BRAF-mutant Melanoma Cell Lines Research Articles

Multi-omics analysis delineates resistance mechanisms associated with BRAF inhibition in melanoma cells

Mutant BRAF is a critical oncogenic driver in melanoma, making it an attractive therapeutic target. However, the success of targeted therapy using BRAF inhibitors vemurafenib and dabrafenib has been limited due to development of resistance, restricting their clinical efficacy. A prior knowledge of resistance mechanisms to BRAFi or any cancer drug can lead to development of drugs that overcome resistance thus improving clinical outcomes. In vitro cellular models are powerful systems that can be utilized to mimic and study resistance mechanisms. In this study, we employed a multi-omics approach to characterize a panel of BRAF mutant melanoma cell lines to develop and systematically characterize BRAFi persister and resistant cells using exome sequencing, proteomics and phosphoproteomics. Our datasets revealed frequently observed intrinsic and acquired, genetic and non-genetic mechanisms of BRAFi resistance that have been studied in patients who developed resistance. In addition, we identified proteins that can be potentially targeted to overcome BRAFi resistance. Overall, we demonstrate that in vitro systems can be utilized not only to predict resistance mechanisms but also to identify putative therapeutic targets.

Combined therapy of dabrafenib and an anti-HER2 antibody–drug conjugate for advanced BRAF-mutant melanoma

BackgroundMelanoma is the most lethal skin cancer characterized by its high metastatic potential. In the past decade, targeted and immunotherapy have brought revolutionary survival benefits to patients with advanced and metastatic melanoma, but these treatment responses are also heterogeneous and/or do not achieve durable responses. Therefore, novel therapeutic strategies for improving outcomes remain an unmet clinical need. The aim of this study was to evaluate the therapeutic potential and underlying molecular mechanisms of RC48, a novel HER2-target antibody drug conjugate, either alone or in combination with dabrafenib, a V600-mutant BRAF inhibitor, for the treatment of advanced BRAF-mutant cutaneous melanoma.MethodsWe evaluated the therapeutic efficacy of RC48, alone or in combination with dabrafenib, in BRAF-mutant cutaneous melanoma cell lines and cell-derived xenograft (CDX) models. We also conducted signaling pathways analysis and global mRNA sequencing to explore mechanisms underlying the synergistic effect of the combination therapy.ResultsOur results revealed the expression of membrane-localized HER2 in melanoma cells. RC48 effectively targeted and inhibited the growth of HER2-positive human melanoma cell lines and corresponding CDX models. When used RC48 and dabrafenib synergically induced tumor regression together in human BRAF-mutant melanoma cell lines and CDX models. Mechanically, our results demonstrated that the combination therapy induced apoptosis and cell cycle arrest while suppressing cell motility in vitro. Furthermore, global RNA sequencing analysis demonstrated that the combination treatment led to the downregulation of several key signaling pathways, including the PI3K-AKT pathway, MAPK pathway, AMPK pathway, and FOXO pathway.ConclusionThese findings establish a preclinical foundation for the combined use of an anti-HER2 drug conjugate and a BRAF inhibitor in the treatment of BRAF-mutant cutaneous melanoma.

Effective Targeting of Melanoma Cells by Combination of Mcl-1 and Bcl-2/Bcl-xL/Bcl-w Inhibitors.

Recent advances in melanoma therapy have significantly improved the prognosis of metastasized melanoma. However, large therapeutic gaps remain that need to be closed by new strategies. Antiapoptotic Bcl-2 proteins critically contribute to apoptosis deficiency and therapy resistance. They can be targeted by BH3 mimetics, small molecule antagonists that mimic the Bcl-2 homology domain 3 (BH3) of proapoptotic BH3-only proteins. By applying in vitro experiments, we aimed to obtain an overview of the possible suitability of BH3 mimetics for future melanoma therapy. Thus, we investigated the effects of ABT-737 and ABT-263, which target Bcl-2, Bcl-xL and Bcl-w as well as the Bcl-2-selective ABT-199 and the Mcl-1-selective S63845, in a panel of four BRAF-mutated and BRAF-WT melanoma cell lines. None of the inhibitors showed significant effectiveness when used alone; however, combination of S63845 with each one of the three ABTs almost completely abolished melanoma cell survival and induced apoptosis in up to 50-90% of the cells. Special emphasis was placed here on the understanding of the downstream pathways involved, which may allow improved applications of these strategies. Thus, cell death induction was correlated with caspase activation, loss of mitochondrial membrane potential, phosphorylation of histone H2AX, and ROS production. Caspase dependency was demonstrated by a caspase inhibitor, which blocked all effects. Upregulation of Mcl-1, induced by S63845 itself, as reported previously, was blocked by the combinations. Indeed, Mcl-1, as well as XIAP (X-linked inhibitor of apoptosis), were strongly downregulated by combination treatments. These findings demonstrate that melanoma cells can be efficiently targeted by BH3 mimetics, but the right combinations have to be selected. The observed pronounced activation of apoptosis pathways demonstrates the decisive role of apoptosis in the loss of cell viability by BH3 mimetics.

