Wild-type Melanoma Research Articles

Targeted Therapy for Melanomas Without BRAF V600 Mutation.

Immune checkpoint inhibitors (ICIs) have revolutionized the treatment paradigm for patients with metastatic melanoma; however, there remains an unmet clinical need for alternative treatment options for those patients who are either intolerant or refractory to immunotherapy. Here we review the role and clinical efficacy of targeted therapies for BRAFV600 wild-type melanoma. Genomic analyses in BRAFV600 wild-type melanoma have previously identified driver mutations along the mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinase (PI3K)-AKT pathways that can be targeted with small molecule inhibitors. New drugs such as bispecific antibodies and antibody drug conjugates may have significant clinical activity even in rare subtypes of melanoma that are less responsive to ICIs. Historically, molecular-targeted therapies have modest clinical success in treating BRAFV600 wild-type melanoma; nevertheless, they may have a significant clinical role in select, genetically distinct groups of patients. Next-generation immunotherapies or immunomodulators may represent the latest breakthrough in the treatment of melanoma. Additional studies are needed to identify novel drug targets and synergistic drug combinations to expand treatment options and optimize clinical outcomes.

Seasonal variation in effect of anti-PD-1 initiation on overall survival among patients with advanced melanoma.

Melanoma is a highly immunogenic cancer, and circannual rhythms influence the activity of the immune system. We retrospectively collected information on all cases with metastatic melanoma (ocular melanoma excluded) that initiated treatment with BRAF-inhibitor-based therapy (BRAFi) or anti-PD-1 monotherapy (PD-1). Cases were divided in two groups based on treatment initiation in the summer half-year (April to September) or winter half-year (October to March). We collected a total of 1054 (BRAF-i) and 1205 (PD-1) patient cases. Median follow-up was 39.7 (BRAFi) and 47.5 (PD-1) months. We did not observe differences in outcomes across patients who were treated in summer versus winter in the BRAFi cohort. Furthermore, we did not observe significant differences in ORR, CRR, and PFS in the PD-1 cohort. However, in patients with BRAF wild-type disease of the PD-1 cohort, treatment initiation in summer was associated with an improved OS (mOS 39.7 months [summer] versus 21.3 months [winter]; HR 0.70, 95% CI 0.57-0.86, p=.0007). This result remained robust to multivariable proportional hazards adjustment (HR 0.70, 95% CI 0.57-0.87, p=.001). Initiation of immunotherapy in summer is associated with prolonged survival in patients with BRAF wild-type melanoma living in Denmark.

Phase 1b study of cobimetinib plus atezolizumab in patients with advanced BRAFV600 wild-type melanoma progressing on prior anti–programmed death-1 therapy

ObjectiveTo evaluate the efficacy and safety of cobimetinib plus atezolizumab in the treatment of patients with advanced BRAFV600 wild-type melanoma who had progressed on prior anti‒programmed death-1 (PD-1) therapy. Patients and methodsThis phase 1b, open-label, international multicentre study enrolled 3 cohorts. Herein, we report on patients in cohorts A and B who had progressed on prior anti‒PD-1 therapy. Patients in cohort A received cobimetinib 60 mg once daily for 21 days followed by a 7-day break and concurrent intravenous atezolizumab 840 mg every 2 weeks. Patients in cohort B received the same dosing regimen as cohort A except for cycle 1 in which patients received cobimetinib only for the first 14 days prior to initiation of atezolizumab on cycle 1 day 15. Coprimary end-points were objective response rate and disease control rate. Secondary end-points were duration of response, progression free survival and overall survival. ResultsBetween 19th June 2017 and 12th December 2018, 103 patients were enrolled. Median follow-up was 6.9 months (interquartile range, 4.8–10.1 months); objective response rate was 14.6% and disease control rate was 38.8% (95% confidence interval, 29.39–48.94). The median duration of response, progression-free survival and overall survival was 12.7 months, 3.8 months and 14.7 months, respectively. The most common adverse events were diarrhoea (75/103; 72.8%), dermatitis acneiform (57/103; 55.3%) and nausea (52/103; 50.5%). Thirty-four patients (33.0%) died: 33 (91.7%) due to progressive disease and one (1%) due to treatment-related oesophagitis. ConclusionsCombination therapy with cobimetinib and atezolizumab in patients with advanced BRAFV600 wild-type melanoma with disease progression on or after prior anti‒PD-1 therapy demonstrated limited activity. Clinical trial registrationThis study is registered with ClinicalTrials.gov; NCT03178851;

Common genetic variants associated with melanoma risk or naevus count in patients with wildtype MC1R melanoma.

Hypomorphic MC1R variants are the most prevalent genetic determinants of melanoma risk in the white population. However, the genetic background of patients with wildtype (WT) MC1R melanoma is poorly studied. To analyse the role of candidate common genetic variants on the melanoma risk and naevus count in Spanish patients with WT MC1R melanoma. We examined 753 individuals with WT MC1R from Spain (497 patients and 256 controls). We used OpenArray reverse-transcriptase polymerase chain reaction to genotype a panel of 221 common genetic variants involved in melanoma, naevogenesis, hormonal pathways and proinflammatory pathways. Genetic models were tested using multivariate logistic regression models. Nonparametric multifactor dimensionality reduction (MDR) was used to detect gene-gene interactions within each biological subgroup of variants. We found that variant rs12913832 in the HERC2 gene, which is associated with blue eye colour, increased melanoma risk in individuals with WT MC1R [odds ratio (OR) 1·97, 95% confidence interval (CI) 1·48-2·63; adjusted P < 0·001; corrected P < 0·001]. We also observed a trend between the rs3798577 variant in the oestrogen receptor alpha gene (ESR1) and a lower naevus count, which was restricted to female patients with WT MC1R (OR 0·51, 95% CI 0·33-0·79; adjusted P = 0·002; corrected P = 0·11). This sex-dependent association was statistically significant in a larger cohort of patients with melanoma regardless of their MC1R status (n = 1497; OR 0·71, 95% CI 0·57-0·88; adjusted P = 0·002), reinforcing the hypothesis of an association between hormonal pathways and susceptibility to melanocytic proliferation. Last, the MDR analysis revealed four genetic combinations associated with melanoma risk or naevus count in patients with WT MC1R. Our data suggest that epistatic interaction among common variants related to melanocyte biology or proinflammatory pathways might influence melanocytic proliferation in individuals with WT MC1R. What is already known about this topic? Genetic variants in the MC1R gene are the most prevalent melanoma genetic risk factor in the white population. Still, 20-40% of cases of melanoma occur in individuals with wildtype MC1R. Multiple genetic variants have a pleiotropic effect in melanoma and naevogenesis. Additional variants in unexplored pathways might also have a role in melanocytic proliferation in these patients. Epidemiological evidence suggests an association of melanocytic proliferation with hormonal pathways and proinflammatory pathways. What does this study add? Variant rs12913832 in the HERC2 gene, which is associated with blue eye colour, increases the melanoma risk in individuals with wildtype MC1R. Variant rs3798577 in the oestrogen receptor gene is associated with naevus count regardless of the MC1R status in female patients with melanoma. We report epistatic interactions among common genetic variants with a role in modulating the risk of melanoma or the number of naevi in individuals with wildtype MC1R. What is the translational message? We report a potential role of hormonal signalling pathways in melanocytic proliferation, providing a basis for better understanding of sex-based differences observed at the epidemiological level. We show that gene-gene interactions among common genetic variants might be responsible for an increased risk for melanoma development in individuals with a low-risk phenotype, such as darkly pigmented hair and skin.

Raman spectroscopy combined with deep learning for rapid detection of melanoma at the single cell level

Melanoma is an aggressive and metastatic skin cancer caused by genetic mutations in melanocytes, and its incidence is increasing year by year. Understanding the gene mutation information of melanoma cases is very important for its precise treatment. The current diagnostic methods for melanoma include radiological, pharmacological, histological, cytological and molecular techniques, but the gold standard for diagnosis is still pathological biopsy, which is time consuming and destructive. Raman spectroscopy is a rapid, sensitive and nondestructive detection method. In this study, a total of 20,000 Surface-enhanced Raman scattering (SERS) spectra of melanocytes and melanoma cells were collected using a positively charged gold nanoparticles planar solid SERS substrate, and a classification network system based on convolutional neural networks (CNN) was constructed to achieve the classification of melanocytes and melanoma cells, wild-type and mutant melanoma cells and their drug resistance. Among them, the classification accuracy of melanocytes and melanoma cells was over 98%. Raman spectral differences between melanocytes and melanoma cells were analyzed and compared, and the response of cells to antitumor drugs were also evaluated. The results showed that Raman spectroscopy provided a basis for the medication of melanoma, and SERS spectra combined with CNN classification model realized classification of melanoma, which is of great significance for rapid diagnosis and identification of melanoma.

Tumor Apolipoprotein E is a key checkpoint blocking anti-tumor immunity in mouse melanoma.

Immunotherapy is a key modality in the treatment of cancer, but many tumors remain immune resistant. The classic mouse model of B16-F10 melanoma is immune resistant even in the face of checkpoint inhibition. Apolipoprotein E (apoE), a known immune suppressant is strikingly elevated in many human tumors, but its role in cancer immunology is not defined. We investigated the role of apoE in the immune micro-environment using a mouse melanoma model. We demonstrate that ApoE is -highly expressed in wild-type B16-F10 melanoma and serum levels progressively increase as tumors grow. The conditioned media from wild type ApoE secreting melanoma cells suppress T-cell activation in vitro while this suppressive effect is absent in conditioned media from ApoE knock out tumor cells. Mechanistically, apoE induces IL-10 secreting dendritic cells and stimulates T-cell apoptosis and arrest partially via the lrp8 receptor. Ablating ApoE in mice inoculated with tumor cells enabled tumor cell rejection and was associated with induction of immune pathway activation and immune cell infiltration. Tumor secreted apoE appears to be a potent immune cell checkpoint and targeting apoE is associated with enhanced tumor immunity in the mouse melanoma model.

108 (PB098) - MYC inhibition by Omomyc unveils a prognostic gene signature in Melanoma

Background: Melanoma is one of the most frequent causes of cancer death and despite significant treatment improvements, non-responder patients and those who develop resistance constitute a significant proportion of patients still in dire need of alternative and improved therapeutic options. Omomyc is a MYC inhibitor designed by Dr. Soucek that is extensively preclinically validated and currently being tested in a Phase I/IIa Clinical Trial. We have seen potent antitumour and antimetastatic effects when Omomyc is transgenically expressed in melanoma cells. Material and methods: We performed microarray analysis on BRAF-mutated A375 and BRAF wild-type SkMel147 human melanoma cells in vivo after one week of Omomyc expression and analysed them in R with Bioconductor. We assessed the overlap on transcriptional reprogramming exerted by Omomyc and investigated how these genes are associated with patient prognosis, by retrieving clinical and transcriptional data from the Skin Cutaneous Melanoma TCGA, PanCancer Atlas study in cBioPortal. Results: We found 620 differentially expressed genes in common, 436 downregulated by Omomyc (DN) and 184 upregulated (UP). DN genes cluster into Cell Cycle, RNA processing, Metabolism and Chromatin modification, organisation, and remodelling, while UP genes are related to Immune Response and several subcellular processes. Expression lower than the median of 45% of the DN genes or higher than the median of 54% of the UP genes are associated with significantly increased survival. We then generated a 6-gene signature combining DN and UP genes, that clearly separates patients in good and bad prognosis cohorts. Those with simultaneous low expression of these DN genes and high expression of these UP genes have significantly increased OS (205 (162-NA) vs. 42 (28– 63) months, p = 7 × 10−7) Additionally, 63% of UP genes are enriched in patients with Good Prognosis whereas 56% of DN genes are enriched in Poor Prognosis patients. Thus, Omomyc in vivo-regulated genes overlap considerably with those genes enriched in patients pointing to a reasonable similarity among in vivo results with Omomyc and clinical and transcriptional data from patients. Finally, pre-ranked GSEA in these patients showed that those with Good Prognosis have lower expression of MYC target genes and genes involved in melanoma relapse and metastases, and increased expression of Adaptive Immune Response. Conclusion: We show that specific gene sets reflecting decreased MYC transcriptional activity, restricted metastatic capacity and increased immune response are identified in Omomyc-expressing melanoma xenografts and indicate patients with Good Prognosis. We can therefore define an Omomyc-derived gene signature that clearly discriminates patients with different prognoses, whose transcriptional signatures resemble those of vehicle or Omomyc-expressing tumours in vivo. Conflict of interest: Ownership: L. Soucek and M. Beaulieu are cofounders and shareholders of Peptomyc, a company focused on developing Myc inhibitors for cancer treatment. M.F. Zacarias Fluck, S. Casacuberta-Serra, and J.R. Whitfield are shareholders of Peptomyc S.L.

Mathematical modeling for the combination treatment of IFN-[formula omitted] and anti-PD-1 in cancer immunotherapy

When the immune-checkpoint programmed death-1 (PD-1) binds to its ligand programmed death ligand 1 (PD-L1) to form the complex PD-1-PD-L1, this complex inactivates immune cells resulting in cell apoptosis, downregulation of immune reaction, and tumor evasion. The antibody, anti-PD-1 or anti-PD-L1, blocks the PD-1-PD-L1 complex formation to restore the functions of T cells. Combination of anti-PD-1 with other treatment shows promising in different types of cancer treatments. Interferon-gamma (IFN-γ) plays an important role in immune responses. It is mainly regarded as a pro-inflammatory cytokine that promotes the proliferation of CD8+ T cell and cytotoxic T cell, enhances the activation of Th1 cells and CD8+ T cells, and enhances tumor elimination. However, recent studies have been discovering many anti-inflammatory functions of IFN-γ, such as promotion of the PD-L1 expression, T cell apoptosis, and tumor metastasis, as well as inhibition of the immune recognition and the killing rates by T cells. In this work, we construct a mathematical model incorporating pro-inflammatory and anti-inflammatory functions of IFN-γ to capture tumor growth under anti-PD-1 treatment in the wild type and IFN-γ null mutant melanoma. Our simulation results qualitatively fit experimental data that IFN-γ null mutant with anti-PD-1 obtains the highest tumor reduction comparing to IFN-γ null mutant without anti-PD-1 and wild type tumor with anti-PD-1 therapy. Moreover, our synergy analysis indicates that, in the combination treatment, the tumor volume decreases as either the dosage of anti-PD-1 increases or the IFN-γ production efficiency decreases. Thus, the combination of anti-PD-1 and IFN-γ blockade improves the tumor reduction comparing to the monotherapy of anti-PD-1 or the monotherapy of IFN-γ blockade. We also find a threshold curve of the minimal dosage of anti-PD-1 corresponding to the IFN-γ production efficiency to ensure the tumor reduction under the presence of IFN-γ.

Combining fiber optical tweezers and Raman spectroscopy for rapid identification of melanoma.

Cutaneous melanoma is a skin tumor with a high degree of malignancy and fatality rate, the incidence of which has increased in recent years. Therefore, a rapid and sensitive diagnostic technique of melanoma cells is urgently needed. In this paper, we present a new approach using fiber optical tweezers to manipulate melanoma cells to measure their Raman spectra. Then, combined with Principal Component Analysis and Support Vector Machines (PCA-SVM) classification model, to achieve the classification of common mutant, wild-type and drug-resistant melanoma cells. A total of 150 Raman spectra of 30 cells were collected from mutant, wild-type and drug-resistant melanoma cell lines, and the classification accuracy was 92%, 94%, 97.5%, respectively. These results suggest that the study of tumor cells based on fiber optical tweezers and Raman spectroscopy is a promising method for early and rapid identification and diagnosis of tumor cells.

848P Second-line systemic treatment for patients with advanced melanoma: Results from the prospective real-world study GEM1801

Information regarding charatcteristics and outcomes of second line treatment for patients with advanced melanoma is scarce since no randomized clinical trials are published in the post-targeted and immunotherapy era. Real-world data studies are capital for benchmarking patients´ outcomes in order to design clinical trials in this scenario. GEM1801 is a prospective observational real-world study that currently has included 551 patients with advanced melanoma treated in 37 centers from the Spanish Melanoma Group (GEM). This is a descriptive analysis of basal characteristics, progression free survival (PFS) and overall survival (OS) from patients that received second line treatment in GEM1801. 186 (33.8%) patients received a second line (2L) treatment. For the 186 patients receiving 2L, with a median follow up of 6.4 months (m) (95% CI: 4.9-9.8), median PFS was 4.7 m (95% CI: 3.5-5.8) and median OS 11.3 m (95% CI: 8.6-13.4) from the beginning of 2L. Additionally, 63 (33.9%) patients received treatment beyond 2L. Table summarizes the patients characteristics and their outcomes according to BRAF status and treatments. For the 365 (66.2%) patients that did not receive 2L, the reasons were: 249 (68.2%) no progression with first line; 110 (30.1%) death before 2L; 6 (1.6%) loss of follow up.Table: 848PCHARACTERISTIC AT 2LBRAF Mutated N= 99BRAF wildtype N= 82SEX female N (%)47 (47.5)33 (40.2)age (median, Rank)59 (23-90)69 (29-93)ECOG 0-1 N (%)87 (87.9)78 (95.1)LDH >UNL N (%)34 (34.3)26 (31.7)M1C-D N (%)62 (62.6)40 (48.8)2L targeted therapy (TT): N, (%)44 (44.4)2 (2.4)TT mPFS, m (95% CI)12.4 (6.1-NR)2 (NA-NA)TT mOS, m (95% CI)20 (11.5-NR)2.7 (NA-NA)2L immunotherapy (IT): N (%)46 (46.5)34 (41.5)IT mPFS, m (95% CI)5.3 (2.9-10.8)3.1 (2.3-8.2)IT mOS, m (95% CI)10.9 (3.7-NR)12.5 (8.1-NR)2L Chemotherapy (CT): N, (%)4 (4)29 (35.4)CT mPFS, m (95% CI)0.4 (0-NR)3.2 (2.7-5.5)CT mOS, m (95% CI)0.6 (0.2-NR)5.6 (3.9-15.2)2L non specified: N (%)5 (5.1)13 (15.9)Treatment beyond 2L N (%)27 (27.8)33 (39.3)There are 5 patients with unknown BRAF status. Open table in a new tab There are 5 patients with unknown BRAF status. Second line treatment is still a difficult clinical scenario, especially for patients with BRAF wildtype melanoma and with BRAF mutated that progress after targeted therapy. The results, remarkably for PFS, need to be improved, thereby clinical trials participation is encouraged.

BRAF and NRAS Mutation Status and Response to Checkpoint Inhibition in Advanced Melanoma.

Little is known about the effect of specific gene mutations on efficacy of immune checkpoint inhibitors in patients with advanced melanoma. All patients with advanced melanoma treated with first-line anti-PD-1 or ipilimumab-nivolumab between 2012 and 2021 in the nationwide Dutch Melanoma Treatment Registry were included in this cohort study. Objective response rate, progression-free survival (PFS), and overall survival (OS) were analyzed according to BRAF and NRAS status. A multivariable Cox model was used to analyze prognostic factors associated with PFS and OS. In total, 1764 patients received anti-PD-1 and 759 received ipilimumab-nivolumab. No significant differences in PFS were found in the anti-PD-1 cohort. In the ipilimumab-nivolumab cohort, median PFS was significantly higher for BRAF-mutant melanoma (9.9 months; 95% CI, 6.8 to 17.2) compared with NRAS-mutant (4.8 months; 95% CI, 3.0 to 7.5) and double wild-type (5.3 months; 95% CI, 3.6 to 7.1). In multivariable analysis, BRAF-mutant melanoma was significantly associated with a lower risk of progression or death in the ipilimumab-nivolumab cohort. Median OS was significantly higher for BRAF-mutant melanoma compared with NRAS-mutant and double wild-type melanoma for both immune checkpoint inhibitor regimens. Ipilimumab-nivolumab-treated patients with BRAF-mutant melanoma display improved PFS and OS compared with patients with NRAS-mutant and double wild-type melanoma. BRAF mutation status is a factor to consider while choosing between mono and dual checkpoint inhibition in advanced melanoma.

Genomic and Transcriptomic Analyses of NF1-Mutant Melanoma Identify Potential Targeted Approach for Treatment

There is currently no targeted therapy to treat NF1-mutant melanomas. In this study, we compared the genomic and transcriptomic signatures of NF1-mutant and NF1 wild-type melanoma to reveal potential treatment targets for this subset of patients. Genomic alterations were verified using qPCR, and differentially expressed genes were independently validated using The Cancer Genome Atlas data and immunohistochemistry. Digital spatial profiling with multiplex immunohistochemistry and immunofluorescence were used to validate the signatures. The efficacy of combinational regimens driven by these signatures was tested through invitro assays using low-passage cell lines. Pathogenic NF1 mutations were identified in 27% of cases. NF1-mutant melanoma expressed higher proliferative markers MK167 and CDC20 than NF1 wild-type (P= 0.008), which was independently validated both in The Cancer Genome Atlas dataset (P= 0.01, P= 0.03) and with immunohistochemistry (P=0.013, P= 0.036), respectively. Digital spatial profiling analysis showed upregulation of LY6E within the tumor cells (false discovery rate < 0.01, log2 fold change > 1), confirmed with multiplex immunofluorescence showing colocalization of LY6E in melanoma cells. The combination of MAPK/extracellular signal‒regulated kinase kinase and CDC20 coinhibition induced both cytotoxic and cytostatic effects, decreasing CDC20 expression in multiple NF1-mutant cell lines. In conclusion, NF1-mutant melanoma is associated with a distinct genomic and transcriptomic profile. Our data support investigating CDC20 inhibition with MAPK pathway inhibitors as a targeted regimen in this melanoma subtype.

Adjuvant Anti-PD-1 Antibody Therapy for Advanced Melanoma: A Multicentre Study of 78 Japanese Cases.

Anti-PD-1 antibodies (Abs) are among the optimal adjuvant therapies for melanoma at high risk of recurrence, especially BRAF wild-type melanoma, but the anti-tumour effects of anti-PD-1 Abs in the adjuvant setting for acral melanoma have not been evaluated previously. The aim of this study was to analyse the efficacy and safety profiles of anti-PD-1 Ab monotherapy in the adjuvant setting in an Asian population including a high ratio of acral melanoma. The efficacy and safety profiles of anti-PD-1 Ab monotherapy in the adjuvant setting were retrospectively analysed in 78 Japanese patients with advanced melanoma, including 31 cases (40%) of acral melanoma. Overall relapse-free survival was 60.3% (47 of 78 cases, 95% confidence interval (CI) 49.2-70.4%), and 39.7% of patients (31 of 78 patients, 95% CI 29.6-50.8%) relapsed during the adjuvant PD-1 Ab treatment. Six cases (7.9%) discontinued the protocol due to serious adverse events. One case (1.3%) discontinued the protocol due to trauma. The relapse-free survival of acral melanoma was 25.8%, whereas that of high cumulative sun damage was 60.0%, and that of low cumulative sun damage was 57.1%. The acral type had a significantly lower 12-month relapse-free survival than other cutaneous types (p = 0.029). The acral type appeared to be an independent prognostic factor on multivariate analysis (p = 0.015). Adverse events due to anti-PD-1 antibody were observed in 37.1% overall. The results of this study suggest that anti-PD-1 Ab therapy in the adjuvant setting is less effective for acral melanoma than for other cutaneous types.

NOTCH4 mutation as predictive biomarker for immunotherapy benefits in NRAS wildtype melanoma.

