Vemurafenib Resistance Research Articles

Neddylation signaling inactivation by tetracaine hydrochloride suppresses cell proliferation and alleviates vemurafenib-resistance of melanoma

Tetracaine, a local anesthetic, exhibits potent cytotoxic effects on multiple cancer; however, the precise underlying mechanisms of its anti-cancer activity remain uncertain. The anti-cancer activity of tetracaine was found to be the most effective among commonly used local anesthetics in this study. After tetracaine treatment, the differentially expressed genes in melanoma cells were identified by the RNAseq technique and enriched in the lysosome signaling pathway, cullin family protein binding, and proteasome signaling pathway through Kyoto Encyclopedia of Genes and Genomes. Additionally, the ubiquitin-like neddylation signaling pathway, which is hyperactivated in melanoma, could be abrogated due to decreased NAE2 expression after tetracaine treatment. The neddylation of the pro-oncogenic Survivin, which enhances its stability, was significantly reduced following treatment with tetracaine. The activation of neddylation signaling by NEDD8 overexpression could reduce the antitumor efficacy of tetracaine in vivo and in vitro. Furthermore, vemurafenib-resistant melanoma cells showed higher level of neddylation, and potential substrate proteins undergoing neddylation modification were identified through immunoprecipitation and mass spectrometry. The tetracaine treatment could reduce drug resistance via neddylation signaling pathway inactivation in melanoma cells. These findings demonstrate that tetracaine effectively inhibits cell proliferation and alleviates vemurafenib resistance in melanoma by suppressing the neddylation signaling pathway, providing a promising avenue for controlling cancer progression.Graphical

ARID1A-Deficient Tumors Acquire Immunogenic Neoantigens during the Development of Resistance to Targeted Therapy.

Neoantigen-based immunotherapy is an attractive potential treatment for previously intractable tumors. To effectively broaden the application of this approach, stringent biomarkers are crucial to identify responsive patients. ARID1A, a frequently mutated subunit of SWI/SNF chromatin remodeling complex, has been reported to determine tumor immunogenicity in some cohorts; however, mutations and deletions of ARID1A are not always linked to clinical responses to immunotherapy. In this study, we investigated immunotherapeutic responses based on ARID1A status in targeted therapy-resistant cancers. Mouse and human BRAFV600E melanomas with or without ARID1A expression were transformed into resistant to vemurafenib, an FDA-approved specific BRAFV600E inhibitor. Anti-PD-1 antibody treatment enhanced antitumor immune responses in vemurafenib-resistant ARID1A-deficient tumors but not in ARID1A-intact tumors or vemurafenib-sensitive ARID1A-deficient tumors. Neoantigens derived from accumulated somatic mutations during vemurafenib resistance were highly expressed in ARID1A-deficient tumors and promoted tumor immunogenicity. Furthermore, the newly generated neoantigens could be utilized as immunotherapeutic targets by vaccines. Finally, targeted therapy resistance-specific neoantigen in experimental human melanoma cells lacking ARID1A were validated to elicit T-cell receptor responses. Collectively, the classification of ARID1A-mutated tumors based on vemurafenib resistance as an additional indicator of immunotherapy response will enable a more accurate prediction to guide cancer treatment. Furthermore, the neoantigens that emerge with therapy resistance can be promising therapeutic targets for refractory tumors. Significance: Chemotherapy resistance promotes the acquisition of immunogenic neoantigens in ARID1A-deficient tumors that confer sensitivity to immune checkpoint blockade and can be utilized for developing antitumor vaccines, providing strategies to improve immunotherapy efficacy.

Targeting PEAK1 sensitizes anaplastic thyroid carcinoma cells harboring BRAFV600E to Vemurafenib by Bim upregulation.