Dataset: Impact of β-Galactosylceramidase Overexpression on the Protein Profile of Braf(V600E) Mutated Melanoma Cells

β-Galactosylceramidase (GALC) is a lysosomal enzyme involved in sphingolipid metabolism by removing β-galactosyl moieties from β-galactosyl ceramide and β-galactosyl sphingosine. Previous observations have shown that GALC exerts a pro-oncogenic activity in human melanoma. Here, the impact of GALC overexpression on the proteomic landscape of BRAF-mutated A2058 and A375 human melanoma cell lines was investigated by liquid chromatography–tandem mass spectrometry analysis of the cell extracts. The results indicate that GALC overexpression causes the upregulation/downregulation of 172/99 proteins in GALC-transduced cells when compared to control cells. Gene ontology categorization of up/down-regulated proteins indicates that GALC may modulate the protein landscape in BRAF-mutated melanoma cells by affecting various biological processes, including RNA metabolism, cell organelle fate, and intracellular redox status. Overall, these data provide further insights into the pro-oncogenic functions of the sphingolipid metabolizing enzyme GALC in human melanoma.

In Vitro Experiments on the Effects of GP-2250 on BRAF-Mutated Melanoma Cell Lines and Benign Melanocytes.

Enhanced glycolysis (Warburg effect) driven by the BRAF oncogene, dysregulated GAPDH expression, and activation of the PI3K/AKT/mTOR signaling pathway may significantly contribute to the resistance-targeted therapy of BRAF-mutated melanomas. Therefore, we aimed to study for the first time the anti-tumor activity of the GAPDH inhibitor GP-2250 in BRAF-mutated melanoma cell lines and benign melanocytes. We employed three melanoma cell lines and one primary melanocyte cell line (Ma-Mel-61a, Ma-Mel-86a, SH-4 and ATCC-PCS-200-013, respectively), which were exposed to different GP-2250 doses. GP-2250's effects on cell proliferation and viability were evaluated by means of the BrdU and MTT assays, respectively. The RealTime-Glo Annexin V Apoptosis and Necrosis Assay was performed for the evaluation of apoptosis and necrosis induction. RT-PCR and western blotting were implemented for the determination of AKT and STAT3 gene and protein expression analyses, respectively. The melanoma cell lines showed a dose-dependent response to GP-2250 during BrDU and MTT testing. The RealTime-Glo Annexin V assay revealed the heterogenous impact of GP-2250 on apoptosis as well as necrosis. With respect to the melanoma cell lines Ma-Mel-86a and SH-4, the responses and dosages were comparable to those used for the MTT viability assay. Using the same dose range of GP-2250 administered to melanoma cells, however, we observed neither the noteworthy apoptosis nor necrosis of GP-2250-treated benign melanocytes. The gene expression profiles in the melanoma cell lines for AKT and STAT3 were heterogenous, whereby AKT as well as STAT3 gene expression were most effectively downregulated using the highest GP-2250 doses. Immunoblotting revealed that there was a time-dependent decrease in protein expression at the highest GP-2250 dose used, whereas a time- as well as dose-dependent AKT decrease was predominantly observed in Ma-Mel-61a. The STAT3 protein expression of Ma-Mel-86a and SH-4 was reduced in a time-dependent pattern at lower and moderate doses. STAT3 expression in Ma-Me-61a was barely altered by GP-2250. In conclusion, GP-2250 has anti-neoplastic effects in BRAF-mutated melanoma cell lines regarding tumor cell viability, proliferation, and apoptosis/necrosis. GP-2250 is able to downregulate the gene and protein expression of aberrant tumorigenic pathways in melanoma cell lines. Since GP-2250 is a GAPDH inhibitor, the substance may be a promising combination therapy for tumors presenting the Warburg effect, such as melanoma.

Hyperforin Enhances Heme Oxygenase-1 Expression Triggering Lipid Peroxidation in BRAF-Mutated Melanoma Cells and Hampers the Expression of Pro-Metastatic Markers.

Hyperforin (HPF) is an acylphloroglucinol compound found abundantly in Hypericum perforatum extract which exhibits antidepressant, anti-inflammatory, antimicrobial, and antitumor activities. Our recent study revealed a potent antimelanoma effect of HPF, which hinders melanoma cell proliferation, motility, colony formation, and induces apoptosis. Furthermore, we have identified glutathione peroxidase-4 (GPX-4), a key enzyme involved in cellular protection against iron-induced lipid peroxidation, as one of the molecular targets of HPF. Thus, in three BRAF-mutated melanoma cell lines, we investigated whether iron unbalance and lipid peroxidation may be a part of the molecular mechanisms underlying the antimelanoma activity of HPF. Initially, we focused on heme oxygenase-1 (HO-1), which catalyzes the heme group into CO, biliverdin, and free iron, and observed that HPF treatment triggered the expression of this inducible enzyme. In order to investigate the mechanism involved in HO-1 induction, we verified that HPF downregulates the BTB and CNC homology 1 (BACH-1) transcription factor, an inhibitor of the heme oxygenase 1 (HMOX-1) gene transcription. Remarkably, we observed a partial recovery of cell viability and an increase in the expression of the phosphorylated and active form of retinoblastoma protein when we suppressed the HMOX-1 gene using HMOX-1 siRNA while HPF was present. This suggests that the HO-1 pathway is involved in the cytostatic effect of HPF in melanoma cells. To explore whether lipid peroxidation is induced, we conducted cytofluorimetric analysis and observed a significant increase in the fluorescence of the BODIPY C-11 probe 48 h after HPF administration in all tested melanoma cell lines. To discover the mechanism by which HPF triggers lipid peroxidation, along with the induction of HO-1, we examined the expression of additional proteins associated with iron homeostasis and lipid peroxidation. After HPF administration, we confirmed the downregulation of GPX-4 and observed low expression levels of SLC7A11, a cystine transporter crucial for the glutathione production, and ferritin, able to sequester free iron. A decreased expression level of these proteins can sensitize cells to lipid peroxidation. On the other hand, HPF treatment resulted in increased expression levels of transferrin, which facilitates iron uptake, and LC3B proteins, a molecular marker of autophagy induction. Indeed, ferritin and GPX-4 have been reported to be digested during autophagy. Altogether, these findings suggest that HPF induced lipid peroxidation likely through iron overloading and decreasing the expression of proteins that protect cells from lipid peroxidation. Finally, we examined the expression levels of proteins associated with melanoma cell invasion and metastatic potential. We observed the decreased expression of CD133, octamer-4, tyrosine-kinase receptor AXL, urokinase plasminogen activator receptor, and metalloproteinase-2 following HPF treatment. These findings provide further support for our previous observations, demonstrating the inhibitory effects of HPF on cell motility and colony formation in soft agar, which are both metastasis-related processes in tumor cells.

Pharmacological induction of membrane lipid poly-unsaturation sensitizes melanoma to ROS inducers and overcomes acquired resistance to targeted therapy

BackgroundOne of the key limitations of targeted cancer therapies is the rapid onset of therapy resistance. Taking BRAF-mutant melanoma as paradigm, we previously identified the lipogenic regulator SREBP-1 as a central mediator of resistance to MAPK-targeted therapy. Reasoning that lipogenesis-mediated alterations in membrane lipid poly-unsaturation lie at the basis of therapy resistance, we targeted fatty acid synthase (FASN) as key player in this pathway to evoke an exquisite vulnerability to clinical inducers of reactive oxygen species (ROS), thereby rationalizing a novel clinically actionable combination therapy to overcome therapy resistance.MethodsUsing gene expression analysis and mass spectrometry-based lipidomics of BRAF-mutant melanoma cell lines, melanoma PDX and clinical data sets, we explored the association of FASN expression with membrane lipid poly-unsaturation and therapy-resistance. Next, we treated therapy-resistant models with a preclinical FASN inhibitor TVB-3664 and a panel of ROS inducers and performed ROS analysis, lipid peroxidation tests and real-time cell proliferation assays. Finally, we explored the combination of MAPK inhibitors, TVB-3664 and arsenic trioxide (ATO, as a clinically used ROS-inducer) in Mel006 BRAF mutant PDX as a gold model of therapy resistance and assessed the effect on tumor growth, survival and systemic toxicity.ResultsWe found that FASN expression is consistently increased upon the onset of therapy resistance in clinical melanoma samples, in cell lines and in Mel006 PDX and is associated with decreased lipid poly-unsaturation. Forcing lipid poly-unsaturation in therapy-resistant models by combining MAPK inhibition with FASN inhibition attenuated cell proliferation and rendered cells exquisitely sensitive to a host of ROS inducers. In particular, the triple combination of MAPK inhibition, FASN inhibition, and the clinical ROS-inducing compound ATO dramatically increased survival of Mel006 PDX models from 15 to 72% with no associated signs of toxicity.ConclusionsWe conclude that under MAPK inhibition the direct pharmacological inhibition of FASN evokes an exquisite vulnerability to inducers of ROS by increasing membrane lipid poly-unsaturation. The exploitation of this vulnerability by combining MAPK and/or FASN inhibitors with inducers of ROS greatly delays the onset of therapy resistance and increases survival. Our work identifies a clinically actionable combinatorial treatment for therapy-resistant cancer.

