A Genome-Wide Screen Identifies PDPK1 as a Target to Enhance the Efficacy of MEK1/2 Inhibitors in NRAS Mutant Melanoma.

Melanomas frequently harbor activating NRAS mutations. However, limited advance has been made in developing targeted therapy options for patients with NRAS mutant melanoma. MEK inhibitors (MEKi) show modest efficacy in the clinic and their actions need to be optimized. In this study, we performed a genome-wide CRISPR-Cas9-based screen and demonstrated that loss of phosphoinositide-dependent kinase-1 (PDPK1) enhances the efficacy of MEKi. The synergistic effects of PDPK1 loss and MEKi was validated in NRAS mutant melanoma cell lines using pharmacologic and molecular approaches. Combined PDPK1 inhibitors (PDPK1i) with MEKi suppressed NRAS mutant xenograft growth and induced gasdermin E-associated pyroptosis. In an immune-competent allograft model, PDPK1i+MEKi increased the ratio of intratumoral CD8+ T cells, delayed tumor growth, and prolonged survival; the combination treatment was less effective against tumors in immune-deficient mice. These data suggest PDPK1i+MEKi as an efficient immunostimulatory strategy against NRAS mutant melanoma. SIGNIFICANCE: Targeting PDPK1 stimulates antitumor immunity and sensitizes NRAS mutant melanoma to MEK inhibition, providing rationale for the clinical development of a combinatorial approach for treating patients with melanoma.

MEK1/2 inhibitors induce class I alcohol dehydrogenase (ADH1) expression by regulating farnesoid X receptor in hepatic cell lines and C57BL/6J mouse.

BACKGROUND: Alcohol is mainly catabolized by class I alcohol dehydrogenase (ADH1) in liver. ADH deficiency can aggravate ethanol-induced tissue injury. Extracellular signal-regulated kinases 1/2 (ERK1/2) is involved in alcohol metabolism. However, the relationship between ERK1/2 and ADH1 remains unclear. METHODS AND RESULTS: To inhibit ERK1/2, HepG2 and BNL cells were treated with mitogen-activated protein kinases 1/2 (MEK1/2) inhibitors (U0126 and PD98059), and C57BL/6J mice were fed U0126. After treatment, the protein and mRNA expression of ADH1 were determined by Western blot and quantitative real time-PCR. The activity of ADH1 promoter was detected using luciferase assay. The results showed MEK1/2 inhibitors significantly increased ADH1 protein expression by inducing its transcription activity. Then we demonstrated a farnesoid X receptor (FXR) response element (FXRE) in ADH1 promoter by ChIP assay. To test whether FXR mediates the induction of MEK1/2 inhibitors on ADH1, HepG2 cells were transfected with FXR siRNA or ADH1 promoters with FXRE mutation. We found both FXR siRNA and FXRE mutation in ADH1 promoter abolished MEK1/2 inhibitors-induced ADH1 expression, indicating the activation of MEK1/2 inhibitors on ADH1 depends on FXR. CONCLUSIONS: Our findings revealed inhibition of ERK1/2 can significantly increase ADH1 expression, indicating MEK1/2 inhibitors may possess potential application in alcohol-related diseases.

Pheophorbide a identified in an Eupatorium perfoliatum extract is a novel lymphatic vascular activator.

The lymphatic vascular system is crucial for maintaining tissue fluid homeostasis and immune surveillance. Promoting lymphatic function represents a new strategy to treat several diseases including lymphedema, chronic inflammation and impaired wound healing. By screening a plant extract library, a petroleum ether extract from the aerial parts of Eupatorium perfoliatum (E. perfoliatum) was found to possess lymphangiogenic properties. With the aid of HPLC activity profiling the active compound was identified as pheophorbide a. Both plant extract and pheophorbide a induced the sprouting and tube formation of human primary lymphatic endothelial cells (LECs). The proliferation of the LECs was increased upon treatment with pheophorbide a but not the E. perfoliatum extract. Treatment with the MEK1/2 inhibitor U0126 reduced the LEC sprouting activity, indicating a potential mechanism of action. These studies suggest that pheophorbide a could represent novel natural therapeutic agent to treat human lymphatic vascular insufficiencies.