BackgroundNRAS wildtype melanoma accounts for approximately 80% of melanomas. Previous studies have shown that NRAS wildtype melanoma had higher response rates and better prognoses than NRAS-mutant patients following immunotherapy, while as major actors in tumor cells and tumor microenvironment (TME), the association between NOTCH family genes and response to immunotherapy in NRAS wildtype melanoma remains indistinct.ObjectiveWe aim to explore whether NOTCH family gene variation is associated with genomic factors in immune checkpoint inhibitor (ICI) response in NRAS wildtype melanoma and with clinical results in these patients.MethodThis research used genomic data of 265 NRAS wildtype ICI-pretreatment samples from five ICI-treated melanoma cohorts to analyze the relationship between NOTCH family gene mutation and the efficacy of ICI therapy.ResultsNRAS wildtype melanomas with NOTCH4-Mut were identified to be associated with prolonged overall survival (OS) in both the discovery (HR: 0.30, 95% CI: 0.11–0.83, P = 0.01) and validation cohorts(HR: 0.21, 95% CI: 0.07–0.68, P = 0.003). Moreover, NOTCH4-Mut melanoma had a superior clinical response in the discovery cohort (ORR, 40.0% vs 13.11%, P = 0.057) and validation cohort (ORR, 68.75% vs 30.07%, P = 0.004). Further exploration found that NOTCH4-Mut tumors had higher tumor mutation burden (TMB) and tumor neoantigen burden (TNB) (P <0.05). NOTCH4-Mut tumors had a significantly increased mutation in the DNA damage response (DDR) pathway. Gene set enrichment analysis revealed NOTCH4-Mut tumor enhanced anti-tumor immunity.ConclusionNOTCH4 mutation may promote tumor immunity and serve as a biomarker to predict good immune response in NRAS wildtype melanoma and guide immunotherapeutic responsiveness.

Ketogenic diets slow melanoma growth in vivo regardless of tumor genetics and metabolic plasticity

BackgroundGrowing evidence supports the use of low-carbohydrate/high-fat ketogenic diets as an adjunctive cancer therapy. However, it is unclear which genetic, metabolic, or immunological factors contribute to the beneficial effect of ketogenic diets. Therefore, we investigated the effect of ketogenic diets on the progression and metabolism of genetically and metabolically heterogeneous melanoma xenografts, as well as on the development of melanoma metastases in mice with a functional immune system.MethodsMice bearing BRAF mutant, NRAS mutant, and wild-type melanoma xenografts as well as mice bearing highly metastatic melanoma allografts were fed with a control diet or ketogenic diets, differing in their triglyceride composition, to evaluate the effect of ketogenic diets on tumor growth and metastasis. We performed an in-depth targeted metabolomics analysis in plasma and xenografts to elucidate potential antitumor mechanisms in vivo.ResultsWe show that ketogenic diets effectively reduced tumor growth in immunocompromised mice bearing genetically and metabolically heterogeneous human melanoma xenografts. Furthermore, the ketogenic diets exerted a metastasis-reducing effect in the immunocompetent syngeneic melanoma mouse model. Targeted analysis of plasma and tumor metabolomes revealed that ketogenic diets induced distinct changes in amino acid metabolism. Interestingly, ketogenic diets reduced the levels of alpha-amino adipic acid, a biomarker of cancer, in circulation to levels observed in tumor-free mice. Additionally, alpha-amino adipic acid was reduced in xenografts by ketogenic diets. Moreover, the ketogenic diets increased sphingomyelin levels in plasma and the hydroxylation of sphingomyelins and acylcarnitines in tumors.ConclusionsKetogenic diets induced antitumor effects toward melanoma regardless of the tumors´ genetic background, its metabolic signature, and the host immune status. Moreover, ketogenic diets simultaneously affected multiple metabolic pathways to create an unfavorable environment for melanoma cell proliferation, supporting their potential as a complementary nutritional approach to melanoma therapy.

Cytotoxicity, acute and sub-chronic toxicities of the fruit extract of Tetrapleura tetraptera (Schumm. & Thonn.) Taub. (Fabaceae)

BackgroundTetrapleura tetraptera is a medicinal spice traditionally used to treat cancer, diabetes, and several other ailments. This study analyzed the cytotoxicity of the dichloromethane methanol extract of T. tetraptera fruits (TTF) and its constituents. The toxicity profile of the TTF extract was also evaluated in rats.MethodsThe Cytotoxicity of this extract was evaluated using the resazurin reduction assay (RRA). Acute and sub-chronic toxicity studies were performed according to the protocol described by the Organisation for Economic Cooperation, and Development (OECD). Hematological, serum, and urine biochemical parameters, as well as histological sections of the liver and kidney, were also evaluated based on standard methods.ResultsThe TTF extract, compound 5, and the reference drug doxorubicin were active in all 9 tested cancer cell lines. The recorded IC50 ranged from 18.32 μM (against B16-F1 murine melanoma cells) to 36.18 μM (against SKMel-505 BRAF wildtype melanoma cells) for TTF, from 10.02 μM (towards MaMel-80a BRAF-V600E homozygous mutant melanoma cells) to 31.73 μM (against SKMel-28 BRAF-V600E homozygous mutant melanoma cells) for compound 5, and from 0.22 μM (against B16-F1 cells) to 9.39 μM (against SKMel-505 cells) for doxorubicin. The study of acute toxicity test showed that the lethal dose (LD50) of this extract was greater than 5000 mg/kg body weight. In the sub-chronic toxicity studies, variations were observed in some biochemical parameters, especially at higher doses.ConclusionTTF and its most active compound (5) are found to be potential cytotoxic agents, meanwhile, TTF was safe when given a single oral dose of 5000 mg/kg. However, caution is necessary in case of prolonged oral administration due to potential alterations of renal function at high doses (> 1000 mg/kg).

