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Immune checkpoint blockade (ICB) targeting the programmed cell death protein 1 (PD-1) and its ligand 1 (PD-L1) fails to provide clinical benefit for most cancer patients due to primary or acquired resistance. Drivers of ICB resistance include tumor antigen processing/presentation machinery (APM) and IFNγ signaling mutations. Thus, there is an unmet clinical need to develop alternative therapies for these patients. To this end, we have developed a CRISPR/Cas9 approach to generate murine tumor models refractory to PD-1/-L1 inhibition due to APM/IFNγ signaling mutations. Guide RNAs were employed to delete B2m, Jak1, or Psmb9 genes in ICB-responsive EMT6 murine tumor cells. B2m was deleted in ICB-responsive MC38 murine colon cancer cells. We report a detailed development and validation workflow including whole exome and Sanger sequencing, western blotting, and flow cytometry to assess target gene deletion. Tumor response to ICB and immune effects of gene deletion were assessed in syngