Non-classical neoantigens from fusion junctions of chimeric RNAs are novel targets for tumor-specific personalized vaccines with low risk of autoimmunity. We present a platform to discover immunogenic neoantigens driving CD8+ T-cell clonotypes from chimeric RNA fusion junctions, with the aim of promoting tumor-reactive T-cell expansion. RNA sequencing data from 1315 patient-derived xenograft (PDX) models harboring 28 different cancer types were analyzed for chimeric RNA. The CD74 [Exon 1-6] | NRG1 [Exon 5-12] fusion was selected based on its established role as an actionable cancer driver and its presence in 4 PDX models with a consistent fusion junction.

We assessed the affinity of 9,10,11mer neopeptides from the CD74-NRG1 fusion to MHC Class I molecules using in silico tools and confirmed these findings in vitro using flow cytometry analyses. Predicted binders were further modeled and ranked by structural features with APEGEN 2.0. Immunogenicity was evaluated via IFN-γ ELISpot assays using HLA-B*07:02-matched PBMCs. Dextramers conjugated with the feature barcode technology were utilized for single-cell 5’ gene expression RNA sequencing and T-cell receptor mapping of antigen-specific activated T cells. This study demonstrates a robust pipeline for identifying immunogenic neoantigens from chimeric RNAs, offering the potential for designing personalized cancer vaccines.

Non-classical neoantigens from fusion junctions of chimeric RNAs are novel targets for tumor-specific personalized vaccines with low risk of autoimmunity. We present a platform to discover immunogenic neoantigens driving CD8+ T-cell clonotypes from chimeric RNA fusion junctions, with the aim of promoting tumor-reactive T-cell expansion. RNA sequencing data from 1315 patient-derived xenograft (PDX) models harboring 28 different cancer types were analyzed for chimeric RNA. The CD74 [Exon 1-6] | NRG1 [Exon 5-12] fusion was selected based on its established role as an actionable cancer driver and its presence in 4 PDX models with a consistent fusion junction.

We assessed the affinity of 9,10,11mer neopeptides from the CD74-NRG1 fusion to MHC Class I molecules using in silico tools and confirmed these findings in vitro using flow cytometry analyses. Predicted binders were further modeled and ranked by structural features with APEGEN 2.0. Immunogenicity was evaluated via IFN-γ ELISpot assays using HLA-B*07:02-matched PBMCs. Dextramers conjugated with the feature barcode technology were utilized for single-cell 5’ gene expression RNA sequencing and T-cell receptor mapping of antigen-specific activated T cells. This study demonstrates a robust pipeline for identifying immunogenic neoantigens from chimeric RNAs, offering the potential for designing personalized cancer vaccines.

Lung adenocarcinoma (LUAD) and pancreatic ductal adenocarcinoma (PDAC) together account for ~15% of all cancer mortalities. Both LUAD and PDAC are commonly driven by KRAS mutations. The first allele-specific KRAS inhibitors were recently approved for LUAD, but clinical benefit is limited by intrinsic and acquired resistance. LUAD predominantly arises from alveolar type 2 (AT2) cells, which function as facultative alveolar stem cells by self-renewing and replacing alveolar type 1 (AT1) cells. Using genetically engineered mouse models, patient-derived xenografts and patient samples, we found inhibition of KRAS promotes transition to a quiescent AT1-like cancer cell state in LUAD tumors. Similarly, suppressing KRAS-induced AT1 differentiation of wild-type AT2 cells upon lung injury. The AT1-like LUAD cells exhibited high growth and differentiation potential upon treatment cessation, whereas ablation of the AT1-like cells robustly improved treatment response to KRAS inhibitors. Intra-tumoral heterogeneity in PDAC is characterized by a balance between basal and classical epithelial cancer cell states, with basal dominance associated with chemoresistance and a dismal prognosis. Using genetically engineered mouse models and patient-derived xenografts, we found that basal PDAC cells are highly sensitive to KRAS inhibitors. Employing fluorescent and bioluminescent reporter systems, we longitudinally tracked cell-state dynamics in vivo and revealed a rapid KRAS inhibitor-induced enrichment of the classical state. Lineage tracing identified these enriched classical PDAC cells as reservoirs for disease relapse. Genetic ablation of the classical cell state is synergistic with KRAS inhibition, providing a pre-clinical proof-of-concept for this strategy. Our results uncover an unexpected role of KRAS in promoting intra-tumoral heterogeneity and suggest targeting alveolar or classical epithelial differentiation may augment KRAS-targeted therapies in LUAD and PDAC, respectively.

