BRAFi-resistant Melanoma Cells Research Articles

Development and in-depth characterization of BRAFi-resistant melanoma cell lines in vitro and in vivo

Regardless of the clinical response and improved patient survival observed following treatment with BRAFi like Vemurafenib (Vem), rapid development of resistance still remains as a major obstacle in melanoma therapy. In this context, we developed and characterized two acquired Vem-resistant melanoma cell lines, A375V and SK-MEL-28V, and an intrinsically Vem-resistant cell line, RPMI-7951. Altered morphology and growth rate of the resistant cell lines displayed spindle-shaped cells with filopodia formation and enhanced proliferation rate as compared to parental cells. Further in vitro characterization in 2D models confirmed the emergence of a resistant phenotype in melanoma cells. To mimic the in vivo tumor microenvironment, spheroids were developed for both parental and resistant cell lines to recognize materialization of invadopodia structures demonstrating elevated invasiveness and proliferation of resistant cells-based spheroids, especially A375V. Importantly, we validated A375V cell line in vivo to prove its tumorigenicity and drug resistance in tumor xenograft model. Taken together, our established clinically relevant Vem-resistant tumor model could be beneficial to elucidate drug resistance mechanisms, screen and identify novel anticancer therapies to overcome BRAFi resistance in melanoma.

Overcoming BRAF and CDK4/6 inhibitor resistance by inhibiting MAP3K3-dependent protection against YAP lysosomal degradation

Transcriptional programs governed by YAP play key roles in conferring resistance to various molecular-targeted anticancer agents. Strategies aimed at inhibiting YAP activity have garnered substantial interest as a means to overcome drug resistance. However, despite extensive research into the canonical Hippo–YAP pathway, few clinical agents are currently available to counteract YAP-associated drug resistance. Here, we present a novel mechanism of YAP stability regulation by MAP3K3 that is independent of Hippo kinases. Furthermore, we identified MAP3K3 as a target for overcoming anticancer drug resistance. Depletion of MAP3K3 led to a substantial reduction in the YAP protein level in melanoma and breast cancer cells. Mass spectrometry analysis revealed that MAP3K3 phosphorylates YAP at serine 405. This MAP3K3-mediated phosphorylation event hindered the binding of the E3 ubiquitin ligase FBXW7 to YAP, thereby preventing its p62-mediated lysosomal degradation. Robust YAP activation was observed in CDK4/6 inhibitor-resistant luminal breast cancer cells. Knockdown or pharmacological inhibition of MAP3K3 effectively suppressed YAP activity and restored CDK4/6 inhibitor sensitivity. Similarly, elevated MAP3K3 expression supported the prosurvival activity of YAP in BRAF inhibitor-resistant melanoma cells. Inhibition of MAP3K3 decreased YAP-dependent cell proliferation and successfully restored BRAF inhibitor sensitivity. In conclusion, our study reveals a previously unrecognized mechanism for the regulation of YAP stability, suggesting MAP3K3 inhibition as a promising strategy for overcoming resistance to CDK4/6 and BRAF inhibitors in cancer treatment.

Delineating effect of headgroup and preparation method on transfection versus toxicity of DNA-loaded lipid nanocarriers.

Aim: To perform a parallel comparison of key parameters affecting the safety and efficiency of lipid-based nanovectors (i.e., complexing headgroups, composition and preparation method). Materials & methods: Various cationic and ionizable headgroups were screened for formulating lipoplexes with GFP-plasmid DNA. Ethanol injection and microfluidics were used to prepare nanoparticles with GFP-plasmid DNA complexed on the surface or within the interior of lipid bilayers. Results: Lipoplexes composed of sphingomyelin 102 exhibited the highest transfection efficiency given their higher cellular uptake in BRAF inhibitor-resistant melanoma cells. Lipid nanoparticlesdemonstrated acceptable transfection efficiency and high spheroid penetration while protecting plasmid DNA under simulated physiological conditions. Conclusion: Selecting the right complexing lipid and preparation method is critical for developing lipid nanocarriers to treat intractable diseases.

Oral lipid nanocomplex of BRD4 PROteolysis TArgeting Chimera and vemurafenib for drug-resistant malignant melanoma

BRAF inhibitors (BRAFi) like vemurafenib (VEM) provide initial regression in mutated melanoma but rapidly develop resistance. Molecular pathways responsible for development of resistance against VEM finally converge towards the activation of oncogenic c-Myc. We identified an epigenetic approach to inhibit the c-Myc expression and resensitize BRAFi-resistant melanoma cells. ARV-825 (ARV) was employed as a BRD4 targeted PROteolysis TArgeting Chimera that selectively degrades the BRD4 to downregulate c-Myc. ARV synergistically enhanced the cytotoxicity of VEM in vitro to overcome its resistance in melanoma. Development of ARV and VEM-loaded lipid nanocomplex (NANOVB) significantly improved their physicochemical properties for oral delivery. Most importantly, oral administration of NANOVB substantially inhibited tumor growth at rate of 41.07 mm3/day in nude athymic mice. NANOVB treatment resulted in prolonged survival with 50% of mice surviving until the experimental endpoint. Histopathological analysis revealed significant tumor necrosis and downregulation of Ki-67 and BRD4 protein in vivo. Promising in vivo antitumor activity and prolonged survival demonstrated by NANOVB signifies its clinical translational potential for BRAFi-resistant melanoma.

Androgen receptor is a determinant of melanoma targeted drug resistance

Melanoma provides a primary benchmark for targeted drug therapy. Most melanomas with BRAFV600 mutations regress in response to BRAF/MEK inhibitors (BRAFi/MEKi). However, nearly all relapse within the first two years, and there is a connection between BRAFi/MEKi-resistance and poor response to immune checkpoint therapy. We reported that androgen receptor (AR) activity is required for melanoma cell proliferation and tumorigenesis. We show here that AR expression is markedly increased in BRAFi-resistant melanoma cells, and in sensitive cells soon after BRAFi exposure. Increased AR expression is sufficient to render melanoma cells BRAFi-resistant, eliciting transcriptional changes of BRAFi-resistant subpopulations, including elevated EGFR and SERPINE1 expression, of likely clinical significance. Inhibition of AR expression or activity blunts changes in gene expression and suppresses proliferation and tumorigenesis of BRAFi-resistant melanoma cells, promoting clusters of CD8+ T cells infiltration and cancer cells killing. Our findings point to targeting AR as possible co-therapeutical approach in melanoma treatment.

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 5822: YAP inhibition via targeting MAP3K3-dependent lysosomal degradation pathway overcomes BRAF and CDK4/6 inhibitor resistance

Abstract The Hippo-yes-associated protein (YAP) signaling pathway activation underlies the resistance to a wide range of molecularly targeted anti-cancer agents. However, strategies to block YAP activity in cancer by promoting YAP degradation have been largely unsuccessful despite rigorous investigations into canonical Hippo-YAP regulatory mechanisms. Here, we report a novel mechanism of YAP stability regulation by MAP3K3 independently of LATS-mediated proteasomal degradation. We observed that MAP3K3 depletion significantly reduced YAP protein levels in both normal and malignant cells, and mass spectrometry analysis revealed that MAP3K3 phosphorylates YAP at its serine 405 residue. MAP3K3-mediated serine 405 YAP phosphorylation polarizes YAP interaction with BTRC while hindering its interaction with FBXW7, collectively preventing both proteasomal degradation and the p62-mediated lysosomal degradation of YAP. We investigated the role of this mechanism in overcoming anti-cancer resistance and observed robust YAP activation in CDK4/6 inhibitor-resistant luminal breast cancer cells. Genetic and pharmacological MAP3K3 inhibition suppressed YAP-induced drug resistance. Moreover, increased MAP3K3 expression supported the pro-survival activity of YAP in BRAF inhibitor-resistant melanoma cells, and MAP3K3 depletion downregulated YAP-dependent cell cycle gene signatures. Accordingly, the combination of vemurafenib and ponatinib was effective in reducing the survival of BRAF inhibitor-resistant melanoma cells. Collectively, our study identified a novel phosphorylation-mediated YAP stability regulation mechanism and underscored MAP3K3 inhibition as a promising strategy to overcome BRAF and CDK4/6 inhibitor resistance by directly promoting YAP degradation. Citation Format: Won-Ji Ryu, Sanghyun Park, Tae Yeong Kim, Yumi Hwang, Hyun Ju Han, Jeong Dong Lee, Gun Min Kim, Joohyuk Sohn, Sang Kyum Kim, Joon Kim, Min Hwan Kim. YAP inhibition via targeting MAP3K3-dependent lysosomal degradation pathway overcomes BRAF and CDK4/6 inhibitor resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5822.

RIDR-PI-103, ROS-activated prodrug PI3K inhibitor inhibits cell growth and impairs the PI3K/Akt pathway in BRAF and MEK inhibitor-resistant BRAF-mutant melanoma cells.

Reactive oxygen species (ROS) levels are elevated after acquisition of resistance to v-raf murine sarcoma viral oncogene homolog B1 (BRAF) inhibitors including dabrafenib and MEK inhibitors such as trametinib in BRAF-mutant melanoma. To circumvent toxicity to PI-103 (a pan PI3K inhibitor), we utilized a novel ROS-induced drug release (RIDR)-PI-103, with a self-cyclizing moiety linked to PI-103. Under high ROS conditions, RIDR-PI-103 releases PI-103, which inhibits conversion of phosphatidylinositol 4,5-bisphosphate (PIP 2 ) to phosphatidylinositol 3,4,5-triphosphate (PIP 3 ). Previous findings demonstrate that trametinib and dabrafenib-resistant (TDR) cells maintain p-Akt levels compared to parental counterparts and have significantly higher ROS. This is a rationale to explore the efficacy RIDR-PI-103 in TDR cells. We tested the effect of RIDR-PI-103 on melanocytes and TDR cells. RIDR-PI-103 exhibited less toxicity compared to PI-103 at 5 µM in melanocytes. RIDR-PI-103 significantly inhibited TDR cell proliferation at 5 and 10 µM. Twenty-four hour treatment with RIDR-PI-103 inhibited p-Akt, p-S6 (Ser240/244) and p-S6 (Ser235/236). We assessed the mechanism of activation of RIDR-PI-103, using glutathione or t-butyl hydrogen peroxide (TBHP) on the TDR cells in the presence or absence of RIDR-PI-103. Addition of the ROS scavenger glutathione to RIDR-PI-103 significantly rescued the cell proliferation in TDR cell lines while addition of the ROS inducer TBHP and RIDR-PI-103 inhibited cell proliferation in WM115 and WM983B TDR cell lines. Examining the efficacy of RIDR-PI-103 on BRAF and MEK inhibitor-resistant cells will expand possible treatment options and open avenues for the development of new ROS-based treatment therapies for BRAF-mutant melanoma patients.

Resistance to BRAF Inhibitors: EZH2 and Its Downstream Targets as Potential Therapeutic Options in Melanoma.

Activating BRAF mutations occurs in 50-60% of malignant melanomas. Although initially treatable, the development of resistance to BRAF-targeted therapies (BRAFi) is a major challenge and limits their efficacy. We have previously shown that the BRAFV600E signaling pathway mediates the expression of EZH2, an epigenetic regulator related to melanoma progression and worse overall survival. Therefore, we wondered whether inhibition of EZH2 would be a way to overcome resistance to vemurafenib. We found that the addition of an EZH2 inhibitor to vemurafenib improved the response of melanoma cells resistant to BRAFi with regard to decreased viability, cell-cycle arrest and increased apoptosis. By next-generation sequencing, we revealed that the combined inhibition of BRAF and EZH2 dramatically suppresses pathways of mitosis and cell cycle. This effect was linked to the downregulation of Polo-kinase 1 (PLK1), a key regulator of cell cycle and proliferation. Subsequently, when we inhibited PLK1, we found decreased cell viability of melanoma cells resistant to BRAFi. When we inhibited both BRAF and PLK1, we achieved an improved response of BRAFi-resistant melanoma cells, which was comparable to the combined inhibition of BRAF and EZH2. These results thus reveal that targeting EZH2 or its downstream targets, such as PLK1, in combination with BRAF inhibitors are potential novel therapeutic options in melanomas with BRAF mutations.

Sestrin2 contributes to BRAF inhibitor resistance via reducing redox vulnerability of melanoma cells

BackgroundTreatment resistance often occurs with BRAF inhibitor (BRAFi) therapy for melanoma, bringing in a great challenge to the treatment of melanoma patients harboring mutant BRAF gene. Recent studies revealed redox vulnerability constitutes a novel opportunity to overcome BRAFi resistance. Previously we found Sestrin2 provided protection to metastatic melanoma cells by detoxifying reactive oxygen species (ROS) induced by anoikis, but its defensive role against redox stimuli elicited by BRAFi was unclear. ObjectiveIn-depth explored the role of Sestrin2 in BRAFi-resistant melanoma. MethodsVemurafenib-resistant melanoma cells were established using 451Lu and UACC62 cell lines carrying BRAFV600E mutation. Mechanistic studies were subsequently performed by transfection of lentiviral vectors encoding an shRNA against SESN2 or embedded with the coding sequences of SESN2 cDNA. ResultsElevated Sestrin2 expression was found in vemurafenib-resistance melanoma cells. Further mechanistic studies revealed that BRAFi-resistant melanoma cells employ Sestrin2 to adapt to higher oxidative stress under vemurafenib exposure. It was also demonstrated that mTOR signaling was significantly activated following Sestrin2 knockdown. Given the known promoting role of active mTOR signaling in melanoma proliferation and survival, the effects of mTOR blocker and Sestrin2 ablation on BRAFi-resistant melanoma cells were further tested, and the combination was found to result in enhanced inhibition of melanoma cell growth. ConclusionsOur findings demonstrated the contribution of Sestrin2 to the development of BRAFi resistance and the fact that the combination of mTOR blocker assisted Sestrein2 ablation in eliminating BRAFi resistance of melanoma. Therefore, mTOR and Sestrin2 may be novel combinatorial therapeutic targets to overcome BRAFi resistance of melanoma.

Increased MARCKS Activity in BRAF Inhibitor-Resistant Melanoma Cells Is Essential for Their Enhanced Metastatic Behavior Independent of Elevated WNT5A and IL-6 Signaling.

Treatment of melanoma with a BRAF inhibitor (BRAFi) frequently initiates development of BRAFi resistance, leading to increased tumor progression and metastasis. Previously, we showed that combined inhibition of elevated WNT5A and IL-6 signaling reduced the invasion and migration of BRAFi-resistant (BRAFi-R) melanoma cells. However, the use of a combined approach per se and the need for high inhibitor concentrations to achieve this effect indicate a need for an alternative and single target. One such target could be myristoylated alanine-rich C-kinase substrate (MARCKS), a downstream target of WNT5A in BRAFi-sensitive melanoma cells. Our results revealed that MARCKS protein expression and activity are significantly elevated in PLX4032 and PLX4720 BRAFi-R A375 and HTB63 melanoma cells. Surprisingly, neither WNT5A nor IL-6 contributed to the increases in MARCKS expression and activity in BRAFi-R melanoma cells, unlike in BRAFi-sensitive melanoma cells. However, despite the above findings, our functional validation experiments revealed that MARCKS is essential for the increased metastatic behavior of BRAFi-R melanoma cells. Knockdown of MARCKS in BRAFi-R melanoma cells caused reductions in the F-actin content and the number of filopodia-like protrusions, explaining the impaired migration, invasion and metastasis of these cells observed in vitro and in an in vivo zebrafish model. In our search for an alternative explanation for the increased activity of MARCKS in BRAFi-R melanoma cells, we found elevated basal activities of PKCα, PKCε, PKCι, and RhoA. Interestingly, combined inhibition of basal PKC and RhoA effectively impaired MARCKS activity in BRAFi-R melanoma cells. Our results reveal that MARCKS is an attractive single antimetastatic target in BRAFi-R melanoma cells.

Patulin and LL-Z1640-2 induce apoptosis of cancer cells by decreasing endogenous protein levels of Zic family member 5.

