Pharmacological Inhibition Of MEK Research Articles

Abstract C022: Elucidation and exploitation of RAS-inhibitor dependent macropinocytic regulation in pancreatic ductal adenocarcinoma

Abstract Alteration of essential metabolic pathways is a major mechanism by which oncogenic KRAS promotes tumor development and growth in pancreatic ductal adenocarcinoma (PDAC). KRAS- driven PDAC is dependent on nutrient scavenging pathways, including macropinocytosis and autophagy to fuel the metabolic demand of rapid proliferation. Thus, these metabolic processes are attractive targets for the development of treatments for PDAC. Acute KRAS loss results in downregulation of macropinocytosis in PDAC. Additionally, our lab demonstrated that KRAS loss or inhibition of ERK MAPK signaling decreased glucose consumption and glycolysis but increased autophagy, thereby enhancing dependency on autophagy for survival and growth. Accordingly, dual ERK MAPK and autophagy inhibition (via chloroquine) synergistically enhanced anti-tumor efficacy in PDAC. However, early clinical data has demonstrated that resistance to this treatment arises over time through unknown mechanisms. We observed that both autophagy induction and macropinocytosis downregulation are transient following RAS, MEK, or ERK inhibition—with autophagic and macropinocytic activity returning to or surpassing basal levels after within two weeks of treatment. Furthermore, we found that prolonged pharmacological inhibition of RAS, MEK, or ERK pathway consistently resulted in significant upregulation of macropinocytosis. We propose that prolonged MAPK inhibition upregulates macropinocytosis over time, consequently abrogating dependency on autophagy and therefore reducing sensitivity to autophagy inhibition. Furthermore, we found that chloroquine, which is commonly thought of as a lysosomotropic drug, does not prevent degradation of proteins engulfed via macropinocytosis. Together our data suggests that upregulation of macropinocytosis may mediate resistance to the combination of ERK MAPK inhibition and chloroquine. With the recent advance in development RAS inhibitors, the resistance mechanisms by which PDAC can overcome RAS inhibitor treatment is a major focus of the field. Given the dependence of PDAC on macropinocytic uptake for tumorigenic growth, it is imperative to understand how long-term treatment with RAS inhibitors regulate macropinocytosis. We demonstrated that PDAC cells with acquired resistance to RAS inhibition exhibit a 2- to 10-fold increase in macropinocytic uptake. We found that upregulated macropinocytosis following long-term RAS or MAPK inhibition is accompanied by increased albumin uptake and sensitivity to albumin-bound paclitaxel (nab-paclitaxel), a therapeutic that is included in the current standard of care for PDAC patients. These results suggest that RAS inhibitor pretreatment or concurrent treatment with nab-paclitaxel may represent a strategy to improve this current PDAC treatment. We conclude that a more complete understanding of the regulation of essential metabolic pathways following both acute and sustained RAS inhibition will better inform future RAS inhibitor combination therapies. Citation Format: Ryan Robb, Sarah Ackermann, Khalilah Taylor, Runying Yang. Elucidation and exploitation of RAS-inhibitor dependent macropinocytic regulation in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C022.

Somatic RIT1 delins in arteriovenous malformations hyperactivate RAS-MAPK signaling amenable to MEK inhibition

Arteriovenous malformations (AVM) are benign vascular anomalies prone to pain, bleeding, and progressive growth. AVM are mainly caused by mosaic pathogenic variants of the RAS-MAPK pathway. However, a causative variant is not identified in all patients. Using ultra-deep sequencing, we identified novel somatic RIT1 delins variants in lesional tissue of three AVM patients. RIT1 encodes a RAS-like protein that can modulate RAS-MAPK signaling. We expressed RIT1 variants in HEK293T cells, which led to a strong increase in ERK1/2 phosphorylation. Endothelial-specific mosaic overexpression of RIT1 delins in zebrafish embryos induced AVM formation, highlighting their functional importance in vascular development. Both ERK1/2 hyperactivation in vitro and AVM formation in vivo could be suppressed by pharmacological MEK inhibition. Treatment with the MEK inhibitor trametinib led to a significant decrease in bleeding episodes and AVM size in one patient. Our findings implicate RIT1 in AVM formation and provide a rationale for clinical trials with targeted treatments.