Combination of the natural product mensacarcin with vemurafenib (Zelboraf) combats BRAF mutant and chemo-resistant melanoma in vitro by affecting cell metabolism and cellular migration

Melanoma is the most prevalent and aggressive form of skin cancer, often with a low survival rate due to rapid metastasis and a high occurrence of multi-drug resistance (MDR). In this study, we examine the combination treatment of the recently discovered cytotoxic bacterial metabolite mensacarcin and FDA approved therapeutic vemurafenib (Zelboraf). Vemurafenib is the standard regiment for BRAF V600E mutation positive patients with unresectable or metastatic malignant melanoma. On its own, menscarcin induces mitochondrial dysfunction in selected BRAF-mutant melanoma cell lines. In combination treatment, mensacarcin enhances vemurafenib's cytotoxicity via metabolic impairment. Specifically, it lowers cellular glucose uptake, decreases the oxygen consumption rate, reduces the abundance of glycolytic enzymes, and halts wound healing. Mensacarcin alone retains strong cytotoxic effects in three vemurafenib resistant cell lines SK-Mel-28PLXr, A375PLXr, and A2058PLXr with an average IC50 of 1 μM, inducing apoptosis via the caspase-3/7 pathway. We also find that mensacarcin lowers metastatic migration in a wound healing assay and it alters the morphology of migrating cells on a nanofiber scaffold. Using extracellular matrix (ECM) mimicking fibers, mensacarcin treated cells exhibit a decrease in single cell maximum coil width and an increase in the protrusion length limiting cellular coiling behavior which may limit metastatic migration. In summary, the bacterial metabolite mensacarcin shows great promise as a melanoma drug lead in vitro: in combination with vemurafenib, the cytotoxic activity is enhanced, and alone, mensacarcin retains activity in BRAF inhibitor chemo-resistant melanoma cell lines, and features a unique, migratory limited single cell phenotype.

The role of altered calcium signaling and handling as an underlying mechanism of drug tolerance in BRAF mutant melanoma

Molecularly‐targeted therapies in patients harboring well‐defined driver mutations have proven to be initially effective for many patients; however, tumor progression is nearly universal several months after initial regression. While it is common in cancer biology to frame treatment failure in terms of genetic mutations that either pre‐exist or are acquired during treatment, a growing body of literature is increasing awareness of non‐genetic processes that play a key role. For example, cancer stem cells, or stem‐like cells that can reversibly shift phenotypes are now a regular topic of investigation within the cancer research community. Recently, we reported that BRAF mutant melanoma cell lines exposed to prolonged treatment (>100 hours) with targeted BRAF inhibitors enter a non‐genetic, reversible state of balanced death and division. It is postulated that this state – that we term idling – acts as a haven for cancer cells to escape the stressful conditions of BRAF inhibition, while allowing for occasional cell division and, hence, DNA replication. Moreover, molecular signatures of error prone DNA replication and repair indicates that this state favors the acquisition of resistance conferring mutations, which lead to recurrence, metastasis, and ultimately death. Intriguingly, because cells are under stress from the initial treatment, it is possible that residual tumors composed of idling cancer cells may be vulnerable to a well‐targeted secondary treatment. However, developing such a treatment will require a detailed understanding of the molecular drivers of the drug tolerant phenotype. Accordingly, we have molecularly profiled cell lines transitioning into idling by RNAseq, ATACseq, and Seahorse metabolic analysis, revealing globally dysregulated ion channel expression and dramatically reduced activity of glycolysis and oxidative phosphorylation. Notably, expression of key calcium signaling and handling genes (i.e. Inositol tri‐phosphate receptors (ITPRs)) were observed to fall significantly during treatment, directing the exploration of calcium signaling and handling in the idling state. This exploration revealed reduced endoplasmic reticulum (ER) calcium content, and reduced store operated calcium entry (SOCE) in the idling state – both of which are well described to be regulators of signaling, metabolism, cell cycle progression, and programmed cell death. However, it is unclear to what extent differential calcium handling properties are responsible for the drug tolerant behavior in the idling state. Therefor this project will explore the role of calcium handling and signaling as an underlying mechanism of the idling state, and how these properties may differ between cells with different fates (i.e. death, division, or neither). In line with observations that death and division rates are low in the idling state, I hypothesize that reduced calcium handling allows cells to avoid programmed cell death, at the expense of reduced proliferative capacity. In contrast, I hypothesize that the rare dividing cells rescue their calcium handling and signaling capabilities, thereby increasing their metabolism and ability to progress through the cell cycle – a hypothesis that is consistent with the preliminary data. Increasing our understanding of the molecular mechanism behind the idling phenotype will shed new light on how BRAF mutant cancer cells survive targeted treatment and potentially provide new treatment strategies for addressing the ultimate failure in the clinic.

Therapeutic Efficacy of Pharmacological Ascorbate on Braf Inhibitor Resistant Melanoma Cells In Vitro and In Vivo.