Zic family member 5 (ZIC5) is a transcription factor that promotes the survival of several cancer cell types. As ZIC5 is expressed at minimal levels in normal human adult tissues, it is a potential therapeutic target. In this study, we screened a chemical library containing 3398 compounds that includes pre-existing drugs and compounds with known effects to identify ZIC5 inhibitors. In the first screening, 18 hit compounds decreased GFP intensity in melanoma A375 cells overexpressing GFP-tagged ZIC5. In the second screening, five compounds that attenuated ZIC5 protein levels in A375 cells were identified. Among them, LL-Z1640-2 and patulin selectively induced apoptosis in melanoma cells expressing ZIC5, while only inducing very low levels of apoptosis in normal human melanocytes, which have no detectable ZIC5 expression. LL-Z1640-2 and patulin also induced apoptosis in BRAF inhibitor-resistant melanoma, pancreatic cancer, cholangiocarcinoma and colorectal cancer cells. LL-Z1640-2- and patulin-mediated suppression of melanoma proliferation were rescued by ZIC5 overexpression. These results suggest that LL-Z1640-2 and patulin are promising compounds that decrease ZIC5 expression to induce apoptosis in cancer cells.

M-CSF as a therapeutic target in BRAFV600E melanoma resistant to BRAF inhibitors.

Disseminated BRAFV600E melanoma responds to BRAF inhibitors (BRAFi) but easily develops resistance with poor prognosis. Secretome plays a pivotal role during tumour progression causing profound effects on therapeutic efficacy. Secreted M-CSF is involved in both cytotoxicity suppression and tumour progression in melanoma. We aimed to analyse the M-CSF contribution in resistant metastatic melanoma to BRAF-targeted therapies. Conditioned media from melanoma cells were analysed by citoarray. Viability and migration/invasion assays were performed with paired melanoma cells and tumour growth in xenografted SCID mice. We evaluated the impact of M-CSF plasma levels with clinical prognosis from 35 metastatic BRAFV600E-mutant melanoma patients. BRAFi-resistant melanoma cells secretome is rich in pro-tumour cytokines. M-CSF secretion is essential to induce a Vemurafenib-resistant phenotype in melanoma cells. Further, we demonstrated that M-CSF mAb in combination with Vemurafenib and autophagy blockers synergistically induce apoptosis, impair migration and reduce tumour growth in BRAFi-resistant melanoma cells. Interestingly, lower M-CSF plasma levels are associated with better prognosis in metastatic melanoma patients. Secreted M-CSF induces a BRAFi-resistant phenotype and means worse prognosis in BRAFV600E metastatic melanoma patients. These results identify secreted M-CSF as a promising therapeutic target toward BRAFi-resistant melanomas.

Abstract 5356: RIDR-PI-103, ROS-activated PI3K inhibitor prodrug inhibits cell growth and impairs the PI3K/Akt pathway in BRAF and MEK inhibitor resistant BRAF-mutant melanoma cells