Pharmacological Inhibition of PAK1 for the Treatment of NF2-Associated Vestibular Schwannoma

Neurofibromatosis Type 2 (NF2) is an autosomal-dominant genetic disorder characterized by loss of Merlin, a tumor suppressor in Schwann cells, resulting in pathognomonic bilateral vestibular schwannoma, among other complications. Depletion of Merlin results in pathologic elevation of p21-activated kinase 1 (PAK1), which promotes tumor cell proliferation, survival, and motility. Previous studies investigating the Nf2flox/flox; PAK1-/-; Postn-Cre+ genetically engineered mouse model (GEMM) revealed that PAK1 genetic knockout in Nf2-cKO mice mitigated tumor size and preserved hearing. This study builds upon this previous work to identify efficacious therapeutic agents that target PAK1. In vitro dose response curves evaluated the efficacy of five PAK inhibitors in merlin-deficient immortalized Schwann cells (MS03). Dose response curves revealed variable IC50s among the five PAK inhibitors tested. Synergy screensrevealed synergy between PAK inhibitors NVS-PAK1-1 or BGJ-398 and selumetinib, a MEK inhibitor FDA-approved for the treatment of NF1. Colony formation assays revealed robust inhibition with NVS-PAK1-1 in combination with VS6766, a RAF/MEK inhibitor currently being evaluated in clinical trial in other cancers. Western blot analysis revealed marked decrease in downstream effectors of PAK1, including phospho-ERK1/2 in cells treated with BGJ-398, NVSPAK1-1, and PAKi plus selumetinib. Together, these findings suggest that inhibition of PAK1 and MEK pathways may be an efficacious therapeutic strategy for the treatment of NF2- associated vestibular schwannoma. Establishing reproducibility of these results in cells derived from human vestibular schwannoma is necessary to continue investigating pharmacological PAK1 and MEK inhibition. Further studies are needed to verify tolerability and therapeuticefficacy of PAK1 and MEK pharmacological inhibition in vivo.

FAK Inhibitor-Based Combinations with MEK or PKC Inhibitors Trigger Synergistic Antitumor Effects in Uveal Melanoma.

Uveal Melanoma (UM) is a rare and malignant intraocular tumor with dismal prognosis. Even if radiation or surgery permit an efficient control of the primary tumor, up to 50% of patients subsequently develop metastases, mainly in the liver. The treatment of UM metastases is challenging and the patient survival is very poor. The most recurrent event in UM is the activation of Gαq signaling induced by mutations in GNAQ/11. These mutations activate downstream effectors including protein kinase C (PKC) and mitogen-activated protein kinases (MAPK). Clinical trials with inhibitors of these targets have not demonstrated a survival benefit for patients with UM metastasis. Recently, it has been shown that GNAQ promotes YAP activation through the focal adhesion kinase (FAK). Pharmacological inhibition of MEK and FAK showed remarkable synergistic growth-inhibitory effects in UM both in vitro and in vivo. In this study, we have evaluated the synergy of the FAK inhibitor with a series of inhibitors targeting recognized UM deregulated pathways in a panel of cell lines. The combined inhibition of FAK and MEK or PKC had highly synergistic effects by reducing cell viability and inducing apoptosis. Furthermore, we demonstrated that these combinations exert a remarkable in vivo activity in UM patient-derived xenografts. Our study confirms the previously described synergy of the dual inhibition of FAK and MEK and identifies a novel combination of drugs (FAK and PKC inhibitors) as a promising strategy for therapeutic intervention in metastatic UM.

Pharmacological inhibition of MEK1/2 signaling disrupts bile acid metabolism through loss of Shp and enhanced Cyp7a1 expression

The RAS-MAPK signaling pathway is one of the most frequently dysregulated pathways in human cancer. Small molecule inhibitors directed against this pathway have clinical activity in patients with various cancer types and can improve patient outcomes. However, the use of these drugs is associated with adverse effects, which can result in dose reduction or treatment interruption. A better molecular understanding of on-target, off-tumor effects may improve toxicity management. In the present study, we aimed to identify early initiating biological changes in the liver upon pharmacological inhibition of the RAS-MAPK signaling pathway. To this end, we tested the effect of MEK inhibitor PD0325901 using mice and human hepatocyte cell lines. Male C57BL/6 mice were treated with either vehicle or PD0325901 for six days, followed by transcriptome analysis of the liver and phenotypic characterization. Pharmacological MEK inhibition altered the expression of 423 genes, of which 78 were upregulated and 345 were downregulated. We identified Shp, a transcriptional repressor, and Cyp7a1, the rate-limiting enzyme in converting cholesterol to bile acids, as the top differentially expressed genes. PD0325901 treatment also affected other genes involved in bile acid regulation, which was associated with changes in the composition of plasma bile acids and composition and total levels of fecal bile acids and elevated predictive biomarkers of early liver toxicity. In conclusion, short-term pharmacological MEK inhibition results in profound changes in bile acid metabolism, which may explain some of the clinical adverse effects of pharmacological inhibition of the RAS-MAPK pathway, including gastrointestinal complications and hepatotoxicity.

