BRAF-mutated Melanoma Patients Research Articles

Адъювантная таргетная терапия и анти-PD-1-иммунотерапия у пациентов с меланомой кожи III стадии с мутацией в гене BRAF. Данные реальной клинической практики

Targeted therapy and anti-PD1 immunotherapy are standard of adjuvant stage III (or equivalent stage III) melanoma treatment. Both treatments significantly improve relapse-free survival compared with placebo and ipilimumab. The impact of adjuvant immunotherapy on overall survival remains unclear. Targeted therapy improved 3-year overall survival, but data didn’t reach statistical significance. There is still a lack of direct comparison in randomized trials between targeted therapy and immunotherapy. Data limited to a few retrospective studies. We conducted a single-center observational retrospective study comparing two adjuvant treatment options in BRAF-mutated high-risk melanoma patients who were treated or consulted at the N.N. Blokhin National Medical Research Center of Oncology.

Efficiency and tolerance of second-line triple BRAF inhibitor/MEK inhibitor/anti-PD1 combined therapy in BRAF mutated melanoma patients with central nervous system metastases occurring during first-line combined targeted therapy: a real-life survey.

Although current systemic therapies significantly improved the outcome of advanced melanoma, the prognosis of patient with central nervous system (CNS) metastases remains poor especially when clinically symptomatic. We aimed to investigate the efficiency of CNS targets and tolerance of second-line combined anti-PD1/dual-targeted anti-BRAF/anti-MEK therapy implemented in patients with CNS progression after initially efficient first-line combined targeted therapy in patients with BRAF-mutated melanoma in a real-life setting. A monocentric retrospective analysis including all such patients treated from January 2017 to January 2022 was conducted in our tertiary referral center. The response of CNS lesions to second-line triple therapy was assessed through monthly clinical and at least quarterly morphological (according to RECIST criteria) evaluation. Tolerance data were also collected. Seventeen patients were included with a mean follow-up of 2.59 (±2.43) months. Only 1 patient displayed a significant clinical and morphological response. No statistically significant difference was observed between patients receiving or not additional local therapy (mainly radiotherapy) as to response achievement. Immunotherapy was permanently discontinued in 1 patient owing to grade 4 toxicity. Mean PFS and OS after CNS progression were 2.59 and 4.12 months, respectively. In this real-life survey, the subsequent addition of anti-PD1 to combined targeted therapy in melanoma patients with upfront CNS metastases did not result in significant response of CNS targets in most BRAF mutated melanoma patients with secondary CNS progression after initially successful first-line combined targeted therapy.

Abstract 4648: Metabolic vulnerabilities of drug-resistant melanoma with therapeutic potential

Abstract Melanoma has been at the center of revolutions in both targeted BRAF therapy and immune therapy. However, the resistance to current therapeutic drugs has been a formidable obstacle to continuing benefit to patients. Overcoming resistance and new therapeutic agents and strategies are the primary goals of current cancer research. We currently found that metabolic pathways altered significantly in BRAFi-resistant melanoma. We hypothesized that BRAFi-resistant melanoma rewires its metabolic routes to reach its needs in energy and biosynthetic precursors, both essential for escaping the BRAFi attack. We found that five amino acids transporters and metabolic enzymes (ASNS, slc7a11, slc7a5, slc3a2, slc4a7) were the significant increase in resistant melanoma. Knockdown by shRNA or knockout by crispr/cas9 of these amino acids transporters or/and metabolic enzymes enhanced the sensitivity of BRAFi resistant melanoma to BRAFi. In contrast, overexpression of these amino acid transporters or/and metabolic enzymes promoted the ability of resistance of BRAFi in parent melanoma cells. Moreover, the inhibition of these amino acid transporters and metabolic enzymes by small molecular inhibitors significantly attenuated the resistance of the BRAFi-resistant melanoma whereas less affected the parent melanoma. Notably, we confirmed that blocking these amino acid transporters and metabolic enzymes by shRNA and/or small molecular inhibitors could rescue the sensitivity of resistant melanoma cells and inhibit resistant-melanoma cell growth in the preclinical mouse model in vivo. Importantly, we found that overexpression of slc7a11, slc7a5 and slc3a2 correlated with worse survival in melanoma in SK-TCGA data. Our findings uncovered the mechanism of how metabolic reprogramming fueled melanoma cell growth and proliferation for escaping drug attacks and offered a rational strategy to guide clinical treatment. The identified novel molecular proteins represented a promising therapeutic target for BRAF mutant melanoma patients with resistance to current therapy. Citation Format: Yanlin Yu, Lei Huang, Weiping Chen, Glenn Merlino. Metabolic vulnerabilities of drug-resistant melanoma with therapeutic potential [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4648.

