RAF Inhibitors Research Articles

Pan-Cancer Efficacy of Vemurafenib in BRAF V600-Mutant Non-Melanoma Cancers.

BRAF V600 mutations occur in a wide range of tumor types, and RAF inhibition has become standard in several of these cancers. Despite this progress, BRAF V600 mutations have historically been considered a clear demonstration of tumor lineage context-dependent oncogene addiction, based predominantly on the insensitivity to RAF inhibition in colorectal cancer. However, the true broader activity of RAF inhibition pan-cancer remains incompletely understood. To address this, we conducted a multicohort "basket" study of the BRAF inhibitor vemurafenib in non-melanoma BRAF V600 mutation-positive solid tumors. In total, 172 patients with 26 unique cancer types were treated, achieving an overall response rate of 33% and median duration of response of 13 months. Responses were observed in 13 unique cancer types, including historically treatment-refractory tumor types such as cholangiocarcinoma, sarcoma, glioma, neuroendocrine carcinoma, and salivary gland carcinomas. Collectively, these data demonstrate that single-agent BRAF inhibition has broader clinical activity than previously recognized. SIGNIFICANCE: These data suggest that BRAF V600 mutations lead to oncogene addiction and are clinically actionable in a broad range of non-melanoma cancers, including tumor types in which RAF inhibition is not currently considered standard of care.See related commentary by Ribas and Lo, p. 640.This article is highlighted in the In This Issue feature, p. 627.

Abstract B31: Combination inhibitor strategies targeting KRAS effector signaling in KRAS-mutant pancreatic cancer

Abstract With KRAS mutations found in 95% of pancreatic ductal adenocarcinoma (PDAC), an effective anti-KRAS therapeutic strategy is anticipated to make a significant impact on the treatment of PDAC. Among the major directions currently being pursued, inhibitors of KRAS effector signaling are believed to be the most promising. However, as monotherapy, effector inhibitors have not demonstrated significant activity and concerns with normal tissue toxicity remain. Combination inhibitor strategies are considered the best approach to overcome these limitations. To identify effector inhibitor-based combinations, we applied a 525-oncology drug screen to identify combinations that enhanced the cytotoxic activity of inhibitors of the RAF-MEK-ERK and the PI3K-AKT-mTOR effector pathways. While many cytotoxic combinations were identified with RAF effector pathway inhibitors, few were identified with PI3K pathway inhibitors. The same classes of inhibitors were identified with RAF, MEK and ERK inhibitors. In addition to inhibitors of the PI3K-AKT-mTOR pathway, microtubule, HDAC and HSP90 inhibitors synergistically enhanced RAF and ERK inhibitor antitumor activity. Addressing a basis for synergy, we found that the different combinations showed enhanced loss of MYC protein, a key ERK substrate. In summary, our studies identify promising effector inhibitor-based combinations for PDAC treatment. Citation Format: Irem Ozkan-Dagliyan, Craig M. Goodwin, Kirsten L. Bryant, Samuel D. George, Kelly Lucas, Prson Gautam, Krister Wennerberg, Adrienne D. Cox, Channing J. Der. Combination inhibitor strategies targeting KRAS effector signaling in KRAS-mutant pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B31.

Hedgehog Signal Inhibitor GANT61 Inhibits the Malignant Behavior of Undifferentiated Hepatocellular Carcinoma Cells by Targeting Non-Canonical GLI Signaling.

The Hedgehog (HH)–GLI pathway plays an important role in cell dedifferentiation and is therefore pivotally involved in the malignant transformation of cancer cells. GANT61, a selective inhibitor of GLI1 and GLI2, was reported as a promising treatment for cancer in various tissues; however, the biological impact of GANT61 in hepatocellular carcinoma (HCC), especially in undifferentiated HCC cells, remains unclear. In this study, we investigated the antitumor effect of GANT61 using two undifferentiated hepatoma cell lines: HLE and HLF. Quantitative PCR and RT-PCR analyses revealed that these cells express GLI transcripts, showing mesenchymal phenotypes characterized by the loss of epithelial and hepatic markers and specific expression of epithelial–mesenchymal transition (EMT)-related genes. GANT61 significantly reduced the proliferation and cell viability after drug treatment using 5-FU and Mitomycin C. We showed that GLI transcript levels were down-regulated by the MEK inhibitor U0126 and the Raf inhibitor sorafenib, suggesting that non-canonical signaling including the Ras–Raf–MEK–ERK pathway is involved. Sphere formation and migration were significantly decreased by GANT61 treatment, and it is suggested that the underlying molecular mechanisms are the down-regulation of stemness-related genes (Oct4, Bmi1, CD44, and ALDH) and the EMT-related gene Snail1. The data presented here showed that direct inhibition of GLI might be beneficial for the treatment of dedifferentiated HCC.