The kinase PLK1 promotes the development of Kras/Tp53-mutant lung adenocarcinoma through transcriptional activation of the receptor RET.

Increased abundance of polo-like kinase 1 (PLK1) is observed in various tumor types, particularly in lung adenocarcinoma (LUAD). Here, we found that PLK1 accelerated the progression of LUAD through a mechanism that was independent of its role in mediating mitotic cell division. Analysis of human tumor databases revealed that increased PLK1 abundance in LUAD correlated with mutations in KRAS and p53, with tumor stage, and with reduced survival in patients. In a mouse model of KRASG12D-driven, p53-deficient LUAD, PLK1 overexpression increased tumor burden, decreased tumor cell differentiation, and reduced animal survival. PLK1 overexpression in cultured cells and mice indirectly increased the expression of the gene encoding the receptor tyrosine kinase RET by phosphorylating the transcription factor TTF-1. Signaling by RET and mutant KRAS in these tumors converged to activate the mitogen-activated protein kinase (MAPK) pathway. Pharmacological inhibition of the MAPK pathway kinase MEK combined with inhibition of either RET or PLK1 markedly suppressed tumor growth. Our findings show that PLK1 can amplify MAPK signaling and reveal a potential target for stemming progression in lung cancers with high PLK1 abundance.

Abstract 1000: E-cadherin and EGFR interactions result in hyper-proliferation via ERK signaling in breast cancer

Abstract The loss of the intercellular adhesion molecule E-cadherin is a hallmark of the epithelial-mesenchymal transition (EMT), during which tumor cells transition into an invasive phenotype. Thus, E-cadherin has long been considered a tumor suppressor gene. However, recent studies have provided evidence that E-cadherin may promote metastasis rather than suppress it, suggesting oncogenic behavior. Here we provide data that E-cadherin plays an oncogenic role in breast cancer by promoting a hyper-proliferative phenotype in breast cancer cells via interaction with EGFR. This interaction results in the activation of the MEK/ERK signaling pathway, leading directly to changes in proliferation via transcription factors such as c-Fos. Pharmacological inhibition of MEK in E-cadherin positive breast cancer cells significantly decreases both tumor growth and macro-metastasis in vivo. This work provides evidence for a novel role of E-cadherin in breast tumor progression and identifies a potential new target to treat hyper-proliferative E-cadherin-positive breast tumors. Citation Format: Gabriella C. Russo, Ashleigh Crawford, David J. Clark, Julie Cui, Ryan Carney, Michelle N. Karl, Boyang Su, Batrholomew Starich, Tung-Shing Lee, Qiming Zhang, Pei-hsun Wu, Meng-Horng Lee, Hon S. Leong, Vito Rebecca, Hui Zhang, Denis Wirtz. E-cadherin and EGFR interactions result in hyper-proliferation via ERK signaling in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1000.

The regulation of CD73 in non-small cell lung cancer

BackgroundLung cancer is the leading cause of global cancer-related mortality. Although immune checkpoint therapy has achieved remarkable results in lung cancer, EGFR-mutant or ALK-positive non-smallcell lung cancer patients show limited benefit. Besides the low tumor mutational burden, PD-L1 expression and CD8+ tumor-infiltrating T cells, upregulation of CD73/adenosine pathway also contributes to the immune-inert microenvironment of EGFR-mutant NSCLC. However, the detailed mechanism underlying the regulation of CD73 is unclear. MethodsTCGA data was used to analyze the CD73 expression in cancer patients. Western blotting, qPCR, and ChIP-PCR were performed in multiple NSCLC cancer cell lines and patient derived organoids were used to explore the regulation of CD73 expression using western blotting. ResultsCD73 expression was highly expressed in multiple cancer types. Pharmacological or genetic inhibition of EGFR, MEK, KRAS, or ALK dramatically reduced the CD73 mRNA and protein expression in NSCLC cancer cells and patient-derived organoids with EGFR mutation, KRAS mutation or ALK-rearrangement. C-Jun overexpression-induced CD73 mRNA and protein expression. ChIP assay showed that c-Jun bind to CD73 genomic regions. ConclusionsHigher CD73 expression in NSCLC cancer cells and patient-derived organoids with EGFR mutation, KRAS mutation or ALK-rearrangement. Mechanistically, CD73 is regulated by ERK-Jun pathway, wherein c-Jun regulates CD73 expression via binding to CD73 genomic regions.

