Sensitivity Of Melanoma Cell Lines Research Articles

Targeting AMP kinase in melanoma: A phase I trial of phenformin with dabrafenib/trametinib.

9536 Background: AMP-activated protein kinase (AMPK) can phosphorylate BRAF and block its activation of MEK. AMPK is activated by biguanides such as metformin and phenformin (phen). Preclinical data indicated that phen can enhance the effects of BRAF inhibitors (BRAFi) in sensitive and BRAFi-resistant melanoma (mel) cell lines whereas metformin does not. Phen also can decrease myeloid-derived suppressor cells (MDSCs) enhancing immune reactivity against mel. For these reasons, we designed a phase I trial combining phen with dabrafenib/trametinib (dab/tram) in patients (pts) with metastatic BRAF V600-mutated mel. Methods: Bulk phen was provided by the Division of Cancer Treatment and Diagnosis of the National Cancer Institute under a materials transfer agreement. Phen was encapsulated at the Pharmaceutical Product Facility at MSKCC under cGMP procedures and used under an IND held by MSKCC. Eligible pts had metastatic BRAF V600-mutated mel. For the dose-escalation phase, prior BRAFi therapy was allowed. A 3+3 dose escalation design was used starting at a phen dose of 50 mg bid and standard dosing of dab/tram (150 mg bid/2 mg qd). Subsequent planned dose levels were 100 mg bid, 200 mg bid, and 300 mg bid with de-escalation to intermediate doses for toxicity. Plasma PK samples were collected and assayed by MRI Global Inc using a validated, GLP-compliant, HPLC-MS/MS assay. PBMC were collected to measure circulating MDSCs. At the recommended phase 2 dose (RP2D), we planned to treat 10 BRAFi-naïve pts. Results: We accrued 18 patients from March 2017 through December 2019. Dose-escalation to 200 mg bid was not tolerable nor was 150 mg bid. Therefore, we determined 100 mg bid to be the RP2D and accrued 2 patients on the dose expansion cohort before further accrual became impossible due to the COVID-19 pandemic. The most common toxicities were gastrointestinal – nausea, vomiting, anorexia, and transaminase and alkaline phosphatase elevation – accounting for 9 DLTs. Two patients were hospitalized due to lactic acidosis. There were also constitutional/inflammatory and skin/mucosal adverse events thought to be related to dab/tram. Weight loss was seen in most patients (median 3.7% at week 8) thought to be related to phen. There were 8 PRs and 2 CRs. Responses were observed in 2/7 pts who had received prior therapy with BRAFi. One CR lasted > 1 year. There were 8/11 (73%) responders among the BRAFi-naïve pts. At the RP2D of 100 mg bid, day 8 median phen plasma level was 73.4 ng/ml (range 32.8-253.2). At the the 50 mg bid dose level, it was 40.8 ng/ml (range 29.7-110.2). MDSC could be analyzed from the 150 mg bid and 200 mg bid dose levels (N = 7 pts). In all but 1 pt, MDSCs decreased while on phen. Conclusions: We observed 10/18 (55.5%) total responses with 2/7 among previously treated pts. Circulating MDSCs decreased on treatment. Phen at 100 mg bid can be combined safely with dab/tram and should be explored in future randomized phase II trials. Clinical trial information: NCT03026517 .

Abstract 2746: PET-CT/FACS platform to detect the biodistribution of radioisotope 89Zr PD-1 inhibitor in syngeneic tumor models

Abstract Cancer treatment has been revolutionized by targeting the interaction between tumors and immune system. For instance, PD-1/PD-L1 mono- or combo-therapy has achieved great success in clinics, and patient responsiveness is closed associated with PD-L1 expression in cancer cells, tumor infiltration leukocytes, and drug bio-distribution in the tumor microenvironment. We established a method to label PD-1 inhibitor with radioisotope 89Zr for in-life imaging (PET-CT). Anti-PD-1 sensitive mouse colorectal cell line MC38 and resistant mouse melanoma cell line B16F10 were implanted to C57BL/6 mice, which were then treated with 89Zr PD-1 inhibitor. PET imaging was performed at multiple time points to detect the bio-distribution of anti-PD-1, while the infiltrated immune cells were identified by FCM and PD-L1 expression measured by IHC. Notable tumor uptake of 89Zr PD-1 inhibitor was observed in mouse liver and tumor grafts. The uptake in tumor increased over time and plateaued 24 hours post-injection. MC38 and B16F10 tumor grafts showed difference in uptake levels. Blood and tumor at both flanks were removed for leucocyte phenotyping, and compared with B16F10, MC-38 tumors exhibited higher percentage of total lymphocytes and cytotoxic T lymphocytes, equivalent number of dendritic cells and less myeloid cells. Further phenotyping indicates that compared to MC-38, B16F10 tumors contain higher percentage of MDSC, most of which are monocytic myeloid derived suppressor cells. With our PET-CT/FACS platform, we can evaluate immunotherapy response and microenvironment changes in different tumors in “one-mouse” system. The unique immune microenvironment (high TIL, high CD8+ T and low MDSC) may contribute to the notable and sustainable uptake of PD-1 inhibitor and the corresponding efficacy. This system can act as a good translational platform to monitor the PK-PD readout of immune therapy. Citation Format: Xuzhen Tang, Fuyang Wang, Junyi Chen, Xuanming Zeng, Zhibo Liu, Qunsheng Ji. PET-CT/FACS platform to detect the biodistribution of radioisotope 89Zr PD-1 inhibitor in syngeneic tumor models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2746.

Abstract B41: BRAF targeting sensitizes resistant melanoma to cytotoxic T cells

Abstract Although treatment of combination therapy with BRAF and MEK inhibitors in patient with melanoma has shown longer duration of response and higher rate of tumor responses, recurrence of tumor from dual targeted therapy in patients with metastatic melanoma has still remained a challenge to overcome. Here, we provide new therapeutic approaches for BRAF inhibitors-resistant melanoma model that upregulation of mannose-6-phosphate receptor (M6PR) on melanoma by treatment of BRAF inhibitors enhances tumor cytotoxicity level via Granzyme B uptake, which is major component of cytotoxic activity of cytotoxic T lymphocytes (CTLs). Our results demonstrate that treatment of BRAF inhibitor increases M6PR expression temporally in both sensitive and resistant BRAFi melanoma cell lines as well as tumor-bearing mouse models (NOD/SCID). Cytotoxic activity of human melanoma cells by co-culture with patient-derived tumor-infiltrating lymphocytes (TILs) shows correlation with M6PR expression level. Furthermore, treatment of mice bearing resistant tumor with BRAFi enhances the antitumor effect of adaptive TILs transfer. In pilot clinical trials of 16 patients with metastatic melanoma, our results support that administration of BRAF inhibitor to patient shows high level of M6PR expression on tumor tissues. Also, combination treatment of BRAF inhibitor and adaptive cell transfer with patient-derived TILs shows promising clinical results in terms of longer response and tumor-free survival. Taken together, BRAF targeted therapy upregulates M6PR level, which enhances activity of TILs to kill resistant melanoma, providing new therapeutic approaches for patients who have recurrence melanoma. Note: This abstract was not presented at the conference. Citation Format: Cigdem Atay, Taekyoung Kwak, Sergio Lavilla-Alonso, Allison Richards, Valerie Moberg, Shari Pilon-Thomas, Michael Schell, Jane L. Messina, Vito W. Rebecca, Gao Zhang, Jeffrey S. Weber, Meenhard Herlyn, Amod A. Sarnaik, Dmitry I. Gabrilovich. BRAF targeting sensitizes resistant melanoma to cytotoxic T cells [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B41.

Imidazothiazole-based potent inhibitors of V600E-B-RAF kinase with promising anti-melanoma activity: biological and computational studies

A series of imidazothiazole derivatives possessing potential activity against melanoma cells were investigated for molecular mechanism of action. The target compounds were tested against V600E-B-RAF and RAF1 kinases. Compound 1zb is the most potent against both kinases with IC50 values 0.978 and 8.2 nM, respectively. It showed relative selectivity against V600E mutant B-RAF kinase. Compound 1zb was also tested against four melanoma cell lines and exerted superior potency (IC50 0.18-0.59 µM) compared to the reference standard drug, sorafenib (IC50 1.95-5.45 µM). Compound 1zb demonstrated also prominent selectivity towards melanoma cells than normal skin cells. It was further tested in whole-cell kinase assay and showed in-cell V600E-B-RAF kinase inhibition with IC50 of 0.19 µM. Compound 1zb induces apoptosis not necrosis in the most sensitive melanoma cell line, UACC-62. Furthermore, molecular dynamic and 3D-QSAR studies were done to investigate the binding mode and understand the pharmacophoric features of this series of compounds.

Inhibition of glucose metabolism through treatment of BRAF mutated metastatic melanoma with vemurafenib.

e21005 Background: Increased glucose metabolism is a hallmark of neoplastic cells that allows self-promotion of growth and survival. The enzyme 6-phosphofructo-2-kinase (PFKFB3) is an integral controller of glycolysis by promoting the synthesis of fructose 2,6-bisphosphonate (F2,6BP) which activates 6-phoshofructo-1-kinase (PFK-1), a rate-limiting enzyme and essential control point in the glycolytic pathway. Additionally, mitogen-activated protein kinase (MAPK) is a key signaling pathway in a number of cancers with mutations of the BRAF component, described most commonly in melanoma, resulting in constitutive activation of the MAPK pathway. We aim to demonstrate that vemurafenib, a BRAF inhibitor, has antiglycolytic activity in sensitive melanoma cell lines which may help guide development of future therapies with specific attention to PFKFB3 as a potential enzymatic target to decrease glycolytic flux thereby inhibiting tumor growth and survival. Methods: Vemurafenib sensitive and resistant variants of two separate human metastatic melanoma cell lines (451Lu and WM983) were treated with 3 mM Vemurafenib for 24 and 48 hours. Additionally, cells from aforementioned lines were probed for PFKFB3 after 24 hours of treatment with vemurafenib. Glycolysis was measured by incubating cells in complete media containing 1 mCi [5-3H]glucose for 60 minutes. [3H]H2O produced by glycolysis through enolase was measured. Results: A decrease in PFKFB3 protein expression was found in vemurafenib sensitive cells compared to controls but not in resistant cells after 24h treatment with 3 mM vemurafenib in both 451Lu and WM983 metastatic melanoma cell lines (n = 2). Treatment with vemurafenib led to decrease in glycolysis compared to untreated controls in both vemurafenib sensitive metastatic melanoma cell lines but not in resistant cell lines (n = 5). Additionally, there was a significant reduction in glycolysis in vemurafenib resistant WM983 at 48 hours compared to resistant untreated control. Conclusions: BRAF mutated metastatic melanoma cells showed decrease in PFKFB3 protein expression and decreased glycolysis after treatment with BRAF inhibitor vemurafenib. Future studies will focus on assessing metastatic melanoma cell viability and glycolytic activity after treatment with combination BRAF inhibition and PFKFB3 specific inhibition.

76P - Targeting p63 upregulation may prevent the development of MAPK inhibitor resistance in melanoma

Background: Melanoma progression is often characterized by mutations in the mitogen-activated protein kinase (MAPK) and phosphoinositol-3-kinase (PI3K) pathways. Understanding regulation of these pathways has led to the development of novel targeted therapies which show high response rates. However, many patients relapse with ensuing resistant disease. P63, a p53 homologue, carries a poorer prognosis when overexpressed. In keratinocytes, degradation could be regulated by two ubiquitin ligases, MDM2 and FBXW7. This project explored the expression of p63, MDM2 and FBXW7 in MAPK-inhibitor (MAPKi) sensitive and resistant melanoma cell lines. Methods: Morphology change associated with the development of resistance was assessed by actin immunofluorescence experiments. Western blot and qRT-PCR enabled analysis of protein and RNA expression of p63, FBXW7 and MDM2, complemented by immunofluorescent staining and confocal microscopy data. Finally, a novel technique of flow cytometry enabled analysis of cell death in MAPKi-resistant melanoma cells treated with Nutlin-3A. Results: MAPKi resistance in melanoma cells is associated with increased p63 expression. Resistance is furthermore associated with reduced FBXW7 expression and enrichment of nuclear MDM2, suggesting a potential nuclear interaction between MDM2 and FBXW7. The resultant inactivation of FBXW7 explains the increased p63 expression upon MAPKi- resistance. Furthermore, by treating MAPKi-resistant cells with the MDM2-inhibitor Nutlin-3A, we were able to restore FBXW7 expression and promote p63-degradation, with resulting increased melanoma cell death. Conclusions: This project identifies FBXW7 and MDM2 as regulators of p63 expression in MAPKi-resistant melanoma and proposes a possible role for Nutlin-3A in treating advanced MAPKi-resistant melanoma. Legal entity responsible for the study: Queen Mary University of London (Blizzard Institute) - Cutaneous Research. Funding: Has not received any funding. Disclosure: All authors have declared no conflicts of interest.

Abstract 5021: Assessment of RAS-RAF-MAPK pathway perturbations and PD-L1 expression in an Isogenic 3D tissue-culture model of drug-resistant melanoma

Abstract Development of the next generation of anti-cancer drugs, biologics, and immunotherapies is currently hampered by extremely poor success rates of seemingly promising experimental therapies in human clinical trials. This poor success rate is partially due to a lack of biologically relevant cancer model systems. The 2D culture system, while relatively inexpensive and easy to use, may not always be adequately representative of the tumor microenvironment. Conversely, the use of animal models is costly and time consuming. The 3D culture system is able to represent biologically relevant complexities such as cell-cell communication, differential proliferation rates, and compound penetration. Combining 3D culture with CRISPR/Cas9 genome editing technologies can generate powerful cell-based model systems with both physiological relevance and well-controlled genetic and drug-susceptibility profiles. These new models will enable the investigation of specific molecular mechanisms, biofunctional outcomes of newly identified genetic alternations, and targeted therapeutic drug responses within a more biologically intricate context. In this study, we use CRISPR/Cas9 to generate isogenic drug-resistant melanoma models that can be used as either 2D or 3D cancer models, as well as for studies of the acquired drug resistance. Two different models were generated starting with the BRAF-inhibitor sensitive A375 melanoma cell line. We introduced either the NRASQ61K or the KRASG13D point mutations, both of which are known to confer BRAF inhibitor resistance and are commonly encountered in BRAF-resistant tumor samples. We then assessed the susceptibility of these new isogenic lines to BRAF inhibitors in both 2D and 3D model systems. We also determined the specific effect of these point mutations on the RAS-RAF-MAPK signaling pathway, a key component of cell-cycle escape and tumor proliferation. Furthermore, we assessed the impact of these mutations on the expression of Programmed Death-Ligand 1 (PD-L1), which recent advances in cancer immunology have directly linked to cancer immune evasion and poor clinical outcomes. Our results show that A375 melanoma isogenic cells carrying KRASG13D have dramatically increased EGFR expression levels, while isogenic cells carrying NRASQ61K have constant activation of the MEK-ERK pathway. We also found a significant increase in PD-L1 expression in KRASG13D, but not in NRASQ61K A375 cells, indicating that expression of PD-L1 is directly linked to KRAS mutation or the downstream effects thereof, rather than to a general increase in RAS-RAF-MAPK signaling. These results have direct implications for cancer immune checkpoint molecule studies, and highlight the utility of these isogenic melanoma models in both 2D and 3D applications in research and development of novel anti-cancer drugs and combination therapies. Citation Format: Elizabeth Turner, Luping Chen, Johnathan Haag, Alexei Miagkov, Lysa-Anne Volpe, Metewo Selase Enuameh, Robert Newman, Fang Tian. Assessment of RAS-RAF-MAPK pathway perturbations and PD-L1 expression in an Isogenic 3D tissue-culture model of drug-resistant melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5021.

High-Speed Live-Cell Interferometry: A New Method for Quantifying Tumor Drug Resistance and Heterogeneity.

We report the development of high-speed live-cell interferometry (HSLCI), a new multisample, multidrug testing platform for directly measuring tumor therapy response via real-time optical cell biomass measurements. As a proof of concept, we show that HSLCI rapidly profiles changes in biomass in BRAF inhibitor (BRAFi)-sensitive parental melanoma cell lines and in their isogenic BRAFi-resistant sublines. We show reproducible results from two different HSLCI platforms at two institutions that generate biomass kinetic signatures capable of discriminating between BRAFi-sensitive and -resistant melanoma cells within 24 h. Like other quantitative phase imaging (QPI) modalities, HSLCI is well-suited to noninvasive measurements of single cells and cell clusters, requiring no fluorescence or dye labeling. HSLCI is substantially faster and more sensitive than field-standard growth inhibition assays, and in terms of the number of cells measured simultaneously, the number of drugs tested in parallel, and temporal measurement range, it exceeds the state of the art by more than 10-fold. The accuracy and speed of HSLCI in profiling tumor cell heterogeneity and therapy resistance are promising features of potential tools to guide patient therapeutic selections.

Identification of coexistence of BRAF V600E mutation and EZH2 gain specifically in melanoma as a promising target for combination therapy

BackgroundCoexistence of enhancer of zeste homolog 2 (EZH2) and BRAF gene aberrations has been described in many cancer types. In this study, we aim to explore the coexistence status of BRAF V600E mutation and the copy number variation of EZH2 and explore the potential of this combination as a therapeutic target.MethodsA total of 138 cases of melanoma samples harboring BRAF V600E mutation were included, and EZH2 copy numbers were examined by QuantiGenePlex DNA Assays. Clinical pathological distinction between patient groups with or without EZH2 amplification (hereafter referred to as EZH2 gain) was statistically analyzed. The sensitivity of melanoma cell lines and patient-derived xenograft (PDX) models containing BRAF V600E mutation with or without EZH2 gain to vemurafenib (BRAF inhibitor), GSK2816126 (EZH2 inhibitor) and a combination of both agents was evaluated.ResultsIn our cohort, the coexistence rate of BRAF V600E mutation and EZH2 gain was up to 29.0%, and significant differences in overall survival and disease-free survival were found between no EZH2 copy number gain and gain groups (P = 0.038, P = 0.030), gain and high EZH2 copy number gain groups (P = 0.006, P = 0.010). Combination with BRAF and EZH2 inhibition showed better inhibitory efficacy in melanoma prevention compared with vemurafenib monotherapy. More importantly, this improved therapeutic effect was observed especially in melanoma cell lines and PDX models containing concurrently BRAF V600E mutation and EZH2 gain.ConclusionsCoexistence of BRAF V600E mutation and EZH2 gain is rather prevalent in melanoma. Our findings provided evidence for the feasibility of combination therapy with EZH2 and BRAF inhibitors in melanoma with concurrent BRAF V600E mutation and EZH2 gain.