Ameloblastic fibrosarcoma: clinicopathological and molecular analysis of seven cases highlighting frequent BRAF and occasional NRAS mutations.

Ameloblastic fibrosarcoma (AFS) is an aggressive odontogenic neoplasm featuring malignant mesenchymal stroma in addition to an ameloblastic epithelial component, and is hence considered to be the malignant counterpart of ameloblastic fibroma (AF). AFS is exceedingly rare, with <110 cases having been reported so far. Although BRAF mutations are recognised driver mutations in ameloblastoma, the molecular pathogenesis of AFS remains elusive. We herein describe seven AFSs that were analysed, for the first time, for mutations in the BRAF-NRAS pathway. The patients were four females and three males aged 23-57years (median, 26years). Three tumours developed after one or multiple recurrences of AF (4-20years after initial diagnosis), two showed transition from AF-like bland areas, and two developed de novo. All patients were treated with surgery; adjuvant chemotherapy was given to one patient. At the last follow-up, five patients were alive and well (19-344months). The remainder were lost to follow-up. Histological examination showed variable sarcomatous overgrowth with varying degrees of atypia and increased mitotic activity. The epithelial component varied greatly according to the degree of sarcomatous overgrowth. Molecular testing revealed BRAF V600E mutations in five cases and NRAS p.Gln61Lys mutation in one case. One tumour was wild-type. To our knowledge, this is the first study on BRAF/NRAS mutations in AFS. Given the activity of RAF and MEK inhibitors across different cancers harbouring V600E mutations, our data strongly suggest that all AFS cases should be genetically tested, and that targeted treatment approaches for this extremely rare sarcoma subtype should be clinically investigated.

RAF kinase dimerization: implications for drug discovery and clinical outcomes

The RAF kinases activated by RAS GTPases regulate cell growth and division by signal transduction through the ERK cascade and mutations leading to constitutive activity are key drivers of human tumors, as are upstream activators including RAS and receptor tyrosine kinases. The development of first-generation RAF inhibitors, including vemurafenib (VEM) and dabrafenib led to initial excitement due to high response rates and profound regression of malignant melanomas carrying BRAFV600E mutations. The excitement about these unprecedented response rates, however, was tempered by tumor unresponsiveness through both intrinsic and acquired drug-resistance mechanisms. In recent years much insight into the complexity of the RAS-RAF axis has been obtained and inactivation and signal transduction mechanisms indicate that RAF dimerization is a critical step in multiple cellular contexts and plays a key role in resistance. Both homo- and hetero-dimerization of BRAF and CRAF can modulate therapeutic response and disease progression in patients treated with ATP-competitive inhibitors and are therefore highly clinically significant. Ten years after the definition of the RAF dimer interface (DIF) by crystallography, this review focuses on the implications of RAF kinase dimerization in signal transduction and for drug development, both from a classical ATP-competitive standpoint and from the perspective of new therapeutic strategies including inhibiting dimer formation. A structural perspective of the DIF, how dimerization impacts inhibitor activation and the structure-based design of next-generation RAF kinase inhibitors with unique mechanisms of action is presented. We also discuss potential fields of application for DIF inhibitors, ranging from non-V600E oncoproteins and BRAF fusions to tumors driven by aberrant receptor tyrosine kinase or RAS signaling.

Temporal activation of WNT/\u03b2-catenin signaling is sufficient to inhibit SOX10 expression and block melanoma growth

Despite advances in the systemic treatment of patients with metastatic melanoma using immune checkpoint and tyrosine kinase inhibitors (TKI), the majority of stage IV melanoma patients eventually succumb to the disease. We have previously identified the transcription factor Sox10 as a crucial player in melanoma, yet the underlying molecular mechanisms mediating Sox10-dependent tumorigenesis remain largely uncharacterized. Here, we show that MEK and RAF inhibitors do not suppress levels of SOX10 protein in patient-derived cells in vitro, as well as in melanoma patients in vivo. In a search for pharmacological inhibitors of SOX10, we performed a mass spectrometry-based screen in human melanoma cells. Subsequent analysis revealed that SOX10 directly interacts with β-catenin, which is a key mediator of canonical Wnt/β-catenin signaling. We demonstrate that inhibitors of glycogen synthase kinase 3 alpha/beta (GSK3α/β) efficiently abrogate SOX10 protein in human melanoma cells in vitro and in melanoma mouse models in vivo. The mechanism of action of GSK3-mediated SOX10 suppression is transcription-independent and relies on the presence of a proteasome degradable form of β-catenin. Taken together, we provide evidence that activation of canonical Wnt signaling has a profound effect on melanoma growth and is able to counteract Sox10-dependent melanoma maintenance both in vitro and in vivo.