T cell differentiation protein 2 facilitates cell proliferation by enhancing mTOR-mediated ribosome biogenesis in non-small cell lung cancer

Dysregulation of T cell differentiation protein 2 (MAL2) has been observed in multiple cancers, but its exact role in lung cancer is poorly understood. Here we report a role of MAL2 in accelerating cell proliferation in non-small cell lung cancer (NSCLC). MAL2 expression enhances cell proliferation in both cell and nude mouse models. Mechanistically, overexpression of MAL2 results in the hyper-activation of the MAPK/mTOR signaling pathway in NSCLC cells which leads to active ribosome biogenesis. Importantly, pharmacological inhibition of mTOR or MEK lowered the abundance of PCNA, a marker of tumor cell proliferation, and subsequently suppressed ribosome biogenesis, cell growth and xenograft growth in mouse model. MAL2 upregulation in clinical tumors is also linked to worse prognosis. Overall our data reveal that MAL2 is a potential diagnostic biomarker and targeting the MAL2/MAPK/mTOR signaling pathway may improve therapeutic strategy and efficacy for this subset of NSCLC patients.

Inhibition of MEK-ERK pathway enhances oncolytic vaccinia virus replication in doxorubicin-resistant ovarian cancer

Oncolytic vaccinia virus (OVV) has been reported to induce cell death in various types of cancer; however, the oncolytic activity of OVV in drug-resistant ovarian cancer remains limited. In the present study, we established doxorubicin-resistant ovarian cancer cells (A2780-R) from the A2780 human ovarian cancer cell line. Both A2780 and A2780-R cells were infected with OVV to explore its anticancer effects. Interestingly, OVV-infected A2780-R cells showed reduced viral replication and cell death compared with A2780 cells, suggesting their resistance against OVV-induced oncolysis; to understand the mechanism underlying this resistance, we explored the involvement of protein kinases. Among protein kinase inhibitors, PD0325901, an MEK inhibitor, significantly augmented OVV replication and cell death in A2780-R cells. PD0325901 treatment increased the phosphorylation of STAT3 in A2780-R cells. Moreover, cryptotanshinone, a STAT3 inhibitor, abrogated PD0325901-stimulated OVV replication. Furthermore, trametinib, a clinically approved MEK inhibitor, increased OVV replication in A2780-R cells. Transcriptomic analysis showed that the MEK inhibitor promoted OVV replication via increasing STAT3 activation and downregulating the cytosolic DNA-sensing pathway. Combined treatment with OVV and trametinib attenuated A2780-R xenograft tumor growth. These results suggest that pharmacological inhibition of MEK reinforces the oncolytic efficacy of OVV in drug-resistant ovarian cancer.

CLL-358: Adaptation of Chronic Lymphocytic Leukemia to Ibrutinib Is Mediated by Epigenetic Plasticity of Residual Disease and Bypass Signaling via the MAPK Pathway

