Multiple clinical trials targeting the gut microbiome are being conducted to optimize treatment outcomes for immune checkpoint blockade (ICB). To improve the success of these interventions, understanding gut microbiome changes during ICB is urgently needed. Here through longitudinal microbiome profiling of 175 patients treated with ICB for advanced melanoma, we show that several microbial species-level genome bins (SGBs) and pathways exhibit distinct patterns from baseline in patients achieving progression-free survival (PFS) of 12 months or longer (PFS ≥12) versus patients with PFS shorter than 12 months (PFS <12). Out of 99 SGBs that could discriminate between these two groups, 20 were differentially abundant only at baseline, while 42 were differentially abundant only after treatment initiation. We identify five and four SGBs that had consistently higher abundances in patients with PFS ≥12 and <12 months, respectively. Constructing a log ratio of these SGBs, we find an association with overall survival. Finally, we find different microbial dynamics in different clinical contexts including the type of ICB regimen, development of immune-related adverse events and concomitant medication use. Insights into the longitudinal dynamics of the gut microbiome in association with host factors and treatment regimens will be critical for guiding rational microbiome-targeted therapies aimed at enhancing ICB efficacy.

Supplementary Figures S1 - S9, Table S1. Supplementary Figure S1 (related to Figure 1) shows TNFR expression in melanoma cells. Supplementary Figure S2 (related to Figure 2) shows an NF-κBα binding site in the MITF promoter and MITF target gene expression. Supplementary Figure S3 (related to Figure 3) shows macrophage differentiation and their effect on melanoma cells, keratinocytes and fibroblasts. Supplementary Figure S4 (related to Figure 3) shows M1 and M2 macrophage marker expression in melanoma. Supplementary Figure S5 (related to Figure 4) shows that MEK inhibition does not affect the protective function of macrophages. Supplementary Figure S6 (related to Figure 4) BRAFi and MEKi induced changes in macrophage marker expression in vivo. Supplementary Figure S7 (related to Figure 6) shows that IKK inhibition and MEK inhibition synergise in vitro. Supplementary FigureS8 (related to Figure 6) shows that TNFα protects from BRAF inhibition and IKK and BRAF inhibition synergise in vitro. Supplementary Figure S9 (related to Figure 7) shows the expression of macrophage markers in IKKi treated allografts. Supplementary Table S1 lists patient details.

Supplementary Figures 1-4 from A Mouse Model of Melanoma Driven by Oncogenic KRAS

Supplementary Figure S1. IGV visualization for whole exome sequencing of codon Q61 of NRAS in the pre-treatment tumor vs. the relapsed tumor in patient 1. The red color indicates the A>G for the p.Q61R mutation in the tumor. Supplementary Figure S2. A. IGV visualization for the targeted sequencing of BRAF and PI3KCA in patient 6. B. IGV visualization for the targeted sequencing of BRAF and NRAS in patient 7. Supplementary Figure S3. Response of patient 5 PDX to PLX4720.

Supplementary Tables 1-2 from A Mouse Model of Melanoma Driven by Oncogenic KRAS

ICIs have transformed the prognosis of MM, but our understanding of mechanisms of response and resistance remains incomplete. The 100,000 Genomes Project presents a melanoma cohort with whole genome sequencing (WGS) and detailed clinical annotation. 97 patients received ICIs in the 1L metastatic disease setting; ipilimumab-nivolumab (60%), anti-PD-1 monotherapy (32%), and ipilimumab (8%). Response rate was 45%. Median follow-up was 30.6 months, median time to treatment failure (TTF) 9.6 months and median overall survival (OS) not reached. From WGS, neoantigen calling, CNA and tumour purity analyses were performed, predictive variables were included in logistic regression. Incorporating MHC-II neoantigen load, CNAs and tumour purity, our multivariate model predicted response with AUC 0.86 Responders were associated with a higher number of predicted clonal neoantigens (p=0.0029), driven by MHC-II neoantigens (p=2e-04), and were more statistically significant biomarkers than TMB (p=0.016). A higher total neoantigen burden (p=0.02) and MHC-II neoantigen burden (p=0.06), but not higher TMB, were associated with superior OS. Independently, low tumour purity was associated with response (p=0.005) and superior OS (p=0.04) Non-responders harboured CNAs in key melanoma driver genes, including CDKN2A loss of function (p=0.04), and TERT gain (p=0.03) which was associated with inferior TTF (p=0.02) Genomic imprinting has established links to cancer pathogenesis, most notably at 11p15.5. In our cohort, loss of heterozygosity (LOH) at 11p.15.5 was associated with response (p=0.007), superior TTF (p=0.05), and higher MHC-II neoantigen load (p=0.04), while non-LOH loss at the same loci was associated with non-response (p=0.003), inferior TTF (p=0.007) and OS (p=0.02), highlighting that different classes of loss can lead to opposing biological sequelae. LOH with loss of the active allele and persistence of the imprinted allele will lead to non-expression of the oncogene. External validation with matched WGS and RNA will be presented at the conference. In our cohort, 1L ICI response in melanoma was driven by MHC-II neoantigens, CNAs and tumour purity.

Background: Immune cell-driven anti-cancer activity is paramount for effective responses to checkpoint inhibitors (ICB). However, the contribution of the different immune cell subsets in the circulation and within the tumour is poorly understood.Materials and methods: To elucidate the role of the different cell subsets in anti-tumour responses elicited by ICB, we performed single-cell analysis of the transcriptome and surface proteome of paired pre-and early on-treatment metastatic melanoma tumour biopsies and matched peripheral blood mononuclear cell samples. We next compared the survival of met-astatic melanoma patients treated with ICB according to the abundance of pre-treatment tumour-infiltrating B cell clonotypes.Results: We identified cell clusters associated with disease control or progression, defined dif-ferential expression of biological pathways likely involved in the immune awakening against the tumour and examined how cell-cell communication patterns between the tumour cell sub-sets change during treatment. Furthermore, we discovered that B cells (immunoglobulin expression and abundance of B cell clonotypes) discriminate the clinical response after ICB and propose that B cells likely contribute to anti-tumour immunity by antigen presentation through major histocompatibility complex molecules. Finally, we demonstrated that the abun-dance of tumour-infiltrating B cell clonotypes at baseline identifies two distinct risk groups, a finding that we confirmed in an independent cohort.Conclusions: Our exploratory translational study provides new insights on the mechanistic role of B cells in anti-melanoma immunity during treatment with ICB. Additionally, we sup-port pre-treatment B cell tumour infiltration as a promising prognostic biomarker to be further validated as a tool for clinical risk stratification.(c) 2022 The Authors. Published by Elsevier Ltd.

The composition of the gut microbiome has been associated with clinical responses to immune checkpoint inhibitor (ICI) treatment, but there is limited consensus on the specific microbiome characteristics linked to the clinical benefits of ICIs. We performed shotgun metagenomic sequencing of stool samples collected before ICI initiation from five observational cohorts recruiting ICI-naive patients with advanced cutaneous melanoma ( n = 165). Integrating the dataset with 147 metagenomic samples from previously published studies, we found that the gut microbiome has a relevant, but cohort-dependent, association with the response to ICIs. A machine learning analysis confirmed the link between the microbiome and overall response rates (ORRs) and progression-free survival (PFS) with ICIs but also revealed limited reproducibility of microbiome-based signatures across cohorts. Accordingly, a panel of species, including Bifidobacterium pseudocatenulatum , Roseburia spp. and Akkermansia muciniphila , associated with responders was identified, but no single species could be regarded as a fully consistent biomarker across studies. Overall, the role of the human gut microbiome in ICI response appears more complex than previously thought, extending beyond differing microbial species simply present or absent in responders and nonresponders. Future studies should adopt larger sample sizes and take into account the complex interplay of clinical factors with the gut microbiome over the treatment course.

Although identified as the key environmental driver of common cutaneous melanoma, the role of ultraviolet radiation (UVR)-induced DNA damage in mucosal melanoma is poorly defined. We analyze 10 mucosal melanomas of conjunctival origin by whole genome sequencing and our data shows a predominance of UVR-associated single base substitution signature 7 (SBS7) in the majority of the samples. Our data shows mucosal melanomas with SBS7 dominance have similar genomic patterns to cutaneous melanomas and therefore this subset should not be excluded from treatments currently used for common cutaneous melanoma.

Tumor infiltration by T cells is paramount for effective anti-cancer immune responses. We hypothesized that the T cell receptor (TCR) repertoire of tumor infiltrating T lymphocytes could therefore be indicative of the functional state of these cells and determine disease course at different stages in cancer progression. Here we show that the diversity of the TCR of tumor infiltrating T cell at baseline is prognostic in various cancers, whereas the TCR clonality of T cell infiltrating metastatic melanoma pre-treatment is predictive for activity and efficacy of PD1 blockade immunotherapy.

Summary In 1967, Sandy Posey pronounced that sunglasses are essential beachwear ( https://www.youtube.com/watch?v=4HVBEb-GA1Y ). Now, whole-genome sequencing reveals that ultraviolet radiation (UVR) can contribute to melanomas in the iris and conjunctiva, data that provide a molecular explanation for why it is important to protect our eyes from exposure to UVR.

BACKGROUND:Precision immuno-oncology approaches are needed to improve cancer care. We recently demonstrated that in patients with metastatic melanoma, an increase of clonality or diversity of the T cell receptor (TCR) repertoire of peripheral T cells following one cycle of immunotherapy is coincident with response to immune-checkpoint blockade (ICB). We also identified a subset of peripheral CD8+ immune-effector memory T cells (TIE cells) whose expansion was associated with response to ICB and increased overall survival. To improve our understanding of peripheral T cell dynamics, we examined the clinical correlates associated with these immune signatures. METHODS:Fifty patients with metastatic melanoma treated with first-line anti-PD-1 ICB were included. We analysed TCR repertoire and peripheral TIE cell dynamics by age before treatment (T0) and after the first cycle of treatment at week 3 (W3). RESULTS:We observed a correlation between TIE abundance and age at T0 (r = 0.40), which reduced following treatment at W3 (r = 0.07). However, at W3, we observed two significantly opposing patterns (p = 0.03) of TCR repertoire rearrangement in patients who responded to treatment, with patients ≥70 years of age showing an increase in TCR clonality and patients <70 years of age showing an increase in TCR diversity. CONCLUSIONS:We demonstrate that immunotherapy-induced immune-awakening patterns in patients with melanoma are age-related and may impact patient response to ICB, and thus have implications for biomarker development and planning of personalised therapeutic strategies.

Precursor melanoma lesions harbor somatic alterations that activate the MAPK signaling pathway, most commonly through the driver oncogenes BRAF and NRAS (Shain et al., 2018Shain A.H. Joseph N.M. Yu R. Benhamida J. Liu S. Prow T. et al.Genomic and transcriptomic analysis reveals incremental disruption of key signaling pathways during melanoma evolution.Cancer Cell. 2018; 34: 45-55.e4Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). The identification of the additional genetic events required to fully trigger malignant progression has been challenging because of the high mutation burden in cutaneous melanomas (Cancer Genome Atlas Network, 2015Cancer Genome Atlas NetworkGenomic classification of cutaneous melanoma.Cell. 2015; 161: 1681-1696Abstract Full Text Full Text PDF PubMed Scopus (1476) Google Scholar). Large-scale sequencing studies revealed that most of the mutations present in cutaneous melanomas carry a signature of UVR-induced DNA damage, consistent with the observation that cutaneous melanoma commonly arises on sun-exposed skin (Shain and Bastian, 2016Shain A.H. Bastian B.C. From melanocytes to melanomas.Nat Rev Cancer. 2016; 16: 345-358Crossref PubMed Scopus (322) Google Scholar). We recently described how different patterns of exposure and UVR wavelengths can affect tumor incidence and survival in a transgenic BRAFV600E melanoma mouse model (Trucco et al., 2019Trucco L.D. Mundra P.A. Hogan K. Garcia-Martinez P. Viros A. Mandal A.K. et al.Ultraviolet radiation–induced DNA damage is prognostic for outcome in melanoma.Nat Med. 2019; 25: 221-224Crossref PubMed Scopus (34) Google Scholar). Whole-exome sequencing of the tumors in our BRAFV600E model revealed recurrent mutations in the Map3k1 gene across all experimental cohorts. Specifically, in 78 melanomas from Braf+/LSL-V600E;Tyr::CreERT2+/o (BrafV600E) mice previously reported (Trucco et al., 2019Trucco L.D. Mundra P.A. Hogan K. Garcia-Martinez P. Viros A. Mandal A.K. et al.Ultraviolet radiation–induced DNA damage is prognostic for outcome in melanoma.Nat Med. 2019; 25: 221-224Crossref PubMed Scopus (34) Google Scholar), we found 37 nonsynonymous Map3k1 mutations in 34 samples (43.6%) (Figure 1a, Supplementary Table S1), including two tumors from non–UVR-exposed mice, three from mice exposed to UVA (350–400 nm), 10 from mice exposed to UVB (310–315 nm), and 19 from mice exposed to broadband UVR (280–380 nm) (Figure 1a). Overall, the tumors with Map3k1 mutations presented a higher number of missense single nucleotide variants than those without Map3k1 mutations (mean: 395.9 vs. 204.9 single nucleotide variants, P = 0.0291; Figure 1a, Supplementary Table S2). A total of 21 Map3k1 mutations (56.8%) in 19 samples were C-to-T nucleotide transitions at dipyrimidines, characteristic of UVR signature mutations (Figure 1a), but we did not find a correlation between the presence of mutations in Map3k1 and exposure to a particular UVR wavelength (P = 0.0549; Supplementary Table S2). MAP3K1 is unique in having both serine-threonine kinase and E3-ligase functions, allowing it to regulate protein phosphorylation and ubiquitin-mediated proteasome degradation (Suddason and Gallagher, 2015Suddason T. Gallagher E. A RING to rule them all? Insights into the Map3k1 PHD motif provide a new mechanistic understanding into the diverse roles of Map3k1.Cell Death Differ. 2015; 22: 540-548Crossref PubMed Scopus (17) Google Scholar). We found that 75.7% (28/37) of the mutations in Map3k1 affected the RING domain of the protein, which contains the E2-binding site for ubiquitin-conjugating enzymes (Figure 1b). The most recurrent codon alterations were at structural residues, including conserved cysteines 438, 454, and 483, involved in the coordination of zinc, and the proline 484, which are essential for function (Figure 1c and d) (Lu et al., 2002Lu Z. Xu S. Joazeiro C. Cobb M.H. Hunter T. The PHD domain of MEKK1 acts as an E3 ubiquitin ligase and mediates ubiquitination and degradation of ERK1/2.Mol Cell. 2002; 9: 945-956Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar). To investigate the role of Map3k1 in melanomagenesis, we generated Tyr::CreERT2+/o;Map3k1fl/fl (Map3k1fl/fl) mice to allow conditional deletion of Map3k1 in melanocytes following topical application of tamoxifen to the dorsal skin of juvenile mice. By itself, Map3k1 deletion did not induce any pigmented lesions or skin tumors in the mice (Figure 2a), indicating that the loss of Map3k1 alone is not sufficient to induce melanocyte proliferation. However, when Map3k1fl/fl mice were crossed to mice carrying a conditional-inducible Braf+/LSL-V600E allele, the topical administration of tamoxifen induced a progressive melanocytic hyperplasia leading to the development of pigmented dermal nevi (Figure 2a). Notably, the nevi from BrafV600E;Map3k1fl/fl mice were more abundant (P = 0.011) and larger (P < 0.0001) than the nevi in BrafV600E mice (Figure 2b and c). Moreover, whereas 72.7% (40/55) of BrafV600E mice developed melanoma with a median latency of 32 weeks, BrafV600E;Map3k1fl/fl mice developed tumors with 95.7% penetrance (22/23) and a median latency of only 8 weeks (P < 0.0001; Figure 2d). These differences were also reflected in tumor burden, with BrafV600E;Map3k1fl/fl mice presenting more melanomas on average than the BrafV600E mice (P < 0.0001; Figure 2e). Note that the tumors from both cohorts shared similar histopathological features and presented as nonpigmented dermal lesions composed of tumor cells with atypical spindle dendritic cell morphology, low cytoplasmic content, and condensed small nuclei that stained positive for S100 (Supplementary Figure S1). Previous studies have indicated that MAP3K1 acts as a negative regulator of the MAPK signaling cascade through MAPK kinase SUMOylation or extracellular signal–regulated kinase (ERK) ubiquitination dependent on its E3-ligase activity (Kubota et al., 2011Kubota Y. O’Grady P. Saito H. Takekawa M. Oncogenic Ras abrogates MEK SUMOylation that suppresses the ERK pathway and cell transformation.Nat Cell Biol. 2011; 13: 282-291Crossref PubMed Scopus (44) Google Scholar, Lu et al., 2002Lu Z. Xu S. Joazeiro C. Cobb M.H. Hunter T. The PHD domain of MEKK1 acts as an E3 ubiquitin ligase and mediates ubiquitination and degradation of ERK1/2.Mol Cell. 2002; 9: 945-956Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar). Consistent with this, when we evaluated the levels of phosphorylated ERK1/2, we observed that the tumors in BrafV600E;Map3k1fl/fl mice and the Map3k1 mutant tumors in BrafV600E mice presented higher phosphorylated ERK1/2 immunostaining than Map3k1 wild-type tumors from BrafV600E mice (P ≤ 0.016; Figure 2f and g). Therefore, our data shows that Map3k1 deletion cooperates with oncogenic BRAFV600E to promote melanomagenesis in mice, and this is associated with enhanced ERK activation. To assess the relevance of our findings for the human disease, we evaluated the expression of MAP3K1 in melanocytic lesions from donor patients and observed decreased MAP3K1 transcript levels in primary melanomas compared with benign nevi (P = 0.0009; Supplementary Figure S2a). Next, we analyzed MAP3K1 in publicly available genomic data from human melanomas. The MAP3K1 mutation rates in melanoma ranged from 1.4% to 5.3% and were scattered throughout the protein (Cancer Genome Atlas Network, 2015Cancer Genome Atlas NetworkGenomic classification of cutaneous melanoma.Cell. 2015; 161: 1681-1696Abstract Full Text Full Text PDF PubMed Scopus (1476) Google Scholar, Catalanotti et al., 2017Catalanotti F. Cheng D.T. Shoushtari A.N. Johnson D.B. Panageas K.S. Momtaz P. et al.PTEN loss-of-function alterations are associated with intrinsic resistance to BRAF inhibitors in metastatic melanoma.JCO Precis Oncol. 