Gene Networks with Transcriptional Bursting Recapitulate Rare Transient Coordinated High Expression States in Cancer.

Non-genetic transcriptional variability is a potential mechanism for therapy resistance in melanoma. Specifically, rare subpopulations of cells occupy a transient pre-resistant state characterized by coordinated high expression of several genes and survive therapy. How might these rare states arise and disappear within the population? It is unclear whether the canonical models of probabilistic transcriptional pulsing can explain this behavior, or if it requires special, hitherto unidentified mechanisms. We show that a minimal model of transcriptional bursting and gene interactions can give rise to rare coordinated high expression states. These states occur more frequently in networks with low connectivity and depend on three parameters. While entry into these states is initiated by a long transcriptional burst that also triggers entry of other genes, the exit occurs through independent inactivation of individual genes. Together, we demonstrate that established principles of gene regulation are sufficient to describe this behavior and argue for its more general existence. A record of this paper's transparent peer review process is included in the Supplemental Information.

Mechanisms of the Cardiac Myocyte-Damaging Effects of Dasatinib.

The anticancer drug dasatinib (Sprycel) is a BCR-ABL1-targeted tyrosine kinase inhibitor used in treating chronic myelogenous leukemia that has been shown in clinical trials to display cardiovascular toxicities. While dasatinib potently inhibits BCR-ABL1, it is not a highly selective kinase inhibitor and may have off-target effects. A neonatal rat cardiac myocyte model was used to investigate potential mechanisms by which dasatinib damaged myocytes. The anthracycline cardioprotective drug dexrazoxane was shown to be ineffective in preventing dasatinib-induced myocyte damage. Dasatinib treatment increased doxorubicin accumulation in myocytes and doxorubicin-induced myocyte damage, likely through its ability to bind to one or more ABC-type efflux transporters. Dasatinib induced myocyte damage either after a brief treatment that mimicked the clinical situation, or more potently after continuous treatment. Dasatinib slightly induced apoptosis in myocytes as evidenced by increases in caspase-3/7 activity. Dasatinib treatment reduced pERK levels in myocytes most likely through inhibition of RAF, which dasatinib strongly inhibits. Thus, inhibition of the RAF/MEK/ERK pro-survival pathway in the heart may be, in part, a mechanism by which dasatinib induces cardiovascular toxicity.

Clinical Application of Next-Generation Sequencing-Based Panel to BRAF Wild-Type Advanced Melanoma Identifies Key Oncogenic Alterations and Therapeutic Strategies.

Molecular profiling with next-generation sequencing (NGS) has been applied in multiple solid cancers to discover potential therapeutic targets. Here, we describe the results of a clinical NGS panel in patients with advanced melanoma. Thirty-six tumor tissues from patients with BRAF wild-type melanoma at Seoul National University Hospital (SNUH; Seoul, Republic of Korea) were collected and deep-sequenced using the SNUH FIRST-Cancer NGS panel to assess single-nucleotide variants, small insertions/deletions, copy number variations, and structural variations to estimate tumor mutation burden (TMB). We discovered 106 oncogenic alterations and most of the patients (n = 33, 92%) harbored at least one oncogenic alteration, including 2 patients who were initially diagnosed as BRAF V600E-negative but were later confirmed to be positive. Altogether, 36 samples were classified into RAS/BRAF/NF1-mutant (n = 14, 39%) or triple wild-type (n = 22, 61%) melanoma subtypes. The estimated median TMB was 8.2 mutations per Mb, ranging from 0 to 146.67 mutations per Mb. Of the 36 patients, 25 (70%) had actionable alterations with currently developed drugs, and 7 (19.4%) were enrolled in clinical trials with an RAF inhibitor, multiple receptor tyrosine kinase inhibitor, and anti-programmed cell death-1 (PD-1) antibody. TMB tended to associate with progression-free survival (PFS) of treatment with anti-PD-1/PDL-1 antibody (HR, 0.96; 95% confidence interval, 0.92-1.00; P = 0.07). High-TMB (≥13) group was associated with longer PFS than the low-TMB group (median 34.0 vs. 11.0 weeks, P = 0.04). Overall, the clinical use of a NGS panel in patients with advanced melanoma shows association with clinical outcomes and several therapeutic strategies.

RAF and MEK inhibitor therapy in adult patients with brain tumors: a case-based overview and practical management of adverse events.

Targeted therapy has gained mainstream attention with notable successes against specific genetic mutations in many cancers. One particular mutation, the BRAF V600E mutation, is present in a small subset of gliomas in adults. Although clinical experience and trial data of RAF-targeted therapy in adults with glioma are lacking at this time, the poor prognosis of adult high-grade glioma has led neuro-oncology practitioners to consider the use of targeted therapy in these patients. In this manuscript, we describe the use of RAF and MEK inhibitors in adults with recurrent glioma. We discuss the utility of these agents, describe their toxicities, and give examples of management strategies. Given the significant toxicities of RAF and MEK inhibitors, along with the long potential duration of treatment, neuro-oncology providers should counsel patients carefully before initiating therapy and monitor them closely while undergoing treatment with RAF-targeted therapy.

BRAF mutant colorectal cancer: ErbB2 expression levels as predictive factor for the response to combined BRAF/ErbB inhibitors

BackgroundColorectal cancer (CRC) is a heterogeneous disease with a complex biology and a wide number of altered genes such as BRAF, KRAS and PIK3CA. Advances with new-targeted therapies have been achieved and available treating options have prolonged patient’s survival. However, BRAF-mutated CRC patients remain unresponsive to available therapies with RAF inhibitors (RAFi) alone or combined with ErbB inhibitors (ErbBi). These unmet needs require further exploitation of oncogenic signaling in order to set up individualized treatments.MethodsTo this end, we tested the efficacy of single agent or combined treatments using the BRAFi, vemurafenib and two different ErbBi: panitumumab and afatinib in CRC cells characterized by different molecular phenotypes.ResultsCombination strategies with BRAFi and ErbBi achieved a better response in BRAFV600E mutated cells expressing high levels of ErbB2.ConclusionsOur findings support the importance of ErbB2 evaluation in BRAF-mutated CRC patients and its role as a positive predictor factor of response to BRAFi/ErbBi combination.

Evidence-Based Network Approach to Recommending Targeted Cancer Therapies.

PURPOSEIn this work, we introduce CDGnet (Cancer-Drug-Gene Network), an evidence-based network approach for recommending targeted cancer therapies. CDGnet represents a user-friendly informatics tool that expands the range of targeted therapy options for patients with cancer who undergo molecular profiling by including the biologic context via pathway information.METHODSCDGnet considers biologic pathway information specifically by looking at targets or biomarkers downstream of oncogenes and is personalized for individual patients via user-inputted molecular alterations and cancer type. It integrates a number of different sources of knowledge: patient-specific inputs (molecular alterations and cancer type), US Food and Drug Administration–approved therapies and biomarkers (curated from DailyMed), pathways for specific cancer types (from Kyoto Encyclopedia of Genes and Genomes [KEGG]), gene-drug connections (from DrugBank), and oncogene information (from KEGG). We consider 4 different evidence-based categories for therapy recommendations. Our tool is delivered via an R/Shiny Web application. For the 2 categories that use pathway information, we include an interactive Sankey visualization built on top of d3.js that also provides links to PubChem.RESULTSWe present a scenario for a patient who has estrogen receptor (ER)–positive breast cancer with FGFR1 amplification. Although many therapies exist for patients with ER-positive breast cancer, FGFR1 amplifications may confer resistance to such treatments. CDGnet provides therapy recommendations, including PIK3CA, MAPK, and RAF inhibitors, by considering targets or biomarkers downstream of FGFR1.CONCLUSIONCDGnet provides results in a number of easily accessible and usable forms, separating targeted cancer therapies into categories in an evidence-based manner that incorporates biologic pathway information.

