BRAFV600E Mutant Melanoma Cell Research Articles

The Oncogenic Signaling Pathways in BRAF-Mutant Melanoma Cells are Modulated by Naphthalene Diimide-Like G-Quadruplex Ligands.

Melanoma is the most aggressive and deadly type of skin cancer. Despite the advent of targeted therapies directed against specific oncogene mutations, melanoma remains a tumor that is very difficult to treat, and ultimately remains incurable. In the past two decades, stabilization of the non-canonical nucleic acid G-quadruplex structures within oncogene promoters has stood out as a promising approach to interfere with oncogenic signaling pathways in cancer cells, paving the way toward the development of G-quadruplex ligands as antitumor drugs. Here, we present the synthesis and screening of a library of differently functionalized core-extended naphthalene diimides for their activity against the BRAFV600E-mutant melanoma cell line. The most promising compound was able to stabilize G-quadruplexes that formed in the promoter regions of two target genes relevant to melanoma, KIT and BCL-2. This activity led to the suppression of protein expression and thus to interference with oncogenic signaling pathways involved in BRAF-mutant melanoma cell survival, apoptosis, and resistance to drugs. This G-quadruplex ligand thus represents a suitable candidate for the development of melanoma treatment options based on a new mechanism of action and could reveal particular significance in the context of resistance to targeted therapies of BRAF-mutant melanoma cells.

1O - Suppression of KRAS-G12D and BRAF-V600E oncogene transcription with PNA-conjugates

BackgroundWe have developed a strategy to block transcription of oncogenes such as BRAF V600E and KRAS G12D directly using modified complementary peptide nucleic acid (PNA) oligomers that target oncogenesis specifically causing inhibition of tumour growth. This proof of principle strategy against BRAF V600E and KRAS G12D in vitro and in vivo should provide a new means to develop PNA-delivery peptide conjugates as targeted drug therapeutics across a broad range of oncogenes that drive cancer cell growth. MethodsObstruction of KRAS G12D and BRAF V600E expression was evaluated through suppression of cell proliferation and specific mRNA transcription. Tumour reduction was assessed through Xenograft mouse models. ResultsExposure of KRAS G12D-dependent cell line to modified PNA-peptide conjugate complementary to KRAS G12D mutation sequence also results in concentration-dependent and time-dependent inhibition of cell growth and specific and complete suppression in mRNA transcription. Cell lines expressing KRAS WT and KRAS G12C, both differing by a single nucleobase, show no suppression. Exposure of the melanoma cell lines to a modified PNA-peptide conjugate complementary to BRAF V600E mutation sequence results in a concentration-dependent and time-dependent inhibition of cell growth that is specific for the BRAF V600E mutant melanoma cell lines with inhibition of mRNA and protein expression. Xenograft mouse trials show tumour growth delay and necrosis compared to PNA controls. This 50mg/kg dose was well tolerated without associated weight loss. By H&E staining, tumor tissue from trials shows ablation and extensive scaring upon exposure to BRAF V600E-complementary PNA-peptide conjugate whereas saline and scramble PNA sequence controls do not. Similarly quantitative measurement shows a 2.5-fold decrease in Ki67 and a 3-fold increase in TUNEL expression. ConclusionsOur results indicate that these PNA-peptide derivatives could represent a novel and promising new therapy for patients with genes specific for and causative of tumorigenesis. This strategy could be applied to a multitude of cancers either with specific translocations or mutations differing from wild-type cells even by only a single base pair. Legal entity responsible for the studyThe authors. FundingCelgene, Fortress Biotech. DisclosureAll authors have declared no conflicts of interest.

Dabrafenib and Trametinib\xa0prolong coagulation through the inhibition of tissue factor in BRAFv600e mutated melanoma cells in vitro

BackgroundNeoplastic cells promote a hypercoagulable state by the expression of cell surface proteins, such as tissue factor. In BRAFv600 mutated melanoma patients upon BRAF inhibitors, a hypercoagulable state correlates with prognosis, while a down-regulation of the hemostatic parameters is observed in patients responders as compared to non responders. The present study was intended to better clarify the strict relationship between coagulation mediators and target therapy in melanoma.MethodsThe expression of tissue factor was investigated after the treatment with the BRAF inhibitor Dabrafenib and the MEK inhibitor Trametinib in the BRAFv600e mutated melanoma cell lines A-375 and SK-MEL-28, together with its ability to activate the coagulation cascade.ResultsDabrafenib and Trametinib caused the down-regulation of TF in both cell lines A-375 and SK-MEL-28. For the cell line A-375 the effect was evident both at RNA and procoagulant activity; for the cell line SK-MEL-28 only at RNA level without any variation of the protein. Interestingly, when in contact with plasma deficient of factor VII, both cell lines were not able to activate the coagulation cascade.ConclusionsThe present study provides the first in vitro observation that tissue factor expressed in melanoma cells may contribute to the modulation of the coagulation state of patients in the treatment with BRAF inhibitors.

