabilities. As proof of concept, we identified differential subtype response to MEK pathway inhibition in a chemical library screen of 89 lung cancer cell lines, which reproduces across model systems and a clinical trial. CONCLUSIONS: Our findings support forward translational relevance of transcriptional subtypes, where further exploration therein may improve lung adenocarcinoma treatment.See related commentary by Skoulidis, p. 913.

Enhanced Protein Damage Clearance Induces Broad Drug Resistance in Multitype of Cancers Revealed by an Evolution Drug-Resistant Model and Genome-Wide siRNA Screening.

Resistance to therapeutic drugs occurs in virtually all types of cancers, and the tolerance to one drug frequently becomes broad therapy resistance; however, the underlying mechanism remains elusive. Combining a whole whole-genome-wide RNA interference screening and an evolutionary drug pressure model with MDA-MB-231 cells, it is found that enhanced protein damage clearance and reduced mitochondrial respiratory activity are responsible for cisplatin resistance. Screening drug-resistant cancer cells and human patient-derived organoids for breast and colon cancers with many anticancer drugs indicates that activation of mitochondrion protein import surveillance system enhances proteasome activity and minimizes caspase activation, leading to broad drug resistance that can be overcome by co-treatment with a proteasome inhibitor, bortezomib. It is further demonstrated that cisplatin and bortezomib encapsulated into nanoparticle further enhance their therapeutic efficacy and alleviate side effects induced by drug combination treatment. These data demonstrate a feasibility for eliminating broad drug resistance by targeting its common mechanism to achieve effective therapy for multiple cancers.

Degradation of 5hmC-marked stalled replication forks by APE1 causes genomic instability.

Synthetic lethality between poly(ADP-ribose) polymerase (PARP) inhibition and BRCA deficiency is exploited to treat breast and ovarian tumors. However, resistance to PARP inhibitors (PARPis) is common. To identify potential resistance mechanisms, we performed a genome-wide RNAi screen in BRCA2-deficient mouse embryonic stem cells and validation in KB2P1.21 mouse mammary tumor cells. We found that resistance to multiple PARPi emerged with reduced expression of TET2 (ten-eleven translocation), which promotes DNA demethylation by oxidizing 5-methylcytosine (5mC) to 5-hydroxymethycytosine (5hmC) and other products. TET2 knockdown in BRCA2-deficient cells protected stalled replication forks (RFs). Increasing 5hmC abundance induced the degradation of stalled RFs in KB2P1.21 and human cancer cells by recruiting the base excision repair-associated apurinic/apyrimidinic endonuclease APE1, independent of the BRCA2 status. TET2 loss did not affect the recruitment of the repair protein RAD51 to sites of double-strand breaks (DSBs) or the abundance of proteins associated with RF integrity. The loss of TET2, of its product 5hmC, and of APE1 recruitment to stalled RFs promoted resistance to the chemotherapeutic cisplatin. Our findings reveal a previously unknown role for the epigenetic mark 5hmC in maintaining the integrity of stalled RFs and a potential resistance mechanism to PARPi and cisplatin.

CDK9 Blockade Exploits Context-dependent Transcriptional Changes to Improve Activity and Limit Toxicity of Mithramycin for Ewing Sarcoma.

There is a need to develop novel approaches to improve the balance between efficacy and toxicity for transcription factor-targeted therapies. In this study, we exploit context-dependent differences in RNA polymerase II processivity as an approach to improve the activity and limit the toxicity of the EWS-FLI1-targeted small molecule, mithramycin, for Ewing sarcoma. The clinical activity of mithramycin for Ewing sarcoma is limited by off-target liver toxicity that restricts the serum concentration to levels insufficient to inhibit EWS-FLI1. In this study, we perform an siRNA screen of the druggable genome followed by a matrix drug screen to identify mithramycin potentiators and a synergistic "class" effect with cyclin-dependent kinase 9 (CDK9) inhibitors. These CDK9 inhibitors enhanced the mithramycin-mediated suppression of the EWS-FLI1 transcriptional program leading to a shift in the IC50 and striking regressions of Ewing sarcoma xenografts. To determine whether these compounds may also be liver protective, we performed a qPCR screen of all known liver toxicity genes in HepG2 cells to identify mithramycin-driven transcriptional changes that contribute to the liver toxicity. Mithramycin induces expression of the BTG2 gene in HepG2 but not Ewing sarcoma cells, which leads to a liver-specific accumulation of reactive oxygen species (ROS). siRNA silencing of BTG2 rescues the induction of ROS and the cytotoxicity of mithramycin in these cells. Furthermore, CDK9 inhibition blocked the induction of BTG2 to limit cytotoxicity in HepG2, but not Ewing sarcoma cells. These studies provide the basis for a synergistic and less toxic EWS-FLI1-targeted combination therapy for Ewing sarcoma.

