BRAFV600-mutant Melanoma Research Articles

Abstract CT069: Randomized phase 3 study (STARBOARD) evaluating encorafenib (enco) + binimetinib (bini) + pembrolizumab (pembro) for first-line treatment of unresectable locally advanced or metastatic BRAF V600-mutant melanoma

Abstract Background BRAF V600 mutations are frequently found in metastatic melanoma. This mutation constitutively activates the MAPK pathway, which leads to melanoma progression. Patients with BRAF V600-mutant metastatic melanoma typically receive BRAF inhibitors (BRAFi) + MEK inhibitors (MEKi), such as enco + bini, or immune checkpoint inhibitors (CPIs; eg, pembro). However, these have limitations. BRAFi and MEKi may increase BRAF V600-mutant tumor sensitivity to CPIs. Previous studies reported improved progression-free survival (PFS) in patients with advanced BRAF V600-mutant melanoma receiving BRAFi + MEKi + CPI compared with targeted therapy alone. This phase 3 trial will evaluate the efficacy and safety of enco + bini + pembro vs placebo + pembro for unresectable locally advanced or metastatic BRAF V600-mutant melanoma. A safety lead-in (SLI) was built in to determine the recommended phase 3 dose (RP3D) prior to starting phase 3. Trial design STARBOARD (NCT04657991) is a randomized, double-blind, placebo-controlled, phase 3 study evaluating approximately 600 patients with BRAF V600 advanced melanoma. Patients will be stratified by prior systemic adjuvant treatment (CPI, BRAFi/MEKi, or none) and by disease stage (per AJCC 8th edition; IIIB, IIIC, IIID, IV M1a[0], and IV M1b[0] vs IV M1a[1], IV M1b[1], IV M1c[0], IV M1c[1], IV M1d[0], and IV M1d[1]). Patients must have measurable disease (per RECIST 1.1); ECOG performance status of 0 or 1; and adequate bone marrow, hepatic, and renal function. Patients must not have received prior systemic therapy for unresectable or metastatic melanoma; adjuvant treatment with BRAFi/MEKi, anti-PD-1, or anti-CTLA-4 is permitted. Patients cannot have symptomatic brain metastases. Study treatments and end points are shown in Table 1. RP3D was established in May 2022; phase 3 enrollment began in June 2022. Table 1. Study treatments and end points Phase 3 Treatment (21-day cycle) RP3D from SLI: enco + bini + pembro Control: placebo + pembro Primary PFS (RP3D vs control; by BICR) Secondary Key: OS (RP3D vs control) Other: PFS by investigator assessment, objective response rate, duration of response, disease control rate, and time to response, all by BICR and investigator assessment, PFS2, quality of life, safety, pharmacokinetics BICR, blinded independent central review; OS, overall survival. Citation Format: Dirk Schadendorf, Reinhard Dummer, Caroline Robert, Antoni Ribas, Ryan J. Sullivan, Timothy Panella, Meredith McKean, Edgardo S. Santos, Jill Clancy, Anna Polli, Alessandra di Pietro, Paolo A. Ascierto. Randomized phase 3 study (STARBOARD) evaluating encorafenib (enco) + binimetinib (bini) + pembrolizumab (pembro) for first-line treatment of unresectable locally advanced or metastatic BRAF V600-mutant melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT069.

Abstract LB251: Blocking genomic instability delays acquired resistance to MAPK inhibitor therapy in melanoma