Abstract 4864: Targeted melanoma therapies as radiosensitizers

Abstract Introduction: In BRAFV600 mutant melanoma combination of BRAF and MEK inhibitors is associated with a rapid response in approximately 70% of patients, and an improvement in survival. Radiotherapy is used in the treatment of melanoma in multiple clinical settings including stereotactic radiosurgery for brain metastasis, oligo-metastatic extracranial disease and palliative radiotherapy to symptomatic lesions. Preclinical studies, and a small number of clinical case reports suggest the BRAF inhibitor vemurafenib and the MEK inhibitor trametinib leads to increased radiosensitivity. Aims: To determine if BRAF Inhibitors dabrafenib, vemurafenib and encorafenib, alone or in combination with their corresponding MEK Inhibitors, are radiation-sensitising in BRAF-mutant treatment naive melanoma cells. Methods: Three melanoma cell lines harbouring a BRAF V600E mutation were studied: A375, SkMel28 and MM200. The half-maximal inhibitory concentration (IC50) for each cell line was determined by MTS assays for vemurafenib, dabrafenib, encorafenib, and the MEK inhibitors trametinib, binimetinib and cobimetinib. Clonogenic assays were conducted on each cell line. The optimal dose of BRAF and MEK inhibitors was determined from MTS assay and single drug clonogenic assays. Cells were treated with BRAF or MEK inhibitor alone and in combination. Cells were also treated with increasing doses of radiation (X-RAD 320 irradiator, PXI). The mode of cell death and changes in cell morphology were determined by live cell imaging (Wide Field Live Cell Observer, Ziess) for five days following the above treatments. Results:Using Clonogenic assays, we confirmed that radiation sensitivity varies across different melanoma cell lines, with MM200 being significantly more sensitive to radiation then SKMel28. Clonogenic assays demonstrated that vemurafenib, dabrafenib, encorafenib, trametinib and binimetinib increased radiosensitivity across all three-cell lines. Further, the level of radiosensitsation was greater in combination across all three sets, than with either BRAF or MEK inhibitor alone. Using live cell imaging, we were able to confirm this increase in cell death, and visualise differences in the mode of cell death and morphology with these therapies. Conclusions: Vemurafenib, dabrafenib, encorafenib, trametinib and binimetinib were shown in this study to be radiosensitisers in BRAFV600Emutant melanoma cell lines. Radiosensitisation was greatest with combined BRAF and MEK inhibition. Further studies are being conducted to determine if this sensitisation continues after BRAF resistance develops. Citation Format: Jessica L. Smith, Cecilia Chang, Tim Wang, Helen Rizos, Han Shen, Matteo Carlino, Eric Hau. Targeted melanoma therapies as radiosensitizers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4864.

Activating PTEN Tumor Suppressor Expression with the CRISPR/dCas9 System

PTEN expression is lost in many cancers, and even small changes in PTEN activity affect susceptibility and prognosis in a range of highly aggressive malignancies, such as melanoma and triple-negative breast cancer (TNBC). Loss of PTEN expression occurs via multiple mechanisms, including mutation, transcriptional repression and epigenetic silencing. Transcriptional repression of PTEN contributes to resistance to inhibitors used in the clinic, such as B-Raf inhibitors in BRAF mutant melanoma. We aimed to activate PTEN expression using the CRISPR system, specifically dead (d) Cas9 fused to the transactivator VP64-p65-Rta (VPR). dCas9-VPR was directed to the PTEN proximal promoter by single-guide RNAs (sgRNAs), in cancer cells that exhibited low levels of PTEN expression. The dCas9-VPR system increased PTEN expression in melanoma and TNBC cell lines, without transcriptional regulation at predicted off-target sgRNA binding sites. PTEN activation significantly repressed downstream oncogenic pathways, including AKT, mTOR, and MAPK signaling. BRAF V600E mutant melanoma cells transduced with dCas9-VPR displayed reduced migration, as well as diminished colony formation in the presence of B-Raf inhibitors, PI3K/mTOR inhibitors, and with combined PI3K/mTOR and B-Raf inhibition. CRISPR-mediated targeted activation of PTEN may provide an alternative therapeutic approach for highly aggressive cancers that are refractory to current treatments.

Analysis of MLN4924 (pevonedistat) as a potential therapeutic agent in malignant melanoma.

The NEDD8 pathway is a known activator of the ubiquitin-protease system, a complex that is partially responsible for the degradation of proteins involved in cell-cycle regulation and neoplastic growth. In this study, we evaluated the antitumor potential of MLN4924 (pevonedistat), a potent NEDD8 inhibitor. We hypothesized that MLN4924 treatment induces apoptosis in human melanoma cells. A375 and Mel39 BRAF V600E mutant melanoma cell lines were treated in vitro with MLN4924 alone or in combination with interferon-α (IFN-α) or vemurafenib - therapeutic agents utilized on melanoma patients. Annexin/propidium iodine flow cytometry analysis showed that treatment with MLN4924 for 72 h induced apoptosis in A375 and Mel39 melanoma cells with an IC50 of 1200 and 143 nmol/l, respectively. Combination therapy of A375 cells with 10 U/ml IFN-α and 1200 nmol/l MLN4924 led to a significant increase in cell death (78.2±3.7%) compared with single-agent treatment by IFN-α (17.5±2.5%) or MLN4924 (50.7±1.0%; P<0.005). Treatment of A375 cells with 1 μmol/l vemurafenib had a notable effect on cell viability. However, the addition of MLN4924 to vemurafenib had an inhibitory effect on apoptosis. Results from MTS proliferation assays indicate that MLN4924 has antiproliferative effects on melanoma cells in vitro, with the addition of IFN-α further inhibiting proliferation. Pretreatment with MLN4924 led to A375 cell sensitization to vemurafenib treatment and immunoblot analysis of MLN4924-treated cells revealed cleavage of caspase-3, caspase-7, caspase-9, and poly-ADP-ribose polymerase. These results show that MLN4924 does have an efficacy in treating melanoma in vitro alone or in combination with IFN-α, and thus it may have potential use in patients with advanced melanoma.