Abstract 1997: The small GTPase ARF6 augments BRAF and PI3K expression to promote melanoma survival

Abstract RAF and MEK inhibitors greatly benefit BRAF-mutant melanoma patients, but eventually, nearly 80% of patients relapse. Reactivation of MAPK signaling and activation of PI3K/AKT signaling play a major role in the development of targeted therapy resistance. Therefore, understanding how tumor cells regulate these signaling pathways may yield new insights into drug resistance. The small GTPase ARF6, a member of the RAS superfamily, influences oncogenic signaling by controlling cytoskeletal rearrangements and through endomembrane trafficking. We previously reported that ARF6 is aberrantly activated in metastatic melanoma and that activation of ARF6 is sufficient to promote invasion and accelerate spontaneous metastasis in vivo through WNT5A/beta-catenin and PI3K/AKT signaling. In contrast to ARF6 activation, herein we show that tumor-specific deletion of Arf6 significantly limits development and progression of BRAFV600E melanoma in vivo. Mechanistically, we discovered these ARF6-dependent phenotypes may be attributed to a previously unknown role for ARF6 in augmenting BRAFV600E and PI3K protein levels. Specifically, pharmacologic or genetic activation ARF6 (ARF6-GTP) is sufficient to increase BRAFV600E and PI3K protein levels in both murine and human melanoma. As a result, ARF6-GTP is also sufficient to enhance activation of MEK, ERK and AKT. Importantly, murine tumors expressing constitutively active ARF6 (ARF6Q67L) show increased expression of anti-apoptotic proteins compared to ARF6WT tumors. Together these data suggest that ARF6 promotes tumor survival through augmentation of the BRAF/MAPK and PI3K/AKT pathways. To test this, we pharmacologically activated ARF6 during serum withdrawal or vemurafenib treatment. In both conditions, ARF6 activation is sufficient to prevent apoptosis. Surprisingly, we found that vemurafenib treatment leads to ARF6 activation, suggesting that ARF6 may be an important component of the cellular machinery that reactivates MAPK signaling after RAF inhibition. Indeed, pharmacologic activation of ARF6 maintains MAPK signaling (pERK levels) in the presence of vemurafenib and enhances pERK and pAKT-mediated suppression of apoptotic signaling. In summary, our data suggest that in addition to well-established roles in tumor cell invasion, ARF6 is a crucial mediator of survival during tumor progression and in the context of BRAF targeted therapy. More work is needed to understand how ARF6 activation increases BRAF and PI3K expression and if ARF6 activation contributes to the development of persister cells in melanoma. Citation Format: Junhua Wang, Coulson Rich, Aaron Rogers, Sheri Holmen, Roger Wolff, Allie Grossmann. The small GTPase ARF6 augments BRAF and PI3K expression to promote melanoma survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1997.

Comparison of Immunotherapy versus Targeted Therapy Effectiveness in BRAF-Mutant Melanoma Patients and Use of cGAS Expression and Aneuploidy as Potential Prognostic Biomarkers.