Pseudopodium-enriched atypical kinase 1 (PEAK1) has been demonstrated to be upregulated in human malignancies and cells. Enhanced PEAK1 expression facilitates tumor cell survival and chemoresistance. However, the role of PEAK1 inhibition to anaplastic thyroid carcinoma cell (ATC) and vemurafenib resistance is still unknown. Here, we observed that targeting PEAK1 inhibited cell viability and colony formation, but not cell apoptosis in both of the 8505C and Hth74 cells in vitro. Targeting PEAK1 sensitized 8505C and Hth74 cells to vemurafenib by inducing cell apoptosis, and thereby decreasing cell viability. Mechanistically, vemurafenib treatment upregulated PEAK1 expression. Combined PEAK1 depletion and Vemurafenib treatment upregulated Bim expression. Targeting PEAK1 sensitized vemurafenib-induced apoptosis by upregulating Bim. In conclusion, vemurafenib resistance in ATC cells harboring BRAFV600E is associated with PEAK1 activation, resulting in the inhibition of pro-apoptotic Bim protein. Therefore, targeting PEAK1 may be an effective strategy to sensitize ATC harboring BRAFV600E to vemurafenib.

Overcoming Vemurafenib Resistance in Metastatic Melanoma: Targeting Integrins to Improve Treatment Efficacy.

Metastatic melanoma, a deadly form of skin cancer, often develops resistance to the BRAF inhibitor drug vemurafenib, highlighting the need for understanding the underlying mechanisms of resistance and exploring potential therapeutic strategies targeting integrins and TGF-β signalling. In this study, the role of integrins and TGF-β signalling in vemurafenib resistance in melanoma was investigated, and the potential of combining vemurafenib with cilengitide as a therapeutic strategy was investigated. In this study, it was found that the transcription of PAI1 and p21 was induced by acquired vemurafenib resistance, and ITGA5 levels were increased as a result of this resistance. The transcription of ITGA5 was mediated by the TGF-β pathway in the development of vemurafenib resistance. A synergistic effect on the proliferation of vemurafenib-resistant melanoma cells was observed with the combination therapy of vemurafenib and cilengitide. Additionally, this combination therapy significantly decreased invasion and colony formation in these resistant cells. In conclusion, it is suggested that targeting integrins and TGF-β signalling, specifically ITGA5, ITGB3, PAI1, and p21, may offer promising approaches to overcoming vemurafenib resistance, thereby improving outcomes for metastatic melanoma patients.

Polyamine and EIF5A hypusination downstream of c-Myc confers targeted therapy resistance in BRAF mutant melanoma

BackgroundBRAF inhibitors are widely employed in the treatment of melanoma with the BRAF V600E mutation. However, the development of resistance compromises their therapeutic efficacy. Diverse genomic and transcriptomic alterations are found in BRAF inhibitor resistant melanoma, posing a pressing need for convergent, druggable target that reverse therapy resistant tumor with different resistance mechanisms.MethodsCRISPR-Cas9 screens were performed to identify novel target gene whose inhibition selectively targets A375VR, a BRAF V600E mutant cell line with acquired resistance to vemurafenib. Various in vitro and in vivo assays, including cell competition assay, water soluble tetrazolium (WST) assay, live-dead assay and xenograft assay were performed to confirm synergistic cell death. Liquid Chromatography-Mass Spectrometry analyses quantified polyamine biosynthesis and changes in proteome in vemurafenib resistant melanoma. EIF5A hypusination dependent protein translation and subsequent changes in mitochondrial biogenesis and activity were assayed by O-propargyl-puromycin labeling assay, mitotracker, mitoSOX labeling and seahorse assay. Bioinformatics analyses were used to identify the association of polyamine biosynthesis with BRAF inhibitor resistance and poor prognosis in melanoma patient cohorts.ResultsWe elucidate the role of polyamine biosynthesis and its regulatory mechanisms in promoting BRAF inhibitor resistance. Leveraging CRISPR-Cas9 screens, we identify AMD1 (S-adenosylmethionine decarboxylase 1), a critical enzyme for polyamine biosynthesis, as a druggable target whose inhibition reduces vemurafenib resistance. Metabolomic and proteomic analyses reveal that polyamine biosynthesis is upregulated in vemurafenib-resistant cancer, resulting in enhanced EIF5A hypusination, translation of mitochondrial proteins and oxidative phosphorylation. We also identify that sustained c-Myc levels in vemurafenib-resistant cancer are responsible for elevated polyamine biosynthesis. Inhibition of polyamine biosynthesis or c-Myc reversed vemurafenib resistance both in vitro cell line models and in vivo in a xenograft model. Polyamine biosynthesis signature is associated with poor prognosis and shorter progression free survival after BRAF/MAPK inhibitor treatment in melanoma cohorts, highlighting the clinical relevance of our findings.ConclusionsOur findings delineate the molecular mechanisms involving polyamine-EIF5A hypusination-mitochondrial respiration pathway conferring BRAF inhibitor resistance in melanoma. These targets will serve as effective therapeutic targets that can maximize the therapeutic efficacy of existing BRAF inhibitors.

