BRAF Melanoma Research Articles

The Investigation of Hsp90C-Terminal Inhibitors Containing Amide Bioisosteres.

Heat Shock Protein 90 (Hsp90) is responsible for the proper folding and maturation of ~400 client protein substrates, many of which are directly associated with the ten hallmarks of cancer. Hsp90 is a great target for cancer therapy including melanoma, since Hsp90 inhibition can disrupt multiple oncogenic pathways simultaneously. In this study, we report the synthesis and anti-proliferative activity manifested by a series of Hsp90 C-terminal inhibitors against mutant BRAF and wild-type BRAF melanoma cells. Furthermore, we explored structure-activity relationships (SAR) for the amide moiety of 6 (B1), a novel Hsp90C-terminal inhibitor via introduction of amide bioisosteres. Compound 6 displayed an IC50 of 1.01 μM, 0.782 μM, 0.607 μM and 1.413 μM against SKMel173, SKMel103, SKMel19 and A375 cells, respectively.

Interaction of DisBa01 peptide from Bothrops alternatus venom with BRAF melanoma receptors: Modeling and molecular docking

Metastatic melanoma is highly aggressive and challenging, often leading to a grim prognosis. Its progression is swift, especially when mutations like BRAFV600E continuously activate pathways vital for cell growth and survival. Although several treatments target this mutation, resistance typically emerges over time. In recent decades, research has underscored the potential of snake venoms and peptides as bioactive substances for innovative drugs, including anti-coagulants, anti-microbial, and anti-cancer agents. Leveraging this knowledge, we propose employing a bioinformatics simulation approach to: a) Predict how well a peptide (DisBa01) from Bothrops alternatus snake venom binds to the melanoma receptor BRAFV600E via Molecular Docking. b) Identify the specific peptide binding sites on receptors and analyze their proximity to active receptor sites. c) Evaluate the behavior of resulting complexes through molecular dynamics simulations. d) Assess whether this peptide qualifies as a candidate for anti-melanoma therapy. Our findings reveal that DisBa01 enhances stability in the BRAFV600E melanoma receptor structure by binding to its RGD motif, an interaction absent in the BRAF WT model. Consequently, both docking and molecular dynamics simulations suggest that DisBa01 shows promise as a BRAFV600E inhibitor.

Prevention of Brain Metastases: A New Frontier.

This review discusses the topic of prevention of brain metastases from the most frequent solid tumor types, i.e., lung cancer, breast cancer and melanoma. Within each tumor type, the risk of brain metastasis is related to disease status and molecular subtype (i.e., EGFR-mutant non-small cell lung cancer, HER2-positive and triple-negative breast cancer, BRAF and NRAF-mutant melanoma). Prophylactic cranial irradiation is the standard of care in patients in small cell lung cancer responsive to chemotherapy but at the price of late neurocognitive decline. More recently, several molecular agents with the capability to target molecular alterations driving tumor growth have proven as effective in the prevention of secondary relapse into the brain in clinical trials. This is the case for EGFR-mutant or ALK-rearranged non-small cell lung cancer inhibitors, tucatinib and trastuzumab-deruxtecan for HER2-positive breast cancer and BRAF inhibitors for melanoma. The need for screening with an MRI in asymptomatic patients at risk of brain metastases is emphasized.

Retraction: Acquired resistance to BRAFi reverses senescence-like phenotype in mutant BRAF melanoma.

Retraction: Acquired resistance to BRAFi reverses senescence-like phenotype in mutant BRAF melanoma.

Comprehensive genomic and immune profiling of non-V600 BRAF-mutated melanomas: Implications for therapeutic strategies beyond BRAF/MEK inhibitors.

