BRAFV600E Mutant Melanoma Cell Research Articles

Abstract 3078: Role of HuR in metabolic reprogramming of BRAF mutant melanoma treated with dabrafenib

Abstract Introduction: Oncogenic BRAF mutations are present in about 50% of melanoma and are clinically targetable by BRAF inhibitors such as dabrafenib. While BRAF inhibitors show high rates of clinical response, that response is transient and resistance inevitably develops. Previously we have demonstrated that BRAF inhibitors induce a metabolic shift towards the mitochondrial oxidative phosphorylation. We have shown that inhibition of wild type isocitrate dehydrogenase 1 (wtIDH1), a vital anaplerotic and anti-ROS enzyme, demonstrates synergy with dabrafenib in vitro and in vivo. IDH1 expression has been found in pancreatic cancer to be posttranscriptionally regulated by the RNA-binding and stress response protein, HuR (ELAVL1) under nutrient and chemotherapy stress. We hypothesize that HuR functions as a survival mechanism in melanoma under BRAF inhibition, regulating metabolic reprogramming including the expression of IDH1. Methods: The human BRAF V600E mutant melanoma cell line, A375, was used for all experiments. HuR and IDH1 expression was suppressed by siRNA oilgos through lipofectamine transfection, as validated by immunoblot analysis. The bioenergetic assay was conducted using Seahorse XFp mini extracellular analyzer to measure the oxygen consumption rate (OCR) under nutrient stress, both with and without dabrafenib exposure. The impact of HuR on IDH1 expression was further explored using qPCR and Immunoblot. Ivosidenib was used for pharmacologic inhibition of wtIDH as previously described. Results: Mitochondrial respiration (OCR) increased in parental A375 BRAF mutant melanoma cells following nutrient stress with low glucose conditions akin to the tumor microenvironment. OCR was further increased after dabrafenib exposure. Immunoblot analysis revealed that dabrafenib treated cells at 6-hour and 12-hour time points displayed HuR translocation from nucleus to cytoplasm, as detected in cytoplasmic lysates. Transient HuR silencing blocked the adaptive OCR response to both low glucose and BRAF inhibition. This suggests that HuR is involved in the metabolic response to acute BRAF inhibition in BRAF mutant melanoma. HuR cytoplasmic translocation is associated with increased IDH1 expression 24 hours after dabrafenib exposure. However, there is no increase in IDH1 levels under nutrient or treatment stress in HuR knockdown cells, indicating that IDH1 is regulated by HuR. Transient IDH1 knockdown, as well as pharmacologic IDH1 inhibition, blocks the OCR response of melanoma cells to BRAF inhibition and low glucose. This indicates that IDH1 is important in the HuR metabolic response to targeted therapy. Conclusion: We find that BRAF mutant melanoma cells adapt to acute dabrafenib exposure via HuR mediated expression of IDH1 to upregulate mitochondrial metabolism. This demonstrates a targeted therapy induced metabolic vulnerability that can be exploited by wtIDH1 inhibition. Citation Format: Alexander Loftus, Mehrdad Zarei, Hallie Graor, Omid Hajihassani, Christina Boutros, Jordan M. Winter, Luke D. Rothermel. Role of HuR in metabolic reprogramming of BRAF mutant melanoma treated with dabrafenib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3078.

USP18 enhances the resistance of BRAF-mutated melanoma cells to vemurafenib by stabilizing cGAS expression to induce cell autophagy

This study aims to discern the possible molecular mechanism of the effect of ubiquitin-specific peptidase 18 (USP18) on the resistance to BRAF inhibitor vemurafenib in BRAF V600E mutant melanoma by regulating cyclic GMP-AMP synthase (cGAS). The cancer tissues of BRAF V600E mutant melanoma patients before and after vemurafenib treatment were collected, in which the protein expression of USP18 and cGAS was determined. A BRAF V600E mutant human melanoma cell line (A2058R) resistant to vemurafenib was constructed with its viability, apoptosis, and autophagy detected following overexpression and depletion assays of USP18 and cGAS. Xenografted tumors were transplanted into nude mice for in vivo validation. Bioinformatics analysis showed that the expression of cGAS was positively correlated with USP18 in melanoma, and USP18 was highly expressed in melanoma. The expression of cGAS and USP18 was up-regulated in cancer tissues of vemurafenib-resistant patients with BRAF V600E mutant melanoma. Knockdown of cGAS inhibited the resistance to vemurafenib in A2058R cells and the protective autophagy induced by vemurafenib in vitro. USP18 could deubiquitinate cGAS to promote its protein stability. In vivo experimentations confirmed that USP18 promoted vemurafenib-induced protective autophagy by stabilizing cGAS protein, which promoted resistance to vemurafenib in BRAF V600E mutant melanoma cells. Collectively, USP18 stabilizes cGAS protein expression through deubiquitination and induces autophagy of melanoma cells, thereby promoting the resistance to vemurafenib in BRAF V600E mutant melanoma.