Context: Ibrutinib inhibits BTK with the most typical response in CLL being partial remission (PR) with measurable minimal residual disease (MRD) in blood. Remission is usually maintained until development of genetically-driven resistance. Objective: Mechanism of adaptation and survival in MRD. Patients and Methods: Pre-treatment CLL cells and under-treatment MRD were systematically collected from 33 high-risk CLL patients. Samples were characterized by high-dimensional, single-cell flow cytometry; bulk genomic, transcriptomic, and chromatin accessibility profiling; and single-cell RNA-seq profiling. Results: The genetic composition of MRD remained constant across timepoints, indicating that ibrutinib did not have any impact in shaping its genomic diversity. Ibrutinib induced chromatin dynamics in MRD resulting in a predominantly closed state. Chromatin regions that became more accessible under ibrutinib were enriched of binding sites for transcription factor that lay downstream ERK signaling. Regions that turned into a less accessible state were enriched for the binding sites of TF emanating from the BCR. At the transcriptomic level, most of the differentially expressed genes between pre- and post-treatment samples were downregulated in MRD, while only a fraction was upregulated. IL4, NF-kB, and metabolism and proliferation signatures were downregulated in MRD, while MAPK and RAS signatures were upregulated. CLL spleen samples from TCL1 mice under ibrutinib treatment showed an upregulation of MAPK pathway genes compared to untreated mice. Single-cell transcriptomes revealed a distinct post-treatment cell population driven by MAPK activity that functionally pre-existed in a fraction of CLL cells of baseline samples before ibrutinib start. At the signaling level, ibrutinib had no effect on the integrity of RAS-BRAF-MAPK-ERK pathway protein expression upon crosslinking of the BCR with anti-IgM but not after stimulation of TLR9 or CD40. Functional validation on primary samples by western blotting shows that RAS/ERK can be activated by BCR stimulation, irrespective of ibrutinib treatment in ex vivo experiments. Pharmacological inhibition of MEK (trametinib) and ERK (ulixertinib) synergized with ibrutinib, leading to decreased cell viability. Conclusions: MRD under ibrutinib adapts its phenotype in an epigenetic way to maintain functional competence of BCR signaling via the MAPK pathway, which turned to be a vulnerability of MRD persisting under ibrutinib.

Pharmacological MEK inhibition promotes polyclonal T-cell reconstitution and suppresses xenogeneic GVHD

Rapid immune reconstitution without developing graft-versus-host disease (GVHD) is required for the success of allogeneic hematopoietic stem cell transplantation. Here, we analyzed the effects of pharmacological MEK inhibition on human polyclonal T-cell reconstitution in a humanized mouse GVHD model utilizing deep sequencing-based T-cell receptor (TCR) repertoire analysis. GVHD mice exhibited a skewed TCR repertoire with a common clone within target organs. The MEK inhibitor trametinib ameliorated GVHD and enabled engraftment of diverse T-cell clones. Furthermore, trametinib also ameliorated GVHD sparing diverse T cell repertoire, even when it was given from day 15 through 28. Although tacrolimus also reduced development of GVHD, it disturbed diverse T cell reconstitution and resulted in skewed TCR repertoire. Thus, trametinib not only suppresses GVHD-inducing T cells but also promotes human T cell reconstitution in vivo, providing a novel rationale for translational studies targeting human GVHD.

1,8-cineole ameliorates ischaemic brain damage via TRPC6/CREB pathways in rats.

A previous in vitro study reported that the monoterpene oxide 1,8-cineole (cineole) attenuates neuronal caused by oxygen-glucose deprivation/reoxygenation in culture. However, to date, there is no in vivo evidence showing neuroprotective effects of cineole against stroke. This study aimed to investigate whether cineole attenuates cerebral ischaemic damage in rats. A rat model of middle cerebral artery occlusion (MCAO) followed by 24 h reperfusion was applied. Male rats were treated with oral cineole (100 mg/kg) for 7 consecutive days, then subjected to MCAO surgery. Infarct volume, neurologic deficits, apoptosis and expression levels of all-spectrin breakdown products of 145 kDa (SBDP145), transient receptor potential canonical (subtype) 6 (TRPC6) and phosphorylated CREB (p-CREB) were measured in ischaemic brain tissues. Cineole treatment significantly reduced infarct volume, neurological dysfunction, neuronal apoptosis, SBDP145 formation and TRPC6 degradation and enhanced p-CREB expression in MCAO rats compared with vehicle treatment. These neuroprotective effects were markedly suppressed by pharmacological inhibition of MEK or CaMKIV signalling. Our study provides in vivo evidence demonstrating that cineole pretreatment attenuates ischaemic stroke-induced brain damage, mainly through blocking calpain-induced TRPC6 degradation and activating CREB via MEK/CREB and CaMKIV/CREB signalling pathways.

