Characterization of two melanoma cell lines resistant to BRAF/MEK inhibitors (vemurafenib and cobimetinib).

BACKGROUND: BRAF (v-raf murine sarcoma viral oncogene homolog B1)/MEK (mitogen-activated protein kinase kinase) inhibitors are used for melanoma treatment. Unfortunately, patients treated with this combined therapy develop resistance to treatment quite quickly, but the mechanisms underlying this phenomenon are not yet fully understood. Here, we report and characterize two melanoma cell lines (WM9 and Hs294T) resistant to BRAF (vemurafenib) and MEK (cobimetinib) inhibitors. METHODS: Cell viability was assessed via the XTT test. The level of selected proteins as well as activation of signaling pathways were evaluated using Western blotting. The expression of the chosen genes was assessed by RT-PCR. The distribution of cell cycle phases was analyzed by flow cytometry, and confocal microscopy was used to take photos of spheroids. The composition of cytokines secreted by cells was determined using a human cytokine array. RESULTS: The resistant cells had increased survival and activation of ERK kinase in the presence of BRAF/MEK inhibitors. The IC50 values for these cells were over 1000 times higher than for controls. Resistant cells also exhibited elevated activation of AKT, p38, and JNK signaling pathways with increased expression of EGFR, ErbB2, MET, and PDGFRß receptors as well as reduced expression of ErbB3 receptor. Furthermore, these cells demonstrated increased expression of genes encoding proteins involved in drug transport and metabolism. Resistant cells also exhibited features of epithelial-mesenchymal transition and cancer stem cells as well as reduced proliferation rate and elevated cytokine secretion. CONCLUSIONS: In summary, this work describes BRAF/MEK-inhibitor-resistant melanoma cells, allowing for better understanding the underlying mechanisms of resistance. The results may thus contribute to the development of new, more effective therapeutic strategies.

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.

Proteogenomics Reveals Perturbed Signaling Networks in Malignant Melanoma Cells Resistant to BRAF Inhibition.

Analysis of nucleotide variants is a cornerstone of cancer medicine. Although only 2% of the genomic sequence is protein coding, mutations occurring in these regions have the potential to influence protein structure or modification status and may have severe impact on disease aetiology. Proteogenomics enables the analysis of sample-specific nonsynonymous nucleotide variants with regard to their effect at the proteome and phosphoproteome levels. Here, we developed a proof-of-concept proteogenomics workflow and applied it to the malignant melanoma cell line A375. Initially, we studied the resistance to serine/threonine-protein kinase B-raf (BRAF) inhibitor (BRAFi) vemurafenib in A375 cells. This allowed identification of several oncogenic nonsynonymous nucleotide variants, including a gain-of-function variant on aurora kinase A (AURKA) at F31I. We also detected significant changes in abundance among (phospho)proteins, which led to reactivation of the MAPK signaling pathway in BRAFi-resistant A375 cells. Upon reconstruction of the multiomic integrated signaling networks, we predicted drug therapies with the potential to disrupt BRAFi resistance mechanism in A375 cells. Notably, we showed that AURKA inhibition is effective and specific against BRAFi-resistant A375 cells. Subsequently, we investigated amino acid variants that interfere with protein posttranslational modification (PTM) status and potentially influence A375 cell signaling irrespective of BRAFi resistance. Mass spectrometry (MS) measurements confirmed variant-driven PTM changes in 12 proteins. Among them was the runt-related transcription factor 1 (RUNX1) displaying a variant on a known phosphorylation site S(Ph)276L. We confirmed the loss of phosphorylation site by MS and demonstrated the impact of this variant on RUNX1 interactome.

Melanoma: BRAFi Rechallenge.