Abstract 4876: A systems pharmacology approach to discover synergistic targeted therapy combinations

Abstract The discovery of oncogenic signal transduction in cancer has enabled the development and clinical use of dozens of kinase-directed targeted therapies, but patient response is often transient and resistance invariably develops. In the setting of BRAF-mutant melanoma, for instance, a majority of patients relapse within one year of initiating targeted therapy. Combination therapies blocking compensatory signaling pathways provide a strategy for boosting treatment efficacy and delaying resistance. To find synergistic kinase inhibitor combinations, we used a multimodal systems pharmacology approach to quantify the early adaptive signaling and gene regulatory dynamics in patient-derived BRAFV600E-mutant melanoma cells under oncogene inhibition. Cells were treated with vemurafenib or vehicle and sampled longitudinally at timepoints spanning minutes to days. To measure signaling dynamics, thousands of protein phosphorylation sites covering commonly altered signaling axes in cancer were motif-enriched by immunoprecipitation, barcoded with isobaric tandem mass tags (TMT), and quantified by high-resolution mass spectrometry (MS). The resulting time-resolved phosphoproteomics data showed rapid (<15 minutes) and potent (>8-fold) downregulation of ERK1/2, confirming strong BRAF inhibition. Statistical integration of signaling measurements with time-series RNA-seq data collected at the same timepoints enabled reconstruction of multi-scale regulatory networks governing the adaptive response to oncogene inhibition. In particular, we observed early induction of SRC-family kinase (SFK) signaling and a broad cytoskeletal signaling module, implicating a compensatory prosurvival signaling program. The induction intensified over the 3-day treatment window and was fully reversed following a 6-day drug holiday, suggesting a reversible nature of drug adaptation. Immunoblotting confirmed sustained loss of ERK1/2 activity and concomitant elevated SFK phosphorylation following drug treatment. Accumulation of reactive oxygen species, a known activator of SFKs, strongly correlated with SFK activity, and both were ablated following treatment with the antioxidant precursor N-acetylcysteine. This adaptive response predicted a significant degree of synergy between vemurafenib and the pan-SFK inhibitor dasatinib, which was validated in a panel of patient-derived melanoma cell lines and in melanoma xenograft mouse models. Targeted MS of phospho-ERK1/2 and canonical SFK substrates, including CTTN, PXN, and PAG1, validated the mechanism of action of this combination. Complementary data from patient-derived non small-cell lung cancer (NSCLC) cells under tyrosine kinase inhibitor treatment demonstrates the generality of our integrative approach, and supports the notion that SFK activation may be a hallmark response to oncogenic RTK-RAS-ERK pathway inhibition. Citation Format: Cameron T. Flower, Chunmei Liu, James R. Heath, Wei Wei, Forest M. White. A systems pharmacology approach to discover synergistic targeted therapy combinations. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4876.

Abstract 396: Role of PHF8 in resistance to melanoma targeted therapies

Abstract Melanoma is a highly prevalent cancer with increasing incidence worldwide. While immune checkpoint inhibitors have drastically improved outcomes for about half of metastatic melanoma patients, many are still resistant to these treatments or develop life-threatening toxicities. Around 50% of melanomas carry BRAF mutations that result in constitutive activation of the MAPK pathway. Therapeutic combinations of BRAF and MEK inhibitors (MAPKi) have proven very efficient against these tumors, but responses are not durable and treatment-resistant recurrence almost inevitably occurs. Our group recently uncovered a role for the histone demethylase PHF8 in melanoma metastasis, and demonstrated that PHF8 directly controls the expression of key components of the TGFβ pathway. Given that TGFβ signaling crosstalks with the MAPK pathway and contributes to resistance to therapy in various tumors, we decided to investigate a potential role for PHF8 in response and resistance to MAPKi therapy in melanoma. To test our hypothesis, we assessed the effect of modulating PHF8 expression in BRAFV600E-mutant cells and their matched MAPKi-resistant derivatives. PHF8 silencing did not affect cell survival or proliferation of parental, MAPKi-sensitive melanoma cell lines, but it re-sensitized resistant cells to MAPKi treatment. Conversely, PHF8 overexpression in melanoma cell lines did not affect their proliferation rate but increased the emergence of resistant clones in BRAFV600E-mutant melanoma cells treated with BRAFi (Dabrafenib) and MEKi (Trametinib) combinations. Next, we assessed the dynamics of PHF8 expression and TGFβ signaling in melanoma cells exposed to MAPKi. Treatment of MAPKi-sensitive cells leads to a fast and complete downregulation of PHF8 protein expression without a change in mRNA levels, paralleled by reduced TGFβ signaling, as evidenced by decreased SMAD2 phosphorylation. However, PHF8 protein expression is restored in MAPKi resistant clones. Therefore, based on our preliminary data, we postulate that PHF8 suppression by the MAPK pathway plays a critical role in melanoma response to targeted therapy and that its restoration contributes to acquisition of resistance. We are currently investigating MAPK-dependent mechanisms controlling PHF8 stability, and testing novel PHF8 inhibitors for their ability to bypass or overcome melanoma resistance to MAPKi. Our studies might provide basis for a novel epigenetic therapy as a strategy to maximize the potential of MAPKi therapy in melanoma treatment. Citation Format: María de los Ángeles Gómez-Muñoz, Nicole Eskow, Ines Delclaux, Rana Moubarak, Eva Hernando-Monge. Role of PHF8 in resistance to melanoma targeted therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 396.

