Acquired Vemurafenib Resistance Research Articles

Vemurafenib and Obinutuzumab as Frontline Therapy for Hairy Cell Leukemia

BackgroundHairy cell leukemia (HCL) is characterized by the underlying genetic lesion of BRAFV600E and responsiveness to BRAF inhibitors. We assessed the safety and activity of the BRAF inhibitor vemurafenib combined with obinutuzumab in patients with previously untreated HCL.MethodsWe conducted a single-arm, multicenter clinical study of vemurafenib plus obinutuzumab. Vemurafenib 960 mg twice daily was administered for four cycles, and obinutuzumab was administered in cycles 2 to 4. The primary end point was complete remission (CR). Secondary end points included assessment of safety, minimal residual disease (MRD), and BRAF allele burden according to digital droplet polymerase chain reaction (ddPCR).ResultsThirty patients were enrolled in the study, and 27 patients completed all four cycles of treatments and achieved CR (90%; 95% confidence interval [CI], 73 to 98). Three patients discontinued the study early because of adverse events and were not evaluable for response. Of the 27 patients who achieved CR, 26 patients (96%; 95% CI, 81 to 99) achieved MRD negativity. BRAFV600E allele was undetectable by ddPCR in all 21 evaluable patients. At a median follow-up of 34.9 months (95% CI, 29.6 to 36.9), no patient experienced disease relapse. The most common vemurafenib-related adverse events were rash and arthralgia. Febrile neutropenia occurred in two patients, and blood or platelet transfusions were required in two patients.ConclusionsCombined time-limited vemurafenib and obinutuzumab achieved CR in more than 90% of patients with previously untreated HCL. In this small study, acquired vemurafenib resistance or dose-limiting toxicity was not observed. Patients were not observed long enough to reveal secondary malignancies. (Funded by the National Cancer Institute and others; ClinicalTrials.gov number, NCT03410875.)

Evolving Acquired Vemurafenib Resistance in a BRAF V600E Mutant Melanoma PDTX Model to Reveal New Potential Targets.

Malignant melanoma is challenging to treat, and metastatic cases need chemotherapy strategies. Targeted inhibition of commonly mutant BRAF V600E by inhibitors is efficient but eventually leads to resistance and progression in the vast majority of cases. Numerous studies investigated the mechanisms of resistance in melanoma cell lines, and an increasing number of in vivo or clinical data are accumulating. In most cases, bypassing BRAF and resulting reactivation of the MAPK signaling, as well as alternative PI3K-AKT signaling activation are reported. However, several unique changes were also shown. We developed and used a patient-derived tumor xenograft (PDTX) model to screen resistance evolution in mice in vivo, maintaining tumor heterogeneity. Our results showed no substantial activation of the canonical pathways; however, RNAseq and qPCR data revealed several altered genes, such as GPR39, CD27, SLC15A3, IFI27, PDGFA, and ABCB1. Surprisingly, p53 activity, leading to apoptotic cell death, was unchanged. The found biomarkers can confer resistance in a subset of melanoma patients via immune modulation, microenvironment changes, or drug elimination. Our resistance model can be further used in testing specific inhibitors that could be used in future drug development, and combination therapy testing that can overcome inhibitor resistance in melanoma.

Identification of Dihydrolipoamide Dehydrogenase as Potential Target of Vemurafenib-Resistant Melanoma Cells.

Despite recent improvements in therapy, the five-year survival rate for patients with advanced melanoma is poor, mainly due to the development of drug resistance. The aim of the present study was to investigate the mechanisms underlying this phenomenon, applying proteomics and structural approaches to models of melanoma cells. Sublines from two human (A375 and SK-MEL-28) cells with acquired vemurafenib resistance were established, and their proteomic profiles when exposed to denaturation were identified through LC-MS/MS analysis. The pathways derived from bioinformatics analyses were validated by in silico and functional studies. The proteomic profiles of resistant melanoma cells were compared to parental counterparts by taking into account protein folding/unfolding behaviors. Several proteins were found to be involved, with dihydrolipoamide dehydrogenase (DLD) being the only one similarly affected by denaturation in all resistant cell sublines compared to parental ones. DLD expression was observed to be increased in resistant cells by Western blot analysis. Protein modeling analyses of DLD's catalytic site coupled to in vitro assays with CPI-613, a specific DLD inhibitor, highlighted the role of DLD enzymatic functions in the molecular mechanisms of BRAFi resistance. Our proteomic and structural investigations on resistant sublines indicate that DLD may represent a novel and potent target for overcoming vemurafenib resistance in melanoma cells.

Quantitative Proteomics Links the Intermediate Filament Nestin to Resistance to Targeted BRAF Inhibition in Melanoma Cells

Targeted inhibition of mutated kinases using selective MAP kinase inhibitors in malignant melanoma often results in temporary improvement of clinical symptoms followed by rapid development of resistance. To gain insights in molecular processes that govern resistance, we performed SILAC-based quantitative proteomics profiling of vemurafenib-resistant and -sensitive melanoma cells. Among downregulated proteins in vemurafenib-resistant cell lines we detected multiple proteins involved in cytoskeletal organization and signaling, including the intermediate filament nestin, which was one of the most downregulated proteins. Previous studies showed that nestin is expressed in various types of solid tumors and its abundance correlates with malignant phenotype of transformed cells. However, the role of nestin in cancer cells regarding acquired resistance is still poorly understood. We performed CRISPR/Cas9 knockout of the nestin gene (NES) in vemurafenib-sensitive cells and showed that loss of nestin leads to increased cellular proliferation and colony formation upon treatment with BRAFV600E and MEK inhibitors. Moreover, nestin depletion led to increased invasiveness and metalloproteinase activity like the phenotype of melanoma cells with acquired resistance to the BRAF inhibitor. Finally, phosphoproteome analysis revealed that nestin depletion influenced signaling through integrin and PI3K/AKT/mTOR pathways and led to increased focal adhesion kinase abundance and phosphorylation. Taken together, our results reveal that nestin is associated with acquired vemurafenib resistance in melanoma cells.

An autophagy-driven pathway of ATP secretion supports the aggressive phenotype of BRAFV600E inhibitor-resistant metastatic melanoma cells

ABSTRACTThe ingrained capacity of melanoma cells to rapidly evolve toward an aggressive phenotype is manifested by their increased ability to develop drug-resistance, evident in the case of vemurafenib, a therapeutic-agent targeting BRAFV600E. Previous studies indicated a tight correlation between heightened melanoma-associated macroautophagy/autophagy and acquired Vemurafenib resistance. However, how this vesicular trafficking pathway supports Vemurafenib resistance remains unclear. Here, using isogenic human and murine melanoma cell lines of Vemurafenib-resistant and patient-derived melanoma cells with primary resistance to the BRAFV600E inhibitor, we found that the enhanced migration and invasion of the resistant melanoma cells correlated with an enhanced autophagic capacity and autophagosome-mediated secretion of ATP. Extracellular ATP (eATP) was instrumental for the invasive phenotype and the expansion of a subset of Vemurafenib-resistant melanoma cells. Compromising the heightened autophagy in these BRAFV600E inhibitor-resistant melanoma cells through the knockdown of different autophagy genes (ATG5, ATG7, ULK1), reduced their invasive and eATP-secreting capacity. Furthermore, eATP promoted the aggressive nature of the BRAFV600E inhibitor-resistant melanoma cells by signaling through the purinergic receptor P2RX7. This autophagy-propelled eATP-dependent autocrine-paracrine pathway supported the maintenance and expansion of a drug-resistant melanoma phenotype. In conclusion, we have identified an autophagy-driven response that relies on the secretion of ATP to drive P2RX7-based migration and expansion of the Vemurafenib-resistant phenotype. This emphasizes the potential of targeting autophagy in the treatment and management of metastatic melanoma.

Preclinical efficacy of a RAF inhibitor that evades paradoxical MAPK pathway activation in protein kinase BRAF-mutant lung cancer

Oncogenic activation of protein kinase BRAF drives tumor growth by promoting mitogen-activated protein kinase (MAPK) pathway signaling. Because oncogenic mutations in BRAF occur in ∼2-7% of lung adenocarcinoma (LA), BRAF-mutant LA is the most frequent cause of BRAF-mutant cancer mortality worldwide. Whereas most tumor types harbor predominantly the BRAFV600E-mutant allele, the spectrum of BRAF mutations in LA includes BRAFV600E (∼60% of cases) and non-V600E mutant alleles (∼40% of cases) such as BRAFG469A and BRAFG466V The presence of BRAFV600E in LA has prompted clinical trials testing selective BRAF inhibitors such as vemurafenib in BRAFV600E-mutant patients. Despite promising clinical efficacy, both innate and acquired resistance often result from reactivation of MAPK pathway signaling, thus limiting durable responses to the current BRAF inhibitors. Further, the optimal therapeutic strategy to block non-V600E BRAF-mutant LA remains unclear. Here, we report the efficacy of the Raf proto-oncogene serine/threonine protein kinase (RAF) inhibitor, PLX8394, that evades MAPK pathway reactivation in BRAF-mutant LA models. We show that PLX8394 treatment is effective in both BRAFV600E and certain non-V600 LA models, in vitro and in vivo. PLX8394 was effective against treatment-naive BRAF-mutant LAs and those with acquired vemurafenib resistance caused by an alternatively spliced, truncated BRAFV600E that promotes vemurafenib-insensitive MAPK pathway signaling. We further show that acquired PLX8394 resistance occurs via EGFR-mediated RAS-mTOR signaling and is prevented by upfront combination therapy with PLX8394 and either an EGFR or mTOR inhibitor. Our study provides a biological rationale and potential polytherapy strategy to aid the deployment of PLX8394 in lung cancer patients.

A Novel Plant Sesquiterpene Lactone Derivative, DETD-35, Suppresses BRAFV600E Mutant Melanoma Growth and Overcomes Acquired Vemurafenib Resistance in Mice.

Acquired resistance to vemurafenib develops through reactivation of RAF/MEK/ERK signaling or bypass mechanisms. Recent combination therapies such as a MEK inhibitor combined with vemurafenib show improvement in major clinical end points, but the percentage of patients with adverse toxic events is higher than with vemurafenib monotherapy and most patients ultimately relapse. Therefore, there is an urgent need to develop new antimelanoma drugs and/or adjuvant agents for vemurafenib therapy. In this study, we created a novel semiorganically modified derivative, DETD-35, from deoxyelephantopin (DET), a plant sesquiterpene lactone demonstrated as an anti-inflammatory and anti-mammary tumor agent. Our results show that DETD-35 inhibited proliferation of a panel of melanoma cell lines, including acquired vemurafenib resistance A375 cells (A375-R) established in this study, with superior activities to DET and no cytotoxicity to normal melanocytes. DETD-35 suppressed tumor growth and reduced tumor mass as effectively as vemurafenib in A375 xenograft study. Furthermore, DETD-35 also reduced tumor growth in both acquired (A375-R) and intrinsic (A2058) vemurafenib resistance xenograft models, where vemurafenib showed no antitumor activity. Notably, the combination of DETD-35 and vemurafenib exhibited the most significant effects in both in vitro and in vivo xenograft studies due to synergism of the compound and the drug. Mechanistic studies suggested that DETD-35 overcame acquired vemurafenib resistance at least in part through deregulating MEK-ERK, Akt, and STAT3 signaling pathways and promoting apoptosis of cancer cells. Overall, our results suggest that DETD-35 may be useful as a therapeutic or adjuvant agent against BRAF(V600E) mutant and acquired vemurafenib resistance melanoma. Mol Cancer Ther; 15(6); 1163-76. ©2016 AACR.

Highlights of This Issue

In cancer treatment, drug combinations can convert weakly efficacious monotherapies into regimens that produce robust antitumor activity. O'Neil, Benita, Feldman, and colleagues performed a high-throughput oncology drug combination screen of 583 doublet combinations and found that synergy is relatively rare. However, both well-known and novel synergistic drug combinations were identified. One novel combination, a Wee1 inhibitor and an mTOR inhibitor, potently and synergistically inhibits cancer cell growth both in vitro and in vivo and is well tolerated in vivo. This approach identified novel combinations with improved efficacy that would be difficult to reliably predict based on our current understanding of cancer cell biology.Durable responses of BRAFV600E inhibitor vemurafenib have been hampered by drug resistance in human melanoma. Here, Feng and colleagues created compound DETD-35, a derivative of plant sesquiterpene lactone deoxyelephantopin, which significantly inhibits intrinsic and acquired vemurafenib resistance tumor growth in xenograft mice through suppression of MEK/ERK signaling reactivation and promoting ROS-induced apoptosis in melanoma cells. The antitumor effect of DETD-35 combined with vemurafenib is superior to vemurafenib or DETD-35 treatment alone. These data suggest the therapeutic potential of DETD-35 in BRAFV600E mutant or acquired vemurafenib resistance melanoma as a single or adjuvant agent.Antibody–drug conjugates (ADC) offer increased therapeutic efficacy with reduced side effects compared with chemotherapy. Currier, Ackerman and colleagues explore the role of alternative tumor targeting agents formed through fusion of a disulfide-rich engineered integrin-binding peptide, known as a knottin, to an antibody Fc domain. Knottin–Fc–drug conjugates (KFDC), attached to monomethyl-auristatin-F through a PEG linker, were synthesized via a non-natural amino acid incorporated during cell-free protein synthesis. The KFDC significantly prolonged tumor regression and survival in a xenograft model compared with a nontargeted conjugate or drug alone, thus providing a basis for further development of KFDCs for targeted drug delivery applications.In the present study, Morrison and colleagues discovered for the first time that the transmembrane protein SLITRK6 is overexpressed in bladder cancer. Moreover, the authors investigated SLITRK6 as a target for antibody–drug conjugate (ADC) treatment of advanced urothelial cancer. AGS15E, a SLITRK6-specific ADC conjugated with Monomethyl Auristatin E turned out to be the most potent among all ADCs generated. When compared with its Momomethyl Auristatin F counterpart, AGS15E showed superior antitumor activity in SLITRK6-highly expressed cancer xenografts as well as others with a broad range of SLITRK6 expression. AGS15E is currently in Phase I clinical trials for advanced urothelial cancer.