Abstract The five-year survival rate for patients diagnosed with metastatic melanoma is only 27%. About half of melanomas have the presence of the BRAFV600E oncoprotein, which leads to hyperactivation of the mitogen activated protein kinase (MAPK) pathway. The MAPK pathway is known to crosstalk with the PI3K/Akt pathway. Treatment with the BRAF inhibitor, Dabrafenib and the MEK inhibitor, Trametinib is limited as patient responses begin to drop due to acquired resistance to these kinase inhibitors. High concentrations of reactive oxygen species (ROS) accompany this resistance. ROS-induced drug release (RIDR)-PI-103 is a novel prodrug with a self-cyclizing moiety linked to PI-103, a PI3K inhibitor. Under high ROS conditions, RIDR-PI-103 releases PI-103, which inhibits conversion of PIP2 to PIP3. We have generated Trametinib and Dabrafenib resistant (TDR) cells for 3 BRAF-mutant cell lines: A375, WM115 and WM983B from parental (drug sensitive) cell lines. Studies in our lab demonstrate that TDR cells maintain p-Akt levels compared to parental counterparts and have significantly higher ROS. This is a rationale to explore the efficacy RIDR-PI-103 in TDR cells. We tested the effect of RIDR-PI-103 on cell viability for 1789C melanocytes, 1788B melanocytes, A375 TDR, WM983B TDR, and WM115 TDR using an MTT proliferation assay. RIDR-PI-103 exhibited less toxicity as compared to PI-103 at 5 µM in normal melanocytes. While RIDR-PI-103 significantly inhibited TDR cell proliferation at 5 and 10 µM compared to cells treated with vehicle DMSO and melanocytes. This effect was consistent when TDR cells were treated with RIDR-PI-103 in long-term crystal violet cell proliferation assays. WM115 TDR and WM983B TDR cells were highly sensitive to RIDR-PI-103 at 5 µM, while A375 TDR cells had only ~50% inhibition at 5 µM in both cell proliferation assays. 24 hour treatment with RIDR-PI-103 inhibited p-Akt (S473) along with downstream p-S6 (S240/244) and p-S6 (235/236). To examine the mechanism of action of RIDR-PI-103 and to test that the drug release is ROS induced, we assessed the effect of glutathione (GSH), an antioxidant on the TDR cells in the presence or absence of RIDR-PI-103. GSH alone did not affect proliferation of TDR cells. Addition of GSH to RIDR-PI-103 significantly rescued the cell proliferation in all three TDR cell lines. We next tested the effect of t-butyl hydrogen peroxide (TBHP), a ROS inducer on TDR cells with lower concentrations of RIDR-PI-103. Addition of TBHP to RIDR-PI-103 significantly induced proliferation in TDR cells. Ongoing experiments are focused on assessing the effect of RIDR-PI-103 on the mTOR pathway. Examining the efficacy of RIDR-PI-103 on BRAF and MEK inhibitor resistant cells will expand possible treatment options and open up avenues for the development of new treatment therapies for BRAF-mutant melanoma patients. Citation Format: Hima Patel, Rosalin Mishra, Adam Wier, Nazanin Mokhtarpour, Edward J. Merino, Joan T. Garrett. RIDR-PI-103, ROS-activated PI3K inhibitor prodrug inhibits cell growth and impairs the PI3K/Akt pathway in BRAF and MEK inhibitor resistant BRAF-mutant melanoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5356.

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.

BRAF Inhibitor Resistance Confers Increased Sensitivity to Mitotic Inhibitors.

Single agent and combination therapy with BRAFV600E/K and MEK inhibitors have remarkable efficacy against melanoma tumors with activating BRAF mutations, but in most cases BRAF inhibitor (BRAFi) resistance eventually develops. One resistance mechanism is reactivation of the ERK pathway. However, only about half of BRAFi resistance is due to ERK reactivation. The purpose of this study is to uncover pharmacological vulnerabilities of BRAFi-resistant melanoma cells, with the goal of identifying new therapeutic options for patients whose tumors have developed resistance to BRAFi/MEKi therapy. We screened a well-annotated compound library against a panel of isogenic pairs of parental and BRAFi-resistant melanoma cell lines to identify classes of compounds that selectively target BRAFi-resistant cells over their BRAFi-sensitive counterparts. Two distinct patterns of increased sensitivity to classes of pharmacological inhibitors emerged. In two cell line pairs, BRAFi resistance conferred increased sensitivity to compounds that share the property of cell cycle arrest at M-phase, including inhibitors of aurora kinase (AURK), polo-like kinase (PLK), tubulin, and kinesin. Live cell microscopy, used to track mitosis in real time, revealed that parental but not BRAFi-resistant melanoma cells were able to exit from compound-induced mitotic arrest through mitotic slippage, thus escaping death. Consistent with the key role of Cyclin B1 levels in regulating mitosis at the spindle checkpoint in arrested cells, we found lower Cyclin B1 levels in parental compared with BRAFi-resistant melanoma cells, suggesting that inability to down-regulate Cyclin B1 expression levels may explain the increased vulnerability of resistant cells to mitotic inhibitors. Another BRAFi-resistant cell line showed increased sensitivity to Chk1/2 inhibitors, which was associated with an accumulation of DNA damage, resulting in mitotic failure. This study demonstrates that BRAFi-resistance, in at least a subset of melanoma cells, confers vulnerability to pharmacological disruption of mitosis and suggests a targeted synthetic lethal approach for overcoming resistance to BRAF/MEK-directed therapies.

Ibrutinib Blocks YAP1 Activation and Reverses BRAF Inhibitor Resistance in Melanoma Cells.