Abstract Background: In cutaneous melanoma, the burden of chromothripsis is high prior to targeted therapy, and additional chromothripsis appears to be a key evolutionary mechanism by which cancer rapidly generates and accumulates highly dynamic structural variants (SVs). Blocking cancer genomic instability may prevent tumor escape from targeted therapies. Methods: We assembled three cohorts of tissues for WGS-based analysis of SVs. The first cohort consisted of patient-matched normal tissues, BRAF V600MUT melanoma tumors before MAPKi therapy and at disease progression (n=10 normal tissues; n=10 pretreatment tumors; n=17 acquired-resistant tumors; n=10 patients). The second cohort consisted of rapid autopsy melanoma tissues (n=3 normal tissues; n=12 acquired-resistant tumors; n=6 metastatic organ sites). The third cohort consisted of cutaneous PDX tumors. To study acquired MAPKi-resistance at the whole-genome level, we subjected PDXs (n=6 models; 1 BRAF MUT and 5 NRAS MUT models) to MAPKi therapy in NSG mice at doses sufficient to elicit tumor regression, and then generated acquired MAPKi-resistant tumors. In total, we used vehicle-treated tumors (n=6), acquired-resistant tumors (n=12), and patient-matched normal tissues (n=6) to generate WGS data. Results: Analysis of genomic amplicons due to intrachromosomal complex genomic rearrangements (CGRs) and extrachromosomal circular DNAs (ecDNAs) uncovered a significant (unpaired Student’s t-test, p=0.0002) association between acquired-resistant tumors and CGRs and/or ecDNAs harboring bona fide MAPKi-resistance genes and revealed copy number amplification of BRAF (range 4.5-27), NRAS (range 5-13), HRAS (range 13-16), MYC (range 12-15) and EGFR (CN 4.6-5), known to drive acquired MAPKi-resistance. Moreover, we validated a recurrent ecDNA by direct isolation and high-depth sequencing using a new approach referred to as CRISPR-CATCH. This alternative technique confirmed the circularized junctions of a 890 kb, driver ecDNA within this acquired-resistant clinical tumor sample. Additionally, resistance-specific (versus sensitivity-specific) chromothriptic single-base substitutions (SBSs) enriched for signatures of defects in base excision repair (BER) and in DNA mismatch repair (MMR) (Wilcoxon rank sum test, p=0.04 and p=0.005 respectively) in 14 of 31 resistant tumors (10 of 16 patients). Moreover, breakpoint-sequence analysis inferred non-homologous end-joining (NHEJ) as critical, and homologous recombination repair (HRR) as adjunctive, to DNA double-stranded break repair underlying CGR and ecDNA formation harboring MAPK-reactivation or MAPKi resistance-driver genes. Inhibition of DNA-PKc or PARP1/2, even only initially during MAPKi treatment, suppressed acquired MAPKi-resistance in melanoma cell lines (BRAF V600MUT n=3 and NRAS Q61MUT n=3) and blunted the expansion of ecDNA + CGR genomic spans. In vivo, DNA-PKi in combination with MAPKi forestalled resistance, reduced ecDNA and CGRs size, and suppressed the contribution of NHEJ in 5 out of 5 cutaneous melanoma PDXs analyzed. Conclusions: Our findings advance the concepts that preventing—instead of reversing—acquired resistant phenotypes may be more clinically impactful and that targeting DNA-PKCS and NHEJ lies at the center of this approach in stabilizing cancer genomes during oncogene-targeted therapies. Citation Format: Prashanthi Dharanipragada, Xiao Zhang, Sixue Liu, Shirley H. Lomeli, Aayoung Hong, Yan Wang, Zhentao Yang, Agustin Vega-Crespo, Antoni Ribas, Stergios J. Moschos, Gatien Moriceau, Roger S. Lo. Blocking genomic instability delays acquired resistance to MAPK inhibitor therapy in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB251.

Abstract 431: FOXD1 promotes dedifferentiation and targeted therapy resistance in melanoma by regulating the expression of connective tissue growth factor