Curcumin increases heat shock protein 70 expression via different signaling pathways in intestinal epithelial cells

Intestinal inflammation is associated with the integrity of the intestinal epithelium, which forms a physical barrier against noxious luminal substances. Heat shock 70 kDa protein 1A (HSP70), a molecular chaperon that exerts a cytoprotective effect, regulates intestinal integrity. This study investigated the modulation of HSP70 expression by dietary polyphenols, with particular reference to curcumin, in human intestinal Caco-2 cells. Immunoblot analysis demonstrated that among the 21 different polyphenols tested, curcumin most potently increased HSP70 levels in Caco-2 cells without affecting cell viability. Curcumin also increased the phosphorylation of heat shock factor 1 (HSF1), a well-known transcription factor of HSP70. Promoter and qRT-PCR assays indicated that curcumin upregulated Hspa1a levels via transcriptional activation. Pharmacological inhibition of MEK, a mechanistic target of rapamycin, p38 mitogen-activated protein kinase, and phosphatidyl 3-inositol kinase suppressed curcumin-mediated HSP70 expression, whereas HSF1 phosphorylation was sensitive only to MEK inhibition. Taken together, curcumin increases the expression of HSP70 in intestinal Caco-2 cells via transcriptional activation, possibly enhancing cell integrity. The effects exerted by curcumin are regulated by various signaling pathways. Our findings will expectedly contribute to a deeper understanding of the regulation of intestinal HSP70 by dietary components.

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

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. 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. 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. 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.

MageC2 protein is upregulated by oncogenic activation of MAPK pathway and causes impairment of the p53 transactivation function

Normal-to-tumor cell transition is accompanied by changes in gene expression and signal transduction that turns the balance toward cancer-cell phenotype, eluding by different mechanisms, the response of tumor-suppressor genes. Here, we observed that MageC2, a MAGE-I protein able to regulate the p53 tumor-suppressor, is accumulated upon MEK/ERK MAPK activation. Overexpression of H-RasV12 oncogene causes an increase in MageC2 protein that is prevented by pharmacologic inhibition of MEK. Similarly, decrease in MageC2 protein levels is shown in A375 melanoma cells (which harbor B-RafV600E oncogenic mutation) treated with MEK inhibitors. MageC2 protein levels decrease when p14ARF is expressed, causing an Mdm2-independent upregulation of p53 transactivation. However, MageC2 is refractory to p14ARF-driven downregulation when H-RasV12 is co-expressed. Using MageC2 knockout A375 cells generated by CRISPR/CAS9 technology, we demonstrated the relevance of MageC2 protein in reducing p53 transcriptional activity in cells containing hyperactive MEK/ERK signaling. Furthermore, gene expression analysis performed in cancer-genomic databases, supports the correlation of reduced p53 transcriptional activity and high MageC2 expression, in melanoma cells containing Ras or B-Raf driver mutations. Data presented here suggest that MageC2 can be a functional target of the oncogenic MEK/ERK pathway to regulate p53.

AXL Inhibition Enhances MEK Inhibitor Sensitivity in Malignant Peripheral Nerve Sheath Tumors.

Dysregulation of the receptor tyrosine kinase AXL is known to promote cancer cell growth and survival in many sarcomas, including the rare subtype, malignant peripheral nerve sheath tumors (MPNST). MPNSTs are largely chemoresistant and carry a poor prognosis. AXL is an attractive potential therapeutic target, as it is aberrantly expressed, and its activation may be an early event in MPNST. However, the effect of AXL inhibition on MPNST development and progression is not known. Here, we investigated the role of AXL in MPNST development and the effects of AXL and MEK1/2 co-inhibition on MPNSTs. We used western blotting to examine AXL expression and activation in MPNST cell lines. We analyzed the effects of exogenous growth arrest-specific 6 (GAS6) expression on downstream signaling and the proliferation, migration, and invasion of MPNST cells. The effect of AXL knockdown with or without mitogen-activated protein kinase (MAPK) inhibition on downstream signal transduction and tumorigenesis was also examined in vivo and in vitro. We found that AXL knockdown increased MAPK pathway signaling. This compensation, in turn, abrogated the antitumorigenic effects linked to AXL knockdown in vivo. AXL knockdown, combined with pharmacological MEK inhibition, reduced the proliferation and increased the apoptosis of MPNST cells both in vitro and in vivo. The pharmacological co-inhibition of AXL and MEK1/2 reduced MPNST volumes. Together these findings suggest that AXL inhibition enhances the sensitivity of MPNST to other small molecule inhibitors. We conclude that combination therapy with AXL inhibitor may be a therapeutic option for MPNST.