Targeting the MAPK Signaling Pathway in Cancer: Promising Preclinical Activity with the Novel Selective ERK1/2 Inhibitor BVD-523 (Ulixertinib).

Aberrant activation of signaling through the RAS-RAF-MEK-ERK (MAPK) pathway is implicated in numerous cancers, making it an attractive therapeutic target. Although BRAF and MEK-targeted combination therapy has demonstrated significant benefit beyond single-agent options, the majority of patients develop resistance and disease progression after approximately 12 months. Reactivation of ERK signaling is a common driver of resistance in this setting. Here we report the discovery of BVD-523 (ulixertinib), a novel, reversible, ATP-competitive ERK1/2 inhibitor with high potency and ERK1/2 selectivity. In vitro BVD-523 treatment resulted in reduced proliferation and enhanced caspase activity in sensitive cells. Interestingly, BVD-523 inhibited phosphorylation of target substrates despite increased phosphorylation of ERK1/2. In in vivo xenograft studies, BVD-523 showed dose-dependent growth inhibition and tumor regression. BVD-523 yielded synergistic antiproliferative effects in a BRAFV600E-mutant melanoma cell line xenograft model when used in combination with BRAF inhibition. Antitumor activity was also demonstrated in in vitro and in vivo models of acquired resistance to single-agent and combination BRAF/MEK-targeted therapy. On the basis of these promising results, these studies demonstrate BVD-523 holds promise as a treatment for ERK-dependent cancers, including those whose tumors have acquired resistance to other treatments targeting upstream nodes of the MAPK pathway. Assessment of BVD-523 in clinical trials is underway (NCT01781429, NCT02296242, and NCT02608229). Mol Cancer Ther; 16(11); 2351-63. ©2017 AACR.

Obstruction of BRAFV600E transcription by complementary PNA oligomers as a means to inhibit BRAF-mutant melanoma growth.

Peptide nucleic acid (PNA) oligomers are DNA mimics, which are capable of binding gene sequences 1000-fold more avidly than complementary native DNA by strand invasion and effectively obstruct transcription. Irreversibly obstructing the transcription or replication of a gene sequence, such as BRAFV600E, offers a potential route to specifically target the cancer cell itself. We have employed PNA oligomers to target BRAFV600E in a sequence-specific complementary manner. These PNAs have been modified by appending configurationally stabilizing cationic peptides in order to improve their cellular delivery and target avidity. Our results indicate that exposure of the melanoma cell lines to a modified PNA-peptide conjugate complementary to BRAFV600E mutation sequence results in a concentration-dependent and time-dependent inhibition of cell growth that is specific for the BRAFV600E-mutant melanoma cell lines with inhibition of mRNA and protein expression. Xenograft mouse trials show increased tumor growth delay and necrosis with the BRAFV600E-complementary PNA-peptide conjugates as compared with the saline and scrambled PNA sequence controls. Similarly, quantitative measurement shows a 2.5-fold decrease in Ki67 and a 3-fold increase in terminal deoxynucleotidyl transferase dUTP nick end labeling expression with this approach. PNA-delivery peptide conjugates represent a novel way to target BRAFV600E and represent a new approach in targeting selective oncogenes that induce tumor growth.

Abstract 365: Cross-talk between BRAF and Hippo/YAP1 signaling in melanoma

Abstract Hippo/YAP1 signaling pathway is a tumor suppressive pathway that controls the organ size by modulating the cell growth, proliferation and apoptosis and is conserved from Drosophila to mammals. In mammals, the Hippo tumor suppressor pathway consists of cascade of kinases in which MST1/2 phosphorylates and activates LATS1/2. The latter phosphorylates the oncogenic transcriptional coactivators YAP1 and TAZ, leading to their cytoplasmic retention by 14-3-3 proteins and/or degradation. Inactivation of MST and LATS kinases allows YAP1 and/ or TAZ nuclear translocation and subsequent activation of their target genes. Deregulation of Hippo pathway can induce tumors in model organisms and occurs in wide range of human cancers including melanoma. Merlin, a key component of this pathway which inhibits YAP1, is mutated/deleted in 8% of melanoma. Majority of uveal melanomas are driven by Gq/11 mutations that trigger YAP1 nuclear translocation, promoting tumor growth. High levels of YAP1 in BRAFV600E mutant tumors confer resistance to RAF- and MEK- targeted therapy in patients. Our results show that YAP1 level is elevated in melanoma and the YAP1 inhibitor verteporfin alone or in combination with B-RAF inhibitor PLX4720 reduces the viability, invasion and anchorage-independent growth of B-RAF V600E mutant SK-MEL-28 and SK-MEL-5 cells. In addition, verteporfin treatment also reduced the viability of PLX4720 resistant 1205 cells. Western blot analysis of verteporfin and PLX4720 treated SK-MEL-28 and SK-MEL-5 cells displayed reduced levels of YAP1, B-RAF, pERK, MEK and pMEK. We also report a novel physical interaction between YAP1 and B-RAF; this could be detected using double immunofluorescence and immunoprecipitation-western blotting techniques in both B-RAF V600E mutant and N-Ras mutant melanoma cells. Proximity ligation assays on tissue microarray showed that YAP1-B-RAF interaction is elevated in metastatic melanoma compared to normal skin. These novel findings highlight the crosstalk between B-RAF and Hippo/YAP1 signaling which might have a potential role in melanoma development and progression. Further, experiments are in progress to elucidate the functional significance of YAP1-B-RAF interaction in melanoma. Citation Format: Mohan Kumar Durai Raj, Jonathan Nguyen, Namrata Bora-singhal, Jane Messina, Geoffrey Gibney, Srikumar Chellappan. Cross-talk between BRAF and Hippo/YAP1 signaling in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 365. doi:10.1158/1538-7445.AM2017-365

Abstract 4023: The SABRE platform: A novel, unbiased technology for drug discovery and prioritization

Abstract Targeted therapies designed to inhibit hyperactive oncogenic signaling have demonstrated some encouraging clinical responses. However, most of the time these responses are not durable as tumors “work around” pathway inhibition, often reactivating the inhibited pathway, leading to drug resistance and disease relapse. New technologies that enable more efficient discovery and development of “suites” of drugs that comprehensively inhibit multiple pathway nodes are needed. The SABRE (Splice Acceptor Brilliant Reporter) platform was developed to provide an unbiased method to screen large compound libraries and dramatically improve the speed and efficiency of which novel targeted therapeutics can be identified. SABRE is built off the premise that oncogenic output is exerted through changes in gene transcription and that such changes can be harnessed as powerful reporters of pathway activation status. SABRE utilizes the power of gene trap technology coupled with a drug selection process to isolate cells that generate a robust “off to on” signal in response to target or pathway inhibition. Via massively parallel comparative analysis, multiple traps let nature provide the best reporter for further analysis and drug discovery. In this proof-of-concept study, we employed the SABRE technology to identify insertion sites that are specifically regulated by the MAPK pathway. Experiments were performed using the human BRAFV600E mutant melanoma cell line A375. SABRE lentiviral transduced A375 cells were treated with trametinib, a MEK inhibitor, and clones were isolated that emitted a positive luciferase signal upon drug treatment. To determine if these reporters were MAPK pathway specific, the platform was miniaturized to a 1536 well format and used to screen a 6000+ compound library. Results from the screen found that 70% (28/40) of the top drug hits were known to directly modulate the MAPK pathway. Since drug resistance is a common occurrence in melanoma, we generated a “resistant” A375 SABRE reporter line to increasing concentrations of vemurafenib and interrogated the top drug candidates identified in the screen. As expected, the resistant SABRE line failed to respond to BRAFV600E specific inhibitors but continued to respond to downstream MEK inhibitors, which is helping to delineate the mechanism of resistance. We were also interested in the screening hits that were not previously known to influence the MAPK pathway including bafetinib and a Tie2 inhibitor. In follow-up testing, we found that these drugs induced robust and titratable reporter signals in both the SABRE A375 vemurafenib sensitive and resistant lines. In addition, treatment of either bafetinib or the Tie2 inhibitor reduced ERK phosphorylation in the vemurafenib resistant cells, suggesting that these drugs could be useful in the treatment of drug resistant melanomas. Studies are currently underway to determine the in vivo efficacy of these drugs in A375 SABRE reporter xenograft models. Citation Format: Shelli M. Morris, Andrew J. Mhyre, Savanna S. Carmack, Connor Burns, Marc Ferrer, Wenjuan Ye, James M. Olson, Richard A. Klinghoffer. The SABRE platform: A novel, unbiased technology for drug discovery and prioritization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4023. doi:10.1158/1538-7445.AM2017-4023

Electrochemotherapy with bleomycin is effective in BRAF mutated melanoma cells and interacts with BRAF inhibitors.

BackgroundThe aim of the study was to explore the effectiveness of electrochemotherapy (ECT) during the treatment of melanoma patients with BRAF inhibitors. Its effectiveness was tested on BRAF mutated and non-mutated melanoma cells in vitro and in combination with BRAF inhibitors.Materials and methodsECT with bleomycin was performed on two human melanoma cell lines, with (SK-MEL-28) or without (CHL-1) BRAF V600E mutation. Cell survival was determined using clonogenic assay to determine the effectiveness of ECT in melanoma cells of different mutation status. Furthermore, the effectiveness of ECT in concomitant treatment with BRAF inhibitor vemurafenib was also determined in BRAF mutated cells SK-MEL-28 with clonogenic assay.ResultsThe survival of BRAF V600E mutated melanoma cells was even lower than non-mutated cells, indicating that ECT is effective regardless of the mutational status of melanoma cells. Furthermore, the synergistic interaction between vemurafenib and ECT with bleomycin was demonstrated in the BRAF V600E mutated melanoma cells.ConclusionsThe effectiveness of ECT in BRAF mutated melanoma cells as well as potentiation of its effectiveness during the treatment with vemurafenib in vitro implies on clinical applicability of ECT in melanoma patients with BRAF mutation and/or during the treatment with BRAF inhibitors.

Abstract 1885: Integrated epigenomic profiling reveals widespread demethylation in melanoma and points to the role of CSF1R-RUNX1 axis in resistance against BRAF inhibition

Abstract Epigenetic changes in cancer are thought to contribute to regulation of invasion and metastasis. To study this at a genome-wide level in melanoma we analyzed the methylome of 44 cases of malignant melanoma with the HELP (HpaII tiny fragment enriched by LM-PCR) assay and compared it to melanocyte controls. We saw widespread demethylation in melanoma occurring preferentially outside of CpG islands. Comparison of primary and metastatic lesions demonstrated that demethylation occurs early during carcinogenesis with few additional alterations in advanced tumors. Parallel transcriptomic analysis revealed many known and novel oncogenic pathways aberrantly expressed and regulated by loss of DNA methylation. The colony stimulating factor-1 receptor (CSF1R) was aberrantly expressed and hypomethylated in nearly all cases. The expression of CSF1R was validated by immunohistochemistry on primary tumors and by Western blotting in BRAF V600E mutant and WT melanoma cell lines. Expression of its ligand IL34, but not of CSF1 was also shown in the melanoma cells by both ELISA and qPCR. The effects of a small molecule inhibitor, PLX3397 as well as shRNA-mediated knockdown of the receptor were investigated in traditional and 3D cell culture. We saw inhibition of cell growth, smaller colony size, increased apoptosis and decreased invasiveness - suggesting a functional role for CSF1R in melanoma. Treatment of melanoma with small molecule inhibitors of BRAF V600E is effective for a time, but resistance invariably develops. The feedback activation of EGFR, BRAF amplification, BRAF splice variants and others are known to aid in the acquisition of resistance and lead to rebound activation of the MAPK-pathway. In Western blotting experiments, the rebound of ERK phosphorylation after BRAF inhibitor treatment was accelerated with the addition of the CSF1R ligands CSF1 and IL34, or delayed with PLX3397, also attenuating AKT phosphorylation. Melanoma cells stably expressing CSF1R shRNA recapitulated the effects of the inhibitor. Assaying the cells at different time points during a long-term V600E inhibitory experiment, we saw increasing levels of the transcription factor RUNX1, followed by increasing levels of IL34 and of the CSF1R protein, as well as its maturation, evidenced by the appearance of the high MW form. Utilizing shRNA-mediated knockdown of RUNX1 resulted in lower levels of the CSF1R and IL34 transcripts and delayed the rebound. Analysis of primary RNA-Seq data showed an increase in RUNX1, CSF1R and IL34 expression as resistance was acquired. Co-inhibition of CSF1R and BRAF was also tested and resulted in synergistic blockade of cell growth in vitro and xenograft growth in vivo. The CSF1R inhibitor, PLX3397, is in clinical trials for breast and other cancers, and these data present a preclinical rationale for its study in malignant melanoma. Citation Format: Orsolya Giricz, Yongkai Mo, Caroline H. Hu, Kimberly Dahlman, Nandini Ramachandra, Matthias Bartenstein, Kith Pradhan, Tushar Bhagat, Yiting Yu, Hoa Nguyen, Elizabeth Burton, Bernice Matusow, Gaston Habets, Rafe Shellooe, Gideon Bollag, Brian West, John Greally, Jeffrey Sosman, Paraic Kenny, Amit Verma. Integrated epigenomic profiling reveals widespread demethylation in melanoma and points to the role of CSF1R-RUNX1 axis in resistance against BRAF inhibition. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1885.

Abstract 2365: Checkpoint inhibitors and a multivalent melanoma vaccine as a novel combinatorial therapy

Abstract Over 1 million cases of skin cancer are diagnosed in the US every year, of which 75% deaths result due to melanoma, one of the deadliest forms of skin cancer, arising from melanocytes. We have isolated five patient derived melanoma cell lines (MEL-V, 3MM, GLM2, Mel2, KFM) and characterized the expression of various melanoma associated antigens (MAAs). The presence of a multitude repertoire of common MAAs (Gp100, MART-1, MAGE-A1, NY-ESO-1, Tyrosinase, TRP-1 (Gp75), TRP-2, Melanotransferrin, CD71, CD146) enabled us to use composite cell membrane preparations of these cells as a multivalent vaccine. To this end, we designed a vaccinia virus vaccine that was composed of membrane lysate preparations of the five primary cells and recombinant IL-2. When tested in a pilot study, delayed type hypersensitivity and a multivalent immune response were noted. In an effort to correlate genetic lesions with MAA expression we observed that in BRAF mutant melanoma cells, treatment with a BRAFv600E inhibitor, PLX4032, enhanced the expression of MAAs making the melanoma cells more visible to the vaccination driven immune response. We also observed that melanoma cells constitutively expressed CTLA-4 as determined by Immunofluorescence and Western Blots. This expression was found on the membranes as well as in the cytoplasm in comparatively high levels on wild type as well as the BRAFV600E mutant melanoma cell lines. Further our results suggest that CTLA-4 expression on melanoma cells promotes the immunosuppressive tumor microenvironment enabling tumor evasion. Given that CTLA-4 monoclonal antibodies such as Ipilimumab are approved for clinical treatment, this study opens up a new avenue into a plausible combinatorial therapy for our vaccinia virus based vaccine with Ipilimumab for BRAF mutant as well as wild type melanoma cells. Citation Format: Rachana R. Maniyar, Neha Tuli, Robert Suriano, Jan Geliebter, Marc Wallack, Raj K. Tiwari. Checkpoint inhibitors and a multivalent melanoma vaccine as a novel combinatorial therapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2365.

Actin remodeling confers BRAF inhibitor resistance to melanoma cells through YAP/TAZ activation.

The activation of transcriptional coactivators YAP and its paralog TAZ has been shown to promote resistance to anti-cancer therapies. YAP/TAZ activity is tightly coupled to actin cytoskeleton architecture. However, the influence of actin remodeling on cancer drug resistance remains largely unexplored. Here, we report a pivotal role of actin remodeling in YAP/TAZ-dependent BRAF inhibitor resistance in BRAF V600E mutant melanoma cells. Melanoma cells resistant to the BRAF inhibitor PLX4032 exhibit an increase in actin stress fiber formation, which appears to promote the nuclear accumulation of YAP/TAZ. Knockdown of YAP/TAZ reduces the viability of resistant melanoma cells, whereas overexpression of constitutively active YAP induces resistance. Moreover, inhibition of actin polymerization and actomyosin tension in melanoma cells suppresses both YAP/TAZ activation and PLX4032 resistance. Our siRNA library screening identifies actin dynamics regulator TESK1 as a novel vulnerable point of the YAP/TAZ-dependent resistance pathway. These results suggest that inhibition of actin remodeling is a potential strategy to suppress resistance in BRAF inhibitor therapies.

Abstract A91: BGB-283 exhibits potentials in enhancing tumor immune sensitization and lymphocyte function

Abstract Oncogenic B-RAF, which drives cell transformation and proliferation, has been detected in a variety of human malignancies. Prior studies suggested that B-RAF-targeting therapies could increase antigen expression, decrease immune suppressive factors in tumor microenvironment, and improve homing of T effector cells to the tumors. Thus, selective BRAF inhibitors were suggested to possess immunosensitization properties and could be used in combination with immunotherapies to achieve more durable disease control/response in treating BRAF V600E melanomas. BGB-283 is a novel inhibitor of the RAF dimer with potent and reversible inhibitory activities against RAF family kinases including wild type A-RAF, B-RAF, C-RAF and B-RAF V600E. In addition, BGB-283 has potent inhibitory activity against EGFR (Mol. Can. Ther. onlinefirst 2015; doi: 10.1158/1535-7163). BGB-283 is currently under Phase 1 clinical investigations. In the present study, BGB-283 showed significant stimulation of melanocyte-differentiation antigens and HLA-A expression in BRAF V600E melanoma cell lines. Intermediate level of BGB-283 was found to stimulate proliferation of human peripheral blood mononuclear cell (PBMC) and isolated human CD4+ and CD8+ T cells. In addition, BGB-283 potentiated IFNγ releasing from PBMCs at concentration range of 30∼300 nM. In newly developed the 3D tumor spheroid/PBMC co-culture system, BGB-283 improved PBMC function as measured by cell proliferation and IFNγ production. The presence of PBMC also enhanced the anti-proliferation activity of BGB-283 on A375 BRAF V600E mutant melanoma cells in the co-culture system. Further studies have shed light on the mechanistic underpinning of the immunomodulation effect of BGB-283 and its potential differentiation from vemurafenib. These findings support BGB-283 as a promising clinical stage antitumor drug that could potentially combine with immunotherapies by enhancing immune sensitization and T cell function. Citation Format: Xi Yuan, Mi Lian, Xiaoran Wu, Lusong Luo. BGB-283 exhibits potentials in enhancing tumor immune sensitization and lymphocyte function. [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 A91.

Abstract LB-C12: Functional proteomics elucidates signaling adaptation driven by combination therapy in BRAF mutant melanoma cell line models

Abstract Targeted kinase inhibition is a promising treatment modality for melanoma, which has significantly increased available clinical strategies and improved survival outcomes for patients, whose tumors harbor the BRAF V600E mutation. However, responses are transient and multiple mechanisms for drug resistance can contribute to therapeutic escape. Therefore, a novel multiplexed proteomics approach provides an optimal choice for comprehensively dissecting multiple mechanisms of resistance to BRAFi and combination therapy within one experiment. A large-scale quantitative expression proteomics and phosphoproteomics analysis (SysQuant) was carried out on BRAF V600E mutant melanoma cell lines treated with two different clinically relevant kinase inhibitor combinations: 1) BRAFi/MEKi and 2) BRAFi/PI3Ki. Changes in protein expression and phosphorylation in response to each treatment are determined by comparison to vehicle controls. Cell line models all harbor BRAF V600E mutations, but differ in PTEN status (A375 is WT, while WM793 is PTEN null). Briefly, cells were treated with either drug combination (as above) and harvested at 1hr, 6hrs, 24hrs and 48hrs post-treatment. Control cells were treated with DMSO. Samples were lysed, reduced, alkylated and digested with trypsin. Tryptic peptides from each sample were chemically labeled or “barcoded” with TMT-10plex reagents (4 BRAFi/MEKi time points, 4 BRAFi/PI3Ki time points, 1 DMSO control and a pooled reference sample) and combined for LC-MS/MS. After peptide fractionation with strong cation exchange chromatography and phosphopeptide enrichment, LC-MS/MS peptide sequencing and relative quantification was performed using an Orbitrap Fusion mass spectrometer (Thermo). Raw MS data was searched by Proteome Discoverer and analyzed by in-house R-scripts and Perseus statistical software package. Pathway analysis was done in GeneGO (Metacore). The SysQuant workflow identified &amp;gt;9,000 protein groups and &amp;gt;17,000 unique phosphosites per cell line across different treatments. Principal component analysis of the phosphoproteomics data revealed signaling differences across different treatment conditions and drug combinations are mainly driven by BRAFi and the time post-treatment. K-means clustering was also used to examine trends in the data; as an example, this technique could be used to track signaling changes that correlate with reduction and recovery in ERK signaling. The SysQuant approach provides a systems view of global signaling changes occurring in response to drug treatment. The ability to multiplex samples with TMT allows quantitative deduction of protein expression and phosphorylation patterns that are common or unique in different cell lines, time post-treatment and the effect of combination treatments. This proteomics approach ties protein expression and phosphorylation status in response to combination therapy and generates several hypotheses for further testing with the goal of developing novel combination therapy strategies. Citation Format: Ritin Sharma, Inna Fedorenko, Sasa Koncarevic, Vikram Mitra, Stefan Selzer, Gitte Boehm, Ian Pike, Keiran Smalley, John M. Koomen. Functional proteomics elucidates signaling adaptation driven by combination therapy in BRAF mutant melanoma cell line models. [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 LB-C12.

AM251 induces apoptosis and G2/M cell cycle arrest in A375 human melanoma cells.

Human cutaneous melanoma is an aggressive and chemotherapy-resistant type of cancer. AM251 is a cannabinoid type 1 (CB1) receptor antagonist/inverse agonist with off-target antitumor activity against pancreatic and colon cancer cells. The current study aimed to characterize the in-vitro antimelanoma activity of AM251. The BRAF V600E mutant melanoma cell line, A375, was used as an in-vitro model system. Characterization tools included a cell viability assay, nuclear morphology assessment, gene expression, western blot, flow cytometry with Annexin V-FITC/7-AAD double staining, cell cycle analyses, and measurements of changes in intracellular cAMP and calcium concentrations. AM251 exerted a marked cytotoxic effect against A375 human melanoma cells with potency comparable with that observed for cisplatin without significant changes in the human dermal fibroblasts viability. AM251, at a concentration that approximates the IC50, downregulated genes encoding antiapoptotic proteins (BCL2 and survivin) and increased transcription levels of proapoptotic BAX, induced alteration of Annexin V reactivity, DNA fragmentation, chromatin condensation in the cell nuclei, and G2/M phase arrest.AM251 also induced a 40% increase in the basal cAMP levels, but it did not affect intracellular calcium concentrations. The involvement of GPR55, TRPA1, and COX-2 in the AM251 mechanism of action was excluded. The combination of AM251 with celecoxib produced a synergistic antitumor activity, although the mechanism underlying this effect remains to be elucidated. This study provides the first evidence of a proapoptotic effect and G2/M cell cycle arrest of AM251 on A375 cells. This compound may be a potential prototype for the development of promising diarylpyrazole derivatives to be evaluated in human cutaneous melanoma.

Abstract 2682: Melanoma phenotype switching to adapt to BRAF inhibition

Abstract Melanoma can adapt to several stress factors, such as TNF treatment, by changing its phenotype from a highly differentiated (MITF and MART-1 positive) to undifferentiated (neural crest markers) states. We studied this phenomenon as a mechanism of adaptive resistance to BRAF inhibition since melanomas with both MITF high and low have been reported to result in BRAF inhibitor resistance. We treated 17 BRAFV600E mutant melanoma cell lines with the BRAF inhibitor vemurafenib for short term (3 days) or long term (17 days), and compared them to no treatment or TNF treated conditions. We profiled them phenotypically for MART1 antigen (differentiated phenotype) and nerve growth factor receptor (NGFR, neural crest phenotype) expression by flow cytometry. Results comparing median fluorescence intensity (MFI) showed a wide spectrum of adaptive responses to short term BRAF inhibition, followed by solidification or further evolution of these changes upon long term exposure and development of drug tolerance. According to each cell line distribution, we calculated a (x,y) value that organized the cell lines and conditions in four quadrants according to MART1 (x axis) or NGFR (y axis) expression. Two double negative (MART1-/NGFR-) cell lines that were unaffected by treatment and stayed in the same quadrant. Seven MART1-/NGFR+ and four MART1+/NGFR- cell lines slightly changed phenotype with vemurafenib exposure but stayed in the same quadrant. Four cell lines with MART1+/NGFR+ phenotype were greatly influenced by treatment, showing wide plasticity with cells shifting towards NGFR+/MART1- and others towards NGFR-/MART1+. Changes in position between baseline and tolerance to vemurafenib after 17days treatment were numerically quantified as an inferred number from plasticity. Cell lines with double positive (MART1+/NGFR+) phenotype had a significant change in plasticity compared to the rest of cell lines in other quadrants (p = 0.0004). This arranges cells in a four-quadrant pattern that could be likened to a U-turn toward de-differentiation. Cells that de-differentiated with BRAF inhibition mimicked the effect induced by TNF. This effect was driven by a decrease in MITF activity confirmed with a tyrosinase promoter-luciferase assay. In conclusion, baseline melanoma differentiation phenotypes guide the phenotype switch response, with those double positive for differentiation and neural crest markers being at a semi-stable state that can be prone to different plastic changes upon BRAF inhibition. Citation Format: Lidia Robert, Jennifer Tsoi, Angel Garica Diaz, Blanca Homet Moreno, Nhat Truong, Thomas Graeber, Antoni Ribas. Melanoma phenotype switching to adapt to BRAF inhibition. [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 2682. doi:10.1158/1538-7445.AM2015-2682

A new water soluble MAPK activator exerts antitumor activity in melanoma cells resistant to the BRAF inhibitor vemurafenib

Recovery of mitogen activated protein kinase (MAPK) or activation of alternative pathways, such as the PI3K/AKT/mTOR, are involved in acquired resistance to BRAF inhibitors which represent the first-line treatment of BRAF-mutated metastatic melanoma. We recently demonstrated that 6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)thio)hexan-1-ol (NBDHEX) and its water soluble analog 2-(2-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)thio)ethoxy)ethoxy)ethanol (MC3181) trigger apoptosis in BRAF V600E mutated melanoma cells through activation of the MAPK c-Jun N-terminal kinase (JNK). Herein, we investigated whether NBDHEX and MC3181 might exert antitumor activity against BRAF V600E mutated human melanoma cells rendered resistant to the BRAF inhibitor vemurafenib. To this aim we generated a subline of A375 melanoma resistant in vitro and in vivo to vemurafenib (A375-VR8) and characterized by NRAS G13R mutation, high basal levels of CRAF protein and phospho-activation of AKT. In these cells ERK phosphorylation was not significantly down-modulated by vemurafenib concentrations capable of abrogating ERK phosphorylation in sensitive A375 cells. Both NBDHEX and MC3181 induced marked antiproliferative and apoptotic effects in A375-VR8 cells and, at equitoxic concentrations, caused a strong phosphorylation of JNK, p38, and of the downstream mediators of apoptosis ATF2 and p53. Drug treatment further increased ERK phosphorylation, which was required for the cellular response to the NBD derivatives, as apoptosis was antagonized by the ERK inhibitor FR180204. Finally, in vivo administration of MC3181 provoked JNK activation at the tumor site and markedly reduced A375-VR8 growth. These evidences strongly suggest that the activation of multiple pro-apoptotic MAPK pathways by MC3181 might represent a new strategy for the treatment of melanoma resistant to BRAF inhibitors.

Abstract 5602: BRAF V600 mutated allelic specific imbalance (MASI) impact on sensitivity to vemurafenib

Abstract V600 mutations of BRAF are present in 40% of cutaneous melanoma and are currently targeted for therapy by specific inhibitors (vemurafenib, dabrafenib). While BRAFV600 mutations are mostly heterozygous, the impact of BRAF mutated allelic specific imbalance (MASI) on the sensitivity to BRAF inhibitors and on clinical outcome has not been evaluated so far. Here we compared cell proliferation, tumor growth and vemurafenib sensitivity of heterozygous versus homozygous BRAF V600E mutated melanoma cell lines. We also performed a retrospective study evaluating the impact on overall survival and drug response of BRAF V600E mutated melanoma patients with or without MASI. Methods: We conducted proliferation and cytotoxity assays to compare cell growth and sensitivity to vemurafenib and trametinib in heterozygous, versus homozygous BRAF V600 mutated human melanoma cell lines. Their in vivo tumorigenesis and sensitivity to vemurafenib was also evaluated upon engraftment in SCID mice. A retrospective series of 179 patients with metastatic melanoma harboring a BRAF V600 mutations were analyzed and early first line treatment response was compared between patients with or without MASI. Results: Our results showed that heterozygous BRAF V600 mutated melanoma cell lines proliferate less rapidly than homozygous cell lines in vitro (cell proliferation counts after 7 days range from 3.6 x106 to 7.7x106 cells for homozygote cells, and from 4.8x104 to 5.5x105 for heterozygous cell lines) and in vivo (palpable tumors appeared on average 7 days after inoculation for homozygous cells but after 39 days for heterozygous cells). Heterozygous cell lines are more resistant to BRAF inhibitors either in vitro or in vivo (IC50 of vemurafenib ranged from 0,1 to 0,198 µM for homozygous cells and from 8,62 to 22,65 µM for heterozygous cell lines). We have also shown that patients with BRAF V600 mutated tumors with MASI may have initially a poorer prognosis (median overall survival 733 days IC95% [381; 1762] versus 1011 days IC95%[879;2969] p=0.236) but tend to have better responses to systemic drugs than patients without MASI (percentage of primary resistance 23% versus 34%; p=0.56). In conclusion: This translational study shows that a MASI of BRAF in melanoma is associated with a higher cell proliferation and tumorigenic potential in vitro and in vivo. This mutational status is also associated with a better response to BRAF inhibitor in vitro and in mice xenografts. Clinical relevance of these data should be more thoroughly evaluated on larger and more homogeneous series of patients however our preliminary data show a trend toward a clinical-experimental correlation with similar yet still statistically insignificant results. If confirmed, our data suggest that study of the MASI of BRAF mutation could serve as a predictive marker of patient response to vemurafenib. The role of paradoxical activation of the MAPK signaling pathway in cells without MASI of BRAF needs to be further explored. Citation Format: Amelie Boespflug, Luc Thomas, Pascal Rousset, Charles Dumontet, Julie Caramel, Carole Ferraro-Peyret, Pierre Paul Bringuier, Brigitte Balme, Stephane Dalle. BRAF V600 mutated allelic specific imbalance (MASI) impact on sensitivity to vemurafenib. [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 5602. doi:10.1158/1538-7445.AM2014-5602