Aberrations of protein-coding genes are a focus of cancer genomics; however, the impact of oncogenes on expression of the ∼50% of transcripts without protein-coding potential, including long noncoding RNAs (lncRNAs), has been largely uncharacterized.,Activating mutations in the BRAF oncogene are present in >70% of melanomas, 90% of which produce active mutant BRAFV600E protein.,To define the impacts of oncogenic BRAF on the melanocyte transcriptome, massively parallel cDNA sequencing (RNA-seq) was performed on genetically matched normal human melanocytes with and without BRAFV600E expression.,To enhance potential disease relevance by verifying expression of altered genes in BRAF-driven cancer tissue, parallel RNA-seq was also undertaken of two BRAFV600E-mutant human melanomas.,BRAFV600E regulated expression of 1027 protein-coding transcripts and 39 annotated lncRNAs, as well as 70 unannotated, potentially novel, intergenic transcripts.,These transcripts display both tissue-specific and multi-tissue expression profiles and harbor distinctive regulatory chromatin marks and transcription factor binding sites indicative of active transcription.,Coding potential analysis of the 70 unannotated transcripts suggested that most may represent newly identified lncRNAs.,BRAF-regulated lncRNA 1 (BANCR) was identified as a recurrently overexpressed, previously unannotated 693-bp transcript on chromosome 9 with a potential functional role in melanoma cell migration.,BANCR knockdown reduced melanoma cell migration, and this could be rescued by the chemokine CXCL11.,Combining RNA-seq of oncogene-expressing normal cells with RNA-seq of their corresponding human cancers may represent a useful approach to discover new oncogene-regulated RNA transcripts of potential clinical relevance in cancer.
The paradoxical coexistence of spontaneous tumor antigen-specific immune responses with progressive disease in cancer patients furthers the need to dissect the molecular pathways involved in tumor-induced T cell dysfunction.,In patients with advanced melanoma, we have previously shown that the cancer-germline antigen NY-ESO-1 stimulates spontaneous NY-ESO-1-specific CD8+ T cells that up-regulate PD-1 expression.,We also observed that PD-1 regulates NY-ESO-1-specific CD8+ T cell expansion upon chronic antigen stimulation.,In the present study, we show that a fraction of PD-1+ NY-ESO-1-specific CD8+ T cells in patients with advanced melanoma up-regulates Tim-3 expression and that Tim-3+PD-1+ NY-ESO-1-specific CD8+ T cells are more dysfunctional than Tim-3−PD-1+ and Tim-3−PD-1− NY-ESO-1-specific CD8+ T cells, producing less IFN-γ, TNF, and IL-2.,Tim-3-Tim-3L blockade enhanced cytokine production by NY-ESO-1-specific CD8+ T cells upon short ex vivo stimulation with cognate peptide, thus enhancing their functional capacity.,In addition, Tim-3-Tim-3L blockade enhanced cytokine production and proliferation of NY-ESO-1-specific CD8+ T cells upon prolonged antigen stimulation and acted in synergy with PD-1-PD-L1 blockade.,Collectively, our findings support the use of Tim-3-Tim-3L blockade together with PD-1-PD-L1 blockade to reverse tumor-induced T cell exhaustion/dysfunction in patients with advanced melanoma.