A Kinome-Wide Synthetic Lethal CRISPR/Cas9 Screen Reveals That mTOR Inhibition Prevents Adaptive Resistance to CDK4/CDK6 Blockade in HNSCC.

The comprehensive genomic analysis of the head and neck squamous cell carcinoma (HNSCC) oncogenome revealed the frequent loss of p16INK4A (CDKN2A) and amplification of cyclin D1 genes in most human papillomavirus-negative HNSCC lesions. However, cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors have shown modest effects in the clinic. The aberrant activation of the PI3K/mTOR pathway is highly prevalent in HNSCC, and recent clinical trials have shown promising clinical efficacy of mTOR inhibitors (mTORi) in the neoadjuvant and adjuvant settings but not in patients with advanced HNSCC. By implementing a kinome-wide CRISPR/Cas9 screen, we identified cell-cycle inhibition as a synthetic lethal target for mTORis. A combination of mTORi and palbociclib, a CDK4/6-specific inhibitor, showed strong synergism in HNSCC-derived cells in vitro and in vivo. Remarkably, we found that an adaptive increase in cyclin E1 (CCNE1) expression upon palbociclib treatment underlies the rapid acquired resistance to this CDK4/6 inhibitor. Mechanistically, mTORi inhibits the formation of eIF4G-CCNE1 mRNA complexes, with the consequent reduction in mRNA translation and CCNE1 protein expression. Our findings suggest that mTORi reverts the adaptive resistance to palbociclib. This provides a multimodal therapeutic option for HNSCC by cotargeting mTOR and CDK4/6, which in turn may halt the emergence of palbociclib resistance. SIGNIFICANCE: A kinome-wide CRISPR/Cas9 screen identified cell-cycle inhibition as a synthetic lethal target of mTORis. A combination of mTORi and palbociclib, a CDK4/6-specific inhibitor, showed strong synergistic effects in HNSCC. Mechanistically, mTORis inhibited palbociclib-induced increase in CCNE1.

An allosteric pan-TEAD inhibitor blocks oncogenic YAP/TAZ signaling and overcomes KRAS G12C inhibitor resistance.

The Hippo pathway is a key growth control pathway that is conserved across species. The downstream effectors of the Hippo pathway, YAP (Yes-associated protein) and TAZ (transcriptional coactivator with PDZ-binding motif), are frequently activated in cancers to drive proliferation and survival. Based on the premise that sustained interactions between YAP/TAZ and TEADs (transcriptional enhanced associate domain) are central to their transcriptional activities, we discovered a potent small-molecule inhibitor (SMI), GNE-7883, that allosterically blocks the interactions between YAP/TAZ and all human TEAD paralogs through binding to the TEAD lipid pocket. GNE-7883 effectively reduces chromatin accessibility specifically at TEAD motifs, suppresses cell proliferation in a variety of cell line models and achieves strong antitumor efficacy in vivo. Furthermore, we uncovered that GNE-7883 effectively overcomes both intrinsic and acquired resistance to KRAS (Kirsten rat sarcoma viral oncogene homolog) G12C inhibitors in diverse preclinical models through the inhibition of YAP/TAZ activation. Taken together, this work demonstrates the activities of TEAD SMIs in YAP/TAZ-dependent cancers and highlights their potential broad applications in precision oncology and therapy resistance.

Synthetic Lethal Screens Reveal Cotargeting FAK and MEK as a Multimodal Precision Therapy for GNAQ-Driven Uveal Melanoma.

PURPOSE: Uveal melanoma is the most common eye cancer in adults. Approximately 50% of patients with uveal melanoma develop metastatic uveal melanoma (mUM) in the liver, even after successful treatment of the primary lesions. mUM is refractory to current chemo- and immune-therapies, and most mUM patients die within a year. Uveal melanoma is characterized by gain-of-function mutations in GNAQ/GNA11, encoding Gαq proteins. We have recently shown that the Gαq-oncogenic signaling circuitry involves a noncanonical pathway distinct from the classical activation of PLCß and MEK-ERK. GNAQ promotes the activation of YAP1, a key oncogenic driver, through focal adhesion kinase (FAK), thereby identifying FAK as a druggable signaling hub downstream from GNAQ. However, targeted therapies often activate compensatory resistance mechanisms leading to cancer relapse and treatment failure. EXPERIMENTAL DESIGN: We performed a kinome-wide CRISPR-Cas9 sgRNA screen to identify synthetic lethal gene interactions that can be exploited therapeutically. Candidate adaptive resistance mechanisms were investigated by cotargeting strategies in uveal melanoma and mUM in vitro and in vivo experimental systems. RESULTS: sgRNAs targeting the PKC and MEK-ERK signaling pathways were significantly depleted after FAK inhibition, with ERK activation representing a predominant resistance mechanism. Pharmacologic inhibition of MEK and FAK showed remarkable synergistic growth-inhibitory effects in uveal melanoma cells and exerted cytotoxic effects, leading to tumor collapse in uveal melanoma xenograft and liver mUM models in vivo. CONCLUSIONS: Coupling the unique genetic landscape of uveal melanoma with the power of unbiased genetic screens, our studies reveal that FAK and MEK-ERK cotargeting may provide a new network-based precision therapeutic strategy for mUM treatment.See related commentary by Harbour, p. 2967.

A Platform of Synthetic Lethal Gene Interaction Networks Reveals that the GNAQ Uveal Melanoma Oncogene Controls the Hippo Pathway through FAK.