Exosomal miR-19a derived from melanoma cell promotes the vemurafenib resistance of malignant melanoma through directly targeting LRIG1 to reactivate AKT and MAPK pathway

Exosomes derived from neighboring v-raf murine sarcoma viral oncogene homolog B1 inhibitor (BRAFi)-resistant melanoma cells mediate the formation of resistance in melanoma cells sensitive to BRAFi. The function and molecular mechanisms of exosomal miRNA in BRAFi resistance of melanoma have not been studied. We found that the expression of miR-19a in BRAFi resistant melanoma cells was significantly higher than that in sensitive cells, and miR-19a contributes to the resistance of melanoma cells to BRAFi by targeting immunoglobulin-like domains protein 1 (LRIG1). miR-19a was highly enriched in exosomes secreted from BRAFi resistant melanoma cells, and these exosomal miR-19a promote the spread of BRAFi resistant. The reactivation of Protein kinase B (AKT) and mitogen-activated protein kinase (MAPK) pathways is the main reason for the BRAFi resistant of melanoma cells. We demonstrated that exosomal miR-19a derived from melanoma cell promotes the formation and spread of BRAFi resistant in melanoma through targeting LRIG1 to reactivate AKT and MAPK pathway. Therefore, miR-19a may serve as a potential therapeutic target in melanoma patients with acquired drug resistance.

Autophagy sustains mitochondrial respiration and determines resistance to BRAFV600E inhibition in thyroid carcinoma cells

ABSTRACT BRAFV600E is the most prevalent mutation in thyroid cancer and correlates with poor prognosis and therapy resistance. Although selective inhibitors of BRAFV600E have been developed, more advanced tumors such as anaplastic thyroid carcinomas show a poor response in clinical trials. Therefore, the study of alternative survival mechanisms is needed. Since metabolic changes have been related to malignant progression, in this work we explore metabolic dependencies of thyroid tumor cells to exploit them therapeutically. Our results show that respiration of thyroid carcinoma cells is highly dependent on fatty acid oxidation and, in turn, fatty acid mitochondrial availability is regulated through macroautophagy/autophagy. Furthermore, we show that both lysosomal inhibition and the knockout of the essential autophagy gene, ATG7, lead to enhanced lipolysis; although this effect is not essential for survival of thyroid carcinoma cells. We also demonstrate that following inhibition of either autophagy or fatty acid oxidation, thyroid tumor cells compensate oxidative phosphorylation deficiency with an increase in glycolysis. In contrast to lipolysis induction, upon autophagy inhibition, glycolytic boost in autophagy-deficient cells is essential for survival and, importantly, correlates with a higher sensitivity to the BRAFV600E selective inhibitor, vemurafenib. In agreement, downregulation of the glycolytic pathway results in enhanced mitochondrial respiration and vemurafenib resistance. Our work provides new insights into the role of autophagy in thyroid cancer metabolism and supports mitochondrial targeting in combination with vemurafenib to eliminate BRAFV600E-positive thyroid carcinoma cells. Abbreviations: AMP: adenosine monophosphate; ATC: anaplastic thyroid carcinoma; ATG: autophagy related; ATP: adenosine triphosphate; BRAF: B-Raf proto-oncogene, serine/threonine kinase; Cas9: CRISPR-associated protein; CREB: cAMP responsive element binding protein; CRISPR: clustered regularly interspaced short palindromic repeats; 2DG: 2-deoxyglucose; FA: fatty acid; FAO: fatty acid oxidation; FASN: fatty acid synthase; FCCP: trifluoromethoxy carbonyl cyanide phenylhydrazone; LAMP1: lysosomal associated membrane protein 1; LIPE/HSL: lipase E, hormone sensitive type; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; OCR: oxygen consumption rate; OXPHOS: oxidative phosphorylation; PRKA/PKA: protein kinase cAMP-activated; PTC: papillary thyroid carcinoma; SREBF1/SREBP1: sterol regulatory element binding transcription factor 1.

HGF facilitates methylation of MEG3, potentially implicated in vemurafenib resistance in melanoma.