562 Sensitivity of melanoma cell lines to combination of mefloquine and vemurafenib

562 Sensitivity of melanoma cell lines to combination of mefloquine and vemurafenib

Silencing FLI or targeting CD13/ANPEP lead to dephosphorylation of EPHA2, a mediator of BRAF inhibitor resistance, and induce growth arrest or apoptosis in melanoma cells

A majority of patients with BRAF-mutated metastatic melanoma respond to therapy with BRAF inhibitors (BRAFi), but relapses are common owing to acquired resistance. To unravel BRAFi resistance mechanisms we have performed gene expression and mass spectrometry based proteome profiling of the sensitive parental A375 BRAF V600E-mutated human melanoma cell line and of daughter cell lines with induced BRAFi resistance. Increased expression of two novel resistance candidates, aminopeptidase-N (CD13/ANPEP) and ETS transcription factor FLI1 was observed in the BRAFi-resistant daughter cell lines. In addition, increased levels of the previously reported resistance mediators, receptor tyrosine kinase ephrine receptor A2 (EPHA2) and the hepatocyte growth factor receptor MET were also identified. The expression of these proteins was assessed in matched tumor samples from melanoma patients obtained before BRAFi and after disease progression. MET was overexpressed in all progression samples while the expression of the other candidates varied between the individual patients. Targeting CD13/ANPEP by a blocking antibody induced apoptosis in both parental A375- and BRAFi-resistant daughter cells as well as in melanoma cells with intrinsic BRAFi resistance and led to dephosphorylation of EPHA2 on S897, previously demonstrated to cause inhibition of the migratory capacity. AKT and RSK, both reported to induce EPHA2 S897 phosphorylation, were also dephosphorylated after inhibition of CD13/ANPEP. FLI1 silencing also caused decreases in EPHA2 S897 phosphorylation and in total MET protein expression. In addition, silencing of FLI1 sensitized the resistant cells to BRAFi. Furthermore, we show that BRAFi in combination with the multi kinase inhibitor dasatinib can abrogate BRAFi resistance and decrease both EPHA2 S897 phosphorylation and total FLI1 protein expression. This is the first report presenting CD13/ANPEP and FLI1 as important mediators of resistance to BRAF inhibition with potential as drug targets in BRAFi refractory melanoma.

Abstract B026: Ubiquitin-conjugating enzyme E2C: a potential therapeutic target for primary and metastatic melanoma by microarray gene expression

Abstract Purpose: Melanoma cells are resistant to endoplasmic reticulum (ER) stress-induced apoptosis. Ubiquitin proteasome pathway is vital for cells to restore normal ER function. Ubiquitin-conjugating enzyme E2C (UBE2C) participates in cell cycle progression and checkpoint control by degradation of mitotic cyclins. We examined UBE2C's biological function in melanoma cell growth. Experimental Design: Tissues from patients with primary and metastatic melanoma were collected. BRAF and NRAS mutation status were determined. We examined the gene expression profiles using cDNA microarray. 8261 differentially expressed genes were identified between primary and metastatic melanoma and were submitted to Panther classification system analysis for cluster analysis. The genes involved in the ubiquitin proteasome pathway were of most significant interest due to its role in the mediation of p53 degradation and ER stress mechanism. RNA was extracted from primary and metastatic melanoma tissue samples where cDNA was synthesized. Expression levels of UBE2C gene were determined based on the levels of the target message relative to the house keeping gene, GAPDH. Gene silencing was performed using UBE2C siRNA method. Growth inhibition was examined by treating melanoma cell lines with UBE2C siRNA and subsequently treating with PLX4032 at various concentrations. Results: The microarray analysis showed the basal level of UBE2C mRNA was highly expressed in several melanoma cell lines. UBE2C gene expression was higher in metastatic melanoma than that in primary melanoma of any tumor thickness ((1.23±0.14 vs 0.64±0.07, p<0.01). Microarray analysis from human melanoma cell lines showed that UBE2C basal expression was high cross the melanoma panel and the expression level was different among melanoma cell lines. UBE2C gene silencing significantly inhibited melanoma cell growth at day 4 (73.8%) and day 6 (83.8%) after UBE2C siRNA treatment. Cell death study demonstrated that the percent of apoptosis in cell lines treated with UBE2C siRNA_8 (22.1±0.89 vs 9.2±1.7, p<0.05) was significantly higher than cells treated with siRNA control oligos. Furthermore, BRAF inhibitor, PLX4032, decreased UBE2C expression through the MAPK pathway inhibition. UBE2C gene silencing sensitized melanoma cells to BRAF inhibitor and resulted in a higher level of apoptotic cells and cell cycle G2-phase arrest. The combination of PLX4032 and UBE2C silence has an additive effect on growth inhibition. Combination of UBE2C gene silencing and PLX4032 significantly inhibited cell growth (74.8±1.1% inhibition). Microarray data showed that UBE2C mRNA level was less down-regulated in BRAF inhibitor- resistant melanoma cell lines than that in BRAF inhibitor- sensitive melanoma cell lines (0.756±0.073 vs 0.393±0.138, p<0.05). Conclusions: Our data suggests that UBE2C may be a valuable target for primary and metastatic melanoma and those resistant to BRAF blockage associated with the MAPK pathway. The combination of a BRAF inhibitor and UBE2C silencing has a significant additive effect on the inhibition of melanoma cell growth. UBE2C gene silencing also sensitized the melanoma cells to BRAF inhibitor. UBE2C may be a powerful therapeutic target for melanoma therapy. Citation Format: Jenny Jimmy Hong, Kewei Gong, David Kaufman, Hsiaowang Chen, Richard Essner. Ubiquitin-conjugating enzyme E2C: a potential therapeutic target for primary and metastatic melanoma by microarray gene expression. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B026.

New Functional Signatures for Understanding Melanoma Biology from Tumor Cell Lineage-Specific Analysis.

SUMMARYMolecular signatures specific to particular tumor types are required to design treatments for resistant tumors. However, it remains unclear whether tumors and corresponding cell lines used for drug development share such signatures. We developed similarity core analysis (SCA), a universal and unsupervised computational framework for extracting core molecular features common to tumors and cell lines. We applied SCA to mRNA/miRNA expression data from various sources, comparing melanoma cell lines and metastases. The signature obtained was associated with phenotypic characteristics in vitro, and the core genes CAPN3 and TRIM63 were implicated in melanoma cell migration/invasion. About 90% of the melanoma signature genes belong to an intrinsic network of transcription factors governing neural development (TFAP2A, DLX2, ALX1, MITF, PAX3, SOX10, LEF1, and GAS7) and miRNAs (211-5p, 221-3p, and 10a-5p). The SCA signature effectively discriminated between two subpopulations of melanoma patients differing in overall survival, and classified MEKi/BRAFi-resistant and -sensitive melanoma cell lines.

Abstract 3089: CDKN2A and p53 status predicts response to CDK4/6 inhibition in melanoma

Abstract The p16-CyclinD1-CDK4-RB pathway regulates G1-S cell cycle transition. This pathway is deregulated in 90% of melanomas, through amplification or increased expression of CDK4 or Cyclin D1 (CCND1), or reduced expression of the CDK inhibitor p16INK4A (CDKN2A). To explore if CDK4 may be a viable target for the treatment of human melanoma we screened a panel of melanoma cell lines with the highly selective CDK4/6 inhibitor PD-0332991 (PD-991). A majority of cell lines were found to be sensitive to PD-991, although there was a distinct population of resistant cell lines. In sensitive melanoma cell lines, treatment with PD-991 resulted in loss of hyperphosphorylated RB and a concomitant G0/G1 cell cycle arrest. Treated cells displayed an enlarged flattened morphology and an increase in SA-β-galactosidase staining compared to vehicle controls, indicative of cellular senescence. RB-null cell lines were resistant to CDK4/6 inhibition, and did not display cell cycle arrest or senescence. Expression and mutational analysis was performed to identify additional genomic predictors of sensitivity and resistance. Loss of CDKN2A/p16 correlated with sensitivity to PD-991 (p<0.02), and this was confirmed at the level of protein expression. Mutation of p53 and the corresponding low expression of p53 targets p21 and MDM2 correlated with resistance (p<0.0001). There was no increase in p53 transcriptional targets or evidence of apoptosis in response to PD-991 treatment of sensitive cells, and senescence occurs in both p53 WT and mutant cells, indicating that this response does not require functional p53. However, in p53 WT cells, combination treatment with PD-991 and the p53 pathway activator Nutlin3a results in increased sensitivity to PD-991 compared to treatment with PD-991 alone. This additive effect is not observed in p53 mutant cells, suggesting there is a functional role for WT p53 in the response to PD-991. Conclusions: These preliminary data identify RB, p16 and p53 as predictors of response to PD-991, and support further evaluation of CDK4/6 inhibitors in melanoma. Citation Format: Claire A. Martin, Laura Kirby, Stephen Wong, Grant A. McArthur, Karen E. Sheppard. CDKN2A and p53 status predicts response to CDK4/6 inhibition in melanoma. [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 3089. doi:10.1158/1538-7445.AM2015-3089