The Role of the Mitogen-Activated Protein Kinase (MAPK) Signaling Pathway in Cancer

The Ras-ERK mitogen-activated protein kinase (MAPK) pathway is hyperactive in >30% of all human cancers, prompting the development of RAS, RAF, MEK, and ERK inhibitors. The identification of intracellular signaling cascades, which promote the growth and survival of cancer cells, is critical for developing targeted cancer therapeutics aimed at blocking these signals. Currently, there are various FDA-approved drugs to inhibit RAF and MEK mutations for cancer treatment, but patients rapidly develop resistance to these drugs within several months, necessitating the need to develop new drugs against other targets in the MAPK pathway. Developing RAS inhibitors has been challenging due to the high affinity of RAS for its natural ligands (GDP and GTP) and the lack of a druggable binding cavity. As an alternative to targeting RAS, ERK inhibitors, which have also been shown to work on RAF/MEK-resistant cell lines, can block the activation of ERK and act as an effective cancer treatment, causing tumor regression. However, to maximize therapeutic effectiveness, it seems unlikely that any monotherapy would be particularly useful. Future treatment strategies should be designed on a patient-by-patient basis to ensure the most rapid reduction in tumor growth and the minimization of off-target effects by using a combination of two (or more) inhibitors within this MAPK pathway that lead to tumor regression and positive patient outcomes.

Abstract C48: Defining mechanisms of adaptation to KRAS G12C inhibitors: Using quantitative proteomics to design combinatorial strategies in pancreatic cancer

Abstract KRAS is mutated in 95% of pancreatic ductal adenocarcinoma (PDAC) tumors and is a critical driver of PDAC initiation, survival, and proliferation. Therapeutic efforts to target KRAS directly have been largely unsuccessful, leading to the thought that KRAS was “undruggable.” Recently, KRAS G12C inhibitors (KRASi) that covalently bind the mutant cysteine have been reported and are currently being tested in clinical trials. Unfortunately, as has been demonstrated with other targeted therapies, the efficacy of targeting an oncogenic driver can be limited by both tumor heterogeneity and development of resistance. Here, we have used a mass spectrometry-based quantitative proteomics workflow to identify pathways of adaptation to KRASi and to predict cytotoxic drug combinations both in 2D and 3D cell culture conditions. We treated multiple KRAS G12C mutant tumor lines (pancreatic, lung) with KRASi, which induced a cytostatic response with subsequent re-establishment of proliferation at longer time points. We profiled the proteomic adaptations acutely and at long term using a multiplexed quantitative proteomics workflow and identified and quantified over 8000 proteins. Pathway analysis by Gene Set Enrichment Analysis (GSEA) identified common mechanisms of acute drug response among KRAS G12C mutant tumor lines such as downregulation of cell cycle/transcription and increased lipid metabolism. Long-term adaptation was associated with increased DNA repair and oxidative metabolism, but further adaptations differed between tumor cell lines, suggesting selective programs are required to reactivate proliferation. Connectivity map (Cmap) analysis identified 30 perturbagen classes (connectivity score of Cmap class&amp;gt;90) that positively correlated with the MiaPaCa-2 KRASi profile at 24h. We identified correlations with elements in the KRAS pathway (MEK, RAF inhibitors) and with pathways previously described to synergize with KRAS inhibition (PI3K inhibitors), validating the utility of our approach. In addition, we identified combinations with HSP90, MET and EGFR inhibitors that, when co-targeted with KRASi, were able to suppress growth in long-term assays and induced cytotoxicity. Given that recent results suggest that 3D culture conditions may be most predictive of in vivo efficacy, we selected cell lines with greater KRASi sensitivity in 3D growth conditions and performed proteomic analysis in 3D versus 2D culture. Through GSEA analysis we determined differences in the 3D versus 2D proteome basally as well as after KRASi. Finally, we identified combinations by Cmap analysis that differentially correlated with the 3D signature, including CDK4/6 inhibitors, which reduced growth in 3D conditions. Overall, we employed a proteomics platform to characterize adaptation to KRASi and identified combinatorial regimens that induce cytotoxicity with potential therapeutic utility. Citation Format: Naiara Santana-Codina, Amrita Singh Chandhoke, Qijia Yu, Beata Malachowska, Miljan Kuljanin, Mark P. Jedrychowski, Ajami Gikandi, Marcin Stanczak, David A. Scott, Wojciech Fendler, Nathanael S. Gray, Joseph D. Mancias. Defining mechanisms of adaptation to KRAS G12C inhibitors: Using quantitative proteomics to design combinatorial strategies in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr C48.

