Time-lapse imaging of A375(Fucci2)-BR cells treated with the control.

A. heat map of complete RPPA data median centered, all tumor grafts untreated. Data are mean of 3 biological replicates, red is up, green is down. B. Principal component analysis (PCA) of median centered data C. heat map of fold change between untreated tumor grafts and on chronic BRAF inhibitor therapy. D. Principal component analysis (PCA) of fold change data.

Time-lapse imaging of A375(Fucci2)-CR cells treated with the control.

Time-lapse imaging of A375(Fucci2)-BR cells treated with 6-thio-dG.

All growth curves of first implantation and subsequent expansions with and without continuous BRAF inhibitor (PLX4720) diet. Error bars are SEM.

Time-lapse imaging of A375(Fucci2) cells treated with 6-thio-dG.

A. PCA of protein expression profiles of WM3965 control and progression tumor grafts. B. unsupervised hierarchical clustering of proteins identified in the PCA.

IHC staining of WM3965 PDX tumors treated with indicated compounds after 3 day of dosing and 4 hours post last dose for Ki67 (upper row) and cleaved caspase 3 (lower row).

Time-lapse imaging of A375(Fucci2)-CR cells treated with 6-thio-dG.

Table S1 Mean density values of crystal violet staining of 16 melanoma cell lines treated with 6-thio-dG and PLX4720. Table S2 RNA-seq data related to A375 cells treated with 6-thio-dG. Table S3 RNA-seq data related to A375 cells treated with BIBR 1532. Table S4 Apoptotic index and ssGSEA scores of 11 gene sets in 18 CCLE melanoma cell lines. Table S5 RPPA data related to A375 cells treated with 6-thio-dG. Table S6 Mean density values of crystal violet staining of 6 therapy-resistant melanoma cell lines treated with 6-thio-dG. Table S7 RPPA data related to the comparison between LOX-IMVI parental and BR cells. Table S8 RPPA data related to LOX-IMVI parental cells treated with 6-thio-dG. Table S9 RPPA data related to LOX-IMVI BR cells treated with 6-thio-dG. Table S10 RPPA data related to LOX-IMVI BR xenografts treated with 6-thio-dG. Table S11 The clinical information from patients with metastatic melanoma treated with immunotherapies.

Supplementary Table S1: Summary of melanoma patients with clinical follow-up. Figure Legends

Time-lapse imaging of A375(Fucci2) cells treated with the control.

A. immunoblot for BRAF splicing variants B. PCR with primers for BRAF_F/R (Exon1-11).

complete targeted sequencing results

H&E staining of all patients and PDX; MP1: first mouse passage; MP3+: min. of 3 and max. of 5 serial transplantations; on BRAFi: mice were chronically dosed with PLX4720-additive diet.

BackgroundImmune cell expression profiling from patient samples is critical for the successful development of immuno-oncology agents and is useful to understand mechanism-of-action, to identify exploratory biomarkers predictive of response, and to guide treatment selection and combination therapy strategies. LAG-3 is an inhibitory immune checkpoint that can suppress antitumor T-cell responses and targeting LAG-3, in combination with PD-1, is a rational approach to enhance antitumor immunity that has recently demonstrated clinical success. Here, we sought to identify human immune cell subsets that express LAG-3 and its ligands, to characterize the marker expression profile of these subsets, and to investigate the potential relationship between LAG-3 expressing subsets and clinical outcomes to immuno-oncology therapies.MethodsComprehensive high-parameter immunophenotyping was performed using mass and flow cytometry of tumor-infiltrating lymphocytes (TILs) and peripheral blood mononuclear cells (PBMCs) from two independent cohorts of samples from patients with various solid tumor types. Profiling of circulating immune cells by single cell RNA-seq was conducted on samples from a clinical trial cohort of melanoma patients treated with immunotherapy.ResultsLAG-3 was most highly expressed by subsets of tumor-infiltrating CD8 T central memory (TCM) and effector memory (TEM) cells and was frequently co-expressed with PD-1. We determined that these PD-1+ LAG-3+ CD8 memory T cells exhibited a unique marker profile, with greater expression of activation (CD69, HLA-DR), inhibitory (TIM-3, TIGIT, CTLA-4) and stimulatory (4-1BB, ICOS) markers compared to cells that expressed only PD-1 or LAG-3, or that were negative for both checkpoints. In contrast to tumors, LAG-3 expression was more limited in circulating immune cells from healthy donors and solid tumor patients. Additionally, we found abundant expression of the LAG-3 ligands MHC-II and galectin-3 in diverse immune cell types, whereas FGL1 and LSECtin were minimally expressed by immune cells in the tumor microenvironment (TME). Lastly, we found an inverse relationship between baseline and on-treatment levels of circulating LAG3 transcript-expressing CD8 memory T cells and response to combination PD-1 and CTLA-4 blockade in a clinical trial cohort of melanoma patients profiled by scRNAseq.ConclusionsThese results provide insights into the nature of LAG-3- and ligand-expressing immune cells within the TME, and suggest a biological basis for informing mechanistic hypotheses, treatment selection strategies, and combination immunotherapy approaches to support continued development of dual PD-1 and LAG-3 blockade.