Abstract Metastatic melanoma is one of the most aggressive skin cancers and is associated with poor prognosis. BRAF and MEK inhibitors are used to treat patients with BRAFV600E-mutated advanced melanoma. However, the development of resistances to these treatments compromises therapeutic success. Our lab previously demonstrated that forkhead box D1 (FOXD1) plays a critical role in melanoma migration and invasion. Here, we found that FOXD1 was highly expressed in melanoma cells. Immunohistochemical assessment of 105 samples from patients with metastatic melanoma revealed that high FOXD1 expression in tumors was associated with poor survival and correlated with low MITF, SOX10 and high AXL expression. Upregulation of FOXD1 expression enhanced the resistance of melanoma to vemurafenib (BRAF inhibitor) or combinatorial treatment with vemurafenib and cobimetinib (MEK inhibitor). On the other hand, loss of FOXD1 increased the sensitivity of naïve melanoma cells towards vemurafenib or combinatorial treatment with vemurafenib and cobimetinib. Furthermore, high FOXD1 expression levels were found in BRAF inhibitor (BRAFi)-resistant cells. Downregulation of FOXD1 resulted in a resensitization of BRAFi-resistant cells to vemurafenib. By using microarray analysis, connective tissue growth factor (CTGF) was found to be one of the most downregulated genes in FOXD1 knockdown (KD) cells while its expression was highly increased upon FOXD1 overexpression. Thus, CTGF was identified as a downstream factor of FOXD1. In addition, in vitro expression analysis and evaluation of clinical samples demonstrated that CTGF and FOXD1 expression were positively correlated. By using a CHIP assay and a dual reporter luciferase assay, we discovered that FOXD1 could regulate the expression of CTGF by directly binding to the CTGF promoter. This result was confirmed with RT-PCR and western blot. In addition, we found that the protein level of CTGF was highly increased in BRAFi-resistant cells. Similar to FOXD1 knockdown, the knockdown of CTGF resensitized BRAFi-resistant cells to vemurafenib. FOXD1 KD cells treated with recombinant CTGF protein were less sensitive towards vemurafenib compared to untreated FOXD1 KD cells. Based on these findings, we conclude that the transcription factor FOXD1 could promote dedifferentiation and targeted therapy-resistance in melanoma cells by regulating the expression of CTGF. FOXD1 might be a promising new diagnostical marker and a therapeutic target of targeted therapy resistant melanoma. Citation Format: Qian Sun, Daniel Novak, Laura Hüser, Juliane Poelchen, Huizi Wu, Karol Granados, Aniello Federico Federico, Ke Liu, Tamara Steinfass, Marlene Vierthaler, Viktor Umansky, Jochen Utikal. FOXD1 promotes dedifferentiation and targeted therapy resistance in melanoma by regulating the expression of connective tissue growth factor [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 431.

Abstract 6639: Preclinical characterization and clinical biomarker studies with RP3, a novel oncolytic immunotherapy expressing a fusogenic protein, a human anti-CTLA-4 antibody, hCD40L and h4-1BBL

Abstract Introduction: RP3, a tumor-directed oncolytic immunotherapy (TDOI), is a selectively replication-competent HSV-1 that contains the codon-optimized sequence for the fusogenic GALV-GP-R- protein, an anti-CTLA-4 antibody-like molecule, CD40L, and 4-1BBL. The expression of GALV-GP-R- potentiates immunogenic tumor cell death through the induction of cell-to-cell fusion. CD40L enhances dendritic cell and macrophage activation and 4-1BBL promotes CD8+ T-cell activation and co-stimulation. The anti-CTLA-4 antibody-like molecule blocks the interaction of CTLA-4 with its ligands B7.1 and B7.2, further enhancing T cell activation. RP3 is currently being assessed in a Phase 1 clinical trial with or without nivolumab in patients with advanced solid tumors (NCT04735978). Methods: CD1 nude mice with A375 melanoma xenograft tumors (Group 1) were treated with a single intratumoral injection of 1 × 107 PFU/mL of RP3. Tumors and blood samples were collected 3 days later, and immunohistochemistry (IHC) was performed to detect CD40L, 4-1BBL and HSV-1. For anti-CTLA-4 detection, two doses of 107 PFU/mL of RP3 were injected intratumorally into C57BL/6 mice bearing 4434 murine BRAFV600E mutant melanoma tumors (Group 2). Tumors and blood samples were collected at 3, 6, 9, 12 and 15-days post-dosing and anti-CTLA-4 ELISA was performed. For clinical biomarker studies, tumor biopsies were collected pre- and post-RP3 treatment. CD8 and PD-L1 were characterized by IHC, and gene expression analysis (GEA) was performed. TCR analysis was also performed using PBMCs. Results: IHC analysis from Group 1 confirmed abundant and widespread expression of CD40-L and 4-1BBL, which were detected alongside with HSV-1 antigen. In the blood samples collected from Group 2, robust levels of anti-CTLA-4 antibody were detected, which stayed in detectable range up to 15 days after the 2nd RP3 injection. IHC analysis of patient tumor samples demonstrated a marked increase in CD8 T cells and PD-L1 expression. GEA of patient tumor biopsies demonstrated increased levels of genes associated with innate and adaptive immune activation and genes previously reported to be associated with responsiveness to anti-PD1 therapy. TCR sequencing of PBMCs and multiplex IHC analysis are ongoing and updated results will be presented. Conclusion: Preclinical experiments from mice models confirmed the robust expression of CD40-L, 4-1BBL and anti-CTLA-4 in RP3-injected tumors. Biomarker studies from patients demonstrate increases in CD8-T cell infiltration and PD-L1 expression post RP3 +/- nivolumab treatment, including increases in the expression of genes associated with response to anti-PD-1 therapy. These data indicate the potential for broad utility of RP3 in a range of tumor types, including in tumors that are not sensitive to PD-1 blockade. Citation Format: Praveen K. Bommareddy, Kevin Harrington, Victoria Roulstone, Joan Kyula, Aaron Clack, Linta Kuncheria, Sylwia Jones, Robert Coffin. Preclinical characterization and clinical biomarker studies with RP3, a novel oncolytic immunotherapy expressing a fusogenic protein, a human anti-CTLA-4 antibody, hCD40L and h4-1BBL. [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 6639.