Melanoma, a frequently encountered cutaneous malignancy characterized by a poor prognosis, persists in presenting formidable challenges despite the advancement in molecularly targeted drugs designed to improve survival rates significantly. Unfortunately, as more therapeutic choices have developed over time, the gradual emergence of drug resistance has become a notable impediment to the effectiveness of these therapeutic interventions. The hepatocyte growth factor (HGF)/c-met signaling pathway has attracted considerable attention, associated with drug resistance stemming from multiple potential mutations within the c-met gene. The activation of the HGF/c-met pathway operates in an autocrine manner in melanoma. Notably, a key player in the regulatory orchestration of HGF/c-met activation is the long non-coding RNA MEG3. Melanoma tissues were collected to measure MEG3 expression. In vitro validation was performed on MEG3 to prove its oncogenic roles. Bioinformatic analyses were conducted on the TCGA database to build the MEG3-related score. The immune characteristics and mutation features of the MEG3-related score were explored. We revealed a negative correlation between HGF and MEG3. In melanoma cells, HGF inhibited MEG3 expression by augmenting the methylation of the MEG3 promoter. Significantly, MEG3 exhibits a suppressive impact on the proliferation and migration of melanoma cells, concurrently inhibiting c-met expression. Moreover, a predictive model centered around MEG3 demonstrates notable efficacy in forecasting critical prognostic indicators, immunological profiles, and mutation statuses among melanoma patients. The present study highlights the potential of MEG3 as a pivotal regulator of c-met, establishing it as a promising candidate for targeted drug development in the ongoing pursuit of effective therapeutic interventions.

MitoCur-1 induces ferroptosis to reverse vemurafenib resistance in melanoma through inhibition of USP14.

Melanoma is an aggressive malignant tumor with a poor prognosis. Vemurafenib (PLX4032, vem) is applied to specifically treat BRAF V600E-mutated melanoma patients. However, prolonged usage of vem makes patients resistant to the drug and finally leads to clinical failure. We previously tested the combination regimen of tubulin inhibitor VERU-111 with vem, as well as USP14 selective inhibitor b-AP15 in combination with vem, both of which have showed profound therapeutic effects in overcoming vem resistance invitro and invivo. Most importantly, we discovered that vem-resistant melanoma cell lines highly expressed E3 ligase SKP2 and DUB enzyme USP14, and we have demonstrated that USP14 directly interacts and stabilizes SKP2, which contributes to vem resistance. These works give us a clue that USP14 might be a promising target to overcome vem resistance in melanoma. MitoCur-1 is a curcumin derivative, which was originally designed to specifically target tumor mitochondria inducing redox imbalance, thereby promoting tumor cell death. In this study, we have demonstrated that it can work as a novel USP14 inhibitor, and thus bears great potential in providing an anti-tumor effect and sensitizing vem-resistant cells by inducing ferroptosis in melanoma. Application of MitoCur-1 dramatically induces USP14 inhibition and inactivation of GPX4 enzyme, meanwhile, increases the depletion of GSH and decreases SLC7A11 expression level. As a result, ferrous iron-dependent lipid ROS accumulated in the cell, inducing ferroptosis, thus sensitizes the vem-resistant melanoma cell. Interestingly, overexpression of USP14 antagonized all the ferroptosis cascade events induced by MitoCur-1, therefore, we conclude that MitoCur-1 induces ferroptosis through inhibition of USP14. We believe that by inhibition of USP14, vem resistance can be reversed and will finally benefit melanoma patients in future.

Vemurafenib and Obinutuzumab as Frontline Therapy for Hairy Cell Leukemia

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

Ezrin Inhibition Overcomes Acquired Resistance to Vemurafenib in BRAFV600E-Mutated Colon Cancer and Melanoma Cells In Vitro.

