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Supplementary Figure S4. MEKi/FAKi combination reduces B2905 syngeneic cells growth in allograft tumor models.

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 PLCβ 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

Supplementary figure S2. Isobologram analysis.

Supplementary figure S1. Synthetic lethality of FAK and ERK/MAPK pathway inhibition.

Supplementary figure 3. MEKi/FAKi combination reduces UM cells growth in xenograft tumor models with limited toxicity.

International audience; Mdm2 antagonizes the tumor suppressor p53. Targeting the Mdm2-p53 interaction represents an attractive approach for the treatment of cancers with functional p53. Investigating mechanisms underlying Mdm2-p53 regulation is therefore important. The scaffold protein β-arrestin2 (β-arr2) regulates tumor suppressor p53 by counteracting Mdm2. β-arr2 nucleocytoplasmic shuttling displaces Mdm2 from the nucleus to the cytoplasm resulting in enhanced p53 signaling. β-arr2 is constitutively exported from the nucleus, via a nuclear export signal, but mechanisms regulating its nuclear entry are not completely elucidated. β-arr2 can be SUMOylated, but no information is available on how SUMO may regulate β-arr2 nucleocytoplasmic shuttling. While we found β-arr2 SUMOylation to be dispensable for nuclear import, we identified a non-covalent interaction between SUMO and β-arr2, via a SUMO interaction motif (SIM), that is required for β-arr2 cytonuclear trafficking. This SIM promotes association of β-arr2 with the multimolecular RanBP2/RanGAP1-SUMO nucleocytoplasmic transport hub that resides on the cytoplasmic filaments of the nuclear pore complex. Depletion of RanBP2/RanGAP1-SUMO levels result in defective β-arr2 nuclear entry. Mutation of the SIM inhibits β-arr2 nuclear import, its ability to delocalize Mdm2 from the nucleus to the cytoplasm and enhanced p53 signaling in lung and breast tumor cell lines. Thus, a β-arr2 SIM nuclear entry checkpoint, coupled with active β-arr2 nuclear export, regulates its cytonuclear trafficking function to control the Mdm2-p53 signaling axis.

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 PLCβ 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

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 inWe 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 mUMsgRNAs 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 modelsCoupling 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.

Vulvar squamous cell carcinoma associated with lichen sclerosus (VLS-VSCC) are rare tumors but with higher recurrence and worse prognosis than other types of VSCC. Lack of experimental models has limited the search for better understanding of the biology and development of treatment modalities. In this study, we isolated and characterized primary cells from VSCC (n = 7) and normal vulvar tissue adjacent to tumor (n = 7). Detailed characterization of the novel spontaneously immortalized cell line, VCC1 revealed a characteristic epithelial morphology in vitro and a well-differentiated keratinizing SCC histology in vivo, closely resembling the tumor of origin. VCC1 expressed higher levels of epithelial-mesenchymal transition markers and higher clonogenic properties as compared to other established non VLS-VSCC cell lines. In vitro 3D organotypic assays and in vivo xenografts revealed a prominent role of cancer-associated fibroblasts in VCC1 invasion and tumor formation. In conclusion, VCC1 mirrored several major VLS-VSCC features and provided a robust experimental tool for further elucidation of VLS-related oncogenesis and drug testing. publishedVersion

The RAS/RAF/MEK/ERK pathway is the most mutated oncogenic pathway in cancer, and RAS pathway mutations often present with an overall worse prognosis. Although RAF and MEK have been validated as anticancer targets and several BRAF and MEK inhibitors (MEKi) are FDA approved, acquired resistance develops in most patients. Preclinically, inhibition of RAF or MEK has been found to activate focal adhesion kinase (FAK) signaling which may bypass RAS pathway blockade by driving tumor growth through activation of downstream pathways such as RhoA and YAP. VS-6766 is a unique dual RAF/MEK inhibitor which allows VS-6766 to block MEK signaling without the compensatory MEK activation that limits the efficacy of other MEK inhibitors. Defactinib is a selective FAK inhibitor (FAKi). Clinical studies are ongoing evaluating VS-6766 and defactinib for the treatment of various solid tumors. In 3D proliferation assays in vitro, defactinib was synergistic with VS-6766 or trametinib (MEKi) in reducing viability of several human tumor cell lines, including KRAS mutant (mt) ovarian cancer (TOV-21G) and KRAS-G12V mt non-small cell lung cancer (NSCLC; H441). We next investigated whether FAKi augments the efficacy of VS-6766 in solid tumor models. Combination of a FAKi with VS-6766 in a KRAS mt ovarian xenograft model (TOV21G) induced >30% tumor regression in 9/10 mice, whereas each agent alone induced mainly tumor stasis (>30% tumor regression with FAKi monotherapy or VS-6766 monotherapy in 1/10 and 3/10 mice, respectively) following 11 days of treatment. Similar results were observed in KRAS mt NSCLC (H2122) and GNAQ mt uveal melanoma (92.1) models in which the combination of FAKi with VS-6766 or trametinib induced tumor regression. In several patients with KRAS mt tumors, sequential biopsies showed that treatment with VS-6766 induced FAK activation (pY397) as a potential resistance mechanism, and this increased FAK activation was reversed in the presence of the defactinib/VS-6766 combination. Accordingly, the combination of VS-6766 with defactinib showed clinical activity in low grade serous ovarian cancer (LGSOC; ORR = 56% in KRAS-G12 mt and ORR = 41% in all 17 LGSOC patients; 8/17/20 data cut off). Importantly, the combination of defactinib with VS-6766 also induced responses in patients who had progressed on previous MEK inhibitor regimens. VS-6766 with defactinib also showed clinical activity in KRAS-G12V mt NSCLC. Furthermore, this combination regimen of VS-6766 with defactinib exhibited a manageable safety profile with no patients discontinuing for adverse events (NCT03875820). These preclinical and clinical data support the recent initiation of two registration-directed studies evaluating VS-6766 ± defactinib for the treatment of recurrent LGSOC with or without a KRAS mutation (NCT04625270) and recurrent NSCLC with KRAS-G12V or other KRAS mutation (NCT04620330). Citation Format: Silvia Coma, Justine S. Paradis, J Silvio Gutkind, Jonathan A. Pachter. Synergistic antitumor efficacy of the dual RAF/MEK inhibitor VS-6766 with FAK inhibition for treatment of RAS-dependent solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1425.