The MEK1/2-inhibitor ATR-002 efficiently blocks SARS-CoV-2 propagation and alleviates pro-inflammatory cytokine/chemokine responses.

Coronavirus disease 2019 (COVID-19), the illness caused by a novel coronavirus now called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to more than 260 million confirmed infections and 5 million deaths to date. While vaccination is a powerful tool to control pandemic spread, medication to relieve COVID-19-associated symptoms and alleviate disease progression especially in high-risk patients is still lacking. In this study, we explore the suitability of the rapid accelerated fibrosarcoma/mitogen-activated protein kinase/extracellular signal-regulated kinase (Raf/MEK/ERK) pathway as a druggable target in the treatment of SARS-CoV-2 infections. We find that SARS-CoV-2 transiently activates Raf/MEK/ERK signaling in the very early infection phase and that ERK1/2 knockdown limits virus replication in cell culture models. We demonstrate that ATR-002, a specific inhibitor of the upstream MEK1/2 kinases which is currently evaluated in clinical trials as an anti-influenza drug, displays strong anti-SARS-CoV-2 activity in cell lines as well as in primary air-liquid-interphase epithelial cell (ALI) cultures, with a safe and selective treatment window. We also observe that ATR-002 treatment impairs the SARS-CoV-2-induced expression of pro-inflammatory cytokines, and thus might prevent COVID-19-associated hyperinflammation, a key player in COVID-19 progression. Thus, our data suggest that the Raf/MEK/ERK signaling cascade may represent a target for therapeutic intervention strategies against SARS-CoV-2 infections and that ATR-002 is a promising candidate for further drug evaluation.

FGFR2 loss sensitizes MYCN-amplified neuroblastoma CHP134 cells to CHK1 inhibitor-induced apoptosis.

Checkpoint kinase 1 (CHK1) plays a key role in genome surveillance and integrity throughout the cell cycle. Selective inhibitors of CHK1 (CHK1i) are undergoing clinical evaluation for various human malignancies, including neuroblastoma. In this study, one CHK1i-sensitive neuroblastoma cell line, CHP134, was investigated, which characteristically carries MYCN amplification and a chromosome deletion within the 10q region. Among several cancer-related genes in the chromosome 10q region, mRNA expression of fibroblast growth factor receptor 2 (FGFR2) was altered in CHP134 cells and associated with an unfavorable prognosis of patients with neuroblastoma. Induced expression of FGFR2 in CHP134 cells reactivated downstream MEK/ERK signaling and resulted in cells resistant to CHK1i-mediated cell growth inhibition. Consistently, the MEK1/2 inhibitor, trametinib, potentiated CHK1 inhibitor-mediated cell death in these cells. These results suggested that FGFR2 loss might be prone to highly effective CHK1i treatment. In conclusion, extreme cellular dependency of ERK activation may imply a possible application for the MEK1/2 inhibitor, either as a single inhibitor or in combination with CHK1i in MYCN-amplified neuroblastomas.

Activating Immune Recognition in Pancreatic Ductal Adenocarcinoma via Autophagy Inhibition, MEK Blockade, and CD40 Agonism.