Despite the advancements in targeted therapy for BRAFV600E-mutated metastatic colorectal cancer (mCRC), the development of resistance to BRAFV600E inhibition limits the response rate and durability of the treatment. Better understanding of the resistance mechanisms to BRAF inhibitors will facilitate the design of novel pharmacological strategies for BRAF-mutated mCRC. The aim of this study was to identify novel protein candidates involved in acquired resistance to BRAFV600E inhibitor vemurafenib in BRAFV600E-mutated colon cancer cells using an integrated proteomics approach. Bioinformatic analysis of obtained proteomics data indicated actin-cytoskeleton linker protein ezrin as a highly ranked protein significantly associated with vemurafenib resistance whose overexpression in the resistant cells was additionally confirmed at the gene and protein level. Ezrin inhibition by NSC305787 increased anti-proliferative and pro-apoptotic effects of vemurafenib in the resistant cells in an additive manner, which was accompanied by downregulation of CD44 expression and inhibition of AKT/c-Myc activities. We also detected an increased ezrin expression in vemurafenib-resistant melanoma cells harbouring the BRAFV600E mutation. Importantly, ezrin inhibition potentiated anti-proliferative and pro-apoptotic effects of vemurafenib in the resistant melanoma cells in a synergistic manner. Altogether, our study suggests a role of ezrin in acquired resistance to vemurafenib in colon cancer and melanoma cells carrying the BRAFV600E mutation and supports further pre-clinical and clinical studies to explore the benefits of combined BRAF inhibitors and actin-targeting drugs as a potential therapeutic approach for BRAFV600E-mutated cancers.

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

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

Vemurafenib induces a noncanonical senescence-associated secretory phenotype in melanoma cells which promotes vemurafenib resistance

More than one half melanoma patients have BRAF gene mutation. BRAF inhibitor vemurafenib is an effective medication for these patients. However, acquired resistance is generally inevitable, the mechanisms of which are not fully understood. Cell senescence and senescence-associated secretory phenotype (SASP) are involved in extensive biological functions. This study was designed to explore the possible role of senescent cells in vemurafenib resistance. The results showed that vemurafenib treatment induced BRAF-mutant but not wild-type melanoma cells into senescence, as manifested by positive β-galactosidase staining, cell cycle arrest, enlarged cellular morphology, and cyclin D1/p-Rb pathway inhibition. However, the senescent cells induced by vemurafenib (SenV) did not display DNA damage response, p53/p21 pathway activation, reactive oxygen species accumulation, decline of mitochondrial membrane potential, or secretion of canonical SASP cytokines. Instead, SenV released other cytokines, including CCL2, TIMP2, and NGFR, to protect normal melanoma cells from growth inhibition upon vemurafenib treatment. Xenograft experiments further confirmed that vemurafenib induced melanoma cells into senescence in vivo. The results suggest that vemurafenib can induce robust senescence in BRAFV600E melanoma cells, leading to the release of resistance-conferring cytokines. Both the senescent cells and the resistant cytokines could be potential targets for tackling vemurafenib resistance.

Reversing vemurafenib-resistance in primary melanoma cells by combined romidepsin and type I IFN treatment through blocking of tumorigenic signals and induction of immunogenic effects.

BRAFV600 mutations are the most common oncogenic alterations in melanoma cells, supporting proliferation, invasion, metastasis and immune evasion. In patients, these aberrantly activated cellular pathways are inhibited by BRAFi whose potent antitumor effect and therapeutic potential are dampened by the development of resistance. Here, by using primary melanoma cell lines, generated from lymph node lesions of metastatic patients, we show that the combination of two FDA-approved drugs, the histone deacetylate inhibitor (HDCAi) romidepsin and the immunomodulatory agent IFN-α2b, reduces melanoma proliferation, long-term survival and invasiveness and overcomes acquired resistance to the BRAFi vemurafenib (VEM). Targeted resequencing revealed that each VEM-resistant melanoma cell line and the parental counterpart are characterized by a distinctive and similar genetic fingerprint, shaping the differential and specific antitumor modulation of MAPK/AKT pathways by combined drug treatment. By using RNA-sequencing and functional in vitro assays, we further report that romidepsin-IFN-α2b treatment restores epigenetically silenced immune signals, modulates MITF and AXL expression and induces both apoptosis and necroptosis in sensitive and VEM-resistant primary melanoma cells. Moreover, the immunogenic potential of drug-treated VEM-resistant melanoma cells results significantly enhanced, given the increased phagocytosis rate of these cells by dendritic cells, which in turn exhibit also a selective down-modulation of the immune checkpoint TIM-3. Overall, our results provide evidence that combined epigenetic-immune drugs can overcome VEM resistance of primary melanoma cells by oncogenic and immune pathways reprogramming, and pave the way for rapidly exploiting this combination to improve BRAFi-resistant metastatic melanoma treatment, also via reinforcement of immune checkpoint inhibitor therapy.

