Project description:In recent years, the understanding of the immune tumor microenvironment has evolved to include B cells as active participants rather than mere bystanders. Retrospective studies investigating the presence of B cells and B cells-associated signatures within tumors have revealed a significant correlation with therapeutic outcomes. Although conflicting data exist regarding whether B cells act as pro-tumor or anti-tumor agents, it is becoming increasingly clear that understanding B cells as a heterogeneous population, shaped by tumor microenvironment cues, is essential. This study explores the crucial roles played by B cells and their extracellular vesicles (EVs) in shaping responses to immune checkpoint blockade (ICB) therapy. We have independently verified the significant enrichment of B cells in ICB therapy responders in comparison to non-responder in tumors of melanoma patients. B cell depletion experiments in an in vitro tumor-killing assay demonstrate a clear impairment of T cell-mediated anti-tumor activity. To investigate the clinical relevance, EVs were isolated from melanoma patient tumors, and fractioned into subpopulations, including B cell-derived EVs. MiRNA profiling of CD19+ EVs identifies miR-99a-5p as a top candidate, significantly upregulated in responder EVs. In downstream assays, we observed a B cell-dependent phenotype of miR-99a-5p, where its silencing impaired T cell-mediated anti-tumor cytotoxicity in an in vitro tumor-killing assay. Mechanistically, miR-99a-5p appears to regulate B cell cell-cycle, favoring non-homologous end joining DNA repair pathway, and promoting class-switch recombination. Collectively, data reported herein emphasizes the indispensable role of B cells and their derived EVs in shaping cancer immunotherapy outcome.
Project description:Immune checkpoint therapy has improved outcome of patients with advanced melanoma, however the tumor becomes refractory in a large number of patients. Therefore, novel combination therapies that enhance anti-melanoma immunity are highly desirable. We studied the impact of MDM2 (mouse double minute 2) inhibition, leading to p53 activation, on response to anti-PD-1 immunotherapy in melanoma. MDM2 treatment caused upregulation of MHC II and IL-15 in melanoma cells in a p53 dependent manner. Survival of melanoma bearing mice improved upon combining anti-PD1 and MDM2 inhibition in vivo. Lack of IL-15Rα in T cells abrogated the beneficial effect of the combined treatment. This gene expression study was performed to determine potential effects of MDM2 inhibition on genes in the melanoma microenvironment.
Project description:Immune checkpoint blockade (ICB) has revolutionized melanoma therapy, but drug resistance represents a significant limitation. Here we utilize a platform incorporating transcriptomic profiling, high-throughput drug screening (HTDS) and murine models to demonstrate the pre-clinical efficacy of cobimetinib and regorafenib (termed Cobi+Reg) for ICB-resistant melanoma. RNA-Sequencing (RNA-Seq) analysis of ICB-resistant melanomas demonstrated activation of several targetable pathways. HTDS targeting these pathways identified several effective combinations in ICB-resistant patient-derived xenograft (PDX) models. Cobi+Reg emerged as the most promising regimen, with efficacy against distinct molecular melanoma subtypes and following progression on ICB in immunocompetent models. RNA-Seq analysis of Cobi+Reg-treated tumors demonstrated upregulation of antigen presentation machinery, with concomitantly increased activated T cell infiltration. Combining Cobi+Reg with ICB was superior to either treatment in vivo. This analytical platform has identified several effective combinations, presenting Cobi+Reg as a rational therapeutic strategy either following resistance to or combined with ICB for advanced melanoma.
Project description:Immune checkpoint blockade (ICB) therapy provides remarkable clinical gains, where melanoma is at the forefront of its success. However, only a subset of patients with advanced tumors currently benefit from these therapies, which at times incur considerable side-effects and costs. Constructing such predictors of patient’s response has remained a serious challenge due to the complexity of the immune response and the shortage of large ICB-treated patient cohorts including both omics and response data. Here we build IMPRES, a predictor of ICB-response in melanoma which encompasses 15 pairwise transcriptomics relations between immune checkpoint genes. It is based on two key conjectures: (a) immune mechanisms underlining spontaneous regression in neuroblastoma can predict ICB response in melanoma, and (b) key immune interactions can be captured via specific pairwise relations of immune checkpoint genes’ expression. IMPRES is validated on 9 published datasets1–6 and on a newly generated dataset of 31 tumor samples treated with anti-PD-1 and 10 tumor samples treated with anti-CTLA-4 (some of these are treated with both antibodies), spanning 297 samples in total. It achieves an overall accuracy of AUC=0.83, outperforming existing predictors, capturing almost all true responders while misclassifying less than half of the non-responders. Future studies are warranted to determine the value of the approach presented here in other cancer types.
Project description:Genome wide DNA methylation profiling of formalin-fixed paraffin-embedded (FFPE) melanoma tissue samples from patients prior to treatment with anti-PD1 immune checkpoint inhibitor therapy. The MethylationEPIC v1.0 BeadChip array was used to obtain DNA methylation profiles across approximately 850,000 CpGs in bisulfite treated DNAs extracted from formalin-fixed paraffin embedded melanoma tissues prior to treatment with anti-PD1 immune checkpoint inhibitor therapy.
Project description:Extracellular vesicles (EVs) are membranous particles released by all cell types. Their role as functional carriers of bioactive molecules is boosted in cancer where they can be either secreted in biological fluids or found in the intercellular space (interstitial EVs, iEVs). Here we have opti-mised a method for the isolation and characterization of zebrafish iEVs from whole melanoma tissues. Zebrafish melanoma iEVs are in the range of ~ 140 nm by nanoparticle tracking analysis (NTA) and TEM analysis. Western blot revealed enrichment for CD63 and Alix in the iEV frac-tion, but not in melanoma cell lysates. Super resolution and confocal microscopy revealed that purified zebrafish iEVs were GFP+ indicating that they integrate the oncogene, GFP-HRASV12G within their vesicular membrane. Analysis of RNA-Seq data revealed that 118 ncRNAs are differentially distributed between zebrafish melanoma and their iEVs, with only 18 of them be-ing selectively enriched in iEVs. Among these, the RNA components of RNAses MRP and P, which process ribosomal RNA precursors, mitochondrial RNAs and some mRNAs, were enriched in iEVs. We found that melanoma iEVs induce an inflammatory response when injected in larval blood stream with increase of macrophage and induction of Interferon Responsive Genes. To clarify whether MRP and P contribute to the inflammation induced by melanoma iEVs we inject-ed larvae with MRP or P RNAs and found an inflammatory response similar to that induced by melanoma iEVs. This suggests that zebrafish melanoma iEVs are a source of MRP and P RNAs that can trigger inflammation in cells of the tumor-microenvironment.
Project description:Immune checkpoint blockade (ICB) has demonstrated significant promise for the treatment of advanced malignancies. Anti-CTLA4 and ant-PD1 therapy can activate the immune system and result in durable control in diseases such as melanoma and non-small cell lung cancer.