Overexpression of pyruvate dehydrogenase kinase supports dichloroacetate as a candidate for cutaneous melanoma therapy

Objective: We aimed to verify if there is evidence to consider dichloroacetate (DCA), which inhibits the pyruvate dehydrogenase kinase (PDK) and reverts the metabolic shift of cancer cells from glycolysis to oxidative phosphorylation, as a promising drug for therapy of cutaneous melanoma (CM) patients.Research design and methods: We assessed the expression profile of PDK 1, 2 and 3 in a series of melanoma samples, to verify if melanoma tumors express the DCA targets, if this expression correlates with the activation of important signaling cascades for melanomagenesis and also with the prognosis of melanoma patients. We also established the sensitivity of melanoma cell lines to DCA treatment, by assessing their metabolic alterations, proliferation and survival.Results: We observed that both PDK 1 and 2 isoforms are overexpressed in CM compared to nevi, this expression being associated with the expression of the mTOR pathway effectors and independent of the BRAF mutational status. Melanoma cell lines treated with DCA showed a shift in metabolism, that is, a decrease in glucose consumption and lactate production, downregulation of proliferation, an increase of apoptosis and a decrease in mTOR pathway activation.Conclusion: Our results suggest that PDK expression may play a role in melanoma development and that DCA can be useful for CM therapy, alone or in combination with mTOR inhibitors.

Activating the Wnt/β-Catenin Pathway for the Treatment of Melanoma--Application of LY2090314, a Novel Selective Inhibitor of Glycogen Synthase Kinase-3.

It has previously been observed that a loss of β-catenin expression occurs with melanoma progression and that nuclear β-catenin levels are inversely proportional to cellular proliferation, suggesting that activation of the Wnt/β-catenin pathway may provide benefit for melanoma patients. In order to further probe this concept we tested LY2090314, a potent and selective small-molecule inhibitor with activity against GSK3α and GSK3β isoforms. In a panel of melanoma cell lines, nM concentrations of LY2090314 stimulated TCF/LEF TOPFlash reporter activity, stabilized β-catenin and elevated the expression of Axin2, a Wnt responsive gene and marker of pathway activation. Cytotoxicity assays revealed that melanoma cell lines are very sensitive to LY2090314 in vitro (IC50 ~10nM after 72hr of treatment) in contrast to other solid tumor cell lines (IC50 >10uM) as evidenced by caspase activation and PARP cleavage. Cell lines harboring mutant B-RAF or N-RAS were equally sensitive to LY2090314 as were those with acquired resistance to the BRAF inhibitor Vemurafenib. shRNA studies demonstrated that β-catenin stabilization is required for apoptosis following treatment with the GSK3 inhibitor since the sensitivity of melanoma cell lines to LY290314 could be overcome by β-catenin knockdown. We further demonstrate that in vivo, LY2090314 elevates Axin2 gene expression after a single dose and produces tumor growth delay in A375 melanoma xenografts with repeat dosing. The activity of LY2090314 in preclinical models suggests that the role of Wnt activators for the treatment of melanoma should be further explored.

Protein disulfide isomerases: Impact of thapsigargin treatment on their expression in melanoma cell lines

Anti-cancer treatments usually elevate the content of unfolded or misfolded proteins in the endoplasmic reticulum (ER). Here we aimed to get insights into the relation between sensitivity of melanoma cell lines to the ER stress inducer thapsigargin (THG) and the genetic expression of protein disulfide isomerase family members (PDIs). The expression of PDIs was analysed by flow cytometry and real-time PCR. The results showed that SK-MEL-30, the less THG sensitive cell line, displays higher basal PDIs’ expression levels and the sensitivity is increased by the PDIs inhibitor bacitracin. While SK-MEL-30 PDIs’ expression is not THG dose-dependent, an increase in glucose related protein 78 (GRP78), PDIA5, PDIA6, and thioredoxin-related-transmembrane proteins’ (TMX3 and TMX4) expression, in response to higher drug concentrations, was observed in MNT-1. The differences in PDIs’ gene expression in MNT-1 suggest a different response to ER stress compared to the other cell lines and highlight the importance of understanding the diversity among cancer cells.

