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Summary Combinatorial inhibition of effector and feedback pathways is a promising treatment strategy for KRAS mutant cancers. However, the particular pathways that should be targeted to optimize therapeutic responses are unclear. Using CRISPR/Cas9, we systematically mapped the pathways whose inhibition cooperates with drugs targeting the KRAS effectors MEK, ERK, and PI3K. By performing 70 screens in models of KRAS mutant colorectal, lung, ovarian, and pancreas cancers, we uncovered universal and tissue-specific sensitizing combinations involving inhibitors of cell cycle, metabolism, growth signaling, chromatin regulation, and transcription. Furthermore, these screens revealed secondary genetic modifiers of sensitivity, yielding a SRC inhibitor-based combination therapy for KRAS / PIK3CA double-mutant colorectal cancers (CRCs) with clinical potential. Surprisingly, acquired resistance to combinations of growth signaling pathway inhibitors develops rapidly following treatment, but by targeting signaling feedback or apoptotic priming, it is possible to construct three-drug combinations that greatly delay its emergence. [ABSTRACT FROM AUTHOR]

The Wnt signaling pathways play pivotal roles in carcinogenesis. Modulation of the cell-surface abundance of Wnt receptors is emerging as an important mechanism for regulating sensitivity to Wnt ligands. Endocytosis and degradation of the Wnt receptors Frizzled (Fzd) and lipoprotein-related protein 6 (LRP6) are regulated by the E3 ubiquitin ligases zinc and ring finger 3 (ZNRF3) and ring finger protein 43 (RNF43), which are disrupted in cancer. In a genome-wide small interfering RNA screen, we identified the deubiquitylase ubiquitin-specific protease 6 (USP6) as a potent activator of Wnt signaling. USP6 enhances Wnt signaling by deubiquitylating Fzds, thereby increasing their cell-surface abundance. Chromosomal translocations in nodular fasciitis result in USP6 overexpression, leading to transcriptional activation of the Wnt/β-catenin pathway. Inhibition of Wnt signaling using Dickkopf-1 (DKK1) or a Porcupine (PORCN) inhibitor significantly decreased the growth of USP6-driven xenograft tumors, indicating that Wnt signaling is a key target of USP6 during tumorigenesis. Our study defines an additional route to ectopic Wnt pathway activation in human disease, and identifies a potential approach to modulate Wnt signaling for therapeutic benefit. [ABSTRACT FROM AUTHOR]

Although KRAS has long been considered undruggable, direct KRASG12C inhibitors have shown promising initial clinical efficacy. However, the majority of patients still fail to respond. Adaptive feedback reactivation of RAS-mitogen-activated protein kinase (MAPK) signaling has been proposed by our group and others as a key mediator of resistance, but the exact mechanism driving reactivation and the therapeutic implications are unclear. We find that upstream feedback activation of wild-type RAS, as opposed to a shift in KRASG12C to its active guanosine triphosphate (GTP)-bound state, is sufficient to drive RAS-MAPK reactivation in a KRASG12C-independent manner. Moreover, multiple receptor tyrosine kinases (RTKs) can drive feedback reactivation, potentially necessitating targeting of convergent signaling nodes for more universal efficacy. Even in colorectal cancer, where feedback is thought to be primarily epidermal growth factor receptor (EGFR)-mediated, alternative RTKs drive pathway reactivation and limit efficacy, but convergent upstream or downstream signal blockade can enhance activity. Overall, these data provide important mechanistic insight to guide therapeutic strategies targeting KRAS. [Display omitted] • Adaptive RAS-MAPK feedback reactivation occurs following KRASG12C inhibition • RTK-mediated feedback activation of wild-type NRAS and HRAS bypasses KRASG12C • Inhibitor combinations abrogate RAS reactivation, boosting efficacy in KRASG12C CRC • Targeting convergent signaling nodes overcomes adaptive resistance by multiple RTKs Ryan et al. provide evidence that feedback reactivation of the RAS-MAPK pathway through wild-type NRAS and HRAS, as opposed to a shift in KRASG12C to its active GTP-bound state, can drive adaptive resistance to KRASG12C inhibitors in a KRASG12C-independent manner. [ABSTRACT FROM AUTHOR]