Melanoma is the most aggressive type of skin cancer. Half of melanoma cases are characterized by the mutation BRAF V600. The case presented concerns a 41-year-old patient with locally advanced melanoma, being positive in mutation BRAF V600. The patient underwent surgery and received additional targeted therapy as part of a clinical study. In subsequent disease progression, immunotherapy was used. When the disease progressed again while the patient was in a good performance status, targeted therapy was administered again, and a good response was noted, making the patient reach a statistically significant overall survival, exceeding four years. Targeted therapy has proven to be an important tool in the treatment of melanoma. The use of BRAFi targeted therapy does not exclude the option of readministration at subsequent disease progression (BRAFi rechallenge). Preclinical models suggest that the resistance mechanism of cancer cells to BRAFi therapy bends, as these cell clones lose their evolutionary advantage after stopping BRAFi. Cell clones sensitive to BRAFi may then outcompete, making the treatment effective again. Therapeutical dilemmas in the management of patients with locally advanced melanoma that progresses to metastatic cancer are discussed.

Mefloquine induces ER stress and apoptosis in BRAFi-resistant A375-BRAFV600E /NRASQ61K malignant melanoma cells targeting intracranial tumors in a bioluminescent murine model.

Molecularly targeted therapeutics have revolutionized the treatment of BRAFV600E -driven malignant melanoma, but the rapid development of resistance to BRAF kinase inhibitors (BRAFi) presents a significant obstacle. The use of clinical antimalarials for the investigational treatment of malignant melanoma has shown only moderate promise, attributed mostly to inhibition of lysosomal-autophagic adaptations of cancer cells, but identification of specific antimalarials displaying single-agent antimelanoma activity has remained elusive. Here, we have screened a focused library of clinically used artemisinin-combination therapeutic (ACT) antimalarials for the apoptotic elimination of cultured malignant melanoma cell lines, also examining feasibility of overcoming BRAFi-resistance comparing isogenic melanoma cells that differ only by NRAS mutational status (BRAFi-sensitive A375-BRAFV600E /NRASQ61 vs. BRAFi-resistant A375-BRAFV600E /NRASQ61K ). Among ACT antimalarials tested, mefloquine (MQ) was the only apoptogenic agent causing melanoma cell death at low micromolar concentrations. Comparative gene expression-array analysis (A375-BRAFV600E /NRASQ61 vs. A375-BRAFV600E /NRASQ61K ) revealed that MQ is a dual inducer of endoplasmic reticulum (ER) and redox stress responses that precede MQ-induced loss of viability. ER-trackerTM DPX fluorescence imaging and electron microscopy indicated ER swelling, accompanied by rapid induction of ER stress signaling (phospho-eIF2α, XBP-1s, ATF4). Fluo-4 AM-fluorescence indicated the occurrence of cytosolic calcium overload observable within seconds of MQ exposure. In a bioluminescent murine model employing intracranial injection of A375-Luc2 (BRAFV600E /NRASQ61K ) cells, an oral MQ regimen efficiently antagonized brain tumor growth. Taken together, these data suggest that the clinical antimalarial MQ may be a valid candidate for drug repurposing aiming at chemotherapeutic elimination of malignant melanoma cells, even if metastasized to the brain and BRAFi-resistant.

Acneiform eruptions with combination targeted cancer therapy in colorectal cancer patients.