BACKGROUND AND AIMS: Patients with pancreatic ductal adenocarcinoma (PDA) have not yet benefitted from the revolution in cancer immunotherapy due in large part to a dominantly immunosuppressive tumor microenvironment. MEK inhibition combined with autophagy inhibition leads to transient tumor responses in some patients with PDA. We examined the functional effects of combined MEK and autophagy inhibition on the PDA immune microenvironment and the synergy of combined inhibition of MEK and autophagy with CD40 agonism (aCD40) against PDA using immunocompetent model systems. METHODS: We implanted immunologically "cold" murine PDA cells orthotopically in wide type C57BL/6J mice. We administered combinations of inhibitors of MEK1/2, inhibitors of autophagy, and aCD40 and measured anticancer efficacy and immune sequelae using mass cytometry and multiplexed immunofluorescence imaging analysis to characterize the tumor microenvironment. We also used human and mouse PDA cell lines and human macrophages in vitro to perform functional assays to elucidate the cellular effects induced by the treatments. RESULTS: We find that coinhibition of MEK (using cobimetinib) and autophagy (using mefloquine), but not either treatment alone, activates the STING/type I interferon pathway in tumor cells that in turn activates paracrine tumor associated macrophages toward an immunogenic M1-like phenotype. This switch is further augmented by aCD40. Triple therapy (cobimetinib + mefloquine + aCD40) achieved cytotoxic T-cell activation in an immunologically "cold" mouse PDA model, leading to enhanced antitumor immunity. CONCLUSIONS: MEK and autophagy coinhibition coupled with aCD40 invokes immune repolarization and is an attractive therapeutic approach for PDA immunotherapy development.

Dual Inhibition of DKC1 and MEK1/2 Synergistically Restrains the Growth of Colorectal Cancer Cells.

Colorectal cancer, one of the most commonly diagnosed cancers worldwide, is often accompanied by uncontrolled proliferation of tumor cells. Dyskerin pseudouridine synthase 1 (DKC1), screened using the genome-wide RNAi strategy, is a previously unidentified key regulator that promotes colorectal cancer cell proliferation. Enforced expression of DKC1, but not its catalytically inactive mutant D125A, accelerates cell growth in vitro and in vivo. DKC1 knockdown or its inhibitor pyrazofurin attenuates cell proliferation. Proteomics, RNA immunoprecipitation (RIP)-seq, and RNA decay analyses reveal that DKC1 binds to and stabilizes the mRNA of several ribosomal proteins (RPs), including RPL10A, RPL22L1, RPL34, and RPS3. DKC1 depletion significantly accelerates mRNA decay of these RPs, which mediates the oncogenic function of DKC1. Interestingly, these DKC1-regulated RPs also interact with HRAS and suppress the RAS/RAF/MEK/ERK pathway. Pyrazofurin and trametinib combination synergistically restrains colorectal cancer cell growth in vitro and in vivo. Furthermore, DKC1 is markedly upregulated in colorectal cancer tissues compared to adjacent normal tissues. Colorectal cancer patients with higher DKC1 expression has consistently poorer overall survival and progression-free survival outcomes. Taken together, these data suggest that DKC1 is an essential gene and candidate therapeutic target for colorectal cancer.

Design, synthesis and biological evaluation of imidazopyridazine derivatives containing isoquinoline group as potent MNK1/2 inhibitors.

Mitogen-activated protein kinase (MAPK)-interacting kinases (MNKs) are located at the meeting-point of ERK and p38 MAPK signaling pathways, which can phosphorylate eukaryotic translation initiation factor 4E (eIF4E) at the conserved serine 209 exclusively. MNKs modulate the translation of mRNA involved in tumor-associated signaling pathways. Consequently, selective inhibitors of MNK1/2 could reduce the level of phosphorylated eIF4E. Series of imidazopyrazines, imidazopyridazines and imidazopyridines derivatives were synthesized and evaluated as MNK1/2 inhibitors. Several compounds exhibited great inhibitory activity against MNK1/2 and selected compounds showed moderate to excellent anti-proliferative potency against diffuse large B-cell lymphoma (DLBCL) cell lines. In particular, compound II-5 (MNK1 IC50 = 2.3 nM; MNK2 IC50 = 3.4 nM) exhibited excellent enzymatic inhibitory potency and proved to be the most potent compound against TMD-8 and DOHH-2 cell lines with IC50 value of 0.3896 µM and 0.4092 µM respectively. These results demonstrated that compound II-5 could be considered as a potential MNK1/2 inhibitor for further investigation.