A Novel Plant Sesquiterpene Lactone Derivative, DETD-35, Suppresses BRAF V600E Mutant Melanoma Growth and Overcomes Acquired Vemurafenib Resistance in Mice

A Novel Plant Sesquiterpene Lactone Derivative, DETD-35, Suppresses BRAF V600E Mutant Melanoma Growth and Overcomes Acquired Vemurafenib Resistance in Mice

Abstract B175: A novel plant sesquiterpene lactone derivative DETD suppresses BRAFV600E mutant melanoma growth and overcomes acquired vemurafenib resistance in mice

Abstract Melanoma is the most lethal form of skin cancer and cases are increasing year by year. BRAFV600E mutation is found approximately in 50% of melanoma patients that drives MAPK (RAF/MEK/ERK) signaling for cancer progression. Vemurafenib, a small molecule that specifically targets BRAFV600E mutation, received FDA approval for treatment of late-stage melanoma in 2011. Unfortunately, patients develop acquired vemurafenib resistance via reactivation of MAPK signaling or bypass mechanisms. Recent combination therapy such as MEK inhibitor combined with vemurafenib shows improvement in major clinical end points but percentage of patients with adverse toxic events are higher compared to vemurafenib monotherapy and most patients relapse ultimately. It is therefore an urgent need to develop new anti-melanoma drug and/or adjuvant agent for vemurafenib therapy. In this study, we created a novel semi-organically modified derivative DETD from deoxyelephantopin (DET), a plant sesquiterpene lactone demonstrated as an anti-inflammatory and anti-mammary tumor agent in our laboratory. DETD suppressed both parental human BRAFV600E mutant melanoma (A375) and vemurafenib resistance melanoma (A375-R) cell proliferation in vitro. Furthermore, through compound drug combination assay, DETD and vemurafenib was found to show synergism in suppressing proliferation, conoly formation and inducing apoptosis of A375 melanoma cell. In A375 xenograft study, DETD suppressed tumor growth and reduced tumor mass by 70.5%, as effective as vemurafenib (71.9%), compared to tumor control group. Moreover, compound-drug combinational treatment with alternate administration of DETD and vemurafenib also showed similar tumor growth inhibition efficacy by 72.3%, suggesting in vivo synergistic or additive effect of DETD and vemurafenib. In A375-R xenograft study, vemurafenib treatment showed little or no anti-tumor activity with similar tumor growth rate and sizes relative to the tumor control group, whereas DETD could reduce A375-R tumor growth and mass with 46.9%. Notably, the combination of DETD and vemurafenib treatment exhibited the most significant effect, with a 65.3% reduction in tumor mass. Our mechanistic studies revealed the activation of non-receptor tyrosine kinase Src and its downstream signaling pathways, including MAPK, Akt and STAT3 pathways in the vemurafenib resistant melanoma A375-R, and DETD could de-regulate the activation of all these multi- kinase signaling pathways. Overall, our results offer strong evidence of DETD in inhibiting BRAFV600E melanoma growth and its acquired vemurafenib resistance in mice that suggest DETD might have great therapeutic or adjuvant potential in management of melanoma patients with BRAFV600E mutation. Citation Format: Jia-Hua Feng, Kyoko Nakagawa-Goto, Kuo-Hsiung Lee, Lie-Fen Shyur. A novel plant sesquiterpene lactone derivative DETD suppresses BRAFV600E mutant melanoma growth and overcomes acquired vemurafenib resistance in mice. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B175.

Abstract 703: A novel ATP-competitive MEK/Aurora kinase inhibitor BI-847325 reverses acquired BRAF inhibitor resistance through suppression of Mcl-1 and inhibition of MEK expression