e21544 Background: The development of targeted therapies ( BRAF plus MEK inhibitors) and immune checkpoint inhibitors (ICIs) has significantly improved the outcomes of patients with advanced melanoma, particularly those harboring BRAF V600 mutations (Class 1). However, melanomas with non-V600 BRAF mutations (classes 2 and 3) remain a subset where effective therapeutic options beyond ICIs are lacking. In this study, we investigated the distinct genomic and immune profile of non-V600 BRAF melanomas to further understand the mechanism of resistance to BRAF/MEK inhibitors and explore other pathways of potential therapeutic targets. Methods: A retrospective cohort of 175 melanoma samples was evaluated by comprehensive genomic and immune profiling, including tumor mutational burden (TMB) and the expression of 395 immune genes. Tumor specimens were classified as BRAFClass1, BRAFClass2, and BRAFClass3. TMB (high ≥10 Mut/Mb) was obtained by DNA sequencing. Gene expression signatures of tumor inflammation and cell proliferation were determined by targeted RNA-sequencing. We used chi-square and Wilcoxon rank-sum tests to determine the association of BRAF mutational status to genomic and immune correlates. Results: The median age of diagnosis was 69 years, with 57% of patients being male. A total of 152 (86.9%), 13 (7.4%), and 10 (5.7%) patients had BRAFClass1, BRAFClass2, and BRAFClass3 alterations, respectively. Genomic alterations in TP53, NF1, MET and ROS1 were more prevalent in BRAFClass2 and BRAFClass3 as opposed to BRAFClass1. BRAFClass2 and BRAFClass3 tumors combined had significantly higher tumor inflammation (median = 55.38; p < 0.05) compared to BRAFClass1 (median = 41.79) as well as higher TMB (median = 54.8; p < 0.001) compared to BRAFClass1 (median = 9.75). BRAFClass3 tumors showed significantly higher cellular proliferation (median = 47.5; p < 0.05) compared to BRAFClass1 (median = 33.5) and BRAFClass2 (median = 31.8) tumors, as well as higher TMB (median = 71.5; p < 0.01) compared to BRAFClass1 (median = 10.9). When looking at differentially expressed genes, CDK1 was upregulated in BRAFClass3 compared to BRAFClass1 and BRAFClass2, and IL12B was upregulated in BRAFClass2 compared to BRAFClass1. Conclusions: Our study showed that the non-Class 1 BRAF (V600E or K) mutated tumors have higher immune response suggesting a favorable response to ICIs. Additionally, we found that BRAFClass2 and BRAFClass3 tumors more commonly harbor alterations in NF1 and TP53, supporting the prior understanding that BRAF non-V600 tumors have a more aggressive clinical course. Comprehensive molecular analysis offers insight into the immunogenicity and distinct oncogenic drivers associated with unfavorable clinical outcomes, which could guide therapeutic development beyond BRAF and MEK inhibitors in BRAF non-V600 melanomas.

Abstract 5847: Characterizing mechanisms of BRAF/MEK inhibitor resistance in BRAF V600 mutant colorectal cancer vs. melanoma

Abstract Background: BRAF V600 mutations comprise ~80% of all oncogenic BRAF mutations in metastatic colorectal cancer (mCRC). While most cancers with BRAF V600 mutations can be effectively treated with combined BRAF and MEK inhibition, BRAF V600 mutant mCRC acquire resistance through EGFR-mediated signaling. The profound difference in response rates to BRAF/MEK inhibitors in BRAF V600 mutant mCRC (12%) vs. melanoma (63-68%) is not sufficiently explained by increased EGFR signaling in CRC. Therefore, we sought to characterize the differences that exist between BRAF V600 mutant CRC and melanomas that could explain the differential sensitivity to BRAF/MEK inhibition and identify more effective treatments for BRAF V600 mutant CRC. Methods: We assessed cell survival by clonogenic assay in the presence of small molecule inhibitors. In parallel, we mined TCGA RNAseq data to calculate the MAPK Pathway Activity Score (MPAS). RTK expression was assessed in TCGA data from patients with BRAF V600 mutant CRC vs. melanoma. We developed cells with acquired resistance to BRAF+MEK inhibitors (Encorafenib+Binimetinib; E+B) by culturing cells in increasing concentrations of drug over several months. RNA-sequencing and analysis was performed on parental and resistant cells treated with vehicle control or BRAF+MEK inhibitors. Results: When comparing the efficacy of BRAF+MEK inhibitors across BRAF V600 mutant cells, we found that BRAF V600 mutant CRC cell lines (n=2) were less sensitive than melanoma cell lines (n=2). MPAS scores were significantly lower in BRAF V600 mutant CRC (n=46) vs. melanoma (n=188) tumors. We interrogated protein array data from the DepMap and identified increased activation of several RTKs (EGFR, HER2, HER3) in BRAF V600 CRC (n=8) vs. melanoma (n=39) cell lines. Similarly, we established resistant HT29 (BRAF V600 CRC) cells and observed sustained MAPK pathway activity despite a lack of difference in EGFR activity in the presence of BRAF+MEK inhibitor treatment. Both HT29 parental and resistant cells showed re-establishment of MAPK signaling after 72 hours. Resistant SkMel28 (BRAF V600 melanoma) cells showed increased pERK levels compared to resistant HT29 cells. RNAseq revealed increased expression of multiple RTKs (PDGFRB, ERBB2, ERBB3) and ligands/growth factors for multiple RTKs (FGF3, PDGFA, HB-EGF) in resistant HT29 cells. Shp2 is a protein tyrosine phosphatase that regulates signaling of multiple RTKs to activate the MAPK pathway. Accordingly, we assessed the efficacy of an inhibitor of Shp2 (TNO155) overcoming MAPKi resistance in BRAF V600 CRC cells. We confirmed that E+B+TNO155 more effectively inhibited cell growth and MAPK pathway activity in BRAF V600 CRC cell lines (n=2) in vitro. Conclusion: Together, these data identify key, actionable drivers of MAPK inhibitor resistance in CRC and highlight Shp2 as a potential therapeutic target for BRAF V600 CRC. Citation Format: Jennifer Maxwell, Emmanuelle Rousselle, Lucy An, April Rose. Characterizing mechanisms of BRAF/MEK inhibitor resistance in BRAF V600 mutant colorectal cancer vs. melanoma [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 5847.