Erianin suppresses constitutive activation of MAPK signaling pathway by inhibition of CRAF and MEK1/2

Constitutive activation of RAS-RAF-MEK-ERK signaling pathway (MAPK pathway) frequently occurs in many cancers harboring RAS or RAF oncogenic mutations. Because of the paradoxical activation induced by a single use of BRAF or MEK inhibitors, dual-target RAF and MEK treatment is thought to be a promising strategy. In this work, we evaluated erianin is a novel inhibitor of CRAF and MEK1/2 kinases, thus suppressing constitutive activation of the MAPK signaling pathway induced by BRAF V600E or RAS mutations. KinaseProfiler enzyme profiling, surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), cellular thermal shift assay, computational docking, and molecular dynamics simulations were utilized to screen and identify erianin binding to CRAF and MEK1/2. Kinase assay, luminescent ADP detection assay, and enzyme kinetics assay were investigated to identify the efficiency of erianin in CRAF and MEK1/2 kinase activity. Notably, erianin suppressed BRAF V600E or RAS mutant melanoma and colorectal cancer cell by inhibiting MEK1/2 and CRAF but not BRAF kinase activity. Moreover, erianin attenuated melanoma and colorectal cancer in vivo. Overall, we provide a promising leading compound for BRAF V600E or RAS mutant melanoma and colorectal cancer through dual targeting of CRAF and MEK1/2.

Abstract 415: The characterization of in vitro models demonstrating a significant delay of acquired resistance to ulixertinib (ERK1/2)

Abstract Ulixertinib (BVD-523) is a first-in-class small molecule inhibitor of ERK1/2 currently being investigated in several oncology clinical trials, both as a single agent and in combination with other anti-cancer therapeutics. Clinical acquired resistance has been described by others for numerous efficacious small molecule agents. Relevant to the MAPK pathway, emergence of resistance to BRAF and MEK inhibitors limits their clinical efficacy. In vitro models of ulixertinib resistance were generated in an endeavor to characterize and predict potential clinical mechanisms of resistance and guide rational combination therapies. The BRAF-V600E mutant melanoma cell line, A375, was cultured in progressively increasing concentrations of ulixertinib or other MAPK pathway inhibitors (dabrafenib and/or trametinib). Drug-resistant A375 clones were readily obtained following growth in high concentrations of dabrafenib or trametinib. In contrast, developing resistance to ulixertinib proved challenging. Eventually, ulixertinib resistant clones emerged and demonstrated varying degrees of cross-resistance to other MAPK pathway inhibitors and displayed bell-shaped dose-response curves indicative of drug addiction for optimal proliferation. To elucidate potential mechanisms of resistance, Reverse Phase Protein Array profiling of 306 proteins was performed on parental A375 and ulixertinib-resistant clones following treatment with ulixertinib at varying concentrations and treatment times. Principal component analysis revealed clustering of resistant clones by ulixertinib treatment conditions (concentrations and duration). Differences between parental A375 and ulixertinib resistant clones were revealed, including components of MAPK, HER2, and autophagy markers. This work begins to tease out potential mechanisms of resistance to ulixertinib and guide potential combination partners that could circumvent acquired resistance. Citation Format: Deborah Knoerzer, Anupama Reddy, David Sorrell, Caroline M. Emery. The characterization of in vitro models demonstrating a significant delay of acquired resistance to ulixertinib (ERK1/2) [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 415.

Cytokine and Chemokine Receptor Patterns of Human Malignant Melanoma Cell Lines.