Combination of the MEK Inhibitor Trametinib and Pyrvinium Pamoate Efficiently Targets RAS Pathway-Mutated Acute Myeloid Leukemia in Preclinical Models

While acute myeloid leukemia (AML) is still associated with a low cure rate, recent advances in understanding its molecular complexity have significantly improved therapy for subgroups of patients, including those harboring FLT3, IDH1 or IDH2 mutations1. However, more than half of AML cases still lack a druggable oncogenic target. Human cancers frequently harbor mutations in RAS oncogene family members, including NRAS, KRAS and HRAS, which drive oncogenesis by augmenting cellular proliferation and survival. These are small protein GTPases, regulated by a switch between active GTP-linked and inactive GDP-bound states governed by a complex network of guanine exchange factors (GEFs, favoring RAS-GTP) and GTPase activating factors (GAPs, favoring RAS-GDP). RAS activation - due to either extrinsic recruitment by transmembrane tyrosine kinase receptors or intrinsic mutations - propagates through the downstream RAF/MEK/ERK and PI3K/AKT signaling pathways. Besides RAS-activating mutations conferring independence from physiological regulators, human cancers harbor mutations in other RAS network genes such as NF1 (encoding neurofibromin, a RAS GAP), BRAF or PTPN11 (encoding the SHP2 tyrosine phosphatase involved in RAS activation). Somatic alterations of RAS pathway genes, notably NRAS, KRAS, PTPN11 (missense mutations) and NF1 (mutations and deletions), are reported in up to 20% of AML cases2. Generally arising as late driver events, RAS pathway mutations participate in leukemogenesis through mitogen activated protein kinase (MAPK) activation. The anti-tumor activity of MEK inhibitors in Nras-mutated AML in mice and in some NRAS or KRAS-mutated AML patients suggests that deregulated RAS pathway signaling may represent a bona fide therapeutic target. However, strategies to inhibit RAS - indirectly in most cases - have been hampered by signaling feedback, redundancy and tumor heterogeneity 3. We identified 127 cases of AML with unmet therapeutic need within a cohort from which we excluded those with European leukemia network (ELN) favorable prognosis or FLT3-ITD mutations. Targeted next-generation sequencing revealed RAS pathway alterations in 50 patients (39.3%) and NF1 mutations and deletions, mostly large cytogenetically detected deletions, in 17 (14.8%). NRAS, KRAS, PTPN11, CBL and BRAF variants were detected in 13 (10.4%), 10 (7.9%), 9 (7.2%), 5 (3.9%) and 2 (1.6%) cases, respectively. Mutations in RAF1, RASA1, SOS1 and MAP2K2 were observed in a single case each. RAS pathway alterations appeared in the putative main leukemic clone as well as in subclones inferred from variant allele frequencies. Concurrent RAS pathway mutations were observed in nine cases. Among 79 patients homogeneously treated with intensive induction chemotherapy, RAS pathway alterations correlated with higher clinical proliferation markers (elevated white blood cell count, blast cell percentage and LDH levels) and reduced survival probability, particularly within the ELN intermediate-risk subgroup. We established robust models of RAS/MAPK activation through genetic NF1 disruption or expression of NRASG12D or PTPN11D61Y in growth factor (GF)-dependent cell lines. We assessed oncogenic addiction to the RAS pathway in these cells through GF-independence, increased RAS activity, faster propagation in immunocompromised mice and an exquisite sensitivity to pharmacological MEK inhibition in vitro and in vivo. High-content pharmacological screens with FDA-approved molecules identified pyrvinium pamoate, an anti-helminthic agent, as preferentially active in RAS-activated cells. This compound significantly impaired cell viability and colony formation in primary AML samples with RAS pathway alterations. Moreover, the combination of trametinib and pyrvinium pamoate demonstrated synergy in cell line models and even primary samples. While pyrvinium pamoate strongly inhibited mitochondrial respiration and induced metabolic reprograming towards increased glycolysis, trametinib impaired glycolysis and mitochondrial respiratory capacity, suggesting a mechanistic basis for the synergy observed. These data highlight the translational opportunity in developing pyrvinium pamoate for RAS pathway mutated AML.

BRAFV600E mutation: A promising target in colorectal neuroendocrine carcinoma.