Abstract 5869: Investigation of molecular alterations associated with double resistance to BRAF and MEK inhibition in melanoma

Abstract More than 50% of melanomas harbor activating BRAF kinase mutations, with BRAFV600E representing more than 90% of BRAF mutations. Inhibition of mutant BRAF by BRAF inhibitors (BRAFi) in patients with advanced melanoma yields a remarkable initial clinical response. Limited duration response and disease recurrence of inhibitors occurs due to acquired drug resistance. Recently identified mechanisms of MAPK hyperactivation in acquired resistance to BRAFi have encouraged clinicians to employ a combination of BRAF and MAPK inhibitors to block the MAPK signaling pathway more effectively. However, most patients treated with this combination therapy still relapse due to de novo drug resistance. Therefore, innovative strategies are still necessary to improve the precision and durability of anti-melanoma therapies. To investigate the mechanism of both BRAFi and MEKi resistance (double resistance, DR) in mutant BRAF melanomas, we have developed models from four human melanoma cell lines to explore the hypothesis that molecular alterations are related to double resistance to BRAFi and MEKi in melanoma. We have found that BRAFi-MEKi double-resistant models exhibit distinct differences in morphology and growth kinetics. Specifically, BRAFi-MEKi double-resistant human melanomas express a higher level of FGFb and PAI-1 expression. We confirmed that overexpression of FGFb and PAI-1 in parental cells could increase the ability to resist BRAF and MEK inhibition. Additionally, we discovered that decreased expression of FGFb and PAI-1, via knock-down, inhibition, or CRISPR in double resistant cells, can restore cell sensitivity to treatments of BRAFi and MEKi. To further elucidate the role of FGFb and PAI-1 on resistant melanoma, we have extended our scope to in vivo mouse xenograft models. Our findings uncovered the mechanism of how melanoma cells can escape BRAFi and MEKi attacks and offer a rational strategy to guide future clinical treatments. Citation Format: Armond J. Isaak, GeGe R. Clements, Lei Huang, Yifei Qin, Qiang Zho, Glenn Merlino, Yanlin Yu. Investigation of molecular alterations associated with double resistance to BRAF and MEK inhibition in melanoma [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 5869.

Melanocortin receptor 4 as a new target in melanoma therapy: Anticancer activity of the inhibitor ML00253764 alone and in association with B-raf inhibitor vemurafenib