Abstract BACKGROUND: BRAF/MEK inhibitors have shown promising levels of response in melanomas harboring the BRAF V600E mutation, however responses tend to be short-lived and resistance is a major clinical problem. In the current study, we evaluated the pharmacological activity of BI847325, a dual inhibitor of MEK and Aurora kinases against multiple melanoma cell line models. METHODS: The cytotoxic effect of BI847325 was evaluated in vitro in a panel of selected BRAF-mutant vemurafenib resistant cell lines by Alamar blue and Annexin V binding assay. 3D spheroid model systems and colony formation assays demonstrated the long-term growth inhibitory effect of BI847325. Western blot analysis and qRT-PCR studies were carried out to evaluate the mechanism underlying BI5-mediated cytotoxicity. In vivo studies in Balb SCID mice were performed to assess the suppression of BRAF-mutant xenografts on treatment with BI847325. RESULTS: BI847325 potently reduced the growth and survival of BRAF-mutant melanoma cell lines with acquired and intrinsic BRAF inhibitor resistance (NRAS mutations, BRAF splice forms, Cyclin D1 amplification, RTK upregulation, PTEN loss, COT amplification). We confirmed that BI847325 induced apoptosis through decrease in Mcl-1 mRNA and protein expression and increase in BIM expression. The effects of BI847325 upon BIM and Mcl-1 expression could not be mimicked by the combination of other MEK and aurora kinase inhibitors. For the first time we demonstrated that BI847325 reverses the acquired vemurafenib resistance by inhibiting MEK expression at mRNA and protein level. A strong suppression of MEK expression was observed without recovery following 72 h of washout. In vivo studies revealed complete tumor suppression with no recurrence over a period of 65 days of treatment with 70mg/kg/week dose of BI847325. In contrast, treatment with vemurafenib analog PLX4720 in the same mouse model was associated with tumor relapse after 30 days of treatment. BI847325 also successfully suppressed the long-term growth of xenografts with acquired vemurafenib resistance. Analysis of tumor samples complied with in vitro results demonstrating inhibition of phospho-ERK, phospho-Histone3, Mcl-1 and total MEK. CONCLUSION: In conclusion we report for the first time that BI847325, a novel ATP-competitive MEK/ Aurora kinase inhibitor effectively inhibits BRAF-mutant melanoma and overcomes vemurafenib-resistance by decreasing expression of MEK and Mcl-1; in vitro and in vivo. Further preclinical and clinical investigations towards this would open new avenues in treatment of melanoma. Citation Format: Manali S. Phadke, Patrizia Sini, Keiran Smalley. A novel ATP-competitive MEK/Aurora kinase inhibitor BI-847325 reverses acquired BRAF inhibitor resistance through suppression of Mcl-1 and inhibition of MEK expression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 703. doi:10.1158/1538-7445.AM2015-703

Abstract B19: Discovery of novel tubulin inhibitor ABI-274 whose synergistic combination with vemurafenib overcome acquired vemurafenib resistance in BRAF mutated melanoma

Abstract Acquired clinical resistance to BRAF inhibitors such as vemurafenib or dabrafefnib arises frequently after short term of these targeted therapy. We hypothesized that a combined therapy using vemurafenib with a G2/M phase blocking agent will arrest resistant cells and overcome vemurafenib resistance. To test this hypothesis, we performed combination studies using our recently discovered tubulin inhibitor ABI-274 and vemurafenib on both vemurafenib-sensitive cells and a resistant A375RF21 sub line. The combination showed strong synergy in vitro, synergistically arrested cells in G1/G2/M phase, and significantly enhanced cancer cell apoptosis. In vivo co-administration of vemurafenib with ABI-274 showed strong synergistic efficacy in the vemurafenib-resistant xenograft model in nude mice. Overall, these results offer a rational combination strategy to significantly enhance the therapeutic benefit in melanoma patients who inevitably become resistant to current vemurafenib therapy. This work was supported by NIH grants R01CA148706, 1S10OD010678-01 and 1S10RR026377-01. Citation Format: Jin Wang, Jianjun Chen, Duane D. Miller, Wei Li. Discovery of novel tubulin inhibitor ABI-274 whose synergistic combination with vemurafenib overcome acquired vemurafenib resistance in BRAF mutated melanoma. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr B19.

Vemurafenib resistance signature by proteome analysis offers new strategies and rational therapeutic concepts.

The FDA-approved BRAF inhibitor vemurafenib achieves outstanding clinical response rates in patients with melanoma, but early resistance is common. Understanding the pathologic mechanisms of drug resistance and identification of effective therapeutic alternatives are key scientific challenges in the melanoma setting. Using proteomic techniques, including shotgun analysis and 2D-gel electrophoresis, we identified a comprehensive signature of the vemurafenib-resistant M24met in comparison with the vemurafenib-sensitive A375 melanoma cell line. The resistant cells were characterized by loss of differentiation, induction of transformation, enhanced expression of the lysosomal compartment, increased potential for metastasis, migration, adherence and Ca2(+) ion binding, enhanced expression of the MAPK pathway and extracellular matrix proteins, and epithelial-mesenchymal transformation. The main features were verified by shotgun analysis with QEXACTIVE orbitrap MS, electron microscopy, lysosomal staining, Western blotting, and adherence assay in a VM-1 melanoma cell line with acquired vemurafenib resistance. On the basis of the resistance profile, we were able to successfully predict that a novel resveratrol-derived COX-2 inhibitor, M8, would be active against the vemurafenib-resistant but not the vemurafenib-sensitive melanoma cells. Using high-throughput methods for cell line and drug characterization may thus offer a new way to identify key features of vemurafenib resistance, facilitating the design of effective rational therapeutic alternatives.