A novel 7 RNA-based signature for prediction of prognosis and therapeutic responses of wild-type BRAF cutaneous melanoma

BackgroundThe prognosis of wild-type BRAF cutaneous melanoma (WT Bf-CM) patients remains poor due to the lack of therapeutic options. However, few studies have investigated the factors contributing to the prognosis of WT Bf-CM patients.MethodsIn this paper, we proposed and validated a novel 7-RNA based signature to predict the prognosis of WT Bf-CM by analyzing the information from TCGA database.ResultsDependence of this signature to other clinical factors were verified and a nomogram was also drawn to promote its application in clinical practice. Functional analysis suggested that the predictive function of this signature might attribute to the prediction of the up-regulation of RNA splicing, transcription, and cellular proliferation in the high-risk group, which have been demonstrated to be linked to malignancy of cancer. Moreover, functional analysis and therapy response analysis supported that the prognosis is highly related to PI3K/Akt/mTOR pathway among WT Bf-CM patients.ConclusionCollectively, this study will provide a preliminary bioinformatics evidence for the molecular mechanism and potential drug targets that could improving WT Bf-CM prognosis.

Abstract 4054: Investigation of antitumor activity induced by antifungal agents in human metastatic melanoma cells

Abstract Melanoma remains as the most aggressive and lethal form of skin cancer for which new therapies are still needed. Current treatment options include conventional chemotherapy, immunotherapy, and targeted therapy. Disadvantages of these therapies, as well as other factors such as refractiveness, have contributed to poor outcomes in melanoma patients. Antifungal drugs potentially represent a novel class of anticancer agents. Our objective here was to analyze the ability of these agents to eradicate primary human metastatic melanoma cells. Antitumor effects were investigated using cell growth and proliferation assays, flow cytometry (FACS), and cell death assays. Wild-type human melanoma cell lines or those possessing the BRAF V600E mutation or the NRAS Q61R mutation were analyzed. Clotrimazole treatment led to profound decreases in cell growth in wild-type and NRAS mutant melanoma cells. Significant, but lesser effects were observed in BRAF mutants. However, treatment with econazole caused substantial reductions in cell growth in wild-type and BRAF mutant cells. These results were corroborated by cell death analyses and FACS, where clotrimazole treatment caused greater than 80% cell death in wild-type and NRAS mutant cells, while econazole caused greater than 75% cell death in BRAF mutants. Taken together, these preliminary studies indicated that clotrimazole and econazole were effective in inducing antitumor effects in human metastatic melanoma cell lines, but with differing degrees of efficacy. This data suggests that melanoma tumors of each known genotype can potentially be treated with antifungal agents. In conclusion, this study demonstrated the ability of antifungal agents to successfully treat human metastatic melanoma cells. Since each agent displayed differential effects for each type of mutation, it is possible that these antifungal agents affect different molecular targets in each melanoma cell line. These antifungal agents thus appear to be potential alternative treatments, as well as affordable and widely accessible therapies, for melanoma patients in the future. Citation Format: Lukas R. Jira, Hattie M. Foster, Hannah K. Hall, David W. Koh. Investigation of antitumor activity induced by antifungal agents in human metastatic 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 4054.

Abstract 5460: Preclinical characterization of QLH11906, a novel pan-RAF inhibitor