High-dose ascorbate paradoxically acts as a pro-oxidant causing the formation of hydrogen peroxide in an oxygen dependent manner. Tumor cells (in particular melanoma cells) show an increased vulnerability to ascorbate induced reactive oxygen species (ROS). Therefore, high-dose ascorbate is a promising pharmacological approach to treating refractory melanomas, e.g., with secondary resistance to targeted BRAF inhibitor therapy. BRAF mutated melanoma cells were treated with ascorbate alone or in combination with the BRAF inhibitor vemurafenib. Viability, cell cycle, ROS production, and the protein levels of phospho-ERK1/2, GLUT-1 and HIF-1α were analyzed. To investigate the treatment in vivo, C57BL/6NCrl mice were subcutaneously injected with D4M.3A (BrafV600E) melanoma cells and treated with intraperitoneal injections of ascorbate with or without vemurafenib. BRAF mutated melanoma cell lines either sensitive or resistant to vemurafenib were susceptible to the induction of cell death by pharmacological ascorbate. Treatment of BrafV600E melanoma bearing mice with ascorbate resulted in plasma levels in the pharmacologically active range and significantly improved the therapeutic effect of vemurafenib. We conclude that intravenous high-dose ascorbate will be beneficial for melanoma patients by interfering with the tumor’s energy metabolism and can be safely combined with standard melanoma therapies such as BRAF inhibitors without pharmacological interference.

PPP6C, a serine-threonine phosphatase, regulates melanocyte differentiation and contributes to melanoma tumorigenesis through modulation of MITF activity

It is critical to understand the molecular mechanisms governing the regulation of MITF, a lineage specific transcription factor in melanocytes and an oncogene in melanoma. We identified PPP6C, a serine/threonine phosphatase, as a key regulator of MITF in melanoma. PPP6C is the only recurrently mutated serine/threonine phosphatase across all human cancers identified in sequencing studies and the recurrent R264C mutation occurs exclusively in melanoma. Using a zebrafish developmental model system, we demonstrate that PPP6C expression disrupts melanocyte differentiation. Melanocyte disruption was rescued by engineering phosphomimetic mutations at serine residues on MITF. We developed an in vivo MITF promoter assay in zebrafish and studied the effects of PPP6C(R264C) on regulating MITF promoter activity. Expression of PPP6C(R264C) cooperated with oncogenic NRAS(Q61K) to accelerate melanoma initiation in zebrafish, consistent with a gain of function alteration. Using a human melanoma cell line, we examined the requirement for PPP6C in proliferation and MITF expression. We show that genetic inactivation of PPP6C increases MITF and target gene expression, decreases sensitivity to BRAF inhibition, and increases phosphorylated MITF in a BRAF(V600E) mutant melanoma cell line. Our data suggests that PPP6C may be a relevant drug target in melanoma and proposes a mechanism for its action.

STK11 Prevents Invasion through Signal Transducer and Activator of Transcription 3/5 and FAK Repression in Cutaneous Melanoma

The STK11/LKB1 is a tumor suppressor involved in metabolism and cell motility. In BRAFV600E melanoma, STK11 is inactivated by extracellular signal‒regulated kinase and RSK, preventing it from binding and activating adenosine monophosphate-activated protein kinase and promoting melanoma cell proliferation. Although STK11 mutations occur in 5‒10% of cutaneous melanoma, few functional studies have been performed. By knocking out STK11 with CRISPR/Cas9 in two human BRAF-mutant melanoma cell lines, we found that STK11 loss reduced the sensitivity to a BRAF inhibitor. More strikingly, STK11 loss led to an increased invasive phenotype in both three-dimensional spheroids and invivo zebrafish xenograft models. STK11 overexpression consistently reversed the invasive phenotype. Interestingly, STK11 knockout increased invasion also in an NRAS-mutant melanoma cell line. Furthermore, although STK11 was expressed in primary human melanoma tumors, its expression significantly decreased in melanoma metastases, especially in brain metastases. In the STK11-knockout cells, we observed increased activating phosphorylation of signal transducer and activator of transcription 3/5 and FAK. Using inhibitors of signal transducer and activator of transcription 3/5 and FAK, we reversed the invasive phenotype in both BRAF- and NRAS-mutated cells. Our findings confirm an increased invasive phenotype on STK11 inactivation in BRAF- and NRAS-mutant cutaneous melanoma that can be targeted by signal transducer and activator of transcription 3/5 and FAK inhibition.

RAF-Mutant Melanomas Differentially Depend on ERK2 Over ERK1 to Support Aberrant MAPK Pathway Activation and Cell Proliferation.