BRAF-mutant melanoma patients can be treated with targeted therapy or immunotherapies, and it is not clear which should be provided first. Targeted treatments do not work in up to one-third of cases, while immunotherapies may only be effective in up to 60% and come with a high risk of immune-related side effects. Determining which treatment to provide first is thus of critical importance. Recent studies suggest that chromosomal instability and aneuploidy and cyclic GMP-AMP synthase (cGAS) can act as biomarkers for cancer severity and patient outcome. Neither potential biomarker has been extensively studied in melanoma. We examined 20 BRAF-mutant melanomas treated with immunotherapy or targeted therapy and measured chromosomal aneuploidy and cGAS expression levels. Treatment type, aneuploidy, and cGAS expression were correlated with progression-free survival (PFS) in these patients. Those treated with immunotherapy first had significantly better outcomes than those treated with targeted therapy, suggesting immunotherapy should be strongly considered as the first-line therapy for patients bearing BRAF-mutant melanoma. We found that there was no correlation of aneuploidy with outcome while there was some positive correlation of cGAS levels with PFS. Further studies are needed to confirm these findings and to test other potential biomarkers.

Abstract A010: Longitudinal histology and spatial transcriptomics profiling in targeted treatment of melanoma patient-derived models interrogates persister and resistant cell populations

Abstract Over half of BRAF-mutant melanoma patients with initial response to targeted therapy will recur with resistant disease. It is thought that recurrence arises from the ability of resistant clonal populations to enter and exit a slow-cycling persister state, evading treatment in quiescence before returning to a proliferative state. However, clonal transcriptional profiles and their spatial relationships in the tumor environment are not well characterized. Through longitudinal profiling of tumors from pre-treatment through the growth of resistant tumors, we track clonal lineages and transcriptional state changes to identify recurrent evolutionary patterns. BRAF mutant patient-derived melanoma xenografts were treated with BRAF/MEK inhibitors through maximum tumor size reduction and re-growth. Spatial transcriptomics (ST) and H&E histological staining were performed at five timepoints across 94 and 133 days of PDX models WM4237 and WM4007 treatment, tracking clonal populations with intact tissue structure. Deep learning feature extraction on high-resolution H&E-stained images allowed the detection of histological features that co-localize with expression levels. A novel computational workflow performed integration of samples across time points, pathway enrichment analysis, and pseudotemporal ordering, allowing spatial recreation of copy number variation based clonal phylogenies and cell fate trajectory inference during the treatment course. We characterize the clonal populations by the burden of accumulated copy number variations and establish clonal phylogeny. With the added temporal resolution, ST profiling during melanoma treatment enables observation of a global shift where all lineages begin the transition into the persister transcriptional state. The drug-sensitive cells are eliminated due to treatment, and distinct transcriptional states arise due to metabolic plasticity regardless of the lineage of origin. The clones of the transient persister state constitute the minimal residual disease, where melanoma cells have an increase in invasive capacity and a slowing of the cell cycle. The ability to survive through the persister state and re-emerge into a proliferating clone where the cell cycle is upregulated varies by lineage. We observe that the transcriptional changes during emergence from dormancy show decreased invasiveness and continued reliance on oxidative respiration versus glycolysis. We compare the re-emerged resistant cell populations and pre-treatment samples to pinpoint the changes in the transcriptional state of the MAPK signaling pathway and find that sets of upregulated genes differ by the model, WM4237 and WM4007. We connect deep learning imaging features extracted from longitudinal histology profiling to ST profiles to associate histology data with the cell cycle activation, invasiveness, hypoxia state, and metabolic mode across the timepoints. We observe that the imaging features alone can be potentially used to track the evolution of the tumor cell population phenotypes during drug treatment. Citation Format: Sergii Domanskyi, Todd B. Sheridan, Brian J. Sanderson, SungHee Park, Jessica Kaster, Haiyin Li, Olga Anczukow, Meenhard Herlyn, Jeffrey H. Chuang, Jill C. Rubinstein. Longitudinal histology and spatial transcriptomics profiling in targeted treatment of melanoma patient-derived models interrogates persister and resistant cell populations [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr A010.

Oral Adverse Events Associated with BRAF and MEK Inhibitors in Melanoma Treatment: A Narrative Literature Review.