A Single-Step, High-Dose Selection Scheme Reveals Distinct Mechanisms of Acquired Resistance to Oncogenic Kinase Inhibition in Cancer Cells.

Despite the remarkable clinical efficacy demonstrated by molecularly targeted cancer therapeutics, the benefits are typically temporary due to the emergence of acquired drug resistance. This has spurred a massive effort by the cancer research community to identify mechanisms used by cancer cells to evade treatment. Among the various methodologies developed and employed to identify such mechanisms, the most commonly used approach has been to model acquired resistance by exposing cancer cells in culture to gradually increasing concentrations of drug over an extended period of time. Here, we employed a less commonly used variation on this approach, wherein resistant cells are selected by immediately exposing cancer cells to a continuous, high concentration of drug. Using this approach, we isolated clones representing three distinct mechanisms of resistance to inhibition of MET kinase activity from a single clonally derived cancer cell line. The emergent clones had acquired resistance through engagement of alternative receptor tyrosine kinases either through upregulation of FGF3 or HBEGF or increased MAPK signaling through an activating V600E mutation in BRAF. Importantly, these mechanisms were not identified using the conventional "ramp-up" approach in previous studies that employed the same cell line. These results suggest that the particular nature of the selection scheme employed in cell culture modeling studies can determine which potential resistance mechanisms are identified and which ones may be missed, highlighting the need for careful consideration of the specific approach used to model resistance in cultured cells. SIGNIFICANCE: Through modeling resistance to MET kinase inhibition in cultured cancer cells using single-step, high-dose selection, these findings highlight that the specific nature of the selection protocol impacts which resistance mechanisms are identified.See related commentary by Floros et al., p. 25.

The alpha-1 subunit of the Na+,K+-ATPase (ATP1A1) is required for macropinocytic entry of respiratory syncytial virus (RSV) in human respiratory epithelial cells.

Human respiratory syncytial virus (RSV) is the leading viral cause of acute pediatric lower respiratory tract infections worldwide, with no available vaccine or effective antiviral drug. To gain insight into virus-host interactions, we performed a genome-wide siRNA screen. The expression of over 20,000 cellular genes was individually knocked down in human airway epithelial A549 cells, followed by infection with RSV expressing green fluorescent protein (GFP). Knockdown of expression of the cellular ATP1A1 protein, which is the major subunit of the Na+,K+-ATPase of the plasma membrane, had one of the strongest inhibitory effects on GFP expression and viral titer. Inhibition was not observed for vesicular stomatitis virus, indicating that it was RSV-specific rather than a general effect. ATP1A1 formed clusters in the plasma membrane very early following RSV infection, which was independent of replication but dependent on the attachment glycoprotein G. RSV also triggered activation of ATP1A1, resulting in signaling by c-Src-kinase activity that transactivated epidermal growth factor receptor (EGFR) by Tyr845 phosphorylation. ATP1A1 signaling and activation of both c-Src and EGFR were found to be required for efficient RSV uptake. Signaling events downstream of EGFR culminated in the formation of macropinosomes. There was extensive uptake of RSV virions into macropinosomes at the beginning of infection, suggesting that this is a major route of RSV uptake, with fusion presumably occurring in the macropinosomes rather than at the plasma membrane. Important findings were validated in primary human small airway epithelial cells (HSAEC). In A549 cells and HSAEC, RSV uptake could be inhibited by the cardiotonic steroid ouabain and the digitoxigenin derivative PST2238 (rostafuroxin) that bind specifically to the ATP1A1 extracellular domain and block RSV-triggered EGFR Tyr845 phosphorylation. In conclusion, we identified ATP1A1 as a host protein essential for macropinocytic entry of RSV into respiratory epithelial cells, and identified PST2238 as a potential anti-RSV drug.

ERBB3 and IGF1R Signaling Are Required for Nrf2-Dependent Growth in KEAP1-Mutant Lung Cancer.