Half of advanced human melanomas are driven by mutant BRAF and dependent on MAPK signaling. Interestingly, the results of three independent genetic screens highlight a dependency of BRAF-mutant melanoma cell lines on BRAF and ERK2, but not ERK1. ERK2 is expressed higher in melanoma compared with other cancer types and higher than ERK1 within melanoma. However, ERK1 and ERK2 are similarly required in primary human melanocytes transformed with mutant BRAF and are expressed at a similar, lower amount compared with established cancer cell lines. ERK1 can compensate for ERK2 loss as seen by expression of ERK1 rescuing the proliferation arrest mediated by ERK2 loss (both by shRNA or inhibition by an ERK inhibitor). ERK2 knockdown, as opposed to ERK1 knockdown, led to more robust suppression of MAPK signaling as seen by RNA-sequencing, qRT-PCR, and Western blot analysis. In addition, treatment with MAPK pathway inhibitors led to gene expression changes that closely resembled those seen upon knockdown of ERK2 but not ERK1. Together, these data demonstrate that ERK2 drives BRAF-mutant melanoma gene expression and proliferation as a function of its higher expression compared with ERK1. Selective inhibition of ERK2 for the treatment of melanomas may spare the toxicity associated with pan-ERK inhibition in normal tissues. IMPLICATIONS: BRAF-mutant melanomas overexpress and depend on ERK2 but not ERK1, suggesting that ERK2-selective inhibition may be toxicity sparing.

15604 Resistance to BRAF and MEK inhibitors in BRAF-mutant melanoma

The 5-year survival rate for patients diagnosed with metastatic melanoma is only 23%. Nearly half of patients diagnosed with melanoma have BRAF-mutant melanoma in which there is a driver mutation in the MAPK pathway, leading to over activation. Single-agent BRAF inhibitors had some success, but their long term efficacy is limited due to the development of resistance. Current standard of care includes a BRAF-inhibitor in combination with a MEK inhibitor in order to co-target the downstream MEK protein in the hopes of preventing reactivation of the pathway. It has been shown that this combination therapy has also met some difficulty due to the development of resistance. We generated BRAF inhibitor (dabrafenib) and MEK inhibitor (trametinib) resistant cell lines (TDR) by adding increasing concentration of the drugs to the parental cells (drug sensitive cells) for WM115 and WM983 (BRAF mutant melanoma cell lines) until they were rendered resistant. The TDR cells remained viable over increasing concentration of the inhibitor as assessed by the MTT assay. There was maintenance of p-ERK in the TDR cells, which was not observed for the parental cells under similar conditions. qPCR analysis demonstrated that TDR cells had elevated levels of the signaling molecules such as AXL, EGFR, HER3, and IGF1R. These molecules might be potential mediators of resistance to targeted therapy for BRAF-mutant melanoma. Examining potential mechanisms of resistance to BRAF and MEK inhibitors helps expand possible treatment options to aid in long-term success for BRAF-mutant melanoma patients.

UVA Radiation Enhances Lomefloxacin-Mediated Cytotoxic, Growth-Inhibitory and Pro-Apoptotic Effect in Human Melanoma Cells through Excessive Reactive Oxygen Species Generation.

Melanoma, the most dangerous type of cutaneous neoplasia, contributes to about 75% of all skin cancer-related deaths. Thus, searching for new melanoma treatment options is an important field of study. The current study was designed to assess whether the condition of mild and low-dose UVA radiation augments the lomefloxacin-mediated cytotoxic, growth-inhibitory and pro-apoptotic effect of the drug in melanoma cancer cells through excessive oxidative stress generation. C32 amelanotic and COLO829 melanotic (BRAF-mutant) melanoma cell lines were used as an experimental model system. The combined exposure of cells to both lomefloxacin and UVA irradiation caused higher alterations of redox signalling pathways, as shown by intracellular reactive oxygen species overproduction and endogenous glutathione depletion when compared to non-irradiated but lomefloxacin-treated melanoma cells. The obtained results also showed that lomefloxacin decreased both C32 and COLO829 cells’ viability in a concentration-dependent manner. This effect significantly intensified when melanoma cells were exposed to UVA irradiation and the drug. For melanoma cells exposed to lomefloxacin or lomefloxacin co-treatment with UVA irradiation, the concentrations of the drug that decreased the cells’ viability by 50% (EC50) were found to be 0.97, 0.17, 1.01, 0.18 mM, respectively. Moreover, we found that the redox imbalance, mitochondrial membrane potential breakdown, induction of DNA fragmentation, and changes in the melanoma cells’ cell cycle distribution (including G2/M, S as well as Sub-G1-phase blockade) were lomefloxacin in a dose-dependent manner and were significantly augmented by UVA radiation. This is the first experimental work that assesses the impact of excessive reactive oxygen species generation upon UVA radiation exposure on lomefloxacin-mediated cytotoxic, growth-inhibitory and pro-apoptotic effects towards human melanoma cells, indicating the possibility of the usage of this drug in the photochemotherapy of malignant melanoma as an innovative medical treatment option which could improve the effectiveness of therapy. The obtained results also revealed that the redox imbalance intensification mediated by the phototoxic potential of fluoroquinolones may be considered as a more efficient treatment model of malignant melanoma and may constitute the basis for the development of new compounds with a high ability to excessive oxidative stress generation upon UVA radiation in cancer cells.