PURPOSE: Epidermal growth factor receptor inhibitors (EGFRI) can be used with pathway inhibitors, including mitogen-activated protein kinase kinase inhibitors (MEKIs), BRAF inhibitors (BRAFIs), and checkpoint inhibitors such as programmed death-ligand 1 (PD-L1) and programmed cell death protein 1 (PD-1) to treat colorectal cancer. These can precipitate treatment-resistant acneiform eruptions, prompting dose modification or discontinuation. Predicting the likelihood of severe rash development and crafting effective treatments may promote adherence to life-saving chemotherapy. METHODS: An Institutional Review Board-approved retrospective chart review of patients with colorectal cancer treated with EGFRI or MEKI in combination with HER2, BRAF, PI3K, or checkpoint inhibitors between January 1, 2016, and January 1, 2020, was performed. Surrogates for rash severity were investigated, including lower extremity involvement, utilization of oral steroids or retinoids, dose modification, and incidence of superinfection. RESULTS: Of 122 patients treated with combination therapy, 105 developed a rash, and 87 developed an acneiform eruption. Common combinations included MEKI/PD-LI, EGFRI/MEKI, and MEKI/PD-1I. Patients treated with EGFRI/MEKI developed the most severe rashes (p = 0.02). Lower extremity involvement was more frequent with EGFRI/MEKI compared to alternative combinations (p = 0.05). Drug holiday correlated with all rash severity surrogates, including rash grade, lower extremity involvement, oral steroid or retinoid use, and incidence of superinfection. Use of oral steroids or retinoids was associated with development of superinfection (p = 0.002). Prophylactic tetracycline use did not impact rash severity or rash incidence. CONCLUSION: This is the first descriptive analysis to characterize acneiform eruptions for patients with colorectal cancer on combination cancer therapy. Approximately 85% of patients developed a cutaneous toxicity with what appears to be synergistic effects of EGFRI and MEKI combination therapy causing the most severe eruptions. Superinfection rate correlated to systemic therapy use beyond oral tetracyclines. Further investigation into the utility of prophylactic oral tetracyclines in this population is needed.

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

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

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.

BRAFi induced demethylation of miR-152-5p regulates phenotype switching by targeting TXNIP in cutaneous melanoma.

Treatment of advanced BRAFV600-mutant melanoma using BRAF inhibitors (BRAFi) eventually leads to drug resistance and selects for highly metastatic tumor cells. We compared the most differentially dysregulated miRNA expression profiles of vemurafenib-resistant and highly-metastatic melanoma cell lines obtained from GEO DataSets. We discovered miR-152-5p was a potential regulator mediating melanoma drug resistance and metastasis. Functionally, knockdown of miR-152-5p significantly compromised the metastatic ability of BRAFi-resistant melanoma cells and overexpression of miR-152-5p promoted the formation of slow-cycling phenotype. Furthermore, we explored the cause of how and why miR-152-5p affected metastasis in depth. Mechanistically, miR-152-5p targeted TXNIP which affected metastasis and BRAFi altered the methylation status of MIR152 promoter. Our study highlights the crucial role of miR-152-5p on melanoma metastasis after BRAFi treatment and holds significant implying that discontinuous dosing strategy may improve the benefit of advanced BRAFV600-mutant melanoma patients.

Indoleamine 2,3-dioxygenase in melanoma progression and BRAF inhibitor resistance.

Indoleamine 2,3-dioxygenase (IDO) is associated with the progression of many types of tumors, including melanoma. However, there is limited information about IDO modulation on tumor cell itself and the effect of BRAF inhibitor (BRAFi) treatment and resistance. Herein, IDO expression was analyzed in different stages of melanoma development and progression linked to BRAFi resistance. IDO expression was increased in primary and metastatic melanomas from patients' biopsies, especially in the immune cells infiltrate. Using a bioinformatics approach, we also identified an increase in the IDO mRNA in the vertical growth and metastatic phases of melanoma. Using in silico analyses, we found that IDO mRNA was increased in BRAFi resistance. In an in vitro model, IDO expression and activity induced by interferon-gamma (IFNγ) in sensitive melanoma cells was decreased by BRAFi treatment. However, cells that became resistant to BRAFi presented random IDO expression levels. Also, we identified that treatment with the IDO inhibitor, 1-methyltryptophan (1-MT), was able to reduce clonogenicity for parental and BRAFi-resistant cells. In conclusion, our results support the hypothesis that the decreased IDO expression in tumor cells is one of the many additional outcomes contributing to the therapeutic effects of BRAFi. Still, the IDO production changeability by the BRAFi-resistant cells reiterates the complexity of the response arising from resistance, making it not possible, at this stage, to associate IDO expression in tumor cells with resistance. On the other hand, the maintenance of 1-MT off-target effect endorses its use as an adjuvant treatment of melanoma that has become BRAFi-resistant.

ER stress promotes antitumor effects in BRAFi/MEKi resistant human melanoma induced by natural compound 4-nerolidylcathecol (4-NC).