Abstract 4876: A systems pharmacology approach to discover synergistic targeted therapy combinations

Abstract The discovery of oncogenic signal transduction in cancer has enabled the development and clinical use of dozens of kinase-directed targeted therapies, but patient response is often transient and resistance invariably develops. In the setting of BRAF-mutant melanoma, for instance, a majority of patients relapse within one year of initiating targeted therapy. Combination therapies blocking compensatory signaling pathways provide a strategy for boosting treatment efficacy and delaying resistance. To find synergistic kinase inhibitor combinations, we used a multimodal systems pharmacology approach to quantify the early adaptive signaling and gene regulatory dynamics in patient-derived BRAFV600E-mutant melanoma cells under oncogene inhibition. Cells were treated with vemurafenib or vehicle and sampled longitudinally at timepoints spanning minutes to days. To measure signaling dynamics, thousands of protein phosphorylation sites covering commonly altered signaling axes in cancer were motif-enriched by immunoprecipitation, barcoded with isobaric tandem mass tags (TMT), and quantified by high-resolution mass spectrometry (MS). The resulting time-resolved phosphoproteomics data showed rapid (<15 minutes) and potent (>8-fold) downregulation of ERK1/2, confirming strong BRAF inhibition. Statistical integration of signaling measurements with time-series RNA-seq data collected at the same timepoints enabled reconstruction of multi-scale regulatory networks governing the adaptive response to oncogene inhibition. In particular, we observed early induction of SRC-family kinase (SFK) signaling and a broad cytoskeletal signaling module, implicating a compensatory prosurvival signaling program. The induction intensified over the 3-day treatment window and was fully reversed following a 6-day drug holiday, suggesting a reversible nature of drug adaptation. Immunoblotting confirmed sustained loss of ERK1/2 activity and concomitant elevated SFK phosphorylation following drug treatment. Accumulation of reactive oxygen species, a known activator of SFKs, strongly correlated with SFK activity, and both were ablated following treatment with the antioxidant precursor N-acetylcysteine. This adaptive response predicted a significant degree of synergy between vemurafenib and the pan-SFK inhibitor dasatinib, which was validated in a panel of patient-derived melanoma cell lines and in melanoma xenograft mouse models. Targeted MS of phospho-ERK1/2 and canonical SFK substrates, including CTTN, PXN, and PAG1, validated the mechanism of action of this combination. Complementary data from patient-derived non small-cell lung cancer (NSCLC) cells under tyrosine kinase inhibitor treatment demonstrates the generality of our integrative approach, and supports the notion that SFK activation may be a hallmark response to oncogenic RTK-RAS-ERK pathway inhibition. Citation Format: Cameron T. Flower, Chunmei Liu, James R. Heath, Wei Wei, Forest M. White. A systems pharmacology approach to discover synergistic targeted therapy combinations. [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 4876.