The aim of our study is to investigate in vitro and in vivo MC4R as a novel target in melanoma using the selective antagonist ML00253764 (ML) alone and in combination with vemurafenib, a B-rafV600E inhibitor.The human melanoma B-raf mutated A-2058 and WM 266-4 cell lines were used. An MC4R null A-2058 cell line was generated using a CRISPR/Cas9 system. MC4R protein expression was analysed by western blotting, immunohistochemistry, and immunofluorescence. Proliferation and apoptotic assays were performed with ML00253764, whereas the synergism with vemurafenib was evaluated by the combination index (CI) and Loewe methods. ERK1/2 phosphorylation and BCL-XL expression were quantified by western blot. In vivo experiments were performed in Athymic Nude-Foxn1nu male mice, injecting subcutaneously melanoma cells, and treating animals with ML, vemurafenib and their concomitant combination. Comet and cytome assays were performed.Our results show that human melanoma cell lines A-2058 and WM 266-4, and melanoma human tissue, express functional MC4R receptors on their surface. MC4R receptors on melanoma cells can be inhibited by the selective antagonist ML, causing antiproliferative and proapoptotic activity through the inhibition of phosphorylation of ERK1/2 and a reduction of BCL-XL. The concomitant combination of vemurafenib and ML caused a synergistic effect on melanoma cells in vitro and inhibited in vivo tumor growth in a preclinical model, without causing mouse weight loss or genotoxicity.Our original research contributes to the landscape of pharmacological treatments for melanoma, providing MC4R antagonists as drugs that can be added to established therapies.

Genomic subtypes of cutaneous melanoma have distinct metabolic profiles: A single-cell transcriptomic analysis.

Genomic profiling previously classified melanoma into distinct subtypes based on the presence or absence of mutations in driver genes, but metabolic differences between and within these groups have yet to be thoroughly analyzed. Thus, the objective of the present study is to provide the first effort to holistically characterize the metabolic landscape of qualified melanoma genomic subtypes at single-cell resolution. Expression data for a total of 1145 malignant cells sourced from NRAS(Q61L), BRAF(V600E), and NRAS/BRAF WT melanomas were retrieved from the Broad Single Cell Portal. Metabolic activity was interrogated by pathway scoring and gene set enrichment analysis. A total of 53 metabolic pathways were differentially regulated in at least one melanoma genomic subtype. Some notable findings include: BRAF/NRAS WT cells were enriched for fatty acid biosynthesis and depleted for metabolism of alanine, aspartate, and glutamate; BRAF(V600E) melanoma cells were enriched for beta-alanine metabolism and depleted for phenylalanine metabolism; NRAS(Q61L) melanoma cells were enriched for steroid biosynthesis and depleted for linoleic acid metabolism. Primary limitations include the total quantity of single cells and breadth of available genomic subtypes plus inherent noisiness of the applied methodologies. Nonetheless, these findings nominate novel, testable therapeutic targets.

Real-World Evaluation of the Management, Treatment Pathways and Outcome of Melanoma Patients with Target Therapies in Italy.

In recent years, an increasing trend in the incidence of melanoma has been observed in Europe. Although early diagnosis and prompt intervention with local resection often results in positive outcomes, conversely, metastatic disease is still clinically challenging with a poor prognosis and a 5-year survival of around 30%. The growing awareness of melanoma biology and of antitumor immune responses has allowed the development of novel therapies targeted at specific molecular alterations occurring at advanced stages. This real-world analysis examined patients with melanoma in Italy, focusing on treatment patterns, outcome, time to discontinuation (TTD), and resource consumption. Two retrospective observational analyses were conducted for BRAF+ patients with metastatic melanoma and those with a positive sentinel lymph node biopsy in an adjuvant setting, retrieving data from the administrative databases covering 13.3 million residents. The cohort melanoma BRAF+ in metastatic setting comprised 729 patients with targeted therapy (TT) (n=671 with TT as first line and 79 as second line). Median TTD was 10.6months in first line and 8.1months in second line. Median overall survival from the start of first TT line was 27months and was 11.8months for patients with brain metastasis. In the dabrafenib plus trametinib patients, main healthcare resource consumption tended to increase in the presence of brain metastasis. The cohort with a positive sentinel lymph node biopsy under adjuvant therapy (n=289) included 8% patients treated with dabrafenib plus trametinib or tested BRAF+, 5% BRAF wild-type, and 10% under immunotherapy. Our findings provided an overview on TT utilization on metastatic melanoma patients in real clinical practice and highlighted an increased burden in brain metastatic patients.