Melanoma accounts for only 4% of malignant neoplasms of the skin, but is considered the most serious because it is highly deadly. Mutations in the MAPK (Ras-Raf-MEK-ERK) pathway is closely linked to the lack of control of cell proliferation. Especially in melanoma, this pathway has become a target for the development of oncogene-targeted therapies, such as the potent inhibitors of v-Raf murine sarcoma viral oncogene homolog B (BRAFi) and mitogen-activated protein kinase kinase (MEKi). Very high rates of response have been achieved, but most patients are relapsed due to the development of resistance, justifying the constant search for new therapeutic compounds. Early results from our group indicated that 4-nerolidylcatechol (4-NC), a catechol compound extracted from Pothomorphe umbellata, induces DNA damage, ROS production, increased p53 expression culminating in apoptosis in melanoma but with no data regarding the 4-NC effects in cells resistant to BRAFi or MEKi. Therefore, here we evaluated the role of 4-NC alone or in combination with BRAFi/MEKi in resistant melanoma cells. Double-resistant cells were generated and characterized by MAPK pathway reactivation. 4-NC alone or in combination (30 µM) with MAPK inhibitors was cytotoxic, inhibited colony formation and decreased invasiveness in two and three-dimensional cell culture models of treatment-naïve, BRAFi-resistant and BRAF/MEKi double-resistant melanoma cells. Apoptosis induction was demonstrated in resistant and double-resistant melanoma cell lines after 4-NC treatments. 4-NC showed important ability to induce apoptosis via Endoplasmatic Reticulum (ER) stress and specifically BiP and CHOP that had increased protein expression in all melanoma cell lines proving to be part of the ER stress pathway activation. CHOP knockdown slightly but enough increases cellular viability following 4-NC treatment indicating that apoptosis observed is partially dependent on CHOP. In summary, we show that 4-NC is a compound with activity against cutaneous melanoma, including resistant cells to clinically approved therapies.

ER stress-induced autophagy in melanoma.

The activation of RAF-MEK-extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase cascade by v-raf murine sarcoma viral oncogene homolog B1 (BRAF)(V600E) mutation is a key alteration in melanoma. Although BRAF inhibitor (BRAFi) has achieved remarkable clinical success, the positive response to BRAFi is not sustainable, and the initial clinical benefit is eventually barred by the development of resistance to BRAFi. There is growing evidence to suggest that endoplasmic reticulum (ER) stress-induced autophagy could be a potential pro-survival mechanism that contributes to genesis of melanoma and to the resistance to BRAFi. ER stress-induced autophagy is an evolutionarily conserved membrane process. By degrading and recycling proteins and organelles via the formation of autophagous vesicles and their fusion with lysosomes, the autophagy plays a key role in homeostasis as well as pathological processes. In this review, we examine the autophagy phenomenon in melanocytic nevus, primary and metastatic melanoma, and its significance in BRAFi-resistant melanoma.

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.

Modeling Melanoma Heterogeneity In Vitro: Redox, Resistance and Pigmentation Profiles.

Microenvironment and transcriptional plasticity generate subpopulations within the tumor, and the use of BRAF inhibitors (BRAFis) contributes to the rise and selection of resistant clones. We stochastically isolated subpopulations (C1, C2, and C3) from naïve melanoma and found that the clones demonstrated distinct morphology, phenotypic, and functional profiles: C1 was less proliferative, more migratory and invasive, less sensitive to BRAFis, less dependent on OXPHOS, more sensitive to oxidative stress, and less pigmented; C2 was more proliferative, less migratory and invasive, more sensitive to BRAFis, less sensitive to oxidative stress, and more pigmented; and C3 was less proliferative, more migratory and invasive, less sensitive to BRAFis, more dependent on OXPHOS, more sensitive to oxidative stress, and more pigmented. Hydrogen peroxide plays a central role in oxidative stress and cell signaling, and PRDXs are one of its main consumers. The intrinsically resistant C1 and C3 clones had lower MITF, PGC-1α, and PRDX1 expression, while C1 had higher AXL and decreased pigmentation markers, linking PRDX1 to clonal heterogeneity and resistance. PRDX2 is depleted in acquired BRAFi-resistant cells and acts as a redox sensor. Our results illustrate that decreased pigmentation markers are related to therapy resistance and decreased antioxidant defense.