Mutations in KEAP1 and NFE2L2 (encoding the protein Nrf2) are prevalent in both adeno and squamous subtypes of non-small cell lung cancer, as well as additional tumor indications. The consequence of these mutations is stabilized Nrf2 and chronic induction of a battery of Nrf2 target genes. We show that knockdown of Nrf2 caused modest growth inhibition of cells growing in two-dimension, which was more pronounced in cell lines expressing mutant KEAP1. In contrast, Nrf2 knockdown caused almost complete regression of established KEAP1-mutant tumors in mice, with little effect on wild-type (WT) KEAP1 tumors. The strong dependency on Nrf2 could be recapitulated in certain anchorage-independent growth environments and was not prevented by excess extracellular glutathione. A CRISPR screen was used to investigate the mechanism(s) underlying this dependence. We identified alternative pathways critical for Nrf2-dependent growth in KEAP1-mutant cell lines, including the redox proteins thioredoxin and peroxiredoxin, as well as the growth factor receptors IGF1R and ERBB3. IGF1R inhibition was effective in KEAP1-mutant cells compared with WT, especially under conditions of anchorage-independent growth. These results point to addiction of KEAP1-mutant tumor cells to Nrf2 and suggest that inhibition of Nrf2 or discrete druggable Nrf2 target genes such as IGF1R could be an effective therapeutic strategy for disabling these tumors. SIGNIFICANCE: This study identifies pathways activated by Nrf2 that are important for the proliferation and tumorigenicity of KEAP1-mutant non-small cell lung cancer.

Therapeutic Ligands Antagonize Estrogen Receptor Function by Impairing Its Mobility.

Estrogen receptor-positive (ER+) breast cancers frequently remain dependent on ER signaling even after acquiring resistance to endocrine agents, prompting the development of optimized ER antagonists. Fulvestrant is unique among approved ER therapeutics due to its capacity for full ER antagonism, thought to be achieved through ER degradation. The clinical potential of fulvestrant is limited by poor physicochemical features, spurring attempts to generate ER degraders with improved drug-like properties. We show that optimization of ER degradation does not guarantee full ER antagonism in breast cancer cells; ER "degraders" exhibit a spectrum of transcriptional activities and anti-proliferative potential. Mechanistically, we find that fulvestrant-like antagonists suppress ER transcriptional activity not by ER elimination, but by markedly slowing the intra-nuclear mobility of ER. Increased ER turnover occurs as a consequence of ER immobilization. These findings provide proof-of-concept that small molecule perturbation of transcription factor mobility may enable therapeutic targeting of this challenging target class.

Integrated Genomic and Functional microRNA Analysis Identifies miR-30-5p as a Tumor Suppressor and Potential Therapeutic Nanomedicine in Head and Neck Cancer.

PURPOSE: To identify deregulated and inhibitory miRNAs and generate novel mimics for replacement nanomedicine for head and neck squamous cell carcinomas (HNSCC). EXPERIMENTAL DESIGN: We integrated miRNA and mRNA expression, copy number variation, and DNA methylation results from The Cancer Genome Atlas (TCGA), with a functional genome-wide screen. RESULTS: We reveal that the miR-30 family is commonly repressed, and all 5 members sharing these seed sequence similarly inhibit HNSCC proliferation in vitro. We uncover a previously unrecognized inverse relationship with overexpression of a network of important predicted target mRNAs deregulated in HNSCC, that includes key molecules involved in proliferation (EGFR, MET, IGF1R, IRS1, E2F7), differentiation (WNT7B, FZD2), adhesion, and invasion (ITGA6, SERPINE1). Reexpression of the most differentially repressed family member, miR-30a-5p, suppressed this mRNA program, selected signaling proteins and pathways, and inhibited cell proliferation, migration, and invasion in vitro. Furthermore, a novel miR-30a-5p mimic formulated into a targeted nanomedicine significantly inhibited HNSCC xenograft tumor growth and target growth receptors EGFR and MET in vivo. Significantly decreased miR-30a/e family expression was related to DNA promoter hypermethylation and/or copy loss in TCGA data, and clinically with decreased disease-specific survival in a validation dataset. Strikingly, decreased miR-30e-5p distinguished oropharyngeal HNSCC with poor prognosis in TCGA (P = 0.002) and validation (P = 0.007) datasets, identifying a novel candidate biomarker and target for this HNSCC subset. CONCLUSIONS: We identify the miR-30 family as an important regulator of signal networks and tumor suppressor in a subset of HNSCC patients, which may benefit from miRNA replacement nanomedicine therapy.

Multiple-gene targeting and mismatch tolerance can confound analysis of genome-wide pooled CRISPR screens.

BACKGROUND: Genome-wide loss-of-function screens using the CRISPR/Cas9 system allow the efficient discovery of cancer cell vulnerabilities. While several studies have focused on correcting for DNA cleavage toxicity biases associated with copy number alterations, the effects of sgRNAs co-targeting multiple genomic loci in CRISPR screens have not been discussed. RESULTS: In this work, we analyze CRISPR essentiality screen data from 391 cancer cell lines to characterize biases induced by multi-target sgRNAs. We investigate two types of multi-targets: on-targets predicted through perfect sequence complementarity and off-targets predicted through sequence complementarity with up to two nucleotide mismatches. We find that the number of on-targets and off-targets both increase sgRNA activity in a cell line-specific manner and that existing additive models of gene knockout effects fail at capturing genetic interactions that may occur between co-targeted genes. We use synthetic lethality between paralog genes to show that genetic interactions can introduce biases in essentiality scores estimated from multi-target sgRNAs. We further show that single-mismatch tolerant sgRNAs can confound the analysis of gene essentiality and lead to incorrect co-essentiality functional networks. Lastly, we also find that single nucleotide polymorphisms located in protospacer regions can impair on-target activity as a result of mismatch tolerance. CONCLUSION: We show the impact of multi-target effects on estimating cancer cell dependencies and the impact of off-target effects caused by mismatch tolerance in sgRNA-DNA binding.