Cytokine and chemokine receptors can promote tumor progression, invasion, and metastasis development by inducing different intracellular signaling pathways. The aim of this study was to determine the cytokine and chemokine receptor gene expression patterns in human melanoma cell lines. We found a large set of cytokine and chemokine receptor genes that were significantly differentially expressed between melanoma cell lines that originated from different subtypes of primary melanomas as well as cell lines that originated from melanoma metastases. The relative expressions of two receptor genes (CCR2 and TNFRSF11B) were positively correlated with the invasive potential of the cell lines, whereas a negative correlation was observed for the TNFRSF14 gene expression. We also found a small set of receptor genes that exhibited a significantly decreased expression in association with a BRAFV600E mutation. Based on our results, we assume that the analyzed cytokine and chemokine receptor collection may provide potential to distinguish the different subtypes of melanomas, helping us to understand the biological behavior of BRAFV600E-mutated melanoma cells.

T Cells and CDDO-Me Attenuate Immunosuppressive Activation of Human Melanoma-Conditioned Macrophages.

Melanoma tumors are highly immunogenic, making them an attractive target for immunotherapy. However, many patients do not mount robust clinical responses to targeted therapies, which is attributable, at least in part, to suppression of immune responses by tumor-associated macrophages (TAMs) in the tumor microenvironment (TME). Using a human in vitro tri-culture system of macrophages with activated autologous T cells and BRAFV600E mutant melanoma cells, we now show that activated T cells and the synthetic triterpenoid the methyl ester of 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO-Me) attenuate immune suppression. Surface expression of CD206, CD16 and CD163 on melanoma-conditioned macrophages was inhibited by the addition of T cells, suggesting relief of immuno-suppressive macrophage activation. We also demonstrated that addition of CDDO-Me to tri-cultures enhanced T cell-mediated reductions in CCL2, VEGF and IL-6 production in a contact-independent manner. Because these results suggest CDDO-Me alters melanoma-conditioned macrophage activation, we interrogated CDDO-Me-mediated changes in macrophage signaling pathway activation. Our results indicated that CDDO-Me inhibited phosphorylation of STAT3, a known inducer of TAM activation. Collectively, our studies suggest that activated T cells and CDDO-Me synergistically relieve immune suppression in melanoma cultures and implicate the potential utility of CDDO-Me in the treatment of melanoma.

Quantifying ERK activity in response to inhibition of the BRAFV600E-MEK-ERK cascade using mathematical modelling

BackgroundSimultaneous inhibition of multiple components of the BRAF-MEK-ERK cascade (vertical inhibition) has become a standard of care for treating BRAF-mutant melanoma. However, the molecular mechanism of how vertical inhibition synergistically suppresses intracellular ERK activity, and consequently cell proliferation, are yet to be fully elucidated.MethodsWe develop a mechanistic mathematical model that describes how the mutant BRAF inhibitor, dabrafenib, and the MEK inhibitor, trametinib, affect BRAFV600E-MEK-ERK signalling. The model is based on a system of chemical reactions that describes cascade signalling dynamics. Using mass action kinetics, the chemical reactions are re-expressed as ordinary differential equations that are parameterised by in vitro data and solved numerically to obtain the temporal evolution of cascade component concentrations.ResultsThe model provides a quantitative method to compute how dabrafenib and trametinib can be used in combination to synergistically inhibit ERK activity in BRAFV600E-mutant melanoma cells. The model elucidates molecular mechanisms of vertical inhibition of the BRAFV600E-MEK-ERK cascade and delineates how elevated BRAF concentrations generate drug resistance to dabrafenib and trametinib. The computational simulations further suggest that elevated ATP levels could be a factor in drug resistance to dabrafenib.ConclusionsThe model can be used to systematically motivate which dabrafenib–trametinib dose combinations, for treating BRAFV600E-mutated melanoma, warrant experimental investigation.

FAM129B-dependent activation of NRF2 promotes an invasive phenotype in BRAF mutant melanoma cells.