Visualizing Subcellular Enrichment of Glycogen in Live Cancer Cells by Stimulated Raman Scattering.

Glycogen, a branched glucose polymer, helps regulate glucose homeostasis through immediate storage and release of glucose. Reprogramming of glycogen metabolism has recently been suggested to play an emerging role in cancer progression and tumorigenesis. However, regulation of metabolic rewiring for glycogen synthesis and breakdown in cancer cells remains less understood. Despite the availability of various glycogen detection methods, selective visualization of glycogen in living cells with high spatial resolution has proven to be highly challenging. Here, we present an optical imaging strategy to visualize glycogen in live cancer cells with minimal perturbation by combining stimulated Raman scattering microscopy with metabolic incorporation of deuterium-labeled glucose. We revealed the subcellular enrichment of glycogen in live cancer cells and achieved specific glycogen mapping through distinct spectral identification. Using this method, different glycogen metabolic phenotypes were characterized in a series of patient-derived BRAF mutant melanoma cell lines. Our results indicate that cell lines manifesting high glycogen storage level showed increased tolerance to glucose deficiency among the studied melanoma phenotypes. This method opens up the possibility for noninvasive study of complex glycogen metabolism at subcellular resolution and may help reveal new features of glycogen regulation in cancer systems.

A Feed-Forward Mechanosignaling Loop Confers Resistance to Therapies Targeting the MAPK Pathway in BRAF-Mutant Melanoma

Aberrant extracellular matrix (ECM) deposition and stiffening is a physical hallmark of several solid cancers and is associated with therapy failure. BRAF-mutant melanomas treated with BRAF and MEK inhibitors almost invariably develop resistance that is frequently associated with transcriptional reprogramming and a de-differentiated cell state. Melanoma cells secrete their own ECM proteins, an event that is promoted by oncogenic BRAF inhibition. Yet, the contribution of cancer cell-derived ECM and tumor mechanics to drug adaptation and therapy resistance remains poorly understood. Here, we show that melanoma cells can adapt to targeted therapies through a mechanosignaling loop involving the autocrine remodeling of a drug-protective ECM. Analyses revealed that therapy-resistant cells associated with a mesenchymal dedifferentiated state displayed elevated responsiveness to collagen stiffening and force-mediated ECM remodeling through activation of actin-dependent mechanosensors Yes-associated protein (YAP) and myocardin-related transcription factor (MRTF). Short-term inhibition of MAPK pathway also induced mechanosignaling associated with deposition and remodeling of an aligned fibrillar matrix. This provided a favored ECM reorganization that promoted tolerance to BRAF inhibition in a YAP- and MRTF-dependent manner. Matrix remodeling and tumor stiffening were also observed in vivo upon exposure of BRAF-mutant melanoma cell lines or patient-derived xenograft models to MAPK pathway inhibition. Importantly, pharmacologic targeting of YAP reversed treatment-induced excessive collagen deposition, leading to enhancement of BRAF inhibitor efficacy. We conclude that MAPK pathway targeting therapies mechanically reprogram melanoma cells to confer a drug-protective matrix environment. Preventing melanoma cell mechanical reprogramming might be a promising therapeutic strategy for patients on targeted therapies. SIGNIFICANCE: These findings reveal a biomechanical adaptation of melanoma cells to oncogenic BRAF pathway inhibition, which fuels a YAP/MRTF-dependent feed-forward loop associated with tumor stiffening, mechanosensing, and therapy resistance. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/10/1927/F1.large.jpg.

Abstract B132: Inhibition of Haspin kinase to promote cell-intrinsic and extrinsic anticancer therapy