Dual inhibition of MEK1/2 and MEK5 suppresses the EMT/migration axis in triple-negative breast cancer through FRA-1 regulation.

Triple-negative breast cancer (TNBC) presents a clinical challenge due to the aggressive nature of the disease and a lack of targeted therapies. Constitutive activation of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway has been linked to chemoresistance and metastatic progression through distinct mechanisms, including activation of epithelial-to-mesenchymal transition (EMT) when cells adopt a motile and invasive phenotype through loss of epithelial markers (CDH1), and acquisition of mesenchymal markers (VIM, CDH2). Although MAPK/ERK1/2 kinase inhibitors (MEKi) are useful antitumor agents in a clinical setting, including the Food and Drug Administration (FDA)-approved MEK1,2 dual inhibitors cobimetinib and trametinib, there are limitations to their clinical utility, primarily adaptation of the BRAF pathway and ocular toxicities. The MEK5 (HGNC: MAP2K5) pathway has important roles in metastatic progression of various cancer types, including those of the prostate, colon, bone and breast, and elevated levels of ERK5 expression in breast carcinomas are linked to a worse prognoses in TNBC patients. The purpose of this study is to explore MEK5 regulation of the EMT axis and to evaluate a novel pan-MEK inhibitor on clinically aggressive TNBC cells. Our results show a distinction between the MEK1/2 and MEK5 cascades in maintenance of the mesenchymal phenotype, suggesting that the MEK5 pathway may be necessary and sufficient in EMT regulation while MEK1/2 signaling further sustains the mesenchymal state of TNBC cells. Furthermore, additive effects on MET induction are evident through the inhibition of both MEK1/2 and MEK5. Taken together, these data demonstrate the need for a better understanding of the individual roles of MEK1/2 and MEK5 signaling in breast cancer and provide a rationale for the combined targeting of these pathways to circumvent compensatory signaling and subsequent therapeutic resistance.

MEK1/2 activity modulates TREM2 cell surface recruitment.

Rare sequence variants in the microglial cell surface receptor TREM2 have been shown to increase the risk for Alzheimer's disease (AD). Disease-linked TREM2 mutations seem to confer a partial loss of function, and increasing TREM2 cell surface expression and thereby its function(s) might have therapeutic benefit in AD. However, druggable targets that could modulate microglial TREM2 surface expression are not known. To identify such targets, we conducted a screen of small molecule compounds with known pharmacology using human myeloid cells, searching for those that enhance TREM2 protein at the cell surface. Inhibitors of the kinases MEK1/2 displayed the strongest and most consistent increases in cell surface TREM2 protein, identifying a previously unreported pathway for TREM2 regulation. Unexpectedly, inhibitors of the downstream effector ERK kinases did not have the same effect, suggesting that noncanonical MEK signaling regulates TREM2 trafficking. In addition, siRNA knockdown experiments confirmed that decreased MEK1 and MEK2 were required for this recruitment. In iPSC-derived microglia, MEK inhibition increased cell surface TREM2 only modestly, so various cytokines were used to alter iPSC microglia phenotype, making cells more sensitive to MEK inhibitor-induced TREM2 recruitment. Of those tested, only IFN-gamma priming prior to MEK inhibitor treatment resulted in greater TREM2 recruitment. These data identify the first known mechanisms for increasing surface TREM2 protein and TREM2-regulated function in human myeloid cells and are the first to show a role for MEK1/MEK2 signaling in TREM2 activity.

Dissection of two routes to naïve pluripotency using different kinase inhibitors.