Melanoma cancer represents the most lethal type of skin cancer originating from the malignant transformation of melanocyte cells. Almost 50% of melanomas show the activation of BRAF mutations. The identification and characterization of BRAF mutations led to the development of specific drugs that radically changed the therapeutic approach to melanoma. We conducted a narrative review of the literature according to a written protocol before conducting the study. This article is based on previously conducted studies. We identified articles by searching electronic databases (Medline, Google Scholar and PubMed). We used a combination of "melanoma", "Braf-Mek inhibitors", " targeted therapy" and "oral side effects". Eighteen studies were reported in this article showing the relationship between the use of targeted therapy in melanoma cancer and the development of oral side effects, such as mucositis, hyperkeratosis and cellular proliferation. Targeted therapy plays an important role in the treatment of melanoma cancer, showing a notable increase in response rate, prolonged progression-free survival and overall survival in BRAF-mutated melanoma patients. Oral side effects represent a common finding over the course of treatment. However, these adverse effects can be easily managed in a multidisciplinary approach involving collaboration between medical oncologists and dental doctors.

Combined BET and MEK Inhibition synergistically suppresses melanoma by targeting YAP1.

Rationale: The response rate to the MEK inhibitor trametinib in BRAF-mutated melanoma patients is less than 30%, and drug resistance develops rapidly, but the mechanism is still unclear. Yes1-associated transcriptional regulator (YAP1) is highly expressed in melanoma and may be related to MEK inhibitor resistance. The purpose of this study was to investigate the mechanism of YAP1 in MEK inhibitor resistance in melanoma and to screen YAP1 inhibitors to further determine whether YAP1 inhibition reverses MEK inhibitor resistance. Methods: On the one hand, we analyzed paired melanoma and adjacent tissue samples using RNA-seq and found that the Hippo-YAP1 signaling pathway was the top upregulated pathway. On the other hand, we evaluated the transcriptomes of melanoma samples from patients before and after trametinib treatment and investigated the correlation between YAP1 expression and trametinib resistance. Then, we screened for inhibitors that repress YAP1 expression and investigated the mechanisms. Finally, we investigated the antitumor effect of YAP1 inhibition combined with MEK inhibition both in vitro and in vivo. Results: We found that YAP1 expression levels upon trametinib treatment in melanoma patients were correlated with resistance to trametinib. YAP1 was translocated into the nucleus after trametinib treatment in melanoma cells, which could render resistance to MEK inhibition. Thus, we screened for inhibitors that repress YAP1 expression and identified multiple bromodomain and extra-terminal (BET) inhibitors, including NHWD-870, as hits. BET inhibition repressed YAP1 expression by decreasing BRD4 binding to the YAP1 promoter. Consistently, YAP1 overexpression was sufficient to reverse the proliferation defect caused by BRD4 depletion. In addition, the BET inhibitor NHWD-870 acted synergistically with trametinib to suppress melanoma growth in vitro and in vivo. Conclusions: We identified a new vulnerability for MEK inhibitor-resistant melanomas, which activated Hippo pathway due to elevated YAP1 activity. Inhibition of BRD4 using BET inhibitors suppressed YAP1 expression and led to blunted melanoma growth when combined with treatment with the MEK inhibitor trametinib.

Bioinformatics-based analysis reveals elevated CYTL1 as a potential therapeutic target for BRAF-mutated melanoma.