Incidence of melanoma continues to rise in the United States with ~100,000 new cases diagnosed in 2019. While the 5-year survival rate of melanoma is 99% when localized, the rate of survival drops to 22.5% when distant disease is detected. As such, an area of great interest is understanding the mechanisms that promote melanoma metastasis so that better potential therapeutic targets can be discovered. Herein, we demonstrate that activation of NRF2 by FAM129B contributes to increased metastatic potential of BRAF V600E mutant melanoma cells. Specifically, FAM129B induces NRF2 by competing for Kelch-like ECH-associated protein 1 (KEAP1) binding (the negative regulator of NRF2) via an ETGE motif. Furthermore, we show that phosphorylation of FAM129B plays a role in mediating the interaction between FAM129B and KEAP1, as the phosphorylation status of FAM129B dictates its subcellular localization. When phosphorylated, FAM129B is found primarily in the cytosol where it can bind to KEAP1, but upon inhibition of mitogen-activated protein kinase kinase activity, FAM129B is localized to the cell membrane and no longer interacts with KEAP1. In BRAF V600E mutant melanoma, the mitogen-activated protein kinase pathway leads to hyperphosphorylation of FAM129B, and therefore FAM129B localizes to the cytosol, binds KEAP1, and upregulates NRF2. Importantly, genetic modulation or pharmacological inhibition that results in a decrease in FAM129B protein level or its phosphorylation decreases migration and invasion of mutant melanoma in an NRF2-dependent manner. Overall, these data indicate that phosphorylation of FAM129B plays a significant role in driving the metastatic potential of BRAF V600E melanoma via upregulation of the NRF2 signaling pathway.

Cell Proliferation Is Strongly Associated with the Treatment Conditions of an ER Stress Inducer New Anti-Melanoma Drug in Melanoma Cell Lines.

HA15 is a new anti-melanoma drug that triggers endoplasmic reticulum (ER) stress and causes deleterious effects on melanoma cell viability due to autophagy and apoptosis, regardless of driver mutations or drug resistance. In this study, we investigated the effect of HA15 on the viability/proliferation of BRAFV600E-mutant melanoma cells using different culture conditions. In contrast to the published data, we did not detect significant melanoma cell death under normal culture conditions using HA15 treatment. Indeed, only cells that were cultured under long-term starvation conditions were sensitive to the drug. Quantitative measurements of ER stress and autophagy markers showed that the compound HA15 does not trigger stress alone but synergistically enhances ER stress under starvation conditions. Importantly, we observed that the viability of normal melanocytes decreased significantly with treatment, even at low HA15 concentrations. Finally yet importantly, we were able to generate HA15-resistant cell lines, which failed by Cerezo et al. In summary, HA15 only influences the viability of cells that are starved for several hours before and during treatment. However, this in vitro setting is far from the in vivo conditions. In addition, our data clearly show that melanoma cells can acquire HA15 resistance. Further studies are needed to prove that HA15 is an effective anti-cancer agent.

Single-cell trajectories of melanoma cell resistance to targeted treatment.

Objective:Cellular heterogeneity is regarded as a major factor affecting treatment response and resistance in malignant melanoma. Recent developments in single-cell sequencing technology have provided deeper insights into these mechanisms.Methods:Here, we analyzed a BRAFV600E-mutant melanoma cell line by single-cell RNA-seq under various conditions: cells sensitive to BRAF inhibition with BRAF inhibitor vemurafenib and cells resistant to BRAF inhibition with vemurafenib alone or vemurafenib in combination with the MEK1/2 inhibitors cobimetinib or trametinib. Dimensionality reduction by t-distributed stochastic neighbor embedding and self-organizing maps identified distinct trajectories of resistance development clearly separating the 4 treatment conditions in cell and gene state space.Results:Trajectories associated with resistance to single-agent treatment involved cell cycle, extracellular matrix, and de-differentiation programs. In contrast, shifts detected in double-resistant cells primarily affected translation and mitogen-activated protein kinase pathway reactivation, with a small subpopulation showing markers of pluripotency. These findings were validated in pseudotime analyses and RNA velocity measurements.Conclusions:The single-cell transcriptomic analyses reported here employed a spectrum of bioinformatics methods to identify mechanisms of melanoma resistance to single- and double-agent treatments. This study deepens our understanding of treatment-induced cellular reprogramming and plasticity in melanoma cells and identifies targets of potential relevance to the management of treatment resistance.