Supplementary materials and methods

AbstractBackgroundMelanoma, the deadliest of skin cancers, has a high propensity to form brain metastases that are associated with a markedly worsened prognosis. In spite of recent therapeutic advances, melanoma brain lesions remain a clinical challenge, biomarkers predicting brain dissemination are not clear and differences with other metastatic sites are poorly understood.MethodsWe examined a genetically diverse panel of human-derived melanoma brain metastasis (MBM) and extracranial cell lines using targeted sequencing, a Reverse Phase Protein Array, protein expression analyses, and functional studies in vitro and in vivo.ResultsBrain-specific genetic alterations were not detected; however, MBM cells in vitro displayed lower proliferation rates and MBM-specific protein expression patterns associated with proliferation, DNA damage, adhesion, and migration. MBM lines displayed higher levels of RAC1 expression, involving a distinct RAC1-PAK1-JNK1 signaling network. RAC1 knockdown or treatment with small molecule inhibitors contributed to a less aggressive MBM phenotype in vitro, while RAC1 knockdown in vivo led to reduced tumor volumes and delayed tumor appearance. Proliferation, adhesion, and migration were higher in MBM vs nonMBM lines in the presence of insulin or brain-derived factors and were affected by RAC1 levels.ConclusionsOur findings indicate that despite their genetic variability, MBM engage specific molecular processes such as RAC1 signaling to adapt to the brain microenvironment and this can be used for the molecular characterization and treatment of brain metastases.

Background Characterization of human immune responses by profiling immune cells from patients is critical for the successful development of immuno-oncology agents and is useful to understand mechanism-of-action, identify pharmacodynamic/response biomarkers, and guide patient selection strategies. Extensive immune cell heterogeneity necessitates comprehensive high parameter immunophenotyping to yield these actionable insights. Methods Cytometry by time-of-flight (CyTOF) was performed on homogenates from commercially procured tumors (n=28) and matched PBMCs (n=7) from patients with various solid tumors (colon (n=10), endometrial (n=9), kidney (n=4), liver (n=2), skin (n=1), lung (n=1), and gastro-intestinal (n=1)). Two antibody panels, recognizing a total of 18 lineage and 31 target proteins, were used to profile marker expression among the major lymphocyte and myeloid lineages. Data were analyzed using manual gating and non-linear dimensionality reduction (tSNE and UMAP), and expression was measured by frequency (% gate) and arcsinh-transformed median ion counts. Pairwise Wilcoxon Rank Sum tests were performed on arcsinh-transformed median ion counts to determine statistically significant differences in marker expression, and P values were adjusted using Benjamini-Hochberg correction (p<0.05 considered statistically significant). Cell subpopulation percentages were compared using unpaired two-sided T-tests. Sample populations with less than 150 events were excluded from analysis. The initial analysis focused on CD8 T cells as a primary mediator of antitumor immunity. Results Matched samples revealed enrichment of effector memory (EM) and central memory (CM) CD8 T cells in tumors compared to PBMCs, as expected. EM cells represented on average 63.36% of the CD8 T cells in tumors vs 30.31% in PBMCs (p=0.0067), and CM cells 12.11% vs 5.58% respectively (p=0.1558). Non-linear dimensionality reduction mapping of these CD8 EM and CM cell subtypes among tumors displayed an activated but potentially dysfunctional phenotype, characterized by substantially higher expression of multiple coinhibitory receptors (PD-1, LAG-3, TIM-3, TIGIT) and activation markers (HLA-DR, ICOS) compared to PBMCs. Among these cells, a PD-1+/LAG-3+ subset, observed in 17/28 TIL samples, expressed TIM-3, TIGIT, HLA-DR, and ICOS at significantly higher levels compared to other PD1/LAG3 expression subsets. Interestingly, CD137 (4-1BB), a marker of potentially tumor-reactive cells, is expressed predominantly in PD-1+ memory CD8 T cells, with the most intense expression levels observed in the PD-1+/LAG-3+ subset. Conclusions The present results provide insight into the relative (co)expression of potentially targetable immunological pathways, and suggest a biological basis for informing approaches to combination checkpoint inhibition therapy. Acknowledgements We thank Paul Fischer for his contributions in acquiring the CyTOF data and performing initial data QC and analysis. Ethics Approval This study was approved by Bristol Myers Squibb’s Global Data Repository (Biological Assessment of Risk (BAR) number LVL_2020_12339). Samples were provided by Discovery Life Sciences (CA), MT Group (CA), Avaden BioSciences (WA), or BioOptions (CA). All patients gave written informed consent at the time of sample collection according to the IRB protocols of each provider.

Influenza A H3N2 viruses circulate globally, leading to substantial morbidity and mortality. Commercially available, antigen-matched influenza vaccines must be updated frequently to match dynamic sequence variability in immune epitopes, especially within viral influenza A H3N2 hemagglutinin (H3). In an effort to create comprehensive immune responses against H3N2, four micro-consensus antigens were designed to mimic the sequence and antigenic diversity of H3. Synthetic plasmid DNA constructs were developed to express each micro-consensus immunogen and combined into a multi-antigen DNA vaccine cocktail, pH3HA. Facilitated delivery of pH3HA via intramuscular electroporation in mice induced comprehensive, potent humoral responses against diverse seasonal H3N2 viruses that circulated between 1968 and the present. Vaccination with pH3HA also induced an antigen-specific cellular cytokine response. Mice immunized with pH3HA were protected against lethal challenge using two distinct H3N2 viruses, highlighting the heterologous protection afforded by synthetic micro-consensus immunogens. These findings warrant further study of the DNA vaccine micro-consensus platform for broad protection against influenza viruses.