Activating mutations in GNAQ/GNA11, encoding Gαq G proteins, are initiating oncogenic events in uveal melanoma (UM). However, there are no effective therapies for UM. Using an integrated bioinformatics pipeline, we found that PTK2, encoding focal adhesion kinase (FAK), represents a candidate synthetic lethal gene with GNAQ activation. We show that Gαq activates FAK through TRIO-RhoA non-canonical Gαq-signaling, and genetic ablation or pharmacological inhibition of FAK inhibits UM growth. Analysis of the FAK-regulated transcriptome demonstrated that GNAQ stimulates YAP through FAK. Dissection of the underlying mechanism revealed that FAK regulates YAP by tyrosine phosphorylation of MOB1, inhibiting core Hippo signaling. Our findings establish FAK as a potential therapeutic target for UM and other Gαq-driven pathophysiologies that involve unrestrained YAP function.

Disease-associated extracellular loop mutations in the adhesion G protein-coupled receptor G1 (ADGRG1; GPR56) differentially regulate downstream signaling.

Mutations to the adhesion G protein-coupled receptor ADGRG1 (G1; also known as GPR56) underlie the neurological disorder bilateral frontoparietal polymicrogyria. Disease-associated mutations in G1 studied to date are believed to induce complete loss of receptor function through disruption of either receptor trafficking or signaling activity. Given that N-terminal truncation of G1 and other adhesion G protein-coupled receptors has been shown to significantly increase the receptors' constitutive signaling, we examined two different bilateral frontoparietal polymicrogyria-inducing extracellular loop mutations (R565W and L640R) in the context of both full-length and N-terminally truncated (ΔNT) G1. Interestingly, we found that these mutations reduced surface expression of full-length G1 but not G1-ΔNT in HEK-293 cells. Moreover, the mutations ablated receptor-mediated activation of serum response factor luciferase, a classic measure of Gα12/13-mediated signaling, but had no effect on G1-mediated signaling to nuclear factor of activated T cells (NFAT) luciferase. Given these differential signaling results, we sought to further elucidate the pathway by which G1 can activate NFAT luciferase. We found no evidence that ΔNT activation of NFAT is dependent on Gαq/11-mediated or ß-arrestin-mediated signaling but rather involves liberation of Gßγ subunits and activation of calcium channels. These findings reveal that disease-associated mutations to the extracellular loops of G1 differentially alter receptor trafficking, depending on the presence of the N terminus, and differentially alter signaling to distinct downstream pathways.

Versatile Signaling Activity of Adhesion GPCRs.

The adhesion G protein-coupled receptors (aGPCRs) are a family of 33 receptors in humans that are widely expressed in various tissues and involved in many diverse biological processes. These receptors possess extremely large N-termini (NT) containing a variety of adhesion domains. A distinguishing feature of these receptors is the presence within the NT of a highly conserved GPCR autoproteolysis-inducing (GAIN) domain, which mediates autoproteolysis of the receptors into N-terminal and C-terminal fragments that stay non-covalently associated. The downstream signaling pathways and G protein-coupling preferences of many aGPCRs have recently been elucidated, and putative endogenous ligands for some aGPCRs have also been discovered and characterized in recent years. A pivotal observation for aGPCRs has been that deletion or removal of the NT up the point of GAIN cleavage results in constitutive receptor activation. For at least some aGPCRs, this activation is dependent on the unmasking of specific agonistic peptide sequences within the N-terminal stalk region (i.e., the region between the site of GAIN domain cleavage and the first transmembrane domain). However, the specific peptide sequences involved and the overall importance of the stalk region for activation can vary greatly from receptor to receptor. An emerging theme of work in this area is that aGPCRs are capable of versatile signaling activity that may be fine-tuned to suit the specific physiological roles played by the various members of this family.

Stalk-dependent and Stalk-independent Signaling by the Adhesion G Protein-coupled Receptors GPR56 (ADGRG1) and BAI1 (ADGRB1).

The adhesion G protein-coupled receptors (aGPCRs) are a large yet poorly understood family of seven-transmembrane proteins. A defining characteristic of the aGPCR family is the conserved GAIN domain, which has autoproteolytic activity and can cleave the receptors near the first transmembrane domain. Several aGPCRs, including ADGRB1 (BAI1 or B1) and ADGRG1 (GPR56 or G1), have been found to exhibit significantly increased constitutive activity when truncated to mimic GAIN domain cleavage (ΔNT). Recent reports have suggested that the new N-terminal stalk, which is revealed by GAIN domain cleavage, can directly activate aGPCRs as a tethered agonist. We tested this hypothesis in studies on two distinct aGPCRs, B1 and G1, by engineering mutant receptors lacking the entire NT including the stalk (B1- and G1-SL, with "SL" indicating "stalkless"). These receptors were evaluated in a battery of signaling assays and compared with full-length wild-type and cleavage-mimicking (ΔNT) forms of the two receptors. We found that B1-SL, in multiple assays, exhibited robust signaling activity, suggesting that the membrane-proximal stalk region is not necessary for its activation. For G1, however, the results were mixed, with the SL mutant exhibiting robust activity in several signaling assays (including TGFα shedding, activation of NFAT luciferase, and ß-arrestin recruitment) but reduced activity relative to ΔNT in a distinct assay (activation of SRF luciferase). These data support a model in which the activation of certain pathways downstream of aGPCRs is stalk-dependent, whereas signaling to other pathways is stalk-independent.