LGG-48. PROLIFIC GROWTH OF BRAF V600E MUTANT PILOCYTIC ASTROCYTOMA WHILE ON KETOGENIC DIET: CASE REPORT

Epilepsy is a common diagnosis among pediatric patients with supratentorial tumors, particularly infiltrating gliomas. The ketogenic diet can be a successful antiepileptic therapy for patients with medically intractable epilepsy. Acetoacetate, a main ketone released during ketosis, has been shown to increase BRAFV600E binding to MEK1 and MEK1 phosphorylation in BRAFV600E mutant melanoma cells, thereby promoting proliferation and tumor growth (Kang et al, 2015, Xia et al, 2017). BRAFV600E mutation is common in pediatric low-grade gliomas. Therefore, use of a ketogenic diet to manage coincident epilepsy could, in theory, promote tumor growth. However, this has not been previously reported in a human patient. We present a 3 year-old male with a non-NF1 optic pathway BRAFV600E mutant pilocytic astrocytoma and medically intractable epilepsy. He was treated with carboplatin and bevacizumab for 1 year, with good tumor response and vision recovery. He showed stable, non-progressive disease for several months. He was placed on a ketogenic diet 9 months into treatment and became seizure free. Tumor recurrence occurred at 8 months off therapy, at which time a biopsy demonstrated BRAFV600E mutation. The tumor progressed rapidly despite treatment with Trametinib and Dabrafenib, leading to salvage therapy with carboplatin, vinblastine, bevacizumab, and XRT, without tumor control. We discuss the potential effect of the ketogenic diet on this patient’s outcome.

Abstract PR17: Sleeping Beauty mutagenesis reveals a Src-dependent DBL GEF family signaling mechanism driving MAPK inhibitor resistance in BRAF mutant melanoma

Abstract BRAF/MEK inhibition remains an important treatment option for patients with BRAF V600 mutant melanoma who show disease progression on immunotherapy; however, the majority of patients treated with BRAF/MEK (MAPKi) therapy develop MAPKi-resistant disease within two years of initiating treatment. Genomic analysis of drug-resistant melanomas has identified somatic mutations associated with resistance, including BRAF amplification or truncation and RAS mutation. In addition, other forms of acquired resistance involving adaptation and reprogramming have been described, with different studies identifying different drivers in different melanoma cell lines and patient-derived xenografts. The heterogeneity of acquired resistance mechanisms presents a major clinical challenge for identifying novel drug combinations with potential activity in significant subsets of patients. To enable the rapid identification of context-dependent drivers of MAPKi resistance, we adapted our Sleeping Beauty (SB) transposon mutagenesis for use in cell culture. SB mutagenesis in this setting identifies predominantly gain-of-function mechanisms and thus has the ability to identify both genetic mutations (such as expression of truncated proteins) and genes within alternative acquired resistance pathways whose upregulated expression drives MAPKi resistance. Validating our approach, our screen identified N-terminal truncation of BRAF—a known mechanism of vemurafenib resistance. In addition, we identified DBL family guanine exchange factors (GEFs), MCF2 and VAV1, as drivers of MAPKi resistance that we have functionally validated in multiple BRAF V600E mutant melanoma cell lines. Crucially, many DBL family GEFs are well known to be regulated by Src family kinases (SFKs), and our data indicate that GEF-driven MAPKi resistance can be blocked by combination treatment with vemurafenib and saracatinib, a selective SFK inhibitor. Expansion of resistant cells in the presence of vemurafenib or vemurafenib with cobimetinib can be reversed by switching to vemurafenib plus saracatinib. Consistently, we find that adding saracatinib converts MAPKi-mediated cytostasis into cell killing and blocks spontaneous MAPKi resistance in responsive melanoma cell lines. The GEFs signal downstream to a RAC1-PAK kinase module that is required for GEF-driven resistance. Importantly, the potential utility of both SFK inhibitors and PAK inhibitors for overcoming MAPKi resistance has been proposed, but the connection between SFK-dependent and PAK-dependent resistance mechanisms had not been obvious. We show that combined BRAF/SFK inhibition is effective in cell lines in which the GEF-driven mechanism operates, but not in cell lines in which the GEFs do not drive resistance. Our SB mutagenesis system has the ability to rapidly survey the context-dependent landscape of MAPKi resistance in any transfectable cell system, with the potential to reveal connections between seemingly disparate resistance mechanisms. This abstract is also being presented as Poster B04. Citation Format: Charlotte R. Feddersen, Jacob Schillo, Afshin Varzavand, Hayley Vaughn, Andrew Voight, Eliot Zhu, Jesse D Riordan, Christopher S. Stipp, Adam J. Dupuy. Sleeping Beauty mutagenesis reveals a Src-dependent DBL GEF family signaling mechanism driving MAPK inhibitor resistance in BRAF mutant melanoma [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr PR17.