To determine the role of BRAFV600E mutation and MAPK signaling as well as the effects of BRAF and MEK directed therapy in gastroenteropancreatic neuroendocrine neoplasia (GEP-NEN), with a focus on highly aggressive gastroenteropancreatic neuroendocrine carcinoma (GEP-NEC). Using Sanger sequencing of BRAF exon 15 we determined the frequency of BRAFV600E mutations in 71 primary GEP-NENs. MEK phosphorylation was examined by immunohistochemistry in corresponding tissue samples. To evaluate the biological relevance of BRAFV600E mutation and MAPK signaling in GEP-NECs, effects of a pharmacological BRAF and MEK inhibition were analyzed in NEC cell lines both in vitro and in vivo. BRAFV600E mutation was detected in 9.9% of all GEP-NENs. Interestingly, only NECs of the colon harbored BRAFV600E mutations, leading to a mutation frequency of 46.7% in this subgroup of patients. In addition, a BRAFV600E mutation was significantly associated with high levels of MEK phosphorylation (pMEK) and advanced tumor stages. Pharmacological inhibition of BRAF and MEK abrogated NEC cell growth, inducing G1 cell cycle arrest and apoptosis only in BRAFV600E mutated cells. BRAF inhibitor dabrafenib and MEK inhibitor trametinib prevented growth of BRAFV600E positive NEC xenografts. High frequencies of BRAFV600E mutation and elevated expression levels of pMEK were detected in biologically aggressive and highly proliferative colorectal NECs. We provide evidence that targeting BRAF oncogene may represent a therapeutic strategy for patients with BRAF mutant colorectal NECs.

Abstract 874: Pharmacologically induced RAS-GTP levels and CRAF-BRAF hetero-dimerization drive sensitization to Type II pan-RAF inhibitors in KRAS mutant cancer

Abstract Although effective in BRAF V600 mutant melanoma, Type 1.5 RAF inhibitors such as vemurafinib and dabrafenib have not proven to be successful in KRAS mutant cancers, neither as single agent nor in combination with MEK inhibitors. Through large-scale cellular compound combination screening we found that Type II RAF inhibitors such as AZ-628 do show synergistic activity with MEK inhibitors in multiple KRAS mutant indications, including NSCLC, colorectal cancer and ovarian cancer. The combination of Type II RAF inhibitors and MEK inhibitors demonstrates robust and durable abrogation of MAPK signaling both on canonical markers of MAPK activity such as pERK and pRSC as well as transcriptional output. We also observe synergistic in vivo tumor growth inhibition in two independent models of KRAS mutant cancer by this combination treatment. We found that treatment with MEK inhibitors alone drives the increase of active RAS-GTP levels and induces CRAF:BRAF hetero-dimerization. These induced dimers are active and able to phosphorylate MEK in vitro. This increased dimerization renders cells sensitive to Type 2 RAF inhibitors. We find that this effect is not limited to KRAS mutant cells, as a subset of KRAS wild-type cells show increased RAS-GTP levels upon MEK inhibitor treatment. These cells also show synergistic sensitivity to Type 2 RAF inhibition. Additionally, we observed significantly higher synergy and higher RAS-GTP levels in KRAS G13D mutant cells, which have intrinsically high GDP exchange and low intrinsic GTP hydrolysis. Finally, we show that GDC-0941 and GDC-0032, two broad PI3K inhibitors, also induce RAS-GTP levels in cells independent of PIK3CA or KRAS mutation status. We subsequently observed a synergistic sensitivity to Type 2 RAF inhibitors in these PI3K inhibitor-treated cells. Overall, we demonstrate that pharmacologic inhibition of MEK or PI3K increases RAS-GTP levels and drives increased CRAF:BRAF hetero-dimerization. This in turn sensitizes cells to Type 2 RAF inhibition, leading to a synergistic drug effect. Combination of these inhibitors may be a viable therapeutic approach in KRAS mutant cancer, and may be especially effective in KRAS G13D-carrying tumors. Citation Format: Christiaan N. Klijn, Ivana Yen, Frances Shanahan, Mark Merchant, Christine Orr, Thomas Hunsaker, Matthew Durk, Hank La, Xiaoling Zhang, Scott Martin, Eva Lin, John Chan, Yihong Yu, Dhara Amin, Amy Gustafson, Scott Foster, Joachim Rudolph, Shiva Malek. Pharmacologically induced RAS-GTP levels and CRAF-BRAF hetero-dimerization drive sensitization to Type II pan-RAF inhibitors in KRAS mutant cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 874.