Lung adenocarcinoma (LUAD) and pancreatic ductal adenocarcinoma (PDAC) together account for ~15% of all cancer mortalities. Both LUAD and PDAC are commonly driven by KRAS mutations. The first allele-specific KRAS inhibitors were recently approved for LUAD, but clinical benefit is limited by intrinsic and acquired resistance. LUAD predominantly arises from alveolar type 2 (AT2) cells, which function as facultative alveolar stem cells by self-renewing and replacing alveolar type 1 (AT1) cells. Using genetically engineered mouse models, patient-derived xenografts and patient samples, we found inhibition of KRAS promotes transition to a quiescent AT1-like cancer cell state in LUAD tumors. Similarly, suppressing KRAS-induced AT1 differentiation of wild-type AT2 cells upon lung injury. The AT1-like LUAD cells exhibited high growth and differentiation potential upon treatment cessation, whereas ablation of the AT1-like cells robustly improved treatment response to KRAS inhibitors. Intra-tumoral heterogeneity in PDAC is characterized by a balance between basal and classical epithelial cancer cell states, with basal dominance associated with chemoresistance and a dismal prognosis. Using genetically engineered mouse models and patient-derived xenografts, we found that basal PDAC cells are highly sensitive to KRAS inhibitors. Employing fluorescent and bioluminescent reporter systems, we longitudinally tracked cell-state dynamics in vivo and revealed a rapid KRAS inhibitor-induced enrichment of the classical state. Lineage tracing identified these enriched classical PDAC cells as reservoirs for disease relapse. Genetic ablation of the classical cell state is synergistic with KRAS inhibition, providing a pre-clinical proof-of-concept for this strategy. Our results uncover an unexpected role of KRAS in promoting intra-tumoral heterogeneity and suggest targeting alveolar or classical epithelial differentiation may augment KRAS-targeted therapies in LUAD and PDAC, respectively.

Advancements in next-generation sequencing (NGS) have transformed transcriptome and small RNA profiling, enabling gene expression studies and the identification of splice variants, isoforms and novel transcripts from challenging samples like liquid biopsies, FFPE tumors and exosomes. This enhances our understanding of molecular mechanisms and aids in identifying RNA variants as potential disease biomarkers. 

In this webinar, we will explore a complete workflow for miRNA biomarker discovery and verification using NGS and digital PCR (dPCR). 

Learning objectives

• Set up for miRNA-seq success: Considerations for sample extraction and library preparation for miRNA-seq projects
• Analyzing your data: Bioinformatic pipeline for analysis
• dPCR assay design: How to leverage sequencing data to verify and design sensitive miRNA biomarker assays by dPCR
• Best practices for sequencing projects: Tips for engaging with sequencing cores

When developing your own master mix, choosing between different options for Hot Start Taq Polymerases and Reverse Transcriptases can be daunting. From endpoint PCR to multiplex PCR, you can accelerate your assay development process and save time by minimizing trial and error using trusted and proven products from QIAGEN.

In this webinar, let’s break down the key features and differentiators you should look out for when evaluating which DNA Taq Polymerases and Reverse Transcriptases are best suited for your molecular assay.

Got other plans? You can still register and we’ll send you a link to the recorded webinar.

When developing your own master mix, choosing between different options for Hot Start Taq Polymerases and Reverse Transcriptases can be daunting. From endpoint PCR to multiplex PCR, you can accelerate your assay development process and save time by minimizing trial and error using trusted and proven products from QIAGEN.

In this webinar, let’s break down the key features and differentiators you should look out for when evaluating which DNA Taq Polymerases and Reverse Transcriptases are best suited for your molecular assay.

Got other plans? You can still register and we’ll send you a link to the recorded webinar.