RF05 A national report on incidence, patient demographics and tumour characteristics of melanoma by BRAF, NRAS and KIT genotype: England 2016–2019

Abstract Although studies have shown variation between countries in the incidence and characteristics of patients and tumours associated with melanoma BRAF, NRAS and KIT mutations, no national molecular genetics data from England have been published. In 2022, the National Institute for Health and Care Excellence highlighted the need for national cohort studies to evaluate the role of biomarkers. This descriptive national observational study used National Disease Registration Service (NDRS) data to identify all site melanoma cases that were diagnosed and underwent molecular genetic testing in England between 2016 and 2019. The NDRS collects molecular genetics data from most genomic laboratories nationally but does not routinely receive results for BRAF testing by immunohistochemistry (IHC) done at local pathology laboratories. Molecular genetic testing and incidence of wild-type (WT), BRAF mutant (+), NRAS+ and KIT+ melanomas were described. Demographic (sex, age, ethnicity, deprivation, geographical region) and tumour factors (site, stage) of BRAF+, NRAS+, KIT+ and WT tumours were described. In total, 61 514 new melanomas were registered in England between 2016 and 2019. Thirteen per cent (n = 7925), 5.0% (n = 3084) and 1.2% (n = 743) of tumours underwent BRAF, NRAS and KIT testing, respectively. BRAF testing rates varied across regions from 19.9% (n = 1040/5217) in the West Midlands to 5.5% (n = 227/4114) in the North East. Thirty-four per cent (n = 2674/7925), 30.5% (n = 942/3084) and 8.6% (n = 64/743) of tumours were BRAF+, NRAS+ and KIT+, respectively. Thirty-four per cent (n = 1523/4479) of males and 37.6% (n = 1132/3014) of females with cutaneous melanomas were BRAF+. Forty-six per cent (n = 1693/3712) of patients aged < 70 years and 20.5% (n = 332/1622) aged ≥ 80 years were BRAF+. The highest proportion of BRAF+ cutaneous melanoma were identified in the North East (46.7%, n = 106/227) and the lowest in the West Midlands (32.6%, n = 339/1040). Thirty-two per cent (n = 560/1760) of males and 30.0% (n = 349/1167) of females with cutaneous melanoma were NRAS+. Twenty-six per cent (n = 356/1354) of patients aged < 70 years and 31.8% (n = 229/718) aged ≥ 80 years were NRAS+. The highest proportion of NRAS+ cutaneous melanoma were in the West Midlands (40%, n = 14/35) and the lowest in the North East (28.3%, n = 36/127). We report the largest and most complete dataset for national molecular genetics of melanoma ever published. Regional differences in BRAF testing may be explained by variations in local practice and methods used, and differences in population age, but could impact on treatment options. Lack of IHC, testing nonuniformity and lack of whole melanoma population data (early-stage nonrecurrent tumours and tumours in frail elderly patients may be excluded) were limitations.

Neoadjuvant Immune Checkpoint Inhibition in Metastatic Conjunctival Melanoma.

The development of immune checkpoint inhibitors (ICI) has transformed the treatment of advanced-stage cutaneous melanoma; however, most trials did not include patients with conjunctival melanoma. Herein the authors describe a patient with recurrent conjunctival melanoma who developed locally advanced, b-raf and v-raf murine sarcoma viral oncogene homolog B1-negative melanoma in her nasal cavity and extensive, metabolically active, bilateral lymphadenopathy in her thorax. Her nasal mass measured 4.3 × 1.7 cm and was determined to be unresectable. She was treated with 4 cycles of combination ipilimumab and nivolumab therapy followed by maintenance nivolumab. She experienced a dramatic treatment response with a reduction in the size of her nasal mass to 3.0 × 1.1 cm and a complete resolution of her adenopathy. She then underwent complete surgical resection of her residual mass (approximately 75% of her original tumor size) and remains melanoma-free at 1 year of follow-up. Given the underlying genetic similarities of conjunctival melanoma to cutaneous melanoma, providers should consider the use of neoadjuvant immune checkpoint inhibitors for patients with locally advanced or limited metastatic disease.

The benefit of co-targeting PARP-1 and c-Met on the efficacy of radiotherapy in wild type BRAF melanoma.