Introduction: Despite many recent emerging therapeutic modalities that have prolonged the survival of melanoma patients, the prognosis of melanoma remains discouraging, and further understanding of the mechanisms underlying melanoma progression is needed. Melanoma patients often have multiple genetic mutations, with BRAF mutations being the most common. In this study, public databases were exploited to explore a potential therapeutic target for BRAF-mutated melanoma. Methods: In this study, we analyzed differentially expressed genes (DEGs) in normal tissues and melanomas, Braf wild-type and Braf mutant melanomas using information from TCGA databases and the GEO database. Subsequently, we analyzed the differential expression of CYTL1 in various tumor tissues and its effect on melanoma prognosis, and resolved the mutation status of CYTL1 and its related signalling pathways. By knocking down CYTL1 in melanoma cells, the effects of CYTL1 on melanoma cell proliferation, migration and invasion were further examined by CCK8 assay, Transwell assay and cell migration assay. Results: 24 overlapping genes were identified by analyzing DEGs common to melanoma and normal tissue, BRAF-mutated and BRAF wild-type melanoma. Among them, CYTL1 was highly expressed in melanoma, especially in BRAF-mutated melanoma, and the high expression of CYTL1 was associated with epithelial-mesenchymal transition (EMT), cell cycle, and cellular response to UV. In melanoma patients, especially BRAF-mutated melanoma patients, clinical studies showed a positive correlation between increased CYTL1 expression and shorter overall survival (OS) and disease-free survival (DFS). In vitro experiments further confirmed that the knockdown of CYTL1 significantly inhibited the migration and invasive ability of melanoma cells. Conclusion: CYTL1 is a valuable prognostic biomarker and a potentially effective therapeutic target in melanoma, especially BRAF-mutated melanoma.

Treatment management for BRAF-mutant melanoma patients with tumor recurrence on adjuvant therapy: a multicenter study from the prospective skin cancer registry ADOREG

BackgroundAdjuvant therapy with immune-checkpoint inhibitors (CPI) or BRAF/MEK-directed targeted therapy (TT) improves recurrence-free survival (RFS) for patients with advanced, BRAFV600-mutant (BRAFmut) resected melanoma. However, 40% of these patients will develop...

Single cell profiling of tumour biopsies and heterogeneity in response of dedifferentiated melanoma.

64 Background: Patients with advanced BRAFV600-mutant melanoma can be treated with immune checkpoint inhibitors or combination BRAF/MEK inhibitors. The sequence of these therapies can influence response to second-line treatment. Patients who progress on first-line PD1 blockade have poorer outcomes on BRAF/MEK inhibitors compared to patients who are treatment-naïve. Thus, defining the molecular features that are modified by therapy and that influence subsequent treatment responses require thorough characterization. Methods: In this study, tumor biopsies from five BRAF-mutant melanoma patients progressing on anti-PD1 (PROG) and five BRAF-mutant treatment-naïve (NAÏVE) patients were dissociated and treated ex vivo with BRAF/MEK inhibitors (dabrafenib+trametinib (DT)) before flow cytometry profiling and single cell transcriptome sequencing. Results: DT treatment did not alter tumor or immune cell proportions but effectively inhibited MAPK signaling by decreasing phosphorylated S6 levels in tumor cells within 8/10 melanoma samples. Single cell RNA-sequencing of the tumor dissociates identified four distinct melanoma differentiation states (e.g. melanocytic, transitory, neural crest-like and undifferentiated). The proportions of these cell states differed between tumor specimens, and were not affected by DT treatment. The neural crest-like and undifferentiated melanoma cells were enriched in PROG compared to NAIVE tumor samples and all differentiation states responded, albeit variably. For instance, MAPK inhibition after DT treatment was less pronounced in undifferentiated melanoma cells. Importantly, within the undifferentiated cell state, we identified a proportion of melanoma cells capable of responding to DT, and comparison of responding vs non-responding undifferentiated cells showed enrichment of pro-inflammatory IL-6 and TNFα signaling in the non-responding population. Elevated IL-6 and TNFα pathway activity correlated with decreased potency to MAPK inhibition, indicating that increased IL-6 and TNFα signaling in undifferentiated melanoma may contribute to resistance to MAPK inhibition. Conclusions: Ex vivo treatment of tumor dissociates coupled with single cell transcriptome sequencing identified cell state-specific responses to MAPK inhibition. Resistance to MAPK inhibition may be driven, in part, by tumor-intrinsic pro-inflammatory IL-6 and TNFα signalling.

PLX4032 resistance of patient-derived melanoma cells: crucial role of oxidative metabolism.