Abstract B20: KrasG12D effector dependencies in the maintenance of pancreatic ductal adenocarcinoma

Abstract More than 90% of pancreatic ductal adenocarcinoma (PDAC) tumors in humans feature activating mutations in the proto-oncogene KRAS. In mice, KrasG12D mutation alone is sufficient for PanIN formation; when coupled with mutational inactivation of TP53 (Tp53R172H), PanIN formation is accelerated and these neoplastic lesions progress to invasive and metastatic disease at high frequency. Several groups have demonstrated that inactivation of mutant Kras in established PanIN and PDAC leads to redifferentiation and apoptosis of the tumor, respectively. More recently, several combinatorial genetic and pharmacologic strategies have been employed that highlight RAF kinases as key oncoeffectors for KRAS addiction, but that RAF inhibition alone demonstrates little efficiency against KRAS mutant tumors. Additionally, previous studies suggest a requirement for both C-raf and EGFR blockade or the combination of RAF-MEK or RAF-PI3K inhibitors to inhibit to tumor growth. However, a systematic evaluation of dependencies of Kras effectors in tumor maintenance has not been explicitly performed. To investigate effector dependencies in the KrasG12D setting, we have developed an organoid platform to generate isogenic lines bearing doxycycline-inducible small hairpin (sh)RNAs coupled to GFP inserted into mouse pancreatic tumor organoids derived from KPC mice. These shRNAs are directed against each of the major Kras effector molecules, including B-raf, C-raf, Pi3k, RalA, RalB, and Rac1, with Kras shRNAs and Renilla shRNA used as controls. We identified 2 effective shRNAs per target, with strong and durable inhibition of each protein observed after doxycycline treatment. To investigate the hierarchy of Kras effectors, we employed a competition assay of the shRNA organoids, and observed the drop-out of Kras, C-raf, and Rac1 shRNA-bearing tumor organoid lines. Cell number and volumetric analyses confirmed strong dependencies on Kras, C-raf, and Rac1. Chromatin accessibility and transcriptomic analyses of Kras- , C-raf- , and Rac1-depleted organoids revealed consensus features of abrogated tumor maintenance and key molecular nodes in tumor maintenance. Together, we have employed a mouse organoid platform to create a rigorous map of the requirements for the key effectors of mutant Kras, and to dissect the biologic mechanisms shared by effectors disruptive of the mutant Kras epigenetic and transcriptional landscape. In turn, we anticipate these findings can inform the subsequent development of novel therapies to address these vulnerabilities. Citation Format: Adrien Grimont, Zhen Zhao, Steven D. Leach, Rohit Chandwani. KrasG12D effector dependencies in the maintenance of pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr B20.

Sphingosine-1-phosphate promotes PDGF-dependent endothelial progenitor cell angiogenesis in human chondrosarcoma cells.

The malignant bone tumors that are categorized as chondrosarcomas display a high potential for metastasis in late-stage disease. Higher-grade chondrosarcomas contain higher levels of expression of platelet-derived growth factor (PDGF) and its receptor. The phosphorylation of sphingosine by sphingosine kinase enzymes SphK1 and SphK2 generates sphingosine-1-phosphate (S1P), which inhibits human chondrosarcoma cell migration, while SphK1 overexpression suppresses lung metastasis of chondrosarcoma. We sought to determine whether S1P mediates levels of PDGF-A expression and angiogenesis in chondrosarcoma. Surprisingly, our investigations found that treatment of chondrosarcoma cells with S1P and transfecting them with SphK1 cDNA increased PDGF-A expression and induced angiogenesis of endothelial progenitor cells (EPCs). Ras, Raf, MEK, ERK and AP-1 inhibitors and their small interfering RNAs (siRNAs) inhibited S1P-induced PDGF-A expression and EPC angiogenesis. Our results indicate that S1P promotes the expression of PDGF-A in chondrosarcoma via the Ras/Raf/MEK/ERK/AP-1 signaling cascade and stimulates EPC angiogenesis.

Activation of the RAS/B-RAF-MEK-ERK pathway in satellite glial cells contributes to substance p-mediated orofacial pain.