Melanoma is known to be a radioresistant cancer. Melanoma radioresistance can be due to several factors such as pigmentation, antioxidant defenses and high Deoxyribonucleic acid (DNA) repair efficacy. However, irradiation induces intracellular translocation of RTKs, including cMet, which regulates response to DNA damage activating proteins and promotes DNA repair. Accordingly, we hypothesized that co-targeting DNA repair (PARP-1) and relevant activated RTKs, c-Met in particular, may radiosensitize wild-type B-Raf Proto-Oncogene, Serine/Threonine Kinase (WTBRAF) melanomas where RTKs are often upregulated. Firstly, we found that PARP-1 is highly expressed in melanoma cell lines. PARP-1 inhibition by Olaparib or its KO mediates melanoma cell sensitivity to radiotherapy (RT). Similarly, specific inhibition of c-Met by Crizotinib or its KO radiosensitizes the melanoma cell lines. Mechanistically, we show that RT causes c-Met nuclear translocation to interact with PARP-1 promoting its activity. This can be reversed by c-Met inhibition. Accordingly, RT associated with the inhibition of both c-Met and PARP-1 resulted in a synergistic effect not only on tumor growth inhibition but also on tumor regrowth control in all animals following the stop of the treatment. We thus show that combining PARP and c-Met inhibition with RT appears a promising therapeutic approach in WTBRAF melanoma.

Abstract 5734: Designing RAF and MEK inhibitor combinations based on their biochemical properties to effectively target MAPK-driven cancers

Abstract Oncogenic alterations of the RAS/RAF/MEK/ERK signaling pathway drives growth of over 40% of human tumors, frequently due to activating mutations of components of the pathway, such as RAS or BRAF. Targeting the pathway using BRAF inhibitors (BRAFi) in combination with MEK inhibitors (MEKi) is now the standard of care for metastatic melanoma patients harboring the BRAF(V600E) mutation. However, effective targeted therapies are lacking for most cancers driven by other pathway alterations, including those with RAS activating mutations. The effectiveness of current clinical BRAFi relies on the fact that they bind selectively and inhibit the mutationally activated monomeric form of the BRAF(V600E) kinase in tumors but not wild-type BRAF in normal tissue where BRAF signals in its dimeric form. Yet, the development of adaptive resistance, frequently due to RAF dimerization, limits BRAFi therapeutic effectiveness. Another class of BRAFi equipotent for both monomeric and dimeric BRAF have been developed, but are predicted to have lower therapeutic index due to inhibition of dimeric wild-type BRAF in normal tissues as well. Recently, we identified and characterized a novel class of BRAFi that preferentially binds and inhibits dimeric BRAF over monomeric BRAF(V600E) (i.e. RAF dimer-selective inhibitors). Furthermore, we rationally designed a combinatorial approach utilizing a BRAF monomer-selective plus a BRAF dimer-selective inhibitor plus a MEK inhibitor, that potently disrupts the BRAF/MEK complex, to maximally inhibit BRAF(V600E) signaling in tumors while retaining a broad therapeutic index. This triple combination potently suppressed tumor growth of multiple BRAF(V600E) colorectal cancer and melanoma models resistant to the current clinical BRAFi and MEKi combinations both in vitro and in vivo. To design effective therapeutic approaches for RAS-mutated tumors, we also assessed the effectiveness of RAF and MEK inhibitors belonging to diverse biochemical and structural classes, either alone or in combination in RAS-mutant tumor models and normal cells with wild-type RAS signaling. We found that RAF dimer-selective inhibitors potently antagonized MAPK signaling and growth in various RAS mutant cancer models as compared to normal cells. We found further that RAF and MEK inhibitor combinations belonging to certain biochemical classes resulted in more potent MAPK suppression in RAS-mutant tumor as compared to wild-type RAS normal cells (“therapeutic synergy”) and should be prioritized for clinical testing. In contrast, those combinations which are more potent in normal over tumor cells (“adverse synergy”) are unlikely to be clinically successful. Thus, rationally designed combinations of selected RAFi and MEKi, based on their distinct biochemical properties, can uncover new therapeutic opportunities tailored for more effective targeting of BRAF or RAS mutant tumors. Citation Format: Ana Orive-Ramos, Christos Adamopoulos, Beau Baars, Jason Lam, Ankita Punetha, Jason Huang, Vasileios I. Petrou, Stuart A. Aaronson, Poulikos I. Poulikakos. Designing RAF and MEK inhibitor combinations based on their biochemical properties to effectively target MAPK-driven cancers. [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 5734.

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.

P38 Mediates Tumor Suppression through Reduced Autophagy and Actin Cytoskeleton Changes in NRAS-Mutant Melanoma.