Embryonic stem cells (ESCs) can be maintained in the naïve state through inhibition of Mek1/2 and Gsk3 (2i). A relevant effect of 2i is the inhibition of Cdk8/19, which are negative regulators of the Mediator complex, responsible for the activity of enhancers. Inhibition of Cdk8/19 (Cdk8/19i) stimulates enhancers and, similar to 2i, stabilizes ESCs in the naïve state. Here, we use mass spectrometry to describe the molecular events (phosphoproteome, proteome, and metabolome) triggered by 2i and Cdk8/19i on ESCs. Our data reveal widespread commonalities between these two treatments, suggesting overlapping processes. We find that post-transcriptional de-repression by both 2i and Cdk8/19i might support the mitochondrial capacity of naive cells. However, proteome reprogramming in each treatment is achieved by different mechanisms. Cdk8/19i acts directly on the transcriptional machinery, activating key identity genes to promote the naïve program. In contrast, 2i stabilizes the naïve circuitry through, in part, de-phosphorylation of downstream transcriptional effectors.

Mechanical stiffness augments ligand-dependent progesterone receptor B activation via MEK 1/2 and Rho/ROCK-dependent signaling pathways in uterine fibroid cells.

OBJECTIVE: To test whether mechanical substrate stiffness would influence progesterone receptor B (PRB) signaling in fibroid cells. Uterine fibroids feature an excessive extracellular matrix, increased stiffness, and altered mechanical signaling. Fibroid growth is stimulated by progestins and opposed by anti-progestins, but a functional interaction between progesterone action and mechanical signaling has not been evaluated. DESIGN: Laboratory studies. SETTING: Translational science laboratory. PATIENT(S)/ANIMAL(S): Human fibroid cell lines and patient-matched fibroid and myometrial cell lines. INTERVENTION(S): Progesterone receptor B-dependent reporter assays and messenger RNA quantitation in cells cultured on stiff polystyrene plates (3GPa) or soft silicone plates (930KPa). Pharmacologic inhibitors of extracellular signal-related protein kinase (ERK) kinase 1/2 (MEK 1/2; PD98059), p38 mitogen-activated protein kinase (SB202190), receptor tyrosine kinases (RTKs; nintedanib), RhoA (A13), and Rho-associated coiled-coil kinase (ROCK; Y27632). MAIN OUTCOME MEASURE(S): Progesterone-responsive reporter activation. RESULT(S): Fibroid cells exhibited higher PRB-dependent reporter activity with progesterone (P4) in cells cultured on stiff vs. soft plates. Mechanically induced PRB activation with P4 was decreased 62% by PD98059, 78% by nintedanib, 38% by A13, and 50% by Y27632. Overexpression of the Rho-guanine nucleotide exchange factor (Rho-GEF), AKAP13, significantly increased PRB-dependent reporter activity. Collagen 1 messenger RNA levels were higher in fibroid cells grown on stiff vs. soft plates with P4. CONCLUSION(S): Cells cultured on mechanically stiff substrates had enhanced PRB activation via a mechanism that required MEK 1/2 and AKAP13/RhoA/ROCK signaling pathways. These studies provide a framework to explore the mechanisms by which mechanical stiffness affects progesterone receptor activation.

Research progress of MEK1/2 inhibitors and degraders in the treatment of cancer.

Mitogen-activated protein kinase kinases 1 and 2 (MEK1/2) are the crucial part of the RAS-RAF-MEK-ERK pathway (or ERK pathway), which is involved in the regulation of various cellular processes including proliferation, survival, and differentiation et al. Targeting MEK has become an important strategy for cancer therapy, and 4 MEK inhibitors (MEKis) have been approved by FDA to date. However, the application of MEKis is limited due to acquired resistance under long-term treatment. Fortunately, an emerging technology, named proteolysis targeting chimera (PROTAC), could break through this limitation by inducing MEK1/2 degradation. Compared to MEKis, MEK1/2 PROTAC is rarely studied and only three MEK1/2 PROTAC molecules, have been reported until now. This paper will outline the ERK pathway and the mechanism and research progress of MEK1/2 inhibitors, but focus on the development of MEK degraders and their optimization strategies. PAC-1 strategy which can induce MEK degradation indirectly, other PROTACs on ERK pathway, the advantages and challenges of PROTAC technology will be subsequently discussed.

