Project description:MEL02-1 and MEL02-2 are melanoma cell lines derived from a melanoma patient. MEL02-1 was established from a pre-treatment metastatic lesion. MEL02-2 was established from post-treatment regressing lesion.
Project description:Blocking the PD-1/PD-L1 immunosuppressive pathway has shown promise in the treatment of certain cancers including melanoma. This study investigates differences in the gene expression profiles of human melanomas that do or do not display the immunosuppressive protein PD-L1. Further understanding of genes expressed within the tumor microenvironment of PD-L1+ tumors may lead to improved rationally designed treatments. Gene expression profiling was performed on total RNA extracted by laser capture microdissection from 11 archived formalin-fixed paraffin-embedded (FFPE) melanoma specimens, 5 of which were PD-L1 positive and 6 PD-L1 negative. Details of the design, and the gene signatures found are given in the paper associated with this GEO Series: Janis M. Taube, Geoffrey D. Young, Tracee L. McMiller, Shuming Chen, January T. Salas, Theresa S. Pritchard, Haiying Xu, Alan K. Meeker, Jinshui Fan, Chris Cheadle, Alan E. Berger, Drew M. Pardoll, and Suzanne L. Topalian, Differential expression of immune-regulatory genes associated with PD-L1 display in melanoma: implications for PD-1 pathway blockade, Clin Cancer Res 2015, in press.
Project description:Treatment of advanced V600BRAF mutant melanoma using a BRAF inhibitor (BRAFi) or its combination with a MEKi typically elicits partial responses. We compared the transcriptomes of patient-derived tumors regressing on MAPKi therapy against MAPKi-induced temporal transcriptomic states in human melanoma cell lines or murine melanoma in immune-competent mice. Despite heterogeneous dynamics of clinical tumor regression, residual tumors displayed highly recurrent transcriptomic alterations and enriched processes, which were also observed in MAPKi-selected cell lines (implying tumor cell-intrinsic reprogramming) or in bulk mouse tumors (and the CD45-negative or -positive fractions,, implying tumor cell-intrinsic or stromal/immune alterations, respectively). Tumor cell-intrinsic reprogramming attenuated MAPK-dependency, while enhancing mesenchymal, angiogenic and IFN-inflammatory features and growth/survival dependence on multi-RTKs and PD-L2. In the immune compartment, PD-L2 upregulation in CD11c+ immunocytes drove the loss of T-cell inflammation and promoted BRAFi resistance. Thus, residual melanoma early on MAPKi therapy already displays potentially exploitable adaptive transcriptomic, epigenomic, immune-regulomic alterations.
Project description:LC-MS/MS targeted files submitted as support material for the paper: Quantitative mass spectrometry analysis of PD-L1 protein expression, N-glycosylation and expression stoichiometry with PD-1 and PD-L2 in human melanoma.
Project description:Melanoma is one of the most commonly diagnosed malignancies and serves as a model for studying immunotherapy. The B16 melanoma model is characterized by low T cell infiltration in the tumor, and blocking the PD-1 pathway shows no significant anti-tumor activity, mirroring melanoma patients with a cold tumor immunophenotype. Therefore, understanding the molecular basis that prevents T cell-mediated anti-tumor activity in B16 melanoma is of great significance. In this study, we generated Tyr knockout B16 melanoma cells using CRISPR/Cas9 and discovered that tyrosinase in melanoma significantly inhibits the anti-tumor activity of T cells. Tyrosinase deficiency significantly increases T-cell infiltration and activation within the tumor. Single-cell RNA sequencing reveals an altered cold tumor immunophenotype in tyrosinase-deficient B16 melanoma. In wild-type mice, T cells in tyrosinase-deficient tumors express elevated levels of PD-1 and Foxp3. However, strikingly, in PD-1 deficient mice, the loss of tyrosinase in B16 melanoma unleashes the anti-tumor activity of PD-1 deficient T cells. This enhanced anti-tumor activity is explained by significantly increased tumor T cell infiltration accompanied by reduced frequencies of Tregs in PD-1 knockout mice. Targeting tyrosinase may enhance the anti-tumor efficacy of PD-1 blockade in cold tumors, offering a novel strategy to render cold tumors responsive to immunotherapy. Clinically, higher levels of tyrosinase expression in human melanoma are associated with a poorer prognosis, indicating that our findings could potentially improve the efficacy of immunotherapy in melanoma patients.
Project description:Thirty-eight tumors from 17 patients treated with BRAF inhibitor (n=12) or combination BRAF/MEK inhibitors (n=5) with known PD-L1 expression were analyzed. RNA expression arrays were performed on all pre-treatment (PRE, n=17), early during treatment (EDT, n=8) and progression (PROG, n=13) biopsies. HLA-A/HLA-DPB1 expression was assessed by immunohistochemistry (IHC). Gene set enrichment analysis (GSEA) of PRE, EDT and PROG melanomas revealed that transcriptome signatures indicative of immune cell activation were strongly positively correlated with PD-L1 staining. In contrast, MAPK signaling and canonical Wnt/-ß-catenin activity were negatively associated with PD-L1 melanoma expression. The expression of PD-L1 and immune activation signatures did not simply reflect the degree or type of immune cell infiltration, and was not sufficient for tumor response to MAPK inhibition.
Project description:In order to study the mechanism of nuclear PD-L1 in uveal melanoma, we knocked down PD-L1 and applied RNA-seq and CUT&Tag to identify its target genes in uveal melanoma.
Project description:PD-L1 functions as a co-inhibitory checkpoint ligand constraining anti-tumor immunity by binding to PD-1 on immune cells. Although antibody blockade therapy displays clear therapeutic responses, it is not effective in all cancer patients, indicating modulation by other factors. We applied cell surface proximity biotinylation combined with mass spectrometry in melanoma cells to identify novel proteins regulating PD-L1 function. Our findings reveal that membrane organizing protein Tetraspanin-4 (TSPAN4) interacts with PD-L1, where both proteins colocalize on migrasomes and retraction fibers. We demonstrate that TSPAN4 negatively affects PD-L1 protein levels and inhibits PD-L1 lateral mobility on the plasma membrane. TSPAN4 knockdown results in a reduced PD-L1 degradation rate, and enhanced PD-L1 interaction with CMTM6 as the molecular mechanism. Consequently, increased PD-L1 levels in TSPAN4 knockdown melanoma cells leads to enhanced PD-1 binding, and more efficient immune checkpoint blockade through inhibition of T cell responses. This study identifies TSPAN4 as a negative regulator of PD-L1 at the cell surface of melanoma cells, suggesting that TSPAN4 may represent a novel target for improving immune checkpoint therapy in cancer patients.
Project description:Blocking the PD-1/PD-L1 immunosuppressive pathway has shown promise in the treatment of certain cancers including melanoma. This study investigates differences in the gene expression profiles of human melanomas that do or do not display the immunosuppressive protein PD-L1. Further understanding of genes expressed within the tumor microenvironment of PD-L1+ tumors may lead to improved rationally designed treatments.