Abstract B08: Identification and characterization of Rho family GTPases as drivers of drug resistance in BRAFV600 mutant melanoma

Abstract The serine/threonine protein kinase BRAF is mutated in approximately 50% of cutaneous melanomas, leading to hyperactivation of the MAPK/ERK pathway. The most common mutations, BRAFV600, can be targeted by selective kinase inhibitors, such as vemurafenib. Although initial clinical response to BRAF inhibition (BRAFi) is encouraging, 90% of patients develop drug resistance within a few months. Drug resistance can be delayed, but not prevented, by combining BRAFi with an MEK inhibitor (MEKi), such as cobimetinib. While some resistance mechanisms are known, disease progression on drug cannot be explained in all patients. We performed a gain-of-function mutagenesis screen utilizing the Sleeping Beauty transposon system to identify novel drivers of resistance in BRAFV600E mutant melanoma cells sensitive to current therapies. We chose four of the top candidates from our screen and validated the ability to drive resistance to both vemurafenib and vemurafenib-cobimetinib combination treatment in multiple melanoma cell lines. In an effort to determine the broader role of candidate vemurafenib-resistance drivers, we conducted an additional in vivo mutagenesis screen, of which genetic analysis is ongoing. Our initial cell-based screen identified two members of the Dbl family of guanine nucleotide exchange factors (GEFs), VAV1 and MCF2, as candidate drivers of vemurafenib resistance. A375 melanoma cells overexpressing VAV1 or MCF2 maintain significant growth under vemurafenib treatment, while control cells do not. Functional tests of VAV1 and MCF2 identified that the active form of two Rho family members, RAC1 and CDC42, increases following treatment with vemurafenib, suggesting a PAK-mediated pathway of resistance. In addition, all candidates that were tested elevated ERK signaling in the presence of vemurafenib. Many of the extracellular signaling pathways known to drive increased vemurafenib resistance activate Rho signaling. Our results suggest that Dbl family members may play an important role in this process. Understanding how Rho activation occurs and its consequences for drug resistance in melanoma will provide critical insights into the design and validation of future targeted therapies. Citation Format: Jacob L. Schillo, Charlotte R. Feddersen, Afshin Varzavand, Hayley R. Vaughn, Lexy S. Wadsworth, Andrew P. Voigt, Eliot Y. Zhu, Jesse D. Riordan, Christopher S. Stipp, Adam J. Dupuy. Identification and characterization of Rho family GTPases as drivers of drug resistance in BRAFV600 mutant melanoma [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr B08.

MicroRNA-211 Modulates the DUSP6-ERK5 Signaling Axis to Promote BRAFV600E-Driven Melanoma Growth In Vivo and BRAF/MEK Inhibitor Resistance

MicroRNAs (miRs) are important posttranscriptional regulators of cell fate in both normal and disease states. miR-211 has previously been shown to be a direct regulator of metabolism in BRAFV600E-mutant melanoma cells invitro. Here, we report that miR-211 expression promotes the aggressive growth of BRAFV600E-mutant melanoma xenografts invivo. miR-211 promoted proliferation through the posttranscriptional activation of extracellular signal-regulated kinase (ERK) 5 signaling, which has recently been implicated in the resistance to BRAF and MAPK/ERK kinase inhibitors. We therefore examined whether miR-211 similarly modulated melanoma resistance to the BRAF inhibitor vemurafenib and the MAPK/ERK kinase inhibitor cobimetinib. Consistent with this model, miR-211 expression increased melanoma cell resistance to both the inhibitors, and this resistance was associated with an increased ERK5 phosphorylation. miR-211 mediates these effects by directly inhibiting the expression of DUSP6, an ERK5 pathway-specific phosphatase and now shown to be an miR-211 target gene. These results dissect the role of the miR-211-DUSP6-ERK5 axis in melanoma tumor growth and suggest a mechanism for the development of drug-resistant tumors and a target for overcoming resistance.

Reprograming of Tumor-Associated Macrophages in Human BrafV600EMutant Melanoma

Abstract Melanoma tumors are highly immunogenic, making them an attractive target for immunotherapy. However, many patients do not mount robust clinical responses to targeted therapies, which is attributable, at least in part, to suppression of immune responses by tumor-associated macrophages (TAMs) in the tumor microenvironment (TME). Using a human in vitro culture system, we have shown that the synthetic triterpenoid CDDO-Me enhances immune activation by reprogramming macrophages from immuno-suppressive to immuno-stimulatory. CDDO-Me significantly reduced CCL2, VEGF and IL-6 secretion by macrophages and inhibited surface expression of CD163, a marker associated with poor clinical outcomes. Furthermore, CDDO-Me mediated both contact-dependent and independent effects on macrophage activation in tri-cultures of macrophages with activated autologous T cells and BRAFV600E mutant melanoma cells. Signaling pathway activation was interrogated to identify the potential mechanism by which CDDO-Me attenuates the pro-tumorigenic macrophage activation profile. Our results demonstrated that CDDO-Me inhibited STAT3 phosphorylation, which is a known regulator of TAM activation. Given these results, we hypothesize that, in addition to mediating direct anti-tumorigenic effects, CDDO-Me may also enhance the efficacy of additional immunotherapies, including BRAF inhibition. Collectively, our studies suggest that the redirection of immuno-suppressive myeloid cell activation may provide both a direct means of inhibiting melanoma growth and may enhance the efficacy of additional targeted immunotherapies through relief of immune suppression in the TME.