Hotspot mutations in the NRAS gene are causative genetic events associated with the development of melanoma. Currently, there are no FDA-approved drugs directly targeting NRAS mutations. Previously, we showed that p38 acts as a tumor suppressor in vitro and in vivo with respect to NRAS-mutant melanoma. We observed that because of p38 activation through treatment with the protein synthesis inhibitor, anisomycin leads to a transient upregulation of several targets of the cAMP pathway, representing a stressed cancer cell state that is often observed by therapeutic doses of MAPK inhibitors in melanoma patients. Meanwhile, genetically induced p38 or its stable transduction leads to a distinct cellular transcriptional state. Contrary to previous work showing an association of invasiveness with high p38 levels in BRAF-mutated melanoma, there was no correlation of p38 expression with NRAS-mutant melanoma invasion, highlighting the difference in BRAF and NRAS-driven melanomas. Although the role of p38 has been reported to be that of both tumor suppressor and oncogene, we show here that p38 specifically plays the role of a tumor suppressor in NRAS-mutant melanoma. Both the transient and stable activation of p38 elicits phosphorylation of mTOR, reported to be a master switch in regulating autophagy. Indeed, we observed a correlation between elevated levels of phosphorylated mTOR and a reduction in LC3 conversion (LCII/LCI), indicative of suppressed autophagy. Furthermore, a reduction in actin intensity in p38-high cells strongly suggests a role of mTOR in regulating actin and a remodeling in the NRAS-mutant melanoma cells. Therefore, p38 plays a tumor suppressive role in NRAS-mutant melanomas at least partially through the mechanism of mTOR upregulation, suppressed autophagy, and reduced actin polymerization. One or more combinations of MEK inhibitors with either anisomycin, rapamycin, chloroquine/bafilomycin, and cytochalasin modulate p38 activation, mTOR phosphorylation, autophagy, and actin polymerization, respectively, and they may provide an alternate route to targeting NRAS-mutant melanoma.

Yap governs a lineage-specific neuregulin1 pathway-driven adaptive resistance to RAF kinase inhibitors

BackgroundInactivation of the Hippo pathway promotes Yap nuclear translocation, enabling execution of a transcriptional program that induces tissue growth. Genetic lesions of Hippo intermediates only identify a minority of cancers with illegitimate YAP activation. Yap has been implicated in resistance to targeted therapies, but the mechanisms by which YAP may impact adaptive resistance to MAPK inhibitors are unknown.MethodsWe screened 52 thyroid cancer cell lines for illegitimate nuclear YAP localization by immunofluorescence and fractionation of cell lysates. We engineered a doxycycline (dox)-inducible thyroid-specific mouse model expressing constitutively nuclear YAPS127A, alone or in combination with endogenous expression of either HrasG12V or BrafV600E. We also generated cell lines expressing dox-inducible sh-miR-E-YAP and/or YAPS127A. We used cell viability, invasion assays, immunofluorescence, Western blotting, qRT-PCRs, flow cytometry and cell sorting, high-throughput bulk RNA sequencing and in vivo tumorigenesis to investigate YAP dependency and response of BRAF-mutant cells to vemurafenib.ResultsWe found that 27/52 thyroid cancer cell lines had constitutively aberrant YAP nuclear localization when cultured at high density (NU-YAP), which rendered them dependent on YAP for viability, invasiveness and sensitivity to the YAP-TEAD complex inhibitor verteporfin, whereas cells with confluency-driven nuclear exclusion of YAP (CYT-YAP) were not. Treatment of BRAF-mutant thyroid cancer cells with RAF kinase inhibitors resulted in YAP nuclear translocation and activation of its transcriptional output. Resistance to vemurafenib in BRAF-mutant thyroid cells was driven by YAP-dependent NRG1, HER2 and HER3 activation across all isogenic human and mouse thyroid cell lines tested, which was abrogated by silencing YAP and relieved by pan-HER kinase inhibitors. YAP activation induced analogous changes in BRAF melanoma, but not colorectal cells.ConclusionsYAP activation in thyroid cancer generates a dependency on this transcription factor. YAP governs adaptive resistance to RAF kinase inhibitors and induces a gene expression program in BRAFV600E-mutant cells encompassing effectors in the NRG1 signaling pathway, which play a central role in the insensitivity to MAPK inhibitors in a lineage-dependent manner. HIPPO pathway inactivation serves as a lineage-dependent rheostat controlling the magnitude of the adaptive relief of feedback responses to MAPK inhibitors in BRAF-V600E cancers.