Previous studies have identified interleukin 6 (IL-6) as an important cytokine with prognostic significance in ovarian cancer. Activation of the IL-6-Stat3 pathway contributes to tumor cell growth, survival and drug resistance in several cancers, including ovarian cancer. To explore potential therapeutic strategies for interrupting signaling through this pathway, we assessed the ability of CDDO-Me, a synthetic triterpenoid, to inhibit IL-6 secretion, Stat3 phosphorylation, Stat3 nuclear translocation and paclitaxel sensitivity in several cell line model systems. These studies demonstrated that CDDO-Me significantly inhibits IL-6 secretion in paclitaxel-resistant ovarian cancer cells and specifically suppresses IL-6- or oncostatin M-induced Stat3 nuclear translocation. Treatment with CDDO-Me significantly decreases the levels of Stat3, Jak2, and Src phosphorylation in ovarian and breast cancer cell lines with constitutively activated Stat3. This inhibition of the IL-6-Stat3 pathway correlated with suppression of the anti-apoptotic Stat3 target genes Bcl-XL, survivin, and Mcl-1, and with apoptosis induction as measured by monitoring PARP and its cleavage product, as well as by quantitative measurement of the apoptosis-associated CK18Asp396. Furthermore, CDDO-Me increases the cytotoxic effects of paclitaxel in the paclitaxel-resistant ovarian cancer cell line OVCAR8TR (2 to 5-fold) and of cisplatin in the cisplatin-resistant ovarian cancer cell line A2780cp70 (2 to 4-fold). Our data confirm that CDDO-Me interrupts the signaling of multiple kinases involved in the IL-6-Stat3 and Src signaling pathways. Inhibition is likely achieved through multiple points within these pathways. In a model system of established acquired drug resistance, CCDO-Me is effective at partially reversing the drug-resistance phenotype. [ABSTRACT FROM AUTHOR]

We apply genetic screens to delineate modulators of KRAS mutant pancreatic ductal adenocarcinoma (PDAC) sensitivity to ERK inhibitor treatment, and we identify components of the ATR-CHK1 DNA damage repair (DDR) pathway. Pharmacologic inhibition of CHK1 alone causes apoptotic growth suppression of both PDAC cell lines and organoids, which correlates with loss of MYC expression. CHK1 inhibition also activates ERK and AMPK and increases autophagy, providing a mechanistic basis for increased efficacy of concurrent CHK1 and ERK inhibition and/or autophagy inhibition with chloroquine. To assess how CHK1 inhibition-induced ERK activation promotes PDAC survival, we perform a CRISPR-Cas9 loss-of-function screen targeting direct/indirect ERK substrates and identify RIF1. A key component of non-homologous end joining repair, RIF1 suppression sensitizes PDAC cells to CHK1 inhibition-mediated apoptotic growth suppression. Furthermore, ERK inhibition alone decreases RIF1 expression and phenocopies RIF1 depletion. We conclude that concurrent DDR suppression enhances the efficacy of ERK and/or autophagy inhibitors in KRAS mutant PDAC. [Display omitted] • Pancreatic cancer cells are dependent on CHEK1 and DNA damage response genes • CHK1 inhibition causes compensatory ERK and autophagy activation • Concurrent CHK1 and ERK inhibition causes synergistic loss of MYC • Concurrent CHK1, ERK, and autophagy inhibition synergistically suppresses growth Klomp et al. show that CHK1 is essential for KRAS mutant pancreatic cancer cell growth. CHK1 inhibition causes apoptotic growth suppression, MYC loss, and compensatory ERK and autophagy activation. Concurrent CHK1, ERK, and/or autophagy inhibition enhances apoptotic growth suppression. Additionally, genetic depletion of ERK-regulated DNA damage repair protein RIF1 phenocopies ERK inhibition. [ABSTRACT FROM AUTHOR]

Summary β-Catenin-dependent WNT signal transduction governs development, tissue homeostasis, and a vast array of human diseases. Signal propagation through a WNT-Frizzled/LRP receptor complex requires proteins necessary for clathrin-mediated endocytosis (CME). Paradoxically, CME also negatively regulates WNT signaling through internalization and degradation of the receptor complex. Here, using a gain-of-function screen of the human kinome, we report that the AP2 associated kinase 1 (AAK1), a known CME enhancer, inhibits WNT signaling. Reciprocally, AAK1 genetic silencing or its pharmacological inhibition using a potent and selective inhibitor activates WNT signaling. Mechanistically, we show that AAK1 promotes clearance of LRP6 from the plasma membrane to suppress the WNT pathway. Time-course experiments support a transcription-uncoupled, WNT-driven negative feedback loop; prolonged WNT treatment drives AAK1-dependent phosphorylation of AP2M1, clathrin-coated pit maturation, and endocytosis of LRP6. We propose that, following WNT receptor activation, increased AAK1 function and CME limits WNT signaling longevity. Graphical Abstract Highlights • Gain-of-function kinome screen identifies AAK1 as a repressor of WNT signaling • AAK1 promotes clathrin-mediated endocytosis of LRP6 • Selective AAK1 inhibitor stabilizes β-catenin and activates WNT signaling • WNT induces AAK1-dependent phosphorylation of AP2M1 and LRP6 endocytosis WNT signal transduction is essential for normal development and contributes to many human diseases. Agajanian et al. used a kinase gain-of-function screen to show that WNT activates the AAK1 kinase to promote clathrin-mediated endocytosis of the WNT receptor. This work identifies an AAK-driven negative feedback loop that downregulates WNT signaling. [ABSTRACT FROM AUTHOR]