Abstract 396: Role of PHF8 in resistance to melanoma targeted therapies

Abstract Melanoma is a highly prevalent cancer with increasing incidence worldwide. While immune checkpoint inhibitors have drastically improved outcomes for about half of metastatic melanoma patients, many are still resistant to these treatments or develop life-threatening toxicities. Around 50% of melanomas carry BRAF mutations that result in constitutive activation of the MAPK pathway. Therapeutic combinations of BRAF and MEK inhibitors (MAPKi) have proven very efficient against these tumors, but responses are not durable and treatment-resistant recurrence almost inevitably occurs. Our group recently uncovered a role for the histone demethylase PHF8 in melanoma metastasis, and demonstrated that PHF8 directly controls the expression of key components of the TGFβ pathway. Given that TGFβ signaling crosstalks with the MAPK pathway and contributes to resistance to therapy in various tumors, we decided to investigate a potential role for PHF8 in response and resistance to MAPKi therapy in melanoma. To test our hypothesis, we assessed the effect of modulating PHF8 expression in BRAFV600E-mutant cells and their matched MAPKi-resistant derivatives. PHF8 silencing did not affect cell survival or proliferation of parental, MAPKi-sensitive melanoma cell lines, but it re-sensitized resistant cells to MAPKi treatment. Conversely, PHF8 overexpression in melanoma cell lines did not affect their proliferation rate but increased the emergence of resistant clones in BRAFV600E-mutant melanoma cells treated with BRAFi (Dabrafenib) and MEKi (Trametinib) combinations. Next, we assessed the dynamics of PHF8 expression and TGFβ signaling in melanoma cells exposed to MAPKi. Treatment of MAPKi-sensitive cells leads to a fast and complete downregulation of PHF8 protein expression without a change in mRNA levels, paralleled by reduced TGFβ signaling, as evidenced by decreased SMAD2 phosphorylation. However, PHF8 protein expression is restored in MAPKi resistant clones. Therefore, based on our preliminary data, we postulate that PHF8 suppression by the MAPK pathway plays a critical role in melanoma response to targeted therapy and that its restoration contributes to acquisition of resistance. We are currently investigating MAPK-dependent mechanisms controlling PHF8 stability, and testing novel PHF8 inhibitors for their ability to bypass or overcome melanoma resistance to MAPKi. Our studies might provide basis for a novel epigenetic therapy as a strategy to maximize the potential of MAPKi therapy in melanoma treatment. Citation Format: María de los Ángeles Gómez-Muñoz, Nicole Eskow, Ines Delclaux, Rana Moubarak, Eva Hernando-Monge. Role of PHF8 in resistance to melanoma targeted therapies [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 396.

Deep and lasting response and acquired resistance to BRAFV600E targeting in a low-grade ovarian cancer patient

ABSTRACT The treatment of BRAFV600E mutant melanoma has been revolutionized by BRAF inhibitors. Furthermore, the BRAF/MEK combination has shown further improvement in clinical outcomes in advanced and in adjuvant melanoma patients. In low-grade ovarian tumors, BRAF inhibitor use has been also proposed. Here we present a patient with an excellent, lasting response to BRAF therapy alone. At first progression, after more than two years on BRAF monotherapy, we could not identify any molecular mechanisms explaining resistance. After a switch to dual BRAF/MEK therapy, the patient responded. However, despite the initial response clinical the patient again progressed, this time with the appearance of a KRAS G12C mutation, which could not be overcome by BRAF/MEK therapy. We provide evidence that BRAF inhibitor alone can be highly beneficial in BRAF mutant low-grade ovarian tumors and the resistance mechanisms are similar to that of other BRAF mutant tumors, including in melanoma.

4-year survival and biomarkers evaluation from a phase II study of sequential immunotherapy and targeted therapy for metastatic BRAF V600-mutated melanoma (SECOMBIT)

4-year survival and biomarkers evaluation from a phase II study of sequential immunotherapy and targeted therapy for metastatic BRAF V600-mutated melanoma (SECOMBIT)

4-year survival and biomarkers evaluation from a phase II study of sequential immunotherapy and targeted therapy for metastatic BRAF V600-mutated melanoma (SECOMBIT)

4-year survival and biomarkers evaluation from a phase II study of sequential immunotherapy and targeted therapy for metastatic BRAF V600-mutated melanoma (SECOMBIT)

Erianin suppresses constitutive activation of MAPK signaling pathway by inhibition of CRAF and MEK1/2

Constitutive activation of RAS-RAF-MEK-ERK signaling pathway (MAPK pathway) frequently occurs in many cancers harboring RAS or RAF oncogenic mutations. Because of the paradoxical activation induced by a single use of BRAF or MEK inhibitors, dual-target RAF and MEK treatment is thought to be a promising strategy. In this work, we evaluated erianin is a novel inhibitor of CRAF and MEK1/2 kinases, thus suppressing constitutive activation of the MAPK signaling pathway induced by BRAF V600E or RAS mutations. KinaseProfiler enzyme profiling, surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), cellular thermal shift assay, computational docking, and molecular dynamics simulations were utilized to screen and identify erianin binding to CRAF and MEK1/2. Kinase assay, luminescent ADP detection assay, and enzyme kinetics assay were investigated to identify the efficiency of erianin in CRAF and MEK1/2 kinase activity. Notably, erianin suppressed BRAF V600E or RAS mutant melanoma and colorectal cancer cell by inhibiting MEK1/2 and CRAF but not BRAF kinase activity. Moreover, erianin attenuated melanoma and colorectal cancer in vivo. Overall, we provide a promising leading compound for BRAF V600E or RAS mutant melanoma and colorectal cancer through dual targeting of CRAF and MEK1/2.