PRAME Expression in Cancer. A Systematic Immunohistochemical Study of >5800 Epithelial and Nonepithelial Tumors.

Preferentially expressed antigen in melanoma (PRAME) is considered a useful marker in the differential diagnosis between malignant melanoma and its melanocytic mimics. Recently PRAME expression was documented in nonmelanocytic tumors, but much of the data are based on mRNA studies. This investigation evaluated PRAME expression in the spectrum of normal tissues and >5800 human tumors using immunohistochemistry and EP461 monoclonal antibody. In normal tissues, PRAME was expressed in the testis and proliferative endometrium. In tumors, PRAME was variably expressed in malignancies of different lineages. Among epithelial tumors, >50% of PRAME-positive lesions were found among endometrial carcinomas (82%), uterine serous carcinomas (82%), uterine carcinosarcomas (60%), ovarian clear cell carcinomas (90%), ovarian serous carcinomas (63%), adenoid cystic carcinomas (81%), seminomas (78%), thymic carcinomas (75%), and basal cell carcinomas (62%). In mesenchymal and neuroectodermal malignancies, PRAME was frequently expressed in synovial sarcoma (71%), myxoid liposarcoma (76%), neuroblastoma (61%) and metastatic melanoma (87%). Also, PRAME was consistently expressed in 4 melanomas that lacked all melanoma markers including S100 protein and SOX10 but harbored typical for melanoma BRAF or NRAS driver mutations. However, strong and diffuse PRAME immunoreactivity was seen in many types of nonmelanocytic poorly differentiated carcinomas and sarcomas. Based on this study, PRAME is a relatively unspecific immunohistochemical marker, which limits its use in diagnostic surgical pathology. However, immunohistochemistry is a reliable and unexpensive method useful in detecting PRAME-positive malignancies for potential immunotherapy.

LOCL-05 CEREBRAL METASTATIC LUNG CARCINOMA: EFFECT OF ALK- AND EGFR-MUTATION STATUS AND SURGICAL MANAGEMENT UPON CLINICAL OUTCOME

PURPOSEThere have been many advancements in the surgical and medical treatment of metastatic lung carcinoma. In the post-genomic era, new directed-oncological therapies such as monoclonal antibodies (mAbs) and tyrosine-kinase inhibitors (TKIs) may offer increased survival for lung carcinoma patients with EGFR- and ALK- mutations. No surgical series have investigated the role of these mutations upon patient survival in lung brain metastases (BM). METHODSWe performed a multi-site, retrospective study of all patients who had BM with primary lung cancer undergoing surgical resection at Emory University Hospital between January 2012 and March 2021. Driver mutational statuses were categorized as EGFR-amplified, ALK-rearranged, or wild-type from biopsied brain tissue. Descriptive, univariate, and multivariate survival analyses were performed. RESULTS95 patients (mean age: 65.8 ± 10.6) met the inclusion criteria. 6 (6.3%) had ALK-rearranged mutations and 19 (20.0%) had EGFR-amplified mutations. 9 (9.5%) received second line therapies in the form of TKIs and mAbs. The majority of patients who underwent craniotomies had gross total resection (GTR) (n=72, 79.1%) with 83.5% (95% CI: 71.2-90.8%) and 89.9% (95% CI: 74.9-96.2%) 1-year overall survival (OS) and progression-free survival (PFS), respectively. On univariate analysis, ALK-rearranged (HR: 2.92; 95% CI: 0.57-9.75; p-value = 0.230) and EGFR-amplified (HR: 0.56; 95% CI: 0.15-1.61; p-value = 0.260) mutations were not significantly associated with OS. CONCLUSIONAfter assessing ALK- and EGFR- mutations on OS, we found no benefit with mutational status, unlike other cancer types such as Melanoma BRAF mutations. Our low sample size of patients receiving targeted therapies may bias our measures of association to the null hypothesis. However, the OS and PFS in our cohort were better than earlier trials in literature, demonstrating the improvement in systemic lung metastasis therapy. We suspect that as further targeted therapies become available, OS and PFS for lung BM patients will continue to improve.