Impact of Physical Exercise on Melanoma Hallmarks: Current Status of Preclinical and Clinical Research.

In recent years, accumulating evidence from preclinical and clinical studies consistently indicated that physical activity/exercise plays a crucial role in reducing the incidence and recurrence of various malignancies, by exerting a beneficial modulation of cancer hallmarks. Moreover, physical activity is suggested to attenuate certain adverse effects of anticancer therapy, including the reduction of cardiovascular toxicity and symptoms related to depression and anxiety, among others, while preserving muscular strength. In the case of melanoma, the relationship with physical activity has been critically debated. Historically, several cohort studies and meta-analyses reported a positive association between physical activity/exercise and melanoma risk. This association was primarily attributed to outdoor activities that may expose the skin to UV radiation, a well-known risk factor for melanocyte transformation. However, more recent evidence does not support such association and recognizes physical activity/exercise role in both melanoma prevention and progression. Nevertheless, sun protection is recommended during outdoor training to minimize UV radiation exposure. This narrative review summarizes preclinical and clinical data about physical activity effects on melanoma hallmarks. Specifically, experimental evidence is reported concerning (i) invasion and metastasis, (ii) reprogramming of energy metabolism, (iii) angiogenesis, (iv) resistance to cell death, (v) evasion from immune destruction, and (vi) tumor-promoting inflammation.

Defining melanoma combination therapies that provide senolytic sensitivity in human melanoma cells.

Malignant Melanoma that resists immunotherapy remains the deadliest form of skin cancer owing to poor clinically lasting responses. Alternative like genotoxic or targeted chemotherapy trigger various cancer cell fates after treatment including cell death and senescence. Senescent cells can be eliminated using senolytic drugs and we hypothesize that the targeted elimination of therapy-induced senescent melanoma cells could complement both conventional and immunotherapies. We utilized a panel of cells representing diverse mutational background relevant to melanoma and found that they developed distinct senescent phenotypes in response to treatment. A genotoxic combination therapy of carboplatin-paclitaxel or irradiation triggered a mixed response of cell death and senescence, irrespective of BRAF mutation profiles. DNA damage-induced senescent melanoma cells exhibited morphological changes, residual DNA damage, and increased senescence-associated secretory phenotype (SASP). In contrast, dual targeted inhibition of Braf and Mek triggered a different mixed cell fate response including senescent-like and persister cells. While persister cells could reproliferate, senescent-like cells were stably arrested, but without detectable DNA damage and senescence-associated secretory phenotype. To assess the sensitivity to senolytics we employed a novel real-time imaging-based death assay and observed that Bcl2/Bcl-XL inhibitors and piperlongumine were effective in promoting death of carboplatin-paclitaxel and irradiation-induced senescent melanoma cells, while the mixed persister cells and senescent-like cells resulting from Braf-Mek inhibition remained unresponsive. Interestingly, a direct synergy between Bcl2/Bcl-XL inhibitors and Braf-Mek inhibitors was observed when used out of the context of senescence. Overall, we highlight diverse hallmarks of melanoma senescent states and provide evidence of context-dependent senotherapeutics that could reduce treatment resistance while also discussing the limitations of this strategy in human melanoma cells.

The Evaluation of Immune Checkpoint Inhibitors and BRAF/MEK Inhibitors in Different Therapy Lines for Metastatic Melanoma: A Retrospective Study.