Malignant melanoma is the most lethal form of skin cancer which shows BRAF mutation in 50% of patients. In this context, the identification of BRAFV600E mutation led to the development of specific inhibitors like PLX4032. Nevertheless, although its initial success, its clinical efficacy is reduced after six-months of therapy leading to cancer relapse due to the onset of drug resistance. Therefore, investigating the mechanisms underlying PLX4032 resistance is fundamental to improve therapy efficacy. In this context, several models of PLX4032 resistance have been developed, but the discrepancy between in vitro and in vivo results often limits their clinical translation. The herein reported model has been realized by treating with PLX4032, for six months, patient-derived BRAF-mutated melanoma cells in order to obtain a reliable model of acquired PLX4032 resistance that could be predictive of patient's treatment responses. Metabolic analyses were performed by evaluating glucose consumption, ATP synthesis, oxygen consumption rate, P/O ratio, ATP/AMP ratio, lactate release, lactate dehydrogenase activity, NAD+/NADH ratio and pyruvate dehydrogenase activity in parental and drug resistant melanoma cells. The intracellular oxidative state was analyzed in terms of reactive oxygen species production, glutathione levels and NADPH/NADP+ ratio. In addition, a principal component analysis was conducted in order to identify the variables responsible for the acquisition of targeted therapy resistance. Collectively, our results demonstrate, for the first time in patient-derived melanoma cells, that the rewiring of oxidative phosphorylation and the maintenance of pyruvate dehydrogenase activity and of high glutathione levels contribute to trigger the onset of PLX4032 resistance. Therefore, it is possible to hypothesize that inhibitors of glutathione biosynthesis and/or pyruvate dehydrogenase activity could be used in combination with PLX4032 to overcome drug resistance of BRAF-mutated melanoma patients. However, the identification of new adjuvant targets related to drug-induced metabolic reprogramming could be crucial to counteract the failure of targeted therapy in metastatic melanoma.

Abstract 1164: Spatiotemporal profiling defines persister and resistance signatures in targeted treatment of melanoma

Abstract Over half of BRAF mutant melanoma patients with response to targeted therapy recur within 15 months. Resistance is postulated to arise from certain clonal populations that enter a slow cycling persister state, evade treatment with relative dormancy, and repopulate the tumor when reactivated. Via longitudinal profiling we define expression states of clonal populations and track their evolutionary progression. BRAF mutant patient derived melanoma xenografts were treated with BRAF/MEK inhibitors through maximum tumor size reduction and re-growth. Spatial transcriptomics (ST) was performed at 5 timepoints across 94 days of treatment, tracking clonal populations with intact tissue structure. Deep learning on H&E-stained images automatically detected histological features that co-localize with expression levels. A novel computational pipeline performed clustering, differential expression and pathway enrichment analysis, copy number variation detection, and pseudotemporal ordering, allowing spatial recreation of clonal phylogenies and cell fate trajectory inference during the treatment course. Three clonal categories were defined; Sensitive: predominant in treatment-naïve samples, Persister: remaining clusters in maximally shrunken specimens, Resistant: re-emergent, fast-proliferating. Analysis of clonal composition identified GAPDH and CCND1 as highly expressed in sensitive clones, suppressed in persisters, and reactivated in resistant clones; TYRP1, DCT, MITF, and NGFR showed an opposite pattern. Persister clones evolved from oxidative phosphorylation toward glycolysis and MAPK regulation. Spatial analysis showed increased glycolysis:oxidative phosphorylation ratio in centrally located sensitive clusters, with more balanced peripheral expression. Resistant clones showed upregulated DUSP6 expression and enrichment in Orexin signaling and MAPK regulation. Imaging- and expression-based clones were highly concordant. Spatial profiling during melanoma treatment identified transient persister and emergent resistant clones, defining expression profiles and phenotypic features. Altered ratio of oxidative to glycolytic metabolic pathways was a hallmark of clonal evolution in both space and time. Specific MAPK pathway re-entry points were identified as candidate resistance mechanisms, suggesting potential therapeutic targets. Deep learning features derived from histological images showed good correlation with ST profiles, providing a promising method for clonal tracking via images alone. This longitudinal experiment mimics a tumor’s clinical course, inducing persistence and resistance via treatment that parallels that seen by patients. Combining ST and imaging techniques, we provide insight into clonal dynamics with novel spatiotemporal resolution, defining an evolutionary roadmap to acquired treatment resistance. Citation Format: Jill Carol Rubinstein, Sergii Domanskyi, Todd B. Sheridan, Brian J. Sanderson, SungHee Park, Jessica Kaster, Haiyin Li, Olga Anczukow, Meenhard Herlyn, Jeffrey H. Chuang. Spatiotemporal profiling defines persister and resistance signatures in targeted treatment of 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 1164.