2017; 1: 1-15Google Scholar, Hayward et al., 2017Hayward N.K. Wilmott J.S. Waddell N. Johansson P.A. Field M.A. Nones K. et al.Whole-genome landscapes of major melanoma subtypes.Nature. 2017; 545: 175-180Crossref PubMed Scopus (531) Google Scholar, Hodis et al., 2012Hodis E. Watson I.R. Kryukov G.V. Arold S.T. Imielinski M. Theurillat J.P. et al.A landscape of driver mutations in melanoma.Cell. 2012; 150: 251-263Abstract Full Text Full Text PDF PubMed Scopus (1618) Google Scholar, Hugo et al., 2016Hugo W. Zaretsky J.M. Sun L. Song C. Moreno B.H. Hu-Lieskovan S. et al.Genomic and transcriptomic features of response to anti-PD-1 therapy in metastatic melanoma.Cell. 2016; 165: 35-44Abstract Full Text Full Text PDF PubMed Scopus (1217) Google Scholar, Liang et al., 2017Liang W.S. Hendricks W. Kiefer J. Schmidt J. Sekar S. Carpten J. et al.Integrated genomic analyses reveal frequent TERT aberrations in acral melanoma.Genome Res. 2017; 27: 524-532Crossref PubMed Scopus (59) Google Scholar, Van Allen et al., 2014Van Allen E.M. Wagle N. Sucker A. Treacy D.J. Johannessen C.M. Goetz E.M. et al.The genetic landscape of clinical resistance to RAF inhibition in metastatic melanoma.Cancer Discov. 2014; 4: 94-109Crossref PubMed Google Scholar) (Supplementary Figure S2b and c). In The Cancer Genome Atlas cohort, the patients whose melanomas expressed MAP3K1 in the lowest 25th percentile presented significantly reduced overall survival (P = 0.0029; Figure 2h), independent of disease stage at diagnosis (P = 0.1427; Supplementary Table S3). This trend extended across different genomic subtypes (Supplementary Table S4). Additionally, as in our mice, we observed an inverse correlation between MAP3K1 expression and phosphorylated ERK1/2 (rs = −0.1191, P = 0.0253) and total ERK2 (rs = −0.1432, P = 0.0070) protein levels extracted from RNA sequencing and reverse-phase protein array data, respectively (Supplementary Figure S3). Combined, these results suggest that MAP3K1 plays a negative regulatory role through ERK in melanomagenesis. In summary, we show that Map3k1 is frequently mutated in our BRAFV600E-driven melanoma mouse model and that Map3k1 deletion cooperates with BRAFV600E to induce melanoma. Map3k1 loss is associated with increased ERK phosphorylation, MAP3K1 expression is decreased in melanomas compared with nevi, and low MAP3K1 is associated with poorer survival in patients. Together, our findings show that MAP3K1 deficiency accelerates melanomagenesis in mice and suggests a similar role in humans. Conversely, a transposon mutagenesis screen identified Map3k1 as a melanoma driver independent of BRAF activation (Ni et al., 2013Ni T.K. Landrette S.F. Bjornson R.D. Bosenberg M.W. Xu T. Low-copy piggyBac transposon mutagenesis in mice identifies genes driving melanoma.Proc Natl Acad Sci USA. 2013; 110: E3640-E3649Crossref PubMed Scopus (23) Google Scholar), and MAP3K1 is amplified in desmoplastic melanomas, but the significance of this is unclear (Shain et al., 2015Shain A.H. Garrido M. Botton T. Talevich E. Yeh I. Sanborn J.Z. et al.Exome sequencing of desmoplastic melanoma identifies recurrent NFKBIE promoter mutations and diverse activating mutations in the MAPK pathway.Nat Genet. 2015; 47: 1194-1199Crossref PubMed Scopus (137) Google Scholar). Thus, MAP3K1 may play complex roles in melanoma, but our data show that its loss contributes to cutaneous melanomagenesis, possibly by modulation of BRAF–MAPK kinase–ERK signaling. Further studies are warranted to elucidate the context-dependent role that MAP3K1 plays in different melanoma subtypes. All procedures involving animals were performed in agreement with the Home Office regulations under the Animals Scientific Procedures Act 1986 (license PPLP671A5B06) and reported in accordance with the ARRIVE guidelines. Procedures were approved by the Animal Welfare and Ethical Review Body of the CRUK Manchester Institute, and tumor volumes did not exceed the guidelines set by the Committee of the National Cancer Research Institute (Workman et al., 2010Workman P. Aboagye E.O. Balkwill F. Balmain A. Bruder G. Chaplin D.J. et al.Guidelines for the welfare and use of animals in cancer research.Br J Cancer. 2010; 102: 1555-1577Crossref PubMed Scopus (851) Google Scholar). Whole-exome sequencing data from mouse samples analyzed in this article can be found in the European Nucleotide Archive (https://www.ebi.ac.uk/ena) under accession code PRJEB30941. Lucas D. Trucco: https://orcid.org/0000-0003-2141-7844 Piyushkumar A. Mundra: https://orcid.org/0000-0001-7769-2939 Pablo García-Martínez: https://orcid.org/0000-0003-0074-2672 Kate Hogan: https://orcid.org/0000-0002-3966-4440 Franziska Baenke: https://orcid.org/0000-0001-9389-4688 Nathalie Dhomen: https://orcid.org/0000-0003-1961-2173 Valeria Pavet: https://orcid.org/0000-0002-4472-6503 Richard Marais: https://orcid.org/0000-0001-7484-4183 Richard Marais is a consultant for Pfizer. As a former employee of the Institute of Cancer Research in London, he may benefit financially from any program that is commercialized. All other authors state no conflict of interest. This work was supported by CRUK Manchester Institute (A27412 and A22902) and the European Research Council (ERC Advanced Grant agreement No. 671262) to RM. The authors thank Nic Jones for providing the Map3k1tm1a(KOMP)Wtsi mouse strain and the CRUK Manchester Institute core facilities for their technical assistance. Conceptualization: LDT, KH, RM; Data Curation: LDT, PAM, PGM, KH, FB; Formal Analysis: LDT, PAM, PGM; Funding Acquisition: RM; Investigation: LDT, KH, FB; Methodology: LDT, KH; Project Administration: ND, VP; Supervision: RM; Visualization: LDT; Writing - Original Draft Preparation: LDT; Writing - Review and Editing: LDT, PAM, PGM, KH, FB, ND, VP, RM We filtered the samples carrying mutations in Map3k1 identified by whole-exome sequencing from the melanoma cohorts from Braf+/LSL-V600E;Tyr::CreERT2+/o mice described in our previous study (Trucco et al., 2019Trucco L.D. Mundra P.A. Hogan K. Garcia-Martinez P. Viros A. Mandal A.K. et al.Ultraviolet radiation–induced DNA damage is prognostic for outcome in melanoma.Nat Med. 2019; 25: 221-224Crossref PubMed Scopus (34) Google Scholar). For Sanger sequencing validation, a region containing exons 7 and 8 of Map3k1 was amplified from genomic tumor DNA using Phusion High-Fidelity DNA Polymerase (New England Biolabs, Ipswich, MA) with the following primers: forward 5′-GACTTCAAAAACATCAAAAACTAGACTAAAAAATG-3′ and reverse 5′-CCATGAAGGCCTAGAGGACATC-3′, according to the manufacturer’s protocol. The PCR products were purified using the Zymoclean Gel DNA Recovery Kit (Zymo Research, Irvine, CA) and then sequenced on a 3130xl Genetic Analyzer (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA). Sequences were visualized using SnapGene Viewer v. 4.3.11 (GSL Biotech LLC, Chicago, IL). A homology model of the RING domain of MAP3K1, spanning the amino acid residues from E435 to K489 of the mouse protein, was built using SWISS-MODEL (Waterhouse et al., 2018Waterhouse A. Bertoni M. Bienert S. Studer G. Tauriello G. Gumienny R. et al.SWISS-MODEL: homology modelling of protein structures and complexes.Nucleic Acids Res. 2018; 46: W296-W303Crossref PubMed Scopus (2946) Google Scholar). The crystal structure of the E3 ubiquitin-protein ligase FANCL was selected as a template for modeling based on the Global Model Quality Estimate score, coverage, and sequence identity. The final model including the two zinc ions was built based on the target-template alignment using ProMod3 version 1.3.0. Transcript levels of MAP3K1 in benign nevi and primary malignant melanomas were extracted from RNA sequencing data deposited in the National Center for Biotechnology Information Gene Expression Omnibus under accession number GSE112509 (Kunz et al., 2018Kunz M. Löffler-Wirth H. Dannemann M. Willscher E. Doose G. Kelso J. et al.RNA-seq analysis identifies different transcriptomic types and developmental trajectories of primary melanomas.Oncogene. 