Safety and rapid response of dabrafenib and trametinib therapy during hyperbilirubinemia in metastatic melanoma.

This case report describes the occurrence of hyperbilirubinemia as a complication of metastatic melanoma. A 72-year-old male patient was diagnosed with BRAF V600E-mutated melanoma with metastases in the liver, lymph nodes, lungs, pancreas, and stomach. Due to a lack of clinical data and specific guidelines for the treatment of mutated metastatic melanoma patients with hyperbilirubinemia, a conference of specialists debated between initiating treatment or providing supportive care. Ultimately, the patient was started on the combination therapy of dabrafenib and trametinib. This treatment resulted in a significant therapeutic response via normalization of bilirubin levels and an impressive radiological response of metastases just one month post-treatment initiation.

TRIDENTE trial: A phase II study of rechallenge with encorafenib, binimetinib, and cetuximab in patients with RAS wild-type/BRAF V600E–mutant metastatic colorectal cancer.

TPS264 Background: The BEACON CRC trial demonstrated survival benefit of combination therapy with encorafenib (ENCO) + cetuximab (CET) +/- binimetinib (BINI) in patients with RAS wild-type (WT)/ BRAF V600E mutant metastatic colorectal cancer (mCRC). However, prognosis of those patients still be poor after the refractoriness to the BEACON combination therapy. One of resistant mechanisms to BRAF inhibitor has been reported as MAPK alterations including RAS mutation, similar to those for anti-EGFR antibody in patients with RAS/ BRAF V600E WT mCRC. Promising results of rechallenge therapy with anti-EGFR antibody in patients with RAS/ BRAF V600E WT mCRC and with combination of BRAF inhibitor + MEK inhibitor in patients with BRAF V600 mutant melanoma suggest the treatment strategy of rechallenge therapy with the BEACON triplet therapy in patients with RAS WT/ BRAF V600E mutant mCRC. Methods: TRIDENTE trial is a multicenter phase II trial to assess efficacy and safety of rechallenge therapy with ENCO + BINI + CET in patients with RAS WT/ BRAF V600E mutant mCRC after the refractoriness to either the BEACON doublet or triplet therapy. Key eligibility criteria includes RAS WT/ BRAF V600E mutant mCRC; ≥ 20 years old; ECOG PS 0-1; refractory or intolerant to at least one fluoropyrimidine-based regimen (including irinotecan or oxaliplatin); refractory or intolerant to anti-PD-1 antibody if patients with MSI-high; history of previous combination therapy containing ENCO + CET with at least partial response by RECIST v1.1; confirmed disease progression within 4 weeks after last administration of previous ENCO; ≥ 4 months of period between the last administration of previous ENCO and the start of study treatment. Enrolled patients receive the combination therapy with ENCO (300 mg, QD), BINI (45 mg, BID), and CET (initially 400 mg/m2, and subsequently 250 mg/m2, QW) as the study treatment. Primary endpoint is the objective response rate (ORR) by investigators’ assessment in patients receiving at least one dose of study treatment. A target sample size is calculated to be 21 on the hypothesis that the threshold ORR is 3% and expected ORR is 20%, with a significant level of 5% (one-sided) and power of 80%. Secondary endpoint includes progression-free and overall survivals, disease control rate, and safety. Exploratory molecular analysis is performed using targeted next-generation sequencing in circulating-tumor DNA at the timepoints of baseline and discontinuation of study treatment. As of September 19, 2022, 8 patients have been enrolled. Clinical trial information: jRCTs031210511 .

Survival Mechanisms of Metastatic Melanoma Cells: The Link between Glucocorticoids and the Nrf2-Dependent Antioxidant Defense System.