Inhibition of HSP90 by AT13387 delays the emergence of resistance to BRAF inhibitors and overcomes resistance to dual BRAF and MEK inhibition in melanoma models.

Emergence of clinical resistance to BRAF inhibitors, alone or in combination with MEK inhibitors, limits clinical responses in melanoma. Inhibiting HSP90 offers an approach to simultaneously interfere with multiple resistance mechanisms. Using the HSP90 inhibitor AT13387, which is currently in clinical trials, we investigated the potential of HSP90 inhibition to overcome or delay the emergence of resistance to these kinase inhibitors in melanoma models. In vitro, treating vemurafenib-sensitive cells (A375 or SK-MEL-28) with a combination of AT13387 and vemurafenib prevented colony growth under conditions in which vemurafenib treatment alone generated resistant colonies. In vivo, when AT13387 was combined with vemurafenib in a SK-MEL-28, vemurafenib-sensitive model, no regrowth of tumors was observed over 5 months, although 2 of 7 tumors in the vemurafenib monotherapy group relapsed in this time. Together, these data suggest that the combination of these agents can delay the emergence of resistance. Cell lines with acquired vemurafenib resistance, derived from these models (A375R and SK-MEL-28R) were also sensitive to HSP90 inhibitor treatment; key clients were depleted, apoptosis was induced, and growth in 3D culture was inhibited. Similar effects were observed in cell lines with acquired resistance to both BRAF and MEK inhibitors (SK-MEL-28RR, WM164RR, and 1205LuRR). These data suggest that treatment with an HSP90 inhibitor, such as AT13387, is a potential approach for combating resistance to BRAF and MEK inhibition in melanoma. Moreover, frontline combination of these agents with an HSP90 inhibitor could delay the emergence of resistance, providing a strong rationale for clinical investigation of such combinations in BRAF-mutated melanoma.

Abstract A37: Complex crosstalk between MAPK signaling and energy metabolism in melanoma

Abstract The notoriously high glucose uptake by metastatic melanoma (MM) lesions is commonly exploited clinically using PET/CT imaging to evaluate disease progression and treatment. The purpose of this study was to use patient-derived xenograft (PDX) MM models and a PDX derived (MM8.1) cell line, to determine how driver oncogene mutations and drugs targeting the RAS-RAF-MEK signaling pathways impact glucose uptake, ATP production and mitochondrial plasticity of MM cells both in vivo and in vitro and the implications on drug resistance. MM BRAF V600E+ patients are routinely treated targeting either mutant BRAF kinase (vemurafenib), or the MAPK downstream signaling intermediate; MEK. To begin exploring links between oncogenic BRAF and metabolism, we first conducted differential gene expression (DGE) profiling of 30 different MM lesions (n=15 patients: pre- and post-engraftment). Focusing on PGC1α; a master mitochondrial regulator promoting oxidative phosphorylation (OXPHOS) revealed lower expression levels in BRAF V600E+ tumors compared to BRAF V600V+ tumors. Further analysis of a BRAF V600E+ MM PDX model revealed strong glucose uptake by PET/CT imaging at baseline. However, 48 hrs. after treatment with vemurafenib triggered growth arrest, accompanied by increased PGC1α; mRNA levels and significant reduction in glucose uptake without change in tumor volume. Upon acquisition of acquired vemurafenib resistance in the PDX model (day 70), rapidly growing tumors displayed reversal to high glucose uptake; which was again diminished by targeting MEK (PD0325901). DGE analysis of vehicle vs. 48 hrs. day 50 and day 70 vemurafenib treated tumors revealed oscillation in the expression of genes regulating glycolysis and oxidative phosphorylation (OXPHOS) that is reflected in the PET/CT imaging. To gain deeper insight, a cell line derived from a vehicle treated tumor, was examined using the Seahorse metabolic analyzer. Vehicle treated MM8.1 cells display high glycolytic capacity and predominantly glycolytic-derived ATP production, which is reflected by only minor inhibition of ATP production with oligomycin (an inhibitor of ATP synthase, a component of the OXHPOS system). Upon treatment with PLX4032, there was rapid reduction in glycolytic capacity along with a concurrent increase in maximum oxygen consumption capacity. PLX4032 treatment additionally enhanced MM8.1 sensitivity to oligomycin, but only transiently (24 hrs. pre-treatment), as later exposure times did not induce oligomycin sensitivity despite DGE changes to indicate otherwise. In conclusion, caution is warranted in evaluating drug responses in patients using PET/CT depending on duration of treatment. Cross-talk involving MAPK signaling and energy metabolism is a dynamic process, with more of a rheostat than switch-type properties, requiring further evaluation to identify therapeutic approaches exploiting metabolic events within MM cells. Citation Format: Nathan J. Lanning, Pimiento Jose, Noel R. Monks, Paula J. Davidson, Andrew S. Borgman, Ting-Tung Chang, Anderson S. Peck, David J. Monsma, Chelsea A. Peterson, Mackeigan P. Jeff, Brian J. Nickoloff. Complex crosstalk between MAPK signaling and energy metabolism in melanoma. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr A37. doi: 10.1158/1557-3125.RASONC14-A37

B-Raf inhibitor vemurafenib in combination with temozolomide and fotemustine in the killing response of malignant melanoma cells.