2018; 37: 6136-6151Crossref PubMed Scopus (34) Google Scholar). We searched for mutations within MAP3K1 across six melanoma studies deposited in the cBioPortal online (http://www.cbioportal.org), composed of 708 cases with mutation data (August 2019). Data from an additional 183 cases from the Australian Melanoma Genome Project was obtained from the International Cancer Genome Consortium Data Portal (https://dcc.icgc.org). mRNA expression data by RNA sequencing and normalized protein expression data by RPPA from the Cancer Genome Atlas–Skin Cutaneous Melanoma cohort were downloaded from The Broad Institute Firehose (http://gdac.broadinstitute.org). Clinical information on this cohort was obtained from cBioPortal. All procedures involving animals were performed in agreement with the Home Office regulations under the Animals Scientific Procedures Act 1986 (license PPLP671A5B06) and reported in accordance with the ARRIVE guidelines. Procedures were approved by the Animal Welfare and Ethical Review Body of the CRUK Manchester Institute, and tumor volumes did not exceed the guidelines set by the Committee of the National Cancer Research Institute (Workman et al., 2010Workman P. Aboagye E.O. Balkwill F. Balmain A. Bruder G. Chaplin D.J. et al.Guidelines for the welfare and use of animals in cancer research.Br J Cancer. 2010; 102: 1555-1577Crossref PubMed Scopus (910) Google Scholar). The Braf+/LSL-V600E;Tyr::CreERT2+/o mouse line and its genotyping details have been described previously (Dhomen et al., 2009Dhomen N. Reis-Filho J.S. da Rocha Dias S. Hayward R. Savage K. Delmas V. et al.Oncogenic Braf induces melanocyte senescence and melanoma in mice.Cancer Cell. 2009; 15: 294-303Abstract Full Text Full Text PDF PubMed Scopus (407) Google Scholar). Mice with a conditional allele of Map3k1 were obtained from the Wellcome Trust Sanger Institute Mouse Genetics Project resource (www.sanger.ac.uk/mouseportal) (Skarnes et al., 2011Skarnes W.C. Rosen B. West A.P. Koutsourakis M. Bushell W. Iyer V. et al.A conditional knockout resource for the genome-wide study of mouse gene function.Nature. 2011; 474: 337-342Crossref PubMed Scopus (1012) Google Scholar). Map3k1tm1a(KOMP)Wtsi mice were crossed with Braf+/LSL-V600E;Tyr::CreERT2+/o mice to generate the Map3k1fl/fl and BrafV600E;Map3k1fl/fl mice used in this study. All strains were maintained on a C57BL/6 background. A total of 86 females and 2 males were used for experiments. Tamoxifen (Sigma-Aldrich, St. Louis, MO, T5648) was freshly prepared in 100% ethanol and applied to the shaven backs of 8- to 12-week-old mice to induce the expression of BRAFV600E in melanocytes as previously described (Dhomen et al., 2009Dhomen N. Reis-Filho J.S. da Rocha Dias S. Hayward R. Savage K. Delmas V. et al.Oncogenic Braf induces melanocyte senescence and melanoma in mice.Cancer Cell. 2009; 15: 294-303Abstract Full Text Full Text PDF PubMed Scopus (407) Google Scholar). Mice were monitored for changes in skin appearance and tumor formation. For melanoma-free survival, time to first lesion was used. Animals were euthanized when their cumulative tumor burden exceeded 1,000 mm3 and no more than 1,500 mm3, determined by caliper measurements of tumor length, width, and depth, and calculated using the formula:length × width× depth × π/6; if they showed signs of ill health or distress; or after a maximum of 24 months. The back skin of age-matched mice (experimental endpoint range: 16–44 weeks after tamoxifen application) was photographed with an Olympus TG-5 camera. Quantification of nevi per skin area was determined by counting the number of pigmented lesions using ImageJ software version 1.52k. The nevus area was estimated by measuring the area occupied by pigmented cells composing dermal melanocytic nevi in H&E-stained skin sections using QuPath software version 0.1.2 (Bankhead et al., 2017Bankhead P. Loughrey M.B. Fernández J.A. Dombrowski Y. McArt D.G. Dunne P.D. et al.QuPath: open source software for digital pathology image analysis.Sci Rep. 2017; 7: 16878Crossref PubMed Scopus (827) Google Scholar). Samples were fixed in 10% neutral buffered formalin for 24 hours before processing. Tissues were dehydrated through graded ethanol, cleared in xylene, and embedded in paraffin wax. Sections of 4 μm were prepared, deparaffinized, and stained with H&E. For immunohistochemistry, sections were deparaffinized and rehydrated through graded alcohol. Heat-induced epitope retrieval was performed at 98 °C for 20 minutes, and slides were blocked with appropriate serum. Primary antibodies rabbit anti–phosphorylate p44/42 MAPK (extracellular signal–regulated kinase 1/2) (Cell Signaling Technology, Danvers, MA, 4370) and rabbit anti-S100 (Dako, Carpinteria, CA, Z0311) were incubated for 30 minutes, followed by detection using either a horseradish peroxidase polymer system with a 3,3′-diaminobenzine chromogen (Dako, K3468) or a red alkaline phosphatase substrate kit (Vector Laboratories, Burlingame, CA, SK5100). Images were captured using a Leica SCN400 slide scanner and processed using QuPath software (Bankhead et al., 2017Bankhead P. Loughrey M.B. Fernández J.A. Dombrowski Y. McArt D.G. Dunne P.D. et al.QuPath: open source software for digital pathology image analysis.Sci Rep. 2017; 7: 16878Crossref PubMed Scopus (827) Google Scholar). Staining was quantified based on the H-score method (Hirsch et al., 2003Hirsch F.R. Varella-Garcia M. Bunn Jr., P.A. Di Maria M.V. Veve R. Bremmes R.M. et al.Epidermal growth factor receptor in non-small-cell lung carcinomas: correlation between gene copy number and protein expression and impact on prognosis.J Clin Oncol. 2003; 21: 3798-3807Crossref PubMed Scopus (1149) Google Scholar) in five randomly selected areas measuring 0.25 mm2 each per digital slide. Nonparametric two-tailed Mann-Whitney tests were performed using GraphPad Prism version 7.0. For Kaplan-Meier survival analysis, two-tailed log-rank test was used to determine the difference between groups. P < 0.05 was considered significant. Summary of missense mutations in Map3k1 found in the mouse melanoma cohorts described in Trucco et al., 2019Trucco L.D. Mundra P.A. Hogan K. Garcia-Martinez P. Viros A. Mandal A.K. et al.Ultraviolet radiation–induced DNA damage is prognostic for outcome in melanoma.Nat Med. 2019; 25: 221-224Crossref PubMed Scopus (34) Google Scholar. Abbreviation: SNVs, single nucleotide variants. Number of cases and total number of missense SNVs annotated for mouse melanomas with or without a mutation in Map3k1 classified by experimental cohort described in Trucco et al., 2019Trucco L.D. Mundra P.A. Hogan K. Garcia-Martinez P. Viros A. Mandal A.K. et al.Ultraviolet radiation–induced DNA damage is prognostic for outcome in melanoma.Nat Med. 2019; 25: 221-224Crossref PubMed Scopus (34) Google Scholar. Abbreviations: AJCC, American Joint Committee on Cancer; NA, not applicable; NOS, not otherwise specified; TCGA, The Cancer Genome Atlas. Neoplasm disease stage and Breslow depth of skin melanoma cases from the TCGA dataset divided according to low (bottom 25th percentile) and high (top 75th percentile) MAP3K1 mRNA levels. Abbreviations: SKCM, skin cutaneous melanoma; TCGA, The Cancer Genome Atlas. Overall survival of patients with melanoma in the TCGA according to low (bottom 25th percentile) and high (top 75th percentile) MAP3K1 mRNA expression and genomic subtype.