Circulating glucocorticoids increase during stress. Chronic stress, characterized by a sustained increase in serum levels of cortisol, has been associated in different cases with an increased risk of cancer and a worse prognosis. Glucocorticoids can promote gluconeogenesis, mobilization of amino acids, fat breakdown, and impair the body's immune response. Therefore, conditions that may favor cancer growth and the acquisition of radio- and chemo-resistance. We found that glucocorticoid receptor knockdown diminishes the antioxidant protection of murine B16-F10 (highly metastatic) melanoma cells, thus leading to a drastic decrease in their survival during interaction with the vascular endothelium. The BRAFV600E mutation is the most commonly observed in melanoma patients. Recent studies revealed that VMF/PLX40-32 (vemurafenib, a selective inhibitor of mutant BRAFV600E) increases mitochondrial respiration and reactive oxygen species (ROS) production in BRAFV600E human melanoma cell lines. Early-stage cancer cells lacking Nrf2 generate high ROS levels and exhibit a senescence-like growth arrest. Thus, it is likely that a glucocorticoid receptor antagonist (RU486) could increase the efficacy of BRAF-related therapy in BRAFV600E-mutated melanoma. In fact, during early progression of skin melanoma metastases, RU486 and VMF induced metastases regression. However, treatment at an advanced stage of growth found resistance to RU486 and VMF. This resistance was mechanistically linked to overexpression of proteins of the Bcl-2 family (Bcl-xL and Mcl-1 in different human models). Moreover, melanoma resistance was decreased if AKT and NF-κB signaling pathways were blocked. These findings highlight mechanisms by which metastatic melanoma cells adapt to survive and could help in the development of most effective therapeutic strategies.

An Immunogenic Cell Death-Related Gene Signature Reflects Immune Landscape and Predicts Prognosis in Melanoma Independently of BRAF V600E Status.

Immunogenic cell death (ICD) is a type of regulated cell death that can activate adaptive immune response, and its ability to reshape the tumor microenvironment via multiple mechanisms may contribute to immunotherapy. The treatment options for patients with skin cutaneous melanoma (SKCM) vary based on BRAF V600E statuses. However, all standard treatments include immunotherapy. Therefore, it is critical to identify ICD-associated signatures that can help classify patients according to benefits from ICD immunotherapy. In this study, data on melanoma samples with BRAF V600E mutation (BRAF V600E-mutant melanoma) and melanoma samples with wild-type BRAF V600E alleles (BRAF V600E WT melanoma) were collected from The Cancer Genome Atlas (TCGA) database. The ICD-related (ICD-high and ICD-low) subgroups of patients with BRAF V600E WT melanoma were established via consensus clustering. The analyses of survival, differentially expressed genes (DEGs), functional annotation, and immune landscape were performed in these two subgroups. Results showed that ICD-high subgroup was correlated with a positive overall survival (OS) and active tumor immune landscape. A model comprising seven prognosis ICD-related gene biomarkers was developed. Survival analysis and receiver operating characteristic (ROC) curve evaluation in both cohorts with BRAF V600E WT and BRAF V600E-mutant melanoma showed an accurate prognostic estimation of ICD-related risk signature. There was a correlation between immune cell infiltration and immunotherapy response and risk score. Thus, the ICD risk signature was closely associated with the tumor's immune microenvironment. Our results may provide insights to further individualize and improve precision therapeutic decision-making in BRAF V600E-mutant and WT melanoma.

Triple Combination of Immune Checkpoint Inhibitors and BRAF/MEK Inhibitors in BRAFV600 Melanoma: Current Status and Future Perspectives.

Immune checkpoint inhibitors (ICIs), namely programmed cell death 1 (PD-1) or cytotoxic t-lymphocyte antigen 4 (CTLA-4) inhibitors, are currently the standard of care for the treatment of advanced melanoma, with robust and durable responses in a subset of patients. For BRAFV600-mutant melanoma, treatment with BRAF and MEK inhibitors has resulted in high objective response rates, but most responses are short-lived. Preclinical data suggest that BRAF and MEK inhibitors result in immunomodulatory changes in the tumor microenvironment; early data in murine models further suggest that these changes could enhance sensitivity to ICIs. Subsequently, the notion of combining the two therapy modalities for a more effective response was further evolved in early phase clinical trials. In this review, we analyzed the results of recent phase 2 and 3 clinical trials investigating the combination of ICIs with targeted therapy in BRAFV600-mutated advanced melanoma. Furthermore, we evaluated the results of recent studies investigating the first-line treatment sequencing of ipilimumab/nivolumab and BRAF/MEK inhibitors in these patients. We discussed the study limitations and interpreted how these recent advances could be incorporated into the treatment landscape of advanced BRAFV600-mutant melanoma.

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.