Summary: In human cells, the expression of ∼1,000 genes is modulated throughout the cell cycle. Although some of these genes are controlled by specific transcriptional programs, very little is known about their post-transcriptional regulation. Here, we analyze the expression signature associated with all 687 RNA-binding proteins (RBPs) and identify 39 that significantly correlate with cell cycle mRNAs. We find that NF45 and NF90 play essential roles in mitosis, and transcriptome analysis reveals that they are necessary for the expression of a subset of mitotic mRNAs. Using proteomics, we identify protein clusters associated with the NF45-NF90 complex, including components of Staufen-mediated mRNA decay (SMD). We show that depletion of SMD components increases the binding of mitotic mRNAs to the NF45-NF90 complex and rescues cells from mitotic defects. Together, our results indicate that the NF45-NF90 complex plays essential roles in mitosis by competing with the SMD machinery for a common set of mRNAs.

Uveal melanoma (UM) is characterized by gain-of-function mutations in GNAQ or GNA11, encoding Gα proteins from the Gq/11 family. UM is the most common eye malignancy in adults. Approximately 50% of UM patients develop liver metastasis (mUM) within 5-10 years after diagnosis, independently of the successful treatment of the primary lesions. mUM is refractory to cytotoxic, targeted, and immunotherapies, with most mUM patients dying within a year. Recent information suggests that GNAQ-oncogenic signaling involves a non-canonical pathway distinct from the activation of PLCβ and PKC-MEK-ERK, which may explain the failure of MEK inhibitors (MEKi) in increasing mUM patient survival. Instead, we found that GNAQ promotes the activation of YAP1, a key oncogenic driver, by a mechanism involving the activation of RhoA by the direct association of Gαq to TRIO, a Rho-GEF (Cancer Cell, 2014). In turn, YAP1 is essential for uveal melanoma cell growth, however no effective and safe YAP1 inhibitors are currently available. Using a novel bioinformatics pipeline, we recently found that PTK2, encoding Focal Adhesion Kinase (FAK), is a synthetic lethal gene with GNAQ activation, and uncovered that GNAQ controls YAP1 through FAK (Cancer Cell, 2019). This study identified FAK as a druggable signaling hub downstream from GNAQ in UM. However, activation of compensatory pathways often results in resistance to targeted agents. Here, we combined the use of CRISPR-Cas9 sgRNA screens with a recently described Cancer Signaling Toolkit approach to identify synthetic lethal interactions enhancing the response to FAKi and signaling networks mediating drug resistance, respectively. Remarkably, both approaches converged to reveal that co-targeting FAK and the MEK-ERK pathway would be a promising combination for treatment of UM. Indeed, MEK-ERK pathway inhibition by multiple approved MEKis (e.g., trametinib), combined with FAK inhibition (VS-4718 or defactinib), showed remarkable synergistic growth inhibitory effects in UM cells. Additionally, the novel RAF/MEK inhibitor RO5126766 also showed synergistic anti-proliferative effects with defactinib. Accordingly, FAKi combination with MEKi exerted cytotoxic effects (apoptotic death) leading to rapid tumor shrinkage in UM xenografts, whereas single drugs were primarily cytostatic. Furthermore, the FAKi/MEKi combination was successful at reducing tumor burden in recently developed liver metastasis UM models. By coupling the unique genetic landscape of UM with the power of unbiased computational pipelines and systems biology genetic screens, our studies revealed that FAK and MEK-ERK co-targeting may provide a new network-based precision therapeutic strategy for mUM treatment. Indeed, the combination of defactinib and RO5126766 is currently being evaluated in patients with various solid tumors (NCT03875820), and could be explored in mUM based on these preclinical findings. Citation Format: Justine S. Paradis, Monica Acosta, Nadia Arang, Robert Saddawi-Konefka, Ayush Kishore, Takahito Sugase, Xiaodong Feng, Kris C. Wood, Silvia Coma, Mizue Terai, Takami Sato, Jonathan A. Pachter, J. Silvio Gutkind. FAK and MEK co-targeting: A new multimodal precision therapy for GNAQ-driven uveal melanoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6406.