Our understanding of how checkpoint inhibitors (CPIs) affect T cell evolution is incomplete, limiting our ability to achieve full clinical benefit from these drugs. Here, we analyzed peripheral T cell populations after one cycle of CPI treatment and identified a dynamic awakening of the immune system, as revealed by T cell evolution in response to treatment. We sequenced T cell receptors in plasma cell-free DNA and peripheral blood mononuclear cells and performed phenotypic analysis of peripheral T cell subsets from patients with metastatic melanoma treated with CPIs. We found that early peripheral T cell turnover and T cell receptor repertoire dynamics identified which patients would respond to treatment. Additionally, the expansion of a subset of immune effector peripheral T cells we call T IE cells correlated with response. These events are prognostic and occur within 3 weeks of starting immunotherapy, raising the potential for monitoring patients’ responses by using minimally invasive liquid biopsies.

Background Combination treatments targeting the MEK-ERK pathway and checkpoint inhibitors have improved overall survival in melanoma. Resistance to treatment especially in the brain remains challenging, and rare disease subtypes such as acral melanoma are not typically included in trials. Here we present analyses from longitudinal sampling of a patient with metastatic acral melanoma that became resistant to successive immune and targeted therapies. Methods We performed whole-exome sequencing and RNA sequencing on an acral melanoma that progressed on successive immune (nivolumab) and targeted (dabrafenib) therapy in the brain to identify resistance mechanisms. In addition, we performed growth inhibition assays, reverse phase protein arrays and immunoblotting on patient-derived cell lines using dabrafenib in the presence or absence of cerebrospinal fluid (CSF) in vitro. Patient-derived xenografts were also developed to analyse response to dabrafenib. Results Immune escape following checkpoint blockade was not due to loss of tumour cell recognition by the immune system or low neoantigen burden, but was associated with distinct changes in the microenvironment. Similarly, resistance to targeted therapy was not associated with acquired mutations but upregulation of the AKT/phospho-inositide 3-kinase pathway in the presence of CSF. Conclusion Heterogeneous tumour interactions within the brain microenvironment enable progression on immune and targeted therapies and should be targeted in salvage treatments.

Abstract Metastatic melanoma shows remarkable tropism for the brain, affecting up to 75% of stage 4 patients, causing devastating symptoms and dramatically reducing survival. Patients with brain metastases have limited treatment options and short-lived unpredictable responses to therapy, probably due to our poor understanding of the biology driving this particular metastatic process. In this work we hypothesize that the brain microenvironment induces particular gene expression changes in melanoma cells as compared to melanoma cells colonizing extra-cranial sites, allowing cells to rewire, survive and proliferate in the brain. To elucidate the molecular basis supporting brain metastasis in melanoma, we enriched for cancer cells from patient blood and established a metastatic circulating tumor cells derived explant (CDX) model of cutaneous melanoma. CDX-derived cell lines were injected intra-dermally in the flank of immunocompromised mice and tumor growth as well as metastatic incidence was quantified. Our data shows that both the CDX and its corresponding derived cell line showed tropism for the brain, liver and kidneys, similar to what was observed in the patient. Notably, the intradermal flank-tumors were predominantly highly pigmented, whereas the liver metastases were either pigmented or non-pigmented. In contrast, the brain metastases located in the parenchyma were exclusively non-pigmented. Cell lines derived from either pigmented or non-pigmented liver lesions as well as non-pigmented lesions from the brain maintained their pigmentation and morphological features in vitro. When injected intradermally into immunocompromised mice, the pigmented cells metastasized to the visceral organs and, even though leptomeningeal metastasis may be observed, these cells did not invade brain parenchyma. In contrast, the non-pigmented cells (derived either from the liver or brain) metastasized to the visceral organs and additionally metastasized to the brain parenchyma; feature that was enhanced following successive in vivo passages. Gene expression analysis revealed an enrichment of genes encoding proteins that regulate extracellular matrix reorganization, angiogenesis and epithelial-to-mesenchymal transition in the non-pigmented cells. Moreover, TGF-beta signalling was found to be one of the most enriched pathways in the cells derived from parenchymal brain metastases, suggesting that increased TGF-beta signalling regulates brain tropism in metastatic melanoma. In conclusion, our experimental model recapitulates the propensity of melanoma to metastasize to the brain parenchyma and our preliminary data suggests that TGF-beta signalling play a key role in supporting this process. Further experiments are currently ongoing for testing our hypothesis that, if corroborated, may open new avenues of treatment options for melanoma patients displaying metastases in the brain. Citation Format: Denys Holovanchuk, Rebecca Lee, Alessio Cannistraci, Nathalie Dhomen, Valeria Pavet, Richard Marais. Elucidating genes that mediate brain colonization by metastatic melanoma cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2727.

Background: KRAS is mutated in similar to 90% of pancreatic ductal adenocarcinomas, similar to 35% of colorectal cancers and w20% of non-small-cell lung cancers. There has been recent progress in targeting G12CKRAS specifically, but therapeutic options for other mutant forms of KRAS are limited, largely because the complexity of downstream signaling and feedback mechanisms mean that targeting individual pathway components is ineffective. Design: The protein kinases RAF and SRC are validated therapeutic targets in KRAS-mutant pancreatic ductal adenocarcinomas, colorectal cancers and non-small-cell lung cancers and we show that both must be inhibited to block growth of these cancers. We describe CCT3833, a new drug that inhibits both RAF and SRC, which may be effective in KRAS-mutant cancers. Results: We show that CCT3833 inhibits RAF and SRC in KRAS-mutant tumors in vitro and in vivo, and that it inhibits tumor growth at well-tolerated doses in mice. CCT3833 has been evaluated in a phase I clinical trial (NCT02437227) and we report here that it significantly prolongs progression-free survival of a patient with a G12VKRAS spindle cell sarcoma who did not respond to a multikinase inhibitor and therefore had limited treatment options. Conclusions: New drug CCT3833 elicits significant preclinical therapeutic efficacy in KRAS-mutant colorectal, lung and pancreatic tumor xenografts, demonstrating a treatment option for several areas of unmet clinical need. Based on these preclinical data and the phase I clinical unconfirmed response in a patient with KRAS-mutant spindle cell sarcoma, CCT3833 requires further evaluation in patients with other KRAS-mutant cancers.

Although immune checkpoint inhibitors (ICIs) have achieved unprecedented results in melanoma, the biological features of the durable responses initiated by these drugs remain unknown. Here we show the genetic and phenotypic changes induced by treatment with programmed cell death-1 (PD-1) blockade in a genetically engineered mouse model of melanoma driven by oncogenic BRAF. In this controlled system anti-PD-1 treatment yields responses in ~35% of the tumors, and prolongs survival in ~27% of the animals. We identify increased stroma remodeling and reduced expression of proliferation markers as features associated with prolonged response. These traits are corroborated in two independent early on-treatment anti-PD-1 melanoma patient cohorts. These insights into the biological responses of tumors to ICI provide a strategy for identification of durable response early during the course of treatment and could improve patient stratification for checkpoint inhibitory drugs.