Exosomes as a predictor tool of acquired resistance to melanoma treatment

Background Exosomes are small endosome-derived vesicles ranging from 30-120 nm in diameter secreted from cells through exocytosis and, in cancer context, are able to prepare metastasis niche and suppress host’s immune system. Despite these pro-tumorigenic characteristics, exosomes can be useful for several Biotechnological applications such as biological anticancer vaccines, cancer diagnosis and prognosis. Due to its property of carrying parental cell’s protein and active nucleic acids there is an increasing interest to use exosomes to track cancer metabolism alterations in different conditions, for example, during drug administration [1]. One of the most common types of cancer that develops drug resistance during drug treatment is Melanoma. The majority of drugs developed against melanoma targets the oncogenic mutations in BRAF (BRAF V600E) that are present in nearly 60% of cutaneous melanoma tumors. Resistance to BRAFV600E inhibitors are classified in two main groups, Intrinsic Resistance and Acquired Resistance, the second is developed after treatment starts that happens mainly by MAPK reactivation and RTK upregulation [2]. Various methods are being studied to overcome drug resistance in melanoma, but there is a need to detect when the resistant mechanism is arising to orientate which approach to choose. This early detection has demonstrated to be a difficult task especially during advanced stages where there are several metastatic niches. With this aim, our research project was designed to create a method to follow tumor’s status through melanoma-derived exosomes. As most types of melanoma overexpress MART-1 we checked its expression, as well as PDGFR-B and others proteins, in PLX4032 (V600E mutated BRAF inhibitor) resistant and sensitive melanoma cell lines to start to understand their potential to be exosomes-associated proteins in the development of acquired resistance detection method[2].

Abstract LB-214: The RAC1 P29S hotspot mutation in melanoma confers resistance to pharmacological inhibition of RAF

Abstract Following recurrent mutations in BRAF and NRAS, the RAC1 c.85C > T single nucleotide variant encoding P29S amino acid change represents the next most frequently observed hotspot mutation in melanoma. However, the biological and clinical relevance of the RAC1 P29S somatic mutation in approximately 4-9% of patients is not entirely clear. Here, we demonstrate that melanoma cell lines possessing the RAC1 hotspot variant are resistant to RAF inhibitors (dabrafenib and vemurafenib). Enforced expression of RAC1 P29S in sensitive BRAF mutant melanoma cell lines resulted in increased viability and in vivo tumor growth upon treatment with RAF inhibitors compared to controls. Conversely, knockdown of endogenous RAC1 P29S in a melanoma cell line with a co-occurring BRAF V600 mutation increased sensitivity to dabrafenib and vemurafenib. Our results suggest that RAC1 P29S status in melanoma may be an important predictor of RAF inhibitor resistance in patients, and consequently should be evaluated as a predictive biomarker to such therapies in the clinic. Citation Format: Ian R. Watson, Liren Li, Peter K. Cabeceiras, Mozhdeh Mahdavi, Zhuangna Fang, Katherine Stemke-Hale, Gordon B. Mills, Lynda Chin. The RAC1 P29S hotspot mutation in melanoma confers resistance to pharmacological inhibition of RAF. [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 LB-214. doi:10.1158/1538-7445.AM2014-LB-214

The RAC1 P29S hotspot mutation in melanoma confers resistance to pharmacological inhibition of RAF.

Following mutations in BRAF and NRAS, the RAC1 c.85C>T single-nucleotide variant (SNV) encoding P29S amino acid change represents the next most frequently observed protein-coding hotspot mutation in melanoma. However, the biologic and clinical significance of the RAC1 P29S somatic mutation in approximately 4% to 9% of patients remains unclear. Here, we demonstrate that melanoma cell lines possessing the RAC1 hotspot variant are resistant to RAF inhibitors (vemurafenib and dabrafenib). Enforced expression of RAC1 P29S in sensitive BRAF-mutant melanoma cell lines confers resistance manifested by increased viability, decreased apoptosis, and enhanced tumor growth in vivo upon treatment with RAF inhibitors. Conversely, RNAi-mediated silencing of endogenous RAC1 P29S in a melanoma cell line with a co-occurring BRAF V600 mutation increased sensitivity to vemurafenib and dabrafenib. Our results suggest RAC1 P29S status may offer a predictive biomarker for RAF inhibitor resistance in melanoma patients, where it should be evaluated clinically.