Phase II study of selumetinib, an orally active inhibitor of MEK1 and MEK2 kinases, in KRASG12R-mutant pancreatic ductal adenocarcinoma.

Background Preclinical evidence has suggested that a subset of pancreatic cancers with the G12R mutational isoform of the KRAS oncogene is more sensitive to MAPK pathway blockade than pancreatic tumors with other KRAS isoforms. We conducted a biomarker-driven trial of selumetinib (KOSELUGO™; ARRY-142886), an orally active, allosteric mitogen-activated protein kinase 1 and 2 (MEK1/2) inhibitor, in pancreas cancer patients with somatic KRASG12R mutations. Methods In this two-stage, phase II study (NCT03040986) patients with advanced pancreas cancer harboring somatic KRASG12R variants who had received at least one standard-of-care systemic therapy regimen received 75 mg selumetinib orally twice a day until disease progression or unacceptable toxicity occurred. The primary outcome of the study was best objective response (BOR). Results From August 2017 to February 2018 a total of 8 patients with confirmed somatic KRASG12R mutations and a median age of 61.5 years were treated with selumetinib. Seven out of eight (87.5%) had received two or more lines of prior systemic chemotherapy. After a median follow-up period of 8.5 months (range 2 to 20), three patients had stable disease for more than 6 months while receiving selumetinib. No patients achieved an objective partial response. Median progression-free survival (PFS) was 3.0 months (95% CI, 0.8-8.2) and median overall survival (OS) 9 months (95% CI, 2.5-20.9). Conclusion This study in heavily pre-treated pancreatic adenocarcinoma patients suggests alternative strategies beyond single agent MEK inhibition are required for this unique, molecular subset of pancreatic cancer patients. The trial was registered on February 2nd, 2017 under identifier NCT03040986 with ClinicalTrials.gov .

Potent and Selective Mitogen-Activated Protein Kinase Kinase 1/2 (MEK1/2) Heterobifunctional Small-molecule Degraders.

Previously, we reported a first-in-class von Hippel-Lindau (VHL)-recruiting mitogen-activated protein kinase kinases 1 and 2 (MEK1/2) degrader, MS432. To date, only two MEK1/2 degrader papers have been published and very limited structure-activity relationships (SAR) have been reported. Here, we describe our extensive SAR studies exploring both von Hippel-Lindau (VHL) and cereblon (CRBN) E3 ligase ligands and a variety of linkers, which resulted in two novel, improved VHL-recruiting MEK1/2 degraders, 24 (MS928) and 27 (MS934), and the first CRBN-recruiting MEK1/2 degrader 50 (MS910). These compounds potently and selectively degraded MEK1/2 by hijacking the ubiquitin-proteasome system, inhibited downstream signaling, and suppressed cancer cell proliferation. Furthermore, concurrent inhibition of BRAF or PI3K significantly potentiated the antitumor activity of degrader 27, suggesting that the combination of MEK1/2 degradation with BRAF or PI3K inhibition may provide potential therapeutic benefits. Finally, besides being more potent, degrader 27 displayed improved plasma exposure levels in mice, representing the best MEK1/2 degrader to date for in vivo studies.

ß2-AR blockade potentiates MEK1/2 inhibitor effect on HNSCC by regulating the Nrf2-mediated defense mechanism.