Pharmacological Induction of RAS-GTP Confers RAF Inhibitor Sensitivity in KRAS Mutant Tumors.

Targeting KRAS mutant tumors through inhibition of individual downstream pathways has had limited clinical success. Here we report that RAF inhibitors exhibit little efficacy in KRAS mutant tumors. In combination drug screens, MEK and PI3K inhibitors synergized with pan-RAF inhibitors through an RAS-GTP-dependent mechanism. Broad cell line profiling with RAF/MEK inhibitor combinations revealed synergistic efficacy in KRAS mutant and wild-type tumors, with KRASG13D mutants exhibiting greater synergy versus KRASG12 mutant tumors. Mechanistic studies demonstrate that MEK inhibition induced RAS-GTP levels, RAF dimerization and RAF kinase activity resulting in MEK phosphorylation in synergistic tumor lines regardless of KRAS status. Taken together, our studies uncover a strategy to rewire KRAS mutant tumors to confer sensitivity to RAF kinase inhibition.

Coordination between Two Branches of the Unfolded Protein Response Determines Apoptotic Cell Fate.

The kinases PERK and IRE1 alleviate endoplasmic reticulum (ER) stress by orchestrating the unfolded protein response (UPR). If stress mitigation fails, PERK promotes cell death by activating pro-apoptotic genes, including death receptor 5 (DR5). Conversely, IRE1-which harbors both kinase and endoribonuclease (RNase) modules-blocks apoptosis through regulated IRE1-dependent decay (RIDD) of DR5 mRNA. Under irresolvable ER stress, PERK activity persists, whereas IRE1 paradoxically attenuates, by mechanisms that remain obscure. Here, we report that PERK governs IRE1's attenuation through a phosphatase known as RPAP2 (RNA polymerase II-associated protein 2). RPAP2 reverses IRE1 phosphorylation, oligomerization, and RNase activation. This inhibits IRE1-mediated adaptive events, including activation of the cytoprotective transcription factor XBP1s, and ER-associated degradation of unfolded proteins. Furthermore, RIDD termination by RPAP2 unleashes DR5-mediated caspase activation and drives cell death. Thus, PERK attenuates IRE1 via RPAP2 to abort failed ER-stress adaptation and trigger apoptosis.

ERK Mutations and Amplification Confer Resistance to ERK-Inhibitor Therapy.

Purpose: MAPK pathway inhibitors targeting BRAF and MEK have shown clinical efficacy in patients with RAF- and/or RAS-mutated tumors. However, acquired resistance to these agents has been an impediment to improved long-term survival in the clinic. In such cases, targeting ERK downstream of BRAF/MEK has been proposed as a potential strategy for overcoming acquired resistance. Preclinical studies suggest that ERK inhibitors are effective at inhibiting BRAF/RAS-mutated tumor growth and overcome BRAF or/and MEK inhibitor resistance. However, as observed with other MAPK pathway inhibitors, treatment with ERK inhibitors is likely to cause resistance in the clinic. Here, we aimed to model the mechanism of resistance to ERK inhibitors.Experimental Design: We tested five structurally different ATP-competitive ERK inhibitors representing three different scaffolds on BRAF/RAS-mutant cancer cell lines of different tissue types to generate resistant lines. We have used in vitro modeling, structural biology, and genomic analysis to understand the development of resistance to ERK inhibitors and the mechanisms leading to it.Results: We have identified mutations in ERK1/2, amplification and overexpression of ERK2, and overexpression of EGFR/ERBB2 as mechanisms of acquired resistance. Structural analysis of ERK showed that specific compounds that induced on-target ERK mutations were impaired in their ability to bind mutant ERK. We show that in addition to MEK inhibitors, ERBB receptor and PI3K/mTOR pathway inhibitors are effective in overcoming ERK-inhibitor resistance.Conclusions: These findings suggest that combination therapy with MEK or ERBB receptor or PI3K/mTOR and ERK inhibitors may be an effective strategy for managing the emergence of resistance in the clinic. Clin Cancer Res; 24(16); 4044-55. ©2018 AACR.