Abstract Oncogene-targeted therapies are initially effective in most patients with BRAF-mutated melanoma. However, patients frequently develop resistance during therapy. At the time of therapy resistance, they are also less likely to respond to immune checkpoint inhibitors (ICI) with response rates ranging from 0-12%, compared to 40-60% when used in the first line. This may be in part be due to rapid tumor growth that outpaces pharmacodynamics of ICI. New drugs that directly control tumor growth and effectively enhance ICI activity are therefore needed. We report that the small molecule CX-6258 has potent activity against both RAF/MEK inhibitor (RMS) and resistant (RMR) BRAF-mutant melanoma cell lines. CX-6258 is annotated as a pan-PIM kinase inhibitor, with an EC50 corrected for growth rate (GR50) of ~200 nM; in contrast, other PIM inhibitors had no in vitro activity, suggesting that PIMs may not be the true target in melanoma - we therefore sought to systematically study the targets of this compound. Using an unbiased kinase profiling approach, we identified Haspin Kinase (HASPIN) as the key target of CX-6258 with an IC50~10nM. Haspin kinase (Haspin) is a poorly understood mitotic kinase, whose only well-defined function is phosphorylation of Histon H3 at Thr3 (pH3T3). Treatment with CX-6258 resulted in reduced proliferation, reduced phosphorylation of pH3T3, frequent formation of micronuclei (MN), recruitment of cGAS and activation of the cGAS-STING-pathway. By using CRISPR/CAS9 knock outs of PIM kinase we show that these targets are not required for CX-6258 function. Furthermore, siRNA mediated knock-down of HASPIN phenocopied CX-6258. In murine models, CX-6258 induces a potent cGAS dependent type-I-interferon response in tumor cells, increases IFNγ-producing CD8+ T-cells, reduces Tregs, and enhances responses to ICIs in vivo. HASPIN is more strongly expressed in malignant compared to healthy tissue and its inhibition by CX-6258 has minimal toxicity in ex vivo expanded human tumor-infiltrating-lymphocytes (TILs) and in vitro differentiated human neurons, suggesting a potential therapeutic index for anti-cancer therapy. CX-6258 also shows activity in several Ewing sarcoma and multiple myeloma cell lines with. In summary, HASPIN inhibition may overcome drug resistance in melanoma, synergize with ICIs and target a vulnerability in different cancer lineages. Citation Format: Johannes C. Melms, Sreeram Vallabhaneni, Caitlin E. Mills, Kai Wucherpfennig, Peter K. Sorger, Benjamin Izar. Inhibition of Haspin kinase to promote cell-intrinsic and extrinsic anticancer therapy [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B132. doi:10.1158/1535-7163.TARG-19-B132

Long-Term Vemurafenib Exposure Induced Alterations of Cell Phenotypes in Melanoma: Increased Cell Migration and Its Association with EGFR Expression.

Acquired resistance during BRAF inhibitor therapy remains a major challenge for melanoma treatment. Accordingly, we evaluated the phenotypical and molecular changes of isogeneic human V600E BRAF-mutant melanoma cell line pairs pre- and post-treatment with vemurafenib. Three treatment naïve lines were subjected to in vitro long-term vemurafenib treatment while three pairs were pre- and post-treatment patient-derived lines. Molecular and phenotypical changes were assessed by Sulforhodamine-B (SRB) assay, quantitative RT-PCR (q-RT-PCR), immunoblot, and time-lapse microscopy. We found that five out of six post-treatment cells had higher migration activity than pretreatment cells. However, no unequivocal correlation between increased migration and classic epithelial–mesenchymal transition (EMT) markers could be identified. In fast migrating cells, the microphthalmia-associated transcription factor (MITF) and epidermal growth factor receptor (EGFR) mRNA levels were considerably lower and significantly higher, respectively. Interestingly, high EGFR expression was associated with elevated migration but not with proliferation. Cells with high EGFR expression showed significantly decreased sensitivity to vemurafenib treatment, and had higher Erk activation and FRA-1 expression. Importantly, melanoma cells with higher EGFR expression were more resistant to the EGFR inhibitor erlotinib treatment than cells with lower expression, with respect to both proliferation and migration inhibition. Finally, EGFR-high melanoma cells were characterized by higher PD-L1 expression, which might in turn indicate that immunotherapy may be an effective approach in these cases.

Drug-induced expression of the RNA-binding protein HuR attenuates the adaptive response to BRAF inhibition in melanoma

Strategies that aim to limit the adaptive response to pathway inhibition in BRAF-mutated melanoma face the inherent limit of signaling redundancy and multiplicity of possible bypass mechanisms. Drug-induced expression of selected RNA-binding proteins, like the ubiquitously expressed HuR, has the potential to differentially stabilize the expression of many genes involved in the compensatory mechanisms of adaptive response. Here, we detect in BRAF-mutated melanoma cell lines having a higher propensity for adaptive response and in non-responding melanoma tumors, a larger proportion of HuRLow cells in the expression distribution of HuR. Using knockdown experiments, we demonstrate, through expression profiling and phenotypic assays, that increasing the proportion of HuRLow cells favors the adaptive response to BRAF inhibition, provided that the HuRLow state stays reversible. The MAPK dependency of melanoma cells appears to be diminished as the proportion of HuRLow cells increases. In single-cell assays, we demonstrate that the HuRLow cells display plasticity in their growth expression profile. Importantly, the adaptive over-proliferating cells emerge in the subpopulation containing the HuRLow cells. Therapeutic concentrations of lithium salts, although they moderately increase the global expression of HuR, are sufficient to suppress the HuRLow cells, induce an overall less resistant expression profile and attenuate in a HuR-dependent manner the adaptive response of melanoma cells in ex vivo assays. The therapeutic effectiveness of this approach is also demonstrated in vivo in mice xenografts. This study has immediate clinical relevance for melanoma therapy and opens a new avenue of strategies to prevent the adaptive response to targeted cancer therapy.