The ß2-Adrenergic receptor (ß2-AR) is a G protein-coupled receptor (GPCR), involved in the development of many cancers, among which HNSCC. In this contest, ß2-AR signaling interacts with different pathways, such as PI3K and MAPK, commonly activated by TK receptors. For this reason, TK blockade is one of the most adopted therapeutic strategies in HNSCC patients. In our study we investigated the effects of the ß2-AR blocking in HNSCC cell lines, using the selective inhibitor ICI118,551 (ICI), in combination with the MAPK inhibitor U0126. We found that ICI leads to the blocking of p38 and NF-kB oncogenic pathways, strongly affecting also the ERK and PI3K pathways. Cotreatment with U0126 displays a synergic effect on cell viability and pathway alteration. Interestingly, we found that the ß2-AR blockade affects Nrf2-Keap1 stability and its nuclear translocation leading to a drastic ROS increase and oxidative stress. Our results are confirmed by a TCGA dataset analysis, showing that NFE2L2 gene is commonly overexpressed in HNSC, and correlated with a lower survival rate. In our system, the PI3K pathway inhibition culminated in the blocking of pro-survival autophagy, a mechanism normally adopted by cancer cells to became less responsive to the therapies. The mTOR expression, commonly upregulated in HNSC, was reduced in patients with disease-recurrence. It is well known that mTOR has a strong autophagy inhibition effect, therefore its downregulation promoted pro-survival autophagy, with a related increase recurrence rate. Our findings highlight for the first time the key role of ß2-AR and related pathway in HNSCC cell proliferation and drug resistance, proposing it as a valuable therapeutic molecular target.

Chloroquine Sensitizes GNAQ/11-mutated Melanoma to MEK1/2 Inhibition.

PURPOSE: Mutational activation of GNAQ or GNA11 (GNAQ/11), detected in >90% of uveal melanomas, leads to constitutive activation of oncogenic pathways, including MAPK and YAP. To date, chemo- or pathway-targeted therapies, either alone or in combination, have proven ineffective in the treatment of patients with metastatic uveal melanoma. EXPERIMENTAL DESIGN: We tested the efficacy of chloroquine or hydroxychloroquine, in combination with MAPK pathway inhibition in GNAQ/11-mutated cells in vitro and in vivo and identified mechanisms of MEK1/2 inhibitor plus chloroquine-induced cytotoxicity. RESULTS: Inhibition of GNAQ/11-mediated activation of MAPK signaling resulted in the induction of autophagy. Combined inhibition of Gα and autophagy or lysosome function resulted in enhanced cell death. Moreover, the combination of MEK1/2 inhibition, using trametinib, with the lysosome inhibitor, chloroquine, also increased cytotoxicity. Treatment of mice bearing GNAQ/11-driven melanomas with trametinib plus hydroxychloroquine resulted in inhibition of tumor growth and significantly prolonged survival. Interestingly, lysosomal- and autophagy-specific inhibition with bafilomycin A1 was not sufficient to promote cytotoxicity in combination with trametinib. However, the addition of YAP inhibition with trametinib plus bafilomycin A1 resulted in cell death at comparable levels to trametinib plus chloroquine (T/CQ) treatment. Furthermore, T/CQ-treated cells displayed decreased YAP nuclear localization and decreased YAP transcriptional activity. Expression of a constitutively active YAP5SA mutant conferred resistance to T/CQ-induced cell death. CONCLUSIONS: These results suggest that YAP, MEK1/2, and lysosome function are necessary and critical targets for the therapy of GNAQ/11-driven melanoma, and identify trametinib plus hydroxychloroquine as a potential treatment strategy for metastatic uveal melanoma.

Dabrafenib and Trametinib in Patients With Tumors With BRAFV600E Mutations: Results of the NCI-MATCH Trial Subprotocol H.