Abstract 4997: PLK1 promotes the metastasis and drug resistance in melanoma

Abstract Melanoma is one of the most frequently diagnosed cancers in the caucasian population of both genders. The survival rate of advanced melanoma patients will be exceedingly poor, as low as 27.3%. BRAF-V600E is the most common mutation found clinically, resulting in the constitutive activation of the MAPK signaling pathway. Vemurafenib, a specific inhibitor of mutant BRAF, has shown an impressive response in phase 3 clinical trials and was approved by FDA in 2011. However, due to the rapid development of resistance, the duration of response under a single treatment is frequently short, highlighting the urgent requirement for novel therapy in melanoma treatment. Polo-like kinase 1 (PLK1), a crucial cell cycle regulator, participates in multiple mitotic processes, including centrosome maturation, mitotic entry, spindle assembly, sister chromatid segregation, mitotic exit, and cytokinesis. Compared to normal tissues, the expression level of PLK1 significantly upregulates in multiple cancers. More importantly, the expression level of PLK1 negatively correlates with the melanoma patients’ survival rate based on the TCGA database. Besides, PLK1 is identified as an oncogene to promote proliferation, motility, and resistance to various drugs. In the present study, we found that overexpression of PLK1 promotes tumor growth, metastasis, metabolic reprogramming, and vemurafenib resistance in the BrafCA/Ptenloxp mouse melanoma model. Through phosphorylation, overexpressed PLK1 stabilizes BACH1, a critical transcriptional factor of multiple metastatic and metabolic genes, and protects it from degradation. Subsequently, a higher level of BACH1 leads to increasing metastasis, upregulation of glycolysis, and suppression of OXPHOs. Besides, we have validated the strong synergy between PLK1 inhibitor volasertib and vemurafenib in treating melanoma carrying BRAF-V600E. Our in vitro and in vivo experiments have shown an improved efficacy of this combined therapy on inhibition of cell proliferation, induction of cell death, and suppression of cell metastasis compared to mono-treatment. In short, our data has indicated PLK1 as a potent and promising target in cancer treatment. Citation Format: Fengyi Mao, Yifan Kong, Chaohao Li, Yanquan Zhang, Xiongjian Rao, Qiongsi Zhang, Xinyi Wang, Zhiguo Li, Xiaoqi Liu. PLK1 promotes the metastasis and drug resistance in melanoma. [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 4997.

Targeting ARF1-IQGAP1 interaction to suppress colorectal cancer metastasis and vemurafenib resistance

IntroductionAcquired resistance to BRAF inhibitor vemurafenib is frequently observed in metastatic colorectal cancer (CRC), and it is a thorny issue that results in treatment failure. As adaptive responses for vemurafenib treatment, a series of cellular bypasses are response for the adaptive feedback reactivation of ERK signaling, which warrant further investigation. ObjectivesWe identified ARF1 (ADP-ribosylation factor 1) as a novel regulator of both vemurafenib resistance and cancer metastasis, its molecular mechanism and potential inhibitor were investigated in this study. MethodsDIA-based quantitative proteomics and RNA-seq were performed to systematic analyze the profiling of vemurafenib-resistant RKO cells (RKO-VR) and highly invasive RKO cells (RKO-I8), respectively. Co‑immunoprecipitation assay was performed to detect the interaction of ARF1 and IQGAP1 (IQ-domain GTPase activating protein 1). An ELISA-based drug screen system on FDA-approved drug library was established to screen the compounds against the interaction of ARF1-IQGAP1.The biological functions of ARF1 and LY2835219 were determined by transwell, western blotting, Annexin V-FITC/PI staining and in vivo experimental metastasis assays. ResultsWe found that ARF1 strongly interacted with IQGAP1 to activate ERK signaling in VR and I8 CRC cells. Deletion of IQGAP1 or inactivation of ARF1 (ARF-T48S) restored the invasive ability induced by ARF1. As ARF1-IQGAP1 interaction is essential for ERK activation, we screened LY2835219 as novel inhibitor of ARF1-IQGAP1 interaction, which inactivated ERK signaling and suppressed CRC metastasis and vemurafenib-resistance in vitro and in vivo with no observed side effect. Furthermore, LY2835219 in combined treatment with vemurafenib exerted significantly inhibitory effect on ARF1-mediated cancer metastasis than used independently. ConclusionThis study uncovers that ARF1-IQGAP1 interaction-mediated ERK signaling reactivation is critical for vemurafenib resistance and cancer metastasis, and that LY2835219 is a promising therapeutic agent for CRC both as a single agent and in combination with vemurafenib.