Identification of Small Molecule Enhancers of Immunotherapy for Melanoma

Small molecule based targeted therapies for the treatment of metastatic melanoma hold promise but responses are often not durable, and tumors frequently relapse. Response to adoptive cell transfer (ACT)-based immunotherapy in melanoma patients are durable but patients develop resistance primarily due to loss of antigen expression. The combination of small molecules that sustain T cell effector function with ACT could lead to long lasting responses. Here, we have developed a novel co-culture cell-based high throughput assay system to identify compounds that could potentially synergize or enhance ACT-based immunotherapy of melanoma. A BRAFV600E mutant melanoma cell line, SB-3123p which is resistant to Pmel-1-directed ACT due to low gp100 expression levels was used to develop a homogenous time resolve fluorescence (HTRF), screening assay. This high throughput screening assay quantitates IFNγ released upon recognition of the SB-3123p melanoma cells by Pmel-1 CD8+ T-cells. A focused collection of approximately 500 small molecules targeting a broad range of cellular mechanisms was screened, and four active compounds that increased melanoma antigen expression leading to enhanced IFNγ production were identified and their in vitro activity was validated. These four compounds may provide a basis for enhanced immune recognition and design of novel therapeutic approaches for patients with BRAF mutant melanoma resistant to ACT due to antigen downregulation.

T-Type Calcium Channels as Potential Therapeutic Targets in Vemurafenib-Resistant BRAFV600E Melanoma

Melanoma is a malignant neoplasia that is highly resistant to chemotherapy and radiotherapy and is associated with poor prognosis in advanced stage. Targeting melanoma that harbors the common BRAFV600E mutation with kinase inhibitors, such as vemurafenib, reduces tumor burden, but these tumors frequently acquire resistance to these drugs. We previously proposed that T-type calcium channel (TTCC) expression may serve as a biomarker for melanoma progression and prognosis, and we showed that TTCC blockers reduce migration and invasion rates because of autophagy blockade only in BRAFV600E-mutant melanoma cells. Here, we demonstrated that high expression of the TTCC Cav3.1 isoform is related to autophagic status in vemurafenib-resistant BRAFV600E-mutant melanoma cells and human biopsies, and in silico analysis revealed an enrichment of Cav3.1 expression in post-treatment melanomas. We also demonstrated that the TTCC blocker mibefradil induces apoptosis and impairs migration and invasion via inhibition of autophagy in resistant melanoma cells and mouse xenograft models. Moreover, we identified an association between PTEN status and Cav3.1 expression in these cells as a marker of sensitivity to combination therapy in resistant cells. Together, our results suggest that TTCC blockers offer a potential targeted therapy in resistant BRAFV600E-mutant melanoma and a therapeutic strategy to reduce progression toward BRAF inhibitor resistance.

A Fatty Acid Oxidation-dependent Metabolic Shift Regulates the Adaptation of BRAF-mutated Melanoma to MAPK Inhibitors.

Treatment of BRAFV600E -mutant melanomas with MAPK inhibitors (MAPKi) results in significant tumor regression, but acquired resistance is pervasive. To understand nonmutational mechanisms underlying the adaptation to MAPKi and to identify novel vulnerabilities of melanomas treated with MAPKi, we focused on the initial response phase during treatment with MAPKi. By screening proteins expressed on the cell surface of melanoma cells, we identified the fatty acid transporter CD36 as the most consistently upregulated protein upon short-term treatment with MAPKi. We further investigated the effects of MAPKi on fatty acid metabolism using in vitro and in vivo models and analyzing patients' pre- and on-treatment tumor specimens. Melanoma cells treated with MAPKi displayed increased levels of CD36 and of PPARα-mediated and carnitine palmitoyltransferase 1A (CPT1A)-dependent fatty acid oxidation (FAO). While CD36 is a useful marker of melanoma cells during adaptation and drug-tolerant phases, the upregulation of CD36 is not functionally involved in FAO changes that characterize MAPKi-treated cells. Increased FAO is required for BRAFV600E -mutant melanoma cells to survive under the MAPKi-induced metabolic stress prior to acquiring drug resistance. The upfront and concomitant inhibition of FAO, glycolysis, and MAPK synergistically inhibits tumor cell growth in vitro and in vivo. Thus, we identified a clinically relevant therapeutic approach that has the potential to improve initial responses and to delay acquired drug resistance of BRAFV600E -mutant melanoma.