In the treatment of metastatic melanoma, a highly therapy-refractory cancer, alkylating agents are used and, for the subgroup of BRAFV600E cancers, the B-Raf inhibitor vemurafenib. Although vemurafenib is initially beneficial, development of drug resistance occurs leading to tumor relapse, which necessitates the requirement for combined or sequential therapy with other drugs, including genotoxic alkylating agents. This leads to the question whether vemurafenib and alkylating agents act synergistically and whether chronic vemurafenib treatment alters the melanoma cell response to alkylating agents. Here we show that a) BRAFV600E melanoma cells are killed by vemurafenib, driving apoptosis, b) BRAFV600E melanoma cells are neither more resistant nor sensitive to temozolomide/fotemustine than non-mutant cells, c) combined treatment with vemurafenib plus temozolomide or fotemustine has an additive effect on cell kill, d) acquired vemurafenib resistance of BRAFV600E melanoma cells does not affect MGMT, MSH2, MSH6, PMS2 and MLH1, nor does it affect the resistance to temozolomide and fotemustine, e) metastatic melanoma biopsies obtained from patients prior to and after vemurafenib treatment did not show a change in the MGMT promoter methylation status and MGMT expression level. The data suggest that consecutive treatment with vemurafenib and alkylating drugs is a reasonable strategy for metastatic melanoma treatment.

Mitogen-activated Protein Kinase (MAPK) Hyperactivation and Enhanced NRAS Expression Drive Acquired Vemurafenib Resistance in V600E BRAF Melanoma Cells

Although targeting the V600E activating mutation in the BRAF gene, the most common genetic abnormality in melanoma, has shown clinical efficacy in melanoma patients, response is, invariably, short lived. To better understand mechanisms underlying this acquisition of resistance to BRAF-targeted therapy in previously responsive melanomas, we induced vemurafenib resistance in two V600E BRAF+ve melanoma cell lines, A375 and DM443, by serial in vitro vemurafenib exposure. The resulting approximately 10-fold more vemurafenib-resistant cell lines, A375rVem and D443rVem, had higher growth rates and showed differential collateral resistance to cisplatin, melphalan, and temozolomide. The acquisition of vemurafenib resistance was associated with significantly increased NRAS levels in A375rVem and D443rVem, increased activation of the prosurvival protein, AKT, and the MAPKs, ERK, JNK, and P38, which correlated with decreased levels of the MAPK inhibitor protein, GSTP1. Despite the increased NRAS, whole exome sequencing showed no NRAS gene mutations. Inhibition of all three MAPKs and siRNA-mediated NRAS suppression both reversed vemurafenib resistance significantly in A375rVem and DM443rVem. Together, the results indicate a mechanism of acquired vemurafenib resistance in V600E BRAF+ve melanoma cells that involves increased activation of all three human MAPKs and the PI3K pathway, as well as increased NRAS expression, which, contrary to previous reports, was not associated with mutations in the NRAS gene. The data highlight the complexity of the acquired vemurafenib resistance phenotype and the challenge of optimizing BRAF-targeted therapy in this disease. They also suggest that targeting the MAPKs and/or NRAS may provide a strategy to mitigate such resistance in V600E BRAF+ve melanoma.

Abstract 3710: The role of eIF4E in promoting melanoma cell proliferation and maintaining acquired resistance to Vemurafenib in melanoma