Most B-Raf proto-oncogene (BRAF)-mutant melanoma tumors respond initially to BRAF inhibitor (BRAFi)/mitogen-activated protein kinase kinase 1 inhibitor (MEKi) therapy, although few patients have durable long-term responses to these agents. The goal of this study was to use an unbiased computational approach to identify inhibitors that reverse an experimentally derived BRAFi resistance gene expression signature. Using this approach, we found that ibrutinib effectively reverses this signature, and we demonstrate experimentally that ibrutinib resensitizes a subset of BRAFi-resistant melanoma cells to vemurafenib. Ibrutinib is used clinically as an inhibitor of the Src family kinase Bruton tyrosine kinase (BTK); however, neither BTK deletion nor treatment with acalabrutinib, another BTK inhibitor with reduced off-target activity, resensitized cells to vemurafenib. These data suggest that ibrutinib acts through a BTK-independent mechanism in vemurafenib resensitization. To better understand this mechanism, we analyzed the transcriptional profile of ibrutinib-treated BRAFi-resistant melanoma cells and found that the transcriptional profile of ibrutinib was highly similar to that of multiple Src proto-oncogene kinase inhibitors. Since ibrutinib, but not acalabrutinib, has appreciable off-target activity against multiple Src family kinases, it suggests that ibrutinib may be acting through this mechanism. Furthermore, genes that are differentially expressed in ibrutinib-treated cells are enriched in Yes1-associated transcriptional regulator (YAP1) target genes, and we showed that ibrutinib, but not acalabrutinib, reduces YAP1 activity in BRAFi-resistant melanoma cells. Taken together, these data suggest that ibrutinib, or other Src family kinase inhibitors, may be useful for treating some BRAFi/MEKi-refractory melanoma tumors. SIGNIFICANCE STATEMENT: MAPK-targeted therapies provide dramatic initial responses, but resistance develops rapidly; a subset of these tumors may be rendered sensitive again by treatment with an approved Src family kinase inhibitor-ibrutinub-potentially providing improved clinical outcomes.

Abstract 1116: HDAC8 activity regulates stress induced phenotype switching in melanoma

Abstract Background: Melanoma cells show great plasticity and can switch from a proliferative, drug-sensitive state to an invasive, drug-resistant phenotype through mechanisms that remain poorly defined. Hypothesis: Transcriptional and chromatin regulatory factors alter the gene expression states of cell in response to targeted therapy treatment and allow melanoma cells to persist and grow in the face of initially effective therapy. These factors/pathways may represent additional targets for anti-melanoma therapy. Methods: BRAF mutant melanoma cells were exposed to the BRAF inhibitor vemurafenib to generate cells that persisted and grew in the face of continued drug treatment. RNA-Seq, CHIP seq, ATAC-Seq and mass spectroscopy analyses were performed on persistent/resistant cells in comparison to parental cells. Additionally, histone deacetylase (HDAC) 8 and EP300 expression was enforced in drug naïve melanoma cell lines and the effects determined. Results: HDAC8 (but not other HDACs) was consistently up-regulated in BRAFi resistant melanoma cells. Increased expression of HDAC8 reduced cell growth, increased p53 activation and suppressed microphthalmia-associated transcription factor MITF, suppressing the melanocyte lineage program driven, leading to increased drug resistance. Introduction of HDAC8 or its active mutant (S39A) increased the tolerance of melanoma cells to multiples stresses (UV-irradiation, hypoxia, and BRAF-MEK inhibitor therapy), reduced their proliferation and increased their invasive potential in in vitro and in vivo models. Gene expression analyses indicate genes associated with a proliferative, drug-sensitive phenotype (e.g. DCT, MITF, MLANA, SOX10) are downregulated in HDAC8 active cells while genes correlative with an invasive, drug-resistant phenotype are upregulated (e.g. AXL, EGFR, TGFB1, WNT5A). ATAC-Seq analysis showed that HDAC8 regulates chromatin structure and decreased accessibility in the DNA binding sites of MITF and Sox10 while increasing accessibility in the DNA binding sites of c-Jun and TEADs (e.g. YAP1). Additionally, ChIP-Seq showed an increase in HDAC8 activity decreased H3K27 acetylation in the promoter region of MITF and MITF regulated genes (e.g. DCT, MLANA, PMEL, TYR). Mass spectrometry-based acetylomics and co-immunoprecipitation experiments demonstrated that active HDAC8 associated with and deacetylated the histone acetyltransferase EP300, a known coactivator of MITF in melanocytes. Increased expression of EP300 increased the sensitivity of melanoma cells to UV-irradiation, hypoxia and BRAF-MEK inhibitor therapy. Conclusions: HDAC8 associated with EP300 and mediated the switch to an invasive, stress resistant state in melanoma cells by suppressing MITF and increasing c-Jun driven transcriptional programs. Targeting HDAC8 could be a strategy to limit plasticity in advanced melanoma, increasing the efficacy of current therapies. Citation Format: Michael Emmons, Richard Bennett, Alberto Riva, Bin Fang, Edward Seto, john koomen, Jonathan Licht, Keiran Smalley. HDAC8 activity regulates stress induced phenotype switching in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1116.