Abstract Background: The type 1/2 BRAF inhibitors (e.g., vemurafenib) have been used for patients with BRAFV600E-mutant tumors. But the application of the inhibitors was restricted to a few types of tumors (e.g., melanoma). Common adverse effects such as pruritus and skin papilloma have limited the usage. It could be attributed to the paradoxical activation of the MAPK pathway. The paradoxical activation also leads to tumor promotion in BRAF wild-type melanoma and skin toxicity in 30%-70% patients with KRAS mutations. Therefore, development of type II pan-RAF inhibitors which avoid MAPK paradoxical activation is necessary. Methods: The selective pan-RAF inhibitor QLH11906 was tested on A/B/C RAF kinases and a representative kinase panel. Both 2D and 3D proliferation studies were conducted for cell lines with BRAF and various KRAS mutations, followed by both immunoprecipitation and ERK phosphorylation assays to elucidate the mode of action. Combination studies with MEK inhibitors were implemented in both KRAS-mutant cell lines and BRAF/KRAS-mutant patient derived organoids (PDO). Single-agent and combination studies were also carried out in vivo on both BRAF/KRAS-mutant cell line-derived xenograft (CDX) and KRAS-mutant patient-derived xenograft (PDX) models. Results: QLH11906 inhibited wild-type A/B/C RAF and BRAFV600E with the IC50 of 4.9, 1.9, 0.5, and 1.1 nM, respectively, and showed no inhibition at 1 μM in a selectivity panel of 28 kinases except for DDR1 and DDR2. Under 2D culture condition, QLH11906 inhibited proliferation of various BRAF/KRAS-mutant cell lines (IC50 300-1700 nM) and suppressed ERK phosphorylation (IC50 20-600 nM). In 3D cultures, QLH11906 showed higher activity in proliferation suppression in both HCT116 (KRASG13D, IC50 24 nM 3D vs 1186 nM 2D) and Calu-6 (KRASQ61K, IC50 85 nM 3D vs 1411 nM 2D). In immunoprecipitation studies with HCT116 and Calu-6 cells, both BRAF homodimers and BRAF/CRAF heterodimers could be detected under the treatment of QLH11906. QLH11906 showed synergistic effect when combined with MEK inhibitors trametinib or cobimetinib in KRAS-mutant cell lines, including A549 (KRASG12S), Calu-6, HCT116, NCIH2122 (KRASG12C) and Panc1005 (KRASG12D), and 77%-99% tumor suppression was reached at the expected clinical Ctrough concentrations. Synergistic effects with trametinib were observed in PDO models including pancreatic cancers (KRASG12D and KRASG12V), colon cancers (KRASG12D and KRASG13D) and lung cancers (KRASG12V). QLH11906 also promoted tumor shrinkage in vivo as a single agent in Calu-6, HCT116, and Colo-205 (BRAFV600E) CDX models or combined with trametinib in PDX models (KRASG12C lung cancers and KRASG12C colon cancers) Conclusion: QLH11906 is a highly selective pan-RAF inhibitor with promising antitumor activity both in vitro and in vivo. It is hopeful to be a therapeutic agent for BRAF and KRAS-mutant tumor patients. Citation Format: Zhe Cheng, Fei Chen, Xile Liu, Cong Gao, Linchao Jia, Yuanzhi Lao. Preclinical characterization of QLH11906, a novel pan-RAF inhibitor [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 5460.

Abstract 4000: ERBB4 is sufficient and necessary for proliferation of BRAF WT melanoma cell lines

Abstract Approximately 50% of metastatic melanomas possess wild-type (WT) BRAF alleles. These BRAF-WT tumors are a significant clinical problem, despite the fact that these tumors commonly possess mutations in NF1 or RAS genes, as there are no targeted therapeutics for these tumors. Here we demonstrate that ERBB4 is sufficient and necessary for proliferation of BRAF WT melanoma cell lines, suggesting that disrupting ERBB4 signaling may be effective against BRAF WT melanomas. ERBB4 (HER4) is a member of the ERBB family of receptor tyrosine kinases, which includes the epidermal growth factor receptor (EGFR/ERBB1/HER1), ERBB2 (Neu/HER2), and ERBB3 (HER3). EGFR and ERBB2 are validated targets in a variety of solid tumors, whereas the roles that ERBB4 plays in human malignancies are ambiguous. Our previous in silico analyses of BRAF-WT tumor genomes in The Cancer Genome Atlas skin cutaneous melanoma (TCGA-SKCM) dataset suggest that ERBB4 mutation or overexpression cooperates with NF1 or RAS gene mutations to drive melanomas via activation of the PI3 kinase/AKT signaling pathway. These in silico analyses have also enabled us to prioritize the ERBB4 mutants found in BRAF-WT melanomas. We have tested this hypothesis using a panel of human BRAF-WT melanoma cell lines. We have shown that expression of wild-type ERBB4 causes increased clonogenic proliferation, whereas expression of a dominant-negative ERBB4 mutant (K751M) causes decreased clonogenic proliferation. These data indicate that ERBB4 is sufficient and necessary for clonogenic proliferation of BRAF-WT melanoma cell lines. Analgous studies are testing whether ERBB4 regulates anchorage independence in semi-solid medium and proliferation rates and saturation density in culture dishes. The BRAF-WT melanoma cell lines endogenously express ERBB4 ligands, EGFR, and ERBB2. Moreover, ERBB4 homodimers function as tumor suppressors, whereas ERBB4-EGFR and ERBB4-ERBB2 heterodimers are oncogenic. Thus, we are using gene silencing and pharmacologic tools to determine whether endogenous ERBB4 ligands, endogenous EGFR, or endogenous ERBB2 is required for the oncogenic activity of ERBB4. Furthermore, we are determining whether prioritized ERBB4 mutants found in BRAF-WT melanomas exhibit greater signaling and oncogenic activities than WT ERBB4. Consequently, these experiments will establish a functional role for ERBB4 mutations and expression in BRAF-WT melanomas and will identify potential strategies for disrupting oncogenic ERBB4 signaling in these tumors. Citation Format: Lauren M. Lucas, Vipasha Dwivedi, Rania H. Mohamedelhassan, Erin L. Harrell, Connor M. Kelley, Elizabeth L. Knerr, Jessica A. Markham, David J. Riese. ERBB4 is sufficient and necessary for proliferation of BRAF WT melanoma cell lines [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 4000.