The cross talk between trigeminal ganglion (TG) neurons and satellite glial cells (SGCs) is crucial for the regulation of inflammatory orofacial pain. Substance P (SP) plays an important role by activating neurokinin (NK)-I receptors in this cross talk. The activation of extracellular signal-regulated kinase (ERK) 1/2, protein kinase A (PKA) and protein kinase C (PKC) in neurons and SGCs of peripheral ganglions by peripheral inflammation is associated with inflammatory hypersensitivity. This study tested the hypothesis that SP evoked SP-NK-I receptor positive feedback via the Renin-Angiotensin System/B-Protein Kinase A-Rapidly Accelerates Fibrosarcoma-MEK-Extracellular Signal-Regulated Kinase (RAS/PKA-RAF-MEK-ERK) pathway, which is involved in pain hypersensitivity. Inflammatory models were induced in vivo by injecting Complete Freund's adjuvant (CFA) into the whisker pad of rats. SP was administrated to SGCs in vitro for investigating, whether SP regulates the expression of NK-I receptor in the SGC nucleus. The effects of RAS-RAF-MEK, PKA and PKC pathways in this process were measured by co-incubating SGCs with respective Raf, PKA, PKC and MEK inhibitors in vitro and by pre-injecting these inhibitors into the TG in vivo. SP significantly upregulated NK-I receptor, p-ERK1/2, Ras, B-Raf, PKA and PKC in SGCs under inflammatory conditions. In addition, L703,606 (NK-I receptor antagonist), U0126 (MEK inhibitor), Sorafenib (Raf inhibitor) and H892HCL (PKA inhibitor) but not chelerythrine chloride (PKC inhibitor) significantly decreased NK-I mRNA and protein levels induced by SP. The allodynia-related behavior evoked by CFA was inhibited by pre-injection of L703,606, U0126, Sorafenib and H892HCL into the TG. Overall, SP upregulates NK-I receptor in TG SGCs via PKA/RAS-RAF-MEK-ERK pathway activation, contributing to a positive feedback of SP-NK-I receptor in inflammatory orofacial pain.

Abstract LB-B10: Targeting a new allosteric site modulates selectivity of BRAF dimer inhibition

Abstract BRAF mutants are present in ~50% of melanoma patients as well as in colorectal (~10%), thyroid carcinomas (~40%) and other malignancies. FDA-approved RAF inhibitors Vemurafenib, Dabrafenib and Encorafenib show remarkable responses in melanoma patients with BRAFV600E tumors. However, these drugs ultimately become ineffective due to adaptive and acquired resistance mechanisms, many of which depend on signaling by poorly inhibited BRAF homo- or hetero-dimers. In about 20-30% of melanoma tumors, intrinsic resistance is driven by a constitutive BRAF dimer splice variant, p61-BRAFV600E, while in resistant colorectal and thyroid tumors RAF dimerization is maintained by frequent RAS activation. An adaptive resistance mechanism depends on inhibitor-induced relief of ERK-mediated feedback pathway inhibition, which stimulates RAS-GTP and increases RAF dimers. Finally, oncogenic non-V600E BRAF species in some tumors signal as obligated dimers. Challenged by the clinical relevance of BRAF dimerization, we initiated a systematic quest for novel inhibitors that specifically recognize BRAFV600E dimers. We developed a phenotypic cellular assay to screen for inhibitors of ERK-signaling, using VEM-resistant melanoma cells that express p61-BRAFV600E dimers. We discovered new potent BRAF inhibitors, including a FDA approved kinase inhibitor. We obtained novel crystal structures of this drug with BRAFV600E and BRAFWT. We used these structural insights and scaffold-based drug-design methods to develop PHI1, a first-in-class αC-IN BRAF inhibitor. Remarkably, the crystal structure of BRAFV600E/PHI1 complex revealed that this compound targets a new BRAF allosteric site, which uniquely modulates the αC-helix conformation. In vitro and cellular studies showed that PHI1 is potent and selective against resistant cells with p61-BRAFV600E dimers. In addition, SAR analysis indicated that selectivity for dimer inhibition is lost upon modification of PHI1’s moiety targeting the allosteric pocket, suggesting that this site modulates specificity for BRAF dimers. Co-treatment experiments revealed that PHI1 inhibits the second site within the dimers with positive cooperativity driven from first site occupancy by αC-OUT inhibitors, in contrast to current RAF inhibitors that exhibit no, or negative, cooperativity. Based on these data, co-treatment of PHI1 with various clinical RAF and MEK inhibitors was explored in BRAF dimer-dependent tumors resulting in promising synergistic effects. This work provides a new class of allosteric BRAF inhibitors that may broadly diminish clinically untargeted oncogenic signaling dependent on BRAF dimers. Citation Format: Paul (Bogos) Agianian, Xiomaris M Cotto-Rios, Nadege Gitego, Emmanouil Zachariouadakis, Evripidis Gavathiotis. Targeting a new allosteric site modulates selectivity of BRAF dimer inhibition [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr LB-B10. doi:10.1158/1535-7163.TARG-19-LB-B10