Deregulated FASN Expression in BRAF Inhibitor-Resistant Melanoma Cells Unveils New Targets for Drug Combinations.

Simple SummaryStrategies to overcome resistance to targeted therapy represent a clinical need for metastatic melanoma. Altered lipid metabolism has been identified in the metabolic reprogramming associated with melanoma progression. Lipid metabolism genes such as fatty acid synthase (FASN) and 24-dehydrocholesterol reductase (DHCR24) have been proposed to contribute to tumor aggressiveness, but the therapeutic value of lipid metabolism inhibitors, particularly in drug-resistant melanoma, is unknown. Here, we found that molecular targeting of FASN in melanoma cells resistant to the BRAF inhibitor PLX4032 increased the sensitivity to the drug. Up-regulation of DHCR24 upon FASN targeting revealed the activation of druggable compensatory pathways sustaining the growth of resistant cells.Metabolic changes promoting cell survival are involved in metastatic melanoma progression and in the development of drug resistance. In BRAF-inhibitor resistant melanoma cells, we explored the role of FASN, an enzyme involved in lipogenesis overexpressed in metastatic melanoma. Resistant melanoma cells displaying enhanced migratory and pro-invasive abilities increased sensitivity to the BRAF inhibitor PLX4032 upon the molecular targeting of FASN and upon treatment with the FASN inhibitor orlistat. This behavior was associated with a marked apoptosis and caspase 3/7 activation observed for the drug combination. The expression of FASN was found to be inversely associated with drug resistance in BRAF-mutant cell lines, both in a set of six resistant/sensitive matched lines and in the Cancer Cell Line Encyclopedia. A favorable drug interaction in resistant cells was also observed with U18666 A inhibiting DHCR24, which increased upon FASN targeting. The simultaneous combination of the two inhibitors showed a synergistic interaction with PLX4032 in resistant cells. In conclusion, FASN plays a role in BRAF-mutated melanoma progression, thereby creating novel therapeutic opportunities for the treatment of melanoma.

NECTIN4: A Novel Therapeutic Target for Melanoma.

Malignant melanoma is the most common lethal skin cancer and causes death in a short time when metastasized. Although BRAF inhibitors (BRAFi) have greatly improved the prognosis of BRAF-mutated melanoma, drug resistance is a major concern even when they are combined with MEK inhibitors. Alternative treatments for BRAFi-resistant melanoma are highly anticipated. Nectin cell adhesion molecule 4 (NECTIN4) is highly expressed and associated with progression in tumors. We aimed to investigate the role of NECTIN4 in melanoma and its potency as a therapeutic target using 126 melanoma samples and BRAFi-resistant cells. Immunohistochemically, most of the clinical samples expressed NECTIN4, at least in part. NECTIN4 was highly expressed in BRAF-mutated melanoma and its high expression was associated with disease-free survival. In BRAFi-resistant melanoma cells, NECTIN4 and the PI3K/Akt pathway were upregulated, along with the acquisition of BRAFi resistance. Monomethyl auristatin E, a cytotoxic part of NECTIN4-targeted antibody–drug conjugate, was effective for BRAF-mutated or BRAFi-resistant melanoma cells. NECTIN4 inhibition increased the sensitivity of BRAFi-resistant cells to BRAFi and induced apoptosis. In conclusion, we revealed the expression and roles of NECTIN4 in melanoma. Targeted therapies against NECTIN4 can be a novel treatment strategy for melanoma, even after the acquisition of BRAFi resistance.