Abstract 1012: Detecting and intervening on rare pre-existing resistant subclones to BRAF/MEK inhibitors in metastatic melanoma

Abstract The combination of BRAF and MEK inhibitors (BRAFi/MEKi) in metastatic melanoma patients with BRAF activating mutations have resulted in dramatic responses in most patients, but unfortunately most patients progress after one year and limited subsequent treatment options are available. Although resistance mutations to BRAFi/MEKi mainly occur in the MAPK and PI3K pathways, after progression on BRAFi/MEKi drugs, targeting these mutations have been clinically unsuccessful. One possible cause of relapse is that a resistant subpopulation to BRAFi/MEKi exists prior to treatment, raising the possibility that earlier counter-resistance treatment could be administered and potentially be more successful. However, the prevalence and clinical significance of pre-existing resistance is currently unknown in melanoma. To address this, we used quantitative blocker displacement amplification (qBDA) to quantify the VAF (down to 0.01% resolution) of resistant subclones along the MAPK/PI3K pathways from FFPE biopsies on 149 metastatic melanoma patients. Due to possible intra-tumoral heterogeneity, multiple samples were taken when available per patient. Of 29 patients currently processed, 5/29 (17%) had detectable NRAS Q61L or Q61R, 5/29 (17%) had KRAS G12 or G13 mutations, and 3/29 (10.3%) had PIK3CA H1047R/Y. As expected, BRAF activating mutations were detectable in 29/29 patients, while VAF of the resistant subclones were found to be as low as 0.16%. We expect some of the putative resistance mutations to be artifacts, and all will be validated by ddPCR. To model the behavior of low-VAF resistance mutations, we conducted in vitro spike-in experiments using paired and labeled parental and BRAF/MEK inhibitor-resistant melanoma lines. Our results show that even down to 0.1%, the resistant lines can rapidly emerge as the dominant population while under drug treatment. We also tested the timing of applying a counter-resistance therapy: early vs late. Earlier intervention led to a significant delay in growth of the resistant population, as expected. However, we are currently preparing an experiment that will determine whether an earlier intervention might not simply delay resistance, but also alter its strength, at different % VAFs. We therefore anticipate mechanistic analyses of how %VAF and drug timing can optimize counter-resistance therapies. Overall, our interim data suggest a significant percentage of metastatic BRAF-mutated melanoma patients may harbor pre-existing resistant subclones, and that early treatment with counter-resistance therapy can potentially delay resistance to targeted therapy. Citation Format: Michael Nakazawa, Lauren Cheng, Mishal Ahmed, Rossana Lazcano, Khalida Wani, Omid Veiseh, Lawrence Kwong. Detecting and intervening on rare pre-existing resistant subclones to BRAF/MEK inhibitors in metastatic 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 1012.