Background: Nivolumab plus ipilimumab (nivo/ipi) combination therapy is highly effective in treating advanced melanoma, but serious immune-related adverse events (irAEs) are prevalent. The overall response rate (ORR) of the BRAF inhibitor plus MEK inhibitor (BRAFi/MEKi) combination therapy for BRAFV600-mutant advanced melanoma surpasses that of immune checkpoint inhibitors (ICIs). However, the OS and PFS of BRAFi/MEKi combination therapy are inferior to those of ICIs. Methods: We retrospectively evaluated 22 melanoma patients treated with nivo/ipi therapy and 13 patients treated with encorafenib plus binimetinib (enco/bini) between November 2018 and July 2023. Results: The ORR of nivo/ipi for metastatic melanoma patients was significantly higher in the first-line cohort [60.0% (95% CI: 31.2-83.3%)] than in the second-line or beyond cohort [8.3% (95% CI: 0-37.5%)], whereas the ORR of enco/bini was comparable between the first-line cohort [75.0% (95% CI: 28.9-96.6%)] and the second-line or beyond cohort [77.8% (95% CI: 44.3-94.7%)]. The median PFS of nivo/ipi significantly improved in the first-line cohort [7.7 months (95% CI: 2.0-11.9)] compared to the second-line or beyond cohort [2.3 months (95% CI: 0.5-6.0)] (p = 0.0109). In addition to efficacy, the incidence of grade 3 or greater AEs was comparable in the first-line and second-line or beyond cohorts. Conclusions: Although our present data are based on a small number of cases, they suggest that nivo/ipi should be administered as the first-line therapy for the treatment of BRAFV600-mutant metastatic melanoma, rather than enco/bini, aligning with findings from previous clinical trials.

miR-224-5p acts as a tumour suppressor and reverses the resistance to BRAF inhibitor in melanoma through directly targeting PAK4 to block the MAPK pathway.

miR-224-5p has been shown to play both an oncogene and tumour suppressor role in many human tumours. However, the role and molecular mechanism of miR-224-5p in cutaneous melanoma remains unclear. miR-224-5p levels were downregulated in melanoma tissue, and low miR-224-5p expression was an independent risk factor for melanoma patients. miR-224-5p blocked proliferation, epithelial-to-mesenchymal transition (EMT), invasion, migration in BRAF wild-type melanoma cell, and overcome acquired BRAFi resistance in VMF-resistant melanoma cells. miR-224-5p exerted its role by directly repressing PAK4 to block the downstream CRAF/MEK/ERK pathways. We demonstrated that miR-224-5p inhibited melanoma growth and metastasis in vivo though xenograft tumor and pulmonary metastasis assay. Thus, miR-224-5p/PAK4-mediated CRAF/MEK/ERK pathways have therapeutic potential in melanoma treatment.

Proteasome inhibition potentiates Kv1.3 potassium channel expression as therapeutic target in drug-sensitive and -resistant human melanoma cells.

Primary and acquired therapy resistance is a major problem in patients with BRAF-mutant melanomas being treated with BRAF and MEK inhibitors (BRAFI, MEKi). Therefore, development of alternative therapy regimes is still required. In this regard, new drug combinations targeting different pathways to induce apoptosis could offer promising alternative approaches. Here, we investigated the combination of proteasome and Kv1.3 potassium channel inhibition on chemo-resistant, BRAF inhibitor-resistant as well as sensitive human melanoma cells. Our experiments demonstrated that all analyzed melanoma cell lines were sensitive to proteasome inhibitor treatment at concentrations that are not toxic to primary human fibroblasts. To further reduce proteasome inhibitor-associated side effects, and to foster apoptosis, potassium channels, which are other targets to induce pro-apoptotic effects in cancer cells, were blocked. In support, combined exposure of melanoma cells to proteasome and Kv1.3 channel inhibitor resulted in synergistic effects and significantly reduced cell viability. On the molecular level, enhanced apoptosis correlated with an increase of intracellular Kv1.3 channels and pro-apoptotic proteins such as Noxa and Bak and a reduction of anti-apoptotic proteins. Thus, use of combined therapeutic strategies triggering different apoptotic pathways may efficiently prevent the outgrowth of drug-resistant and -sensitive BRAF-mutant melanoma cells. In addition, this could be the basis for an alternative approach to treat other tumors expressing mutated BRAF such as non-small-cell lung cancer.