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

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

Silencing FOXP2 reverses vemurafenib resistance in BRAFV600E mutant papillary thyroid cancer and melanoma cells

BackgroundVemurafenib (VEM) is a commonly used inhibitor of papillary thyroid cancer (PTC) and melanoma with the BRAFV600E mutation; however, acquired resistance is unavoidable. The present study aimed to identify a potential target to reverse resistance.Materials and methodsA VEM-resistant PTC cell line (B-CPAP/VR) was established by gradually increasing the drug concentration, and a VEM-resistant BRAFV600E melanoma cell line (A375/VR) was also established. RNA sequencing and bioinformatics analyses were conducted to identify dysregulated genes and construct a transcription factor (TF) network. The role of a potential TF, forkhead box P2 (FOXP2), verified by qRT-PCR, was selected for further confirmation.ResultsThe two resistant cell lines were tolerant of VEM and displayed higher migration and colony formation abilities (p < 0.05). RNA sequencing identified 9177 dysregulated genes in the resistant cell lines, and a TF network consisting of 13 TFs and 44 target genes was constructed. Alterations in FOXP2 expression were determined to be consistent between the two VEM-resistant cell lines. Finally, silencing FOXP2 resulted in an increase in drug sensitivity and significant suppression of the migration and colony formation abilities of the two resistant cell lines (p < 0.05).ConclusionsThe present study successfully established two VEM-resistant cell lines and identified a potential target for VEM-resistant PTC or melanoma.

Cooperative induction of receptor tyrosine kinases contributes to adaptive MAPK drug resistance in melanoma through the PI3K pathway.

Vemurafenib-induced drug resistance in melanoma has been linked to receptor tyrosine kinase (RTK) upregulation. The MITF and SOX10 genes play roles as master regulators of melanocyte and melanoma development. Here, we aimed to explore the complex mechanisms behind the MITF/SOX10-controlled RTK-induced drug resistance in melanoma. To achieve this, we used a number of molecular techniques, including melanoma patient data from TCGA, vemurafenib-resistant melanoma cell lines, and knock-down studies. The melanoma cell lines were classified as proliferative or invasive based upon their MITF/AXL expression activity. We measured the change of expression activity for MITF/SOX10 and their receptor (AXL/ERBB3) and ligand (NRG1/GAS6) targets known to be involved in RTK-induced drug resistance after vemurafenib treatment. We find that melanoma cell lines characterized as proliferative (high MITF low AXL), transform into an invasive (low MITF, high AXL) cell state after vemurafenib resistance, indicating novel feedback loops and advanced compensatory regulation mechanisms between the master regulators, receptors, and ligands involved in vemurafenib-induced resistance. Together, our data disclose fine-tuned mechanisms involved in RTK-facilitated vemurafenib resistance that will be challenging to overcome by using single drug targeting strategies against melanoma.

RSK2 promotes melanoma cell proliferation and vemurafenib resistance via upregulating cyclin D1.

BRAF inhibitors are commonly used in targeted therapies for melanoma patients harboring BRAFV600E mutant. Despite the benefit of vemurafenib therapy, acquired resistance during or after treatment remains a major obstacle in BRAFV600E mutant melanoma. Here we found that RSK2 is overexpressed in melanoma cells and the high expression of RSK2 indicates poor overall survival (OS) in melanoma patients. Overexpression of RSK2 leads to vemurafenib resistance, and the deletion of RSK2 inhibits cell proliferation and sensitizes melanoma cells to vemurafenib. Mechanistically, RSK2 enhances the phosphorylation of FOXO1 by interacting with FOXO1 and promoting its subsequent degradation, leading to upregulation of cyclin D1 in melanoma cells. These results not only reveal the presence of a RSK2-FOXO1-cyclin D1 signaling pathway in melanoma, but also provide a potential therapeutic strategy to enhance the efficacy of vemurafenib against cancer.