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

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

Targeting EphA2 and DDR signaling can overcome the BRAF and MEK inhibitors acquired resistance in melanoma cell lines

The BRAF and MEK inhibitors combined strategies have dramatically changed the outcome of BRAF-mutated metastatic melanoma patients. However, despite the initial promising results, the onset of primary or acquired resistance occurs in nearly half of the patients at about one year from the diagnosis. Understanding the mechanisms of resistance to these inhibitors is therefore critical for planning more effective therapeutic strategies able to improve patient outcomes. To this aim we generated BRAF and MEK inhibitors resistant melanoma cells starting from the SAN and A375 lines, both harboring the most common BRAF-V600 mutation and sensitive to these drugs. The obtained double-resistant cell lines were characterized by MTT cell proliferation, migration, invasion assays, phosphoarray and western blot analysis. Here we report that the overexpression of several Tyrosine Kinase Receptors (TKRs), such as EphA2 and DDRs, drives the resistance to these drugs and that this resistance can be overcome by treatment with ALW‑II‑41‑27 multikinase inhibitor. ALW‑II‑41‑27 blocks not only TKRs expression, but also the related downstream AKT and MAPK signaling pathways and its efficacy is documented by decreased cell viability and reduced cell invasion/migration of the resistant cells. Our results can delineate a novel promising therapeutic approach to overcoming the drug resistance occurring in BRAF-mutated metastatic melanoma.

Retrospective review of outcomes associated with metastatic melanoma patients treated with 1st-line BRAF-targeted therapy.

BRAF-mutant melanoma patients can theoretically access both immunotherapy and BRAF-targeted therapy as treatment for metastatic disease. BRAF-targeted therapy is increasingly used 1st line for poorer prognostic patients, so we wanted to assess realistic expectations of these patients accessing 2nd-line immunotherapy. We conducted a retrospective review of clinical outcomes in 25 patients treated over the last 3 years with 1st-line BRAF-targeted therapy in a real-world clinical setting at a UK-based tertiary centre. Compared with the registration trials, our patients receiving 1st-line BRAF-targeted therapy had poorer performance status, higher disease burden, shorter median progression-free survival (5.05 months, 95% CI: 3.96-8.88) and shorter median overall survival (11.5months, 95% CI: 6.24 - not reached). Overall response rate was similar, at 64%. On disease progression, median survival was 2.34 months (95% CI: 1.62 - not reached). Only five patients went on to receive 2nd-line immunotherapy. Metastatic melanoma patients treated with 1st-line BRAF-targeted therapy now have different demographics compared with those recruited to registration trials conducted over the last 10 years. In a modern-day, real-world setting, these patients should be counselled that only 1 in 5 are likely to receive 2nd-line immunotherapy and their survival times are expected to be short.

811P Three-years real-world evidence of adjuvant dabrafenib plus trametinib (DT) in patients with resected melanoma in Spain (GEM 1901 - DESCRIBE-AD)

DT combination has shown efficacy in the adjuvant setting for BRAF-mutated melanoma (BMM) patients (pts) in clinical trials. Previous reports from DESCRIBE-AD resulted in promising overall survival (OS) rates at 12 months.

Clinical characteristics and treatment outcomes of non-V600 E/K BRAF mutant melanoma patients: a single-institution experience.

The widespread use of more sensitive detection tools, such as next-generation sequencing, has increased the identification of a variety of BRAF mutations other than V600E/K in melanoma patients. However, there is a lack of established data regarding the efficacy of BRAF/MEK inhibitors and immune-checkpoint immune inhibitors (ICI) for these patients. We performed a retrospective study, including all the patients diagnosed with stage III or IV melanoma that were referred to the University Hospital of Bologna from 2011 to 2021, carrying a non-V600E or V600K mutation of BRAF and who were started on systemic treatment. We found 14 patients with stage III or IV melanoma harboring the following BRAF mutations: V600R, V600_K601delinsE, K601E, p.T599_V600insT, L597V, G466R, S467L, and A598T. Of note, G466R and A598T BRAF mutations have never been previously reported in melanoma. Four patients received combined BRAF/MEK inhibitors, two patients BRAF inhibitor monotherapy, and six patients were treated with ICI for advanced melanoma; four patients received adjuvant treatment with nivolumab. Given the few cases and the absence of randomized clinical trials, it is important to report clinical experiences, which can guide physicians in the treatment of melanomas harboring rare BRAF mutations.

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

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