In addition to G protein-coupled receptor (GPCR) desensitization and endocytosis, β-arrestin recruitment to ligand-stimulated GPCRs promotes non-canonical signalling cascades. Distinguishing the respective contributions of β-arrestin recruitment to the receptor and β-arrestin-promoted endocytosis in propagating receptor signalling has been limited by the lack of selective analytical tools. Here, using a combination of virtual screening and cell-based assays, we have identified a small molecule that selectively inhibits the interaction between β-arrestin and the β2-adaptin subunit of the clathrin adaptor protein AP2 without interfering with the formation of receptor/β-arrestin complexes. This selective β-arrestin/β2-adaptin inhibitor (Barbadin) blocks agonist-promoted endocytosis of the prototypical β2-adrenergic (β2AR), V2-vasopressin (V2R) and angiotensin-II type-1 (AT1R) receptors, but does not affect β-arrestin-independent (transferrin) or AP2-independent (endothelin-A) receptor internalization. Interestingly, Barbadin fully blocks V2R-stimulated ERK1/2 activation and blunts cAMP accumulation promoted by both V2R and β2AR, supporting the concept of β-arrestin/AP2-dependent signalling for both G protein-dependent and -independent pathways., Beta-arrestins play central roles in the mechanisms regulating GPCR signalling and trafficking. Here the authors identify a selective inhibitor of the interaction between β-arrestin and the β2-adaptin subunit of the clathrin adaptor protein AP-2, which they use to dissect the role of the β-arrestin/β2-adaptin interaction in GPCR signalling.

International audience; MAPKs are activated in response to G protein-coupled receptor (GPCR) stimulation and play essential roles in regulating cellular processes downstream of these receptors. However, very little is known about the reciprocal effect of MAPK activation on GPCRs. To investigate possible crosstalk between the MAPK and GPCRs, we assessed the effect of ERK1/2 on the activity of several GPCR family members. We found that ERK1/2 activation leads to a reduction in the steady-state cell-surface expression of many GPCRs because of their intracellular sequestration. This subcellular redistribution resulted in a global dampening of cell responsiveness, as illustrated by reduced ligand-mediated G-protein activation and second-messenger generation as well as blunted GPCR kinases and β-arrestin recruitment. This ERK1/2-mediated regulatory process was observed for GPCRs that can interact with β-arrestins, such as type-2 vasopressin, type-1 angiotensin, and CXC type-4 chemokine receptors, but not for the prostaglandin F receptor that cannot interact with β-arrestin, implicating this scaffolding protein in the receptor's subcellular redistribution. Complementation experiments in mouse embryonic fibroblasts lacking β-arrestins combined with in vitro kinase assays revealed that β-arrestin-2 phosphorylation on Ser14 and Thr276 is essential for the ERK1/2-promoted GPCR sequestration. This previously unidentified regulatory mechanism was observed after constitutive activation as well as after receptor tyrosine kinase- or GPCR-mediated activation of ERK1/2, suggesting that it is a central node in the tonic regulation of cell responsiveness to GPCR stimulation, acting both as an effector and a negative regulator.

Communication across membranes controls critical cellular processes and is achieved by receptors translating extracellular signals into selective cytoplasmic responses. While receptor tertiary structures can now be readily characterized, receptor associations into quaternary structures are very challenging to study and their implications in signal transduction remain poorly understood. Here, we report a computational approach for predicting membrane receptor self-associations, and designing receptor oligomers with various quaternary structures and signaling properties. Using this approach, we designed chemokine receptor CXCR4 dimers with reprogrammed stabilities, conformations, and abilities to activate distinct intracellular signaling proteins. In agreement with our predictions, the designed CXCR4s dimerized through distinct conformations and displayed different quaternary structural changes upon activation. Consistent with the active state models, all engineered CXCR4 oligomers activated the G protein Gi, but only a few specific dimer structures also recruited β-arrestins. Overall, we demonstrate that quaternary structures represent an important unforeseen mechanism of receptor biased signaling and reveal the existence of a conformational switch at the dimer interface of several G protein-coupled receptors including CXCR4, mu-Opioid and type-2 Vasopressin receptors that selectively control the activation of G proteins vs β-arrestin-mediated pathways. The approach should prove useful for predicting and designing receptor associations to uncover and reprogram selective cellular signaling functions.