Abstract In eukaryotic cells, mRNA translation is restricted by the translation initiation step, in which eIF4E has been regarded as a master regulator. Overexpression of eIF4E has been found in different cancer types; however, whether eIF4E is involved in melanoma tumorigenesis and resistance to standard of care in melanoma remains unclear. We hypothesize that eIF4E promotes melanoma cell proliferation and facilitates the development of acquired resistance to the B-raf inhibitor, Vemurafenib. From our preliminary data, we show that eIF4E is overexpressed in a panel of malignant melanoma cell lines compared to immortalized melanocytes Mel ST. By genetically knocking down eIF4E via siRNA, we can significantly repress the proliferation of a subset of melanoma cell lines and decrease the level of the eIF4E downstream target Cyclin D1. In addition, the B-raf inhibitor Vemurafenib can drastically inhibit 4EBP-1 phosphorylation in BRAFV600E melanoma cell lines, and further cap binding analysis confirmed that Vemurafenib stabilizes the eIF4E - 4EBP-1 association in these cell lines.. In contrast, 4EBP-1 is highly phosphorylated in all Vemurafenib resistant cell lines, compared to the parental A375, even in the presence of Vemurafenib. Furthermore, cap binding assay show that, unlike their parental counterpart, four out of six resistant lines exhibit weak eIF4E - 4EBP-1 association even in the presence of Vemurafenib. This may imply that more free eIF4E is available for cap-dependent translation initiation in Vemu resistant lines compared to the parental A375. Besides, eIF4E activity contributes to the proliferation of some melanoma cell lines with acquired resistance to Vemurafenib, since siRNA-silencing eIF4E decreases their proliferation. In conclusion, our data show that eIF4E promotes proliferation of some melanoma cell lines and may be involved in maintaining the survival of some melanoma cell lines with acquired vemurafenib resistance. These data suggest that therapeutically targeting eIF4E may be a viable means of inhibiting melanoma cell proliferation and overcoming Vemurafenib resistance. Citation Format: Yao Zhan, Michael S. Dahabieh, Filippa Pettersson, Monica C. Dobocan, Marie Noel M. Boutchou, Leon Van Kempen, Sonia V. del Rincon, Wilson H. Miller, Jr.. The role of eIF4E in promoting melanoma cell proliferation and maintaining acquired resistance to Vemurafenib in melanoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3710. doi:10.1158/1538-7445.AM2014-3710

Usp5 links suppression of p53 and FAS levels in melanoma to the BRAF pathway.

Usp5 is a deubiquitinase (DUB) previously shown to regulate unanchored poly-ubiquitin (Ub) chains, p53 transcriptional activity and double-strand DNA repair. In BRAF mutant melanoma cells, Usp5 activity was suppressed by BRAF inhibitor (vemurafenib) in sensitive but not in acquired or intrinsically resistant cells. Usp5 knockdown overcame acquired vemurafenib resistance and sensitized BRAF and NRAS mutant melanoma cells to apoptosis initiated by MEK inhibitor, cytokines or DNA-damaging agents. Knockdown and overexpression studies demonstrated that Usp5 regulates p53 (and p73) levels and alters cell growth and cell cycle distribution associated with p21 induction. Usp5 also regulates the intrinsic apoptotic pathway by modulating p53-dependent FAS expression. A small molecule DUB inhibitor (EOAI3402143) phenocopied the FAS induction and apoptotic sensitization of Usp5 knockdown and fully blocked melanoma tumor growth in mice. Overall, our results demonstrate that BRAF activates Usp5 to suppress cell cycle checkpoint control and apoptosis by blocking p53 and FAS induction; all of which can be restored by small molecule-mediated Usp5 inhibition. These results suggest that Usp5 inhibition can provide an alternate approach in recovery of diminished p53 (or p73) function in melanoma and can add to the targeted therapies already used in the treatment of melanoma.

Evaluating melanoma drug response and therapeutic escape with quantitative proteomics.

The evolution of cancer therapy into complex regimens with multiple drugs requires novel approaches for the development and evaluation of companion biomarkers. Liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM) is a versatile platform for biomarker measurement. In this study, we describe the development and use of the LC-MRM platform to study the adaptive signaling responses of melanoma cells to inhibitors of HSP90 (XL888) and MEK (AZD6244). XL888 had good anti-tumor activity against NRAS mutant melanoma cell lines as well as BRAF mutant cells with acquired resistance to BRAF inhibitors both in vitro and in vivo. LC-MRM analysis showed HSP90 inhibition to be associated with decreased expression of multiple receptor tyrosine kinases, modules in the PI3K/AKT/mammalian target of rapamycin pathway, and the MAPK/CDK4 signaling axis in NRAS mutant melanoma cell lines and the inhibition of PI3K/AKT signaling in BRAF mutant melanoma xenografts with acquired vemurafenib resistance. The LC-MRM approach targeting more than 80 cancer signaling proteins was highly sensitive and could be applied to fine needle aspirates from xenografts and clinical melanoma specimens (using 50 μg of total protein). We further showed MEK inhibition to be associated with signaling through the NFκB and WNT signaling pathways, as well as increased receptor tyrosine kinase expression and activation. Validation studies identified PDGF receptor β signaling as a potential escape mechanism from MEK inhibition, which could be overcome through combined use of AZD6244 and the PDGF receptor inhibitor, crenolanib. Together, our studies show LC-MRM to have unique value as a platform for the systems level understanding of the molecular mechanisms of drug response and therapeutic escape. This work provides the proof-of-principle for the future development of LC-MRM assays for monitoring drug responses in the clinic.