464TiP Phase III study of adjuvant encorafenib plus binimetinib vs placebo in fully resected stage IIB/C BRAFV600-mutated melanoma: COLUMBUS-AD study design

Melanoma treatment has radically changed during the past decade. This is due to the discovery that BRAFV600-mutant directed targeted therapy, next to immune checkpoint blockade. In the Columbus study, encorafenib in combination with binimetinib has shown safety and efficacy in BRAF V600E/K mutant advanced / metastatic stage IV melanoma. Standard of care for stage II melanoma was surveillance follow-up after full resection. Considering that stage IIB/IIC melanoma has an equal or higher risk of recurrence and mortality compared to stage IIIA/B, there is an unmet medical need for stage IIB/C melanoma.

SERPINB8 and furin regulate ITGAX expression and affect the proliferation and invasion of melanoma cells.

In the past 10 years, the systemic treatment of advanced melanoma has undergone tremendous changes through the development of targeted therapy. However, there is still a long way to go. This study aims to characterize the function and interaction of ITGAX, SERPINB8 and furin in BRAF V600E mutant melanoma. Differentially expressed genes related to BRAF V600E mutation and BRAFi treatment were obtained by analysing GSE141484 and GSE22838. two kinds of BRAFi (Vemurafenib, 10μM; Dabrafenib, 1μM) were used to treat A375 and 1205Lu cells, respectively. The expression of ITGAX, SERPINB8 and Furin in A375 and 1205Lu cells was down-regulated by specific siRNAs, and cell proliferation, clone formation and invasion were detected by CCK-8, colony formation and transwell assays. The physical binding of furin and SERPINB8 was detected by immunoprecipitation. BRAFi treatment down-regulated the ITGAX and SERPINB8 expression and did not change furin expression. Knockdown of ITGAX and SERPINB8 both inhibited the proliferation and invasion of A375 and 1205Lu cells. Knocking down SERPINB8 down-regulated the expression of ITGAX. Furin knockdown and inhibitors all up-regulated the protein level of ITGAX. SERPINB8 can physically bind to furin. In summary, SERPINB8 and furin regulate the expression of ITGAX in melanoma cells, and ITGAX significantly promotes the proliferation and invasion of melanoma cells.

4 Oral - Improved outcomes in women with BRAF-mutant melanoma treated with BRAF/MEK-targeted therapy across randomized clinical trials. A systematic review and meta-analysis

Background: Preclinical and clinical evidence suggests that patients’ sex could be associated with the outcome of patients with advanced BRAF V600-mutant melanoma, treated with the combination of B-RAF and MEK-inhibitors. We performed a systematic review and meta-analysis of all RCTs testing the combination of BRAF and MEK inhibitors, to assess the interaction between treatment effect and patients’ sex.

An open label, non-randomised, phase IIIb study of trametinib in combination with dabrafenib in patients with unresectable (stage III) or distant metastatic (stage IV) BRAF V600-mutant melanoma: A subgroup analysis of patients with brain metastases

BackgroundDespite the poor prognosis associated with melanoma brain metastases (BM), data concerning these patients and their inclusion in clinical trials remains scarce. We report here the efficacy results of a subgroup analysis in patients with BRAFV600-mutant melanoma and BM treated with BRAF and MEK inhibitors dabrafenib (D) and trametinib (T). Patients and methodsThis phase IIIb single-arm, open-label, multicenter, French study included patients with unresectable stage IIIc or IV BRAFV600-mutant melanoma with or without BM. The present analysis focuses on patients with BM. Response rates were determined clinically and/or radiologically as per standard clinical practice. Progression-free survival (PFS) was estimated using the Kaplan Meier analysis and modelled with multivariate Cox regression model. Risk subgroups were identified using an exponential regression tree analysis. Significance was set at p < 0.05. ResultsBetween March 2015 and November 2016, 856 patients were included and 275 (32%) patients had BM. Median PFS was 5.68 months (95% confidence interval [CI], 5.29–6.87). Significant independent factors associated with shorter PFS were ECOG ≥1, elevated serum lactate dehydrogenase (LDH), ≥3 metastatic sites, and non-naïve status. The binary-split classification and regression tree modelling identified baseline LDH and ECOG status as major prognostic factors. ConclusionThis is to date the largest, close to real-world, study in advanced BRAFV600-mutant melanoma patients with BM treated with D+T. ECOG >1, ≥3 metastatic sites and elevated LDH were associated with shorter PFS, a finding previously demonstrated only in patients without BM. Further studies are warranted to determine the optimal treatment sequence in this population.