Abstract A137: Drug screening and molecular profiling identifies INKA1 as a predictive biomarker for sensitivity to MAPK inhibition-antimitotic combination treatment in pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive solid tumors and one of the leading causes of cancer death world-wide. PDAC patients have the poorest prognosis with a median survival of less than 6 months and a 5-year survival rate of less than 5%. Despite detailed genetic mapping of PDAC, there are currently no effective targeted therapies for the disease. More than 95% of PDAC harbor an activating mutation in the KRAS gene. Several attempts on targeting each effector molecule in MEK- and PI3K- pathways, which are well-validated effector pathways in KRAS mutant cancer, have been made. However, none of the monotherapies have shown to be efficacious due to resistance and reprogramming. Hence, in this study we used a drug sensitivity based functional approach to identify combination strategies to render RAF/MEK/ERK- and PI3K/AKT/mTOR- targeted therapies efficacious against KRAS-driven PDAC. Thirty KRAS-mutant patient derived cell lines were screened against an in-house drug library of 525 clinically relevant small molecule anti-cancer agents in combination with RAF/MEK/ERK and PI3K/AKT/mTOR inhibitors, to identify synergistic/synthetic lethal partners to LY3009120 (RAF inhibitor), trametinib (MEK inhibitor), SCH772984 (ERK inhibitor), alpelisib (PI3K inhibitor), ipatasertib (AKT inhibitor), everolimus (mTOR inhibitor). Promising drug combinations from the initial screen were re-evaluated in 8X8 dose response matrices. In parallel, RNA sequencing was carried out on the cell lines to stratify the combination treatment strategies against PDAC. One of the most striking synergies seen was between the RAF/MEK/ERK inhibitors and mitosis/ microtubule-targeting agent vincristine. The combination of a MEK inhibitor trametinib and vincristine selectively induced cell death in a subset of the PDAC cell lines (25%). A correlation was observed between a selective effect of the combination and high expression levels of INKA1, a tumor suppressor and endogenous inhibitor of PAK4. The result therefore suggests that PAK4 activity is an important negative regulator of cell death triggered by the trametinib-vincristine combination. In summary, we identified trametinib + vincristine as a selective drug combination for a subset of KRAS-mutant PDAC with a putative predictive biomarker (INKA1 expression), which holds a potential for clinical translation, hence requires further exploration. Citation Format: Prson Gautam, Markus Vähä-Koskela, Max Tomlinson, Adrienne D. Cox, Channing J. Der, Tero Aittokallio, Krister Wennerberg. Drug screening and molecular profiling identifies INKA1 as a predictive biomarker for sensitivity to MAPK inhibition-antimitotic combination treatment in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A137. doi:10.1158/1535-7163.TARG-19-A137

Abstract LB-B09: ERK pulses drive non-genetic resistance in drug-adapted BRAFV600E melanoma cells

Abstract Anti-cancer drugs commonly target signal transduction proteins activated by mutation. In patients with BRAFV600E melanoma, small molecule RAF and MEK kinase inhibitors cause dramatic but often transient tumor regression. Emerging evidence suggests that cancer cells adapting by non-genetic mechanisms constitute a reservoir for the development of drug-resistant tumors. Here, we show that few hours after exposure to RAF/MEK inhibitors, BRAFV600E melanomas undergo adaptive changes involving disruption of negative feedback and sporadic pulsatile reactivation of the MAPK pathway, so that MAPK activity is transiently high enough in some cells to drive proliferation. ERK pulses are locally triggered by autocrine/paracrine factors in the local microenvironment so that ERK activity levels are low on average, but transiently high enough in patches of cells to explain the infrequent division characteristic of persister cells. Quantitative proteomics and computational modeling show that pulsatile MAPK reactivation is possible due to the co-existence in cells of two MAPK cascades: one driven by BRAFV600E that is drug-sensitive and a second driven by receptors that is drug-resistant. Paradoxically, this may account both for the frequent emergence of drug resistance and for the tolerability of RAF/MEK therapy in patients. Citation Format: Luca Gerosa, Christopher Chidley, Fabian Froehlich, Gabriela Sanchez, Sang Kyun Lim, Jeremy Muhlich, Jia-Yun Chen, Gregory J. Baker, Denis Schapiro, Tujin Shi, Lian Yi, Carrie D. Nicora, Wei-Jun Qian, H. Steven Wiley, Peter Sorger. ERK pulses drive non-genetic resistance in drug-adapted BRAFV600E melanoma cells [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr LB-B09. doi:10.1158/1535-7163.TARG-19-LB-B09