PURPOSE: BRAFV600 mutations are commonly found in melanoma and thyroid cancers and to a lesser degree in other tumor types. Subprotocol H (EAY131-H) of the NCI-MATCH platform trial sought to investigate the selective BRAF inhibitor dabrafenib and the MEK1/2 inhibitor trametinib in patients with solid tumors, lymphomas, or multiple myeloma whose tumors harbored a BRAFV600 mutation. PATIENTS AND METHODS: EAY131-H is an open-label, single-arm study. Patients with melanoma, thyroid, or colorectal cancer were excluded; patients with non-small-cell lung cancer were later excluded in an amendment. Patients received dabrafenib 150 mg twice per day and trametinib 2 mg per day continuously until disease progression or intolerable toxicity. The primary end point was centrally assessed objective response rate (ORR); secondary end points included progression-free survival (PFS), 6-month PFS, and overall survival. RESULTS: Thirty-five patients were enrolled, and 29 were included in the primary efficacy analysis as prespecified in the protocol. Median age was 59 years, and 45% of the patients had received ≥ 3 lines of therapy. The confirmed ORR was 38% (90% CI, 22.9% to 54.9%) with P < .0001 against a null rate of 5%, and PFS was 11.4 months (90% CI, 8.4 to 16.3 months); responses were seen in 7 distinct tumor types. Seven patients had a duration of response of > 12 months, including 4 patients with a duration of response of > 24 months. An additional 8 patients had a PFS > 6 months. The median overall survival was 28.6 months. Reported adverse events were comparable to those noted in previously reported profiles of dabrafenib and trametinib. CONCLUSION: This study met its primary end point, with an ORR of 38% (P < .0001) in this mixed histology, pretreated cohort. This promising activity warrants additional investigations in BRAFV600-mutated tumors outside of currently approved indications.

MILO/ENGOT-ov11: Binimetinib Versus Physician's Choice Chemotherapy in Recurrent or Persistent Low-Grade Serous Carcinomas of the Ovary, Fallopian Tube, or Primary Peritoneum.

PURPOSE: Low-grade serous ovarian carcinomas (LGSOCs) have historically low chemotherapy responses. Alterations affecting the MAPK pathway, most commonly KRAS/BRAF, are present in 30%-60% of LGSOCs. The purpose of this study was to evaluate binimetinib, a potent MEK1/2 inhibitor with demonstrated activity across multiple cancers, in LGSOC. METHODS: This was a 2:1 randomized study of binimetinib (45 mg twice daily) versus physician's choice chemotherapy (PCC). Eligible patients had recurrent measurable LGSOC after ≥ 1 prior platinum-based chemotherapy but ≤ 3 prior chemotherapy lines. The primary end point was progression-free survival (PFS) by blinded independent central review (BICR); additional assessments included overall survival (OS), overall response rate (ORR), duration of response (DOR), clinical-benefit rate, biomarkers, and safety. RESULTS: A total of 303 patients were randomly assigned to an arm of the study at the time of interim analysis (January 20, 2016). Median PFS by BICR was 9.1 months (95% CI, 7.3 to 11.3) for binimetinib and 10.6 months (95% CI, 9.2 to 14.5) for PCC (hazard ratio,1.21; 95%CI, 0.79 to 1.86), resulting in early study closure according to a prespecified futility boundary after 341 patients had enrolled. Secondary efficacy end points were similar in the two groups: ORR 16% (complete response [CR]/partial responses[PRs], 32) versus 13% (CR/PRs, 13); median DOR, 8.1 months (range, 0.03 to ≥ 12.0 months) versus 6.7 months (0.03 to ≥ 9.7 months); and median OS, 25.3 versus 20.8 months for binimetinib and PCC, respectively. Safety results were consistent with the known safety profile of binimetinib; the most common grade ≥ 3 event was increased blood creatine kinase level (26%). Post hoc analysis suggests a possible association between KRAS mutation and response to binimetinib. Results from an updated analysis (n = 341; January 2019) were consistent. CONCLUSION: Although the MEK Inhibitor in Low-Grade Serous Ovarian Cancer Study did not meet its primary end point, binimetinib showed activity in LGSOC across the efficacy end points evaluated. A higher response to chemotherapy than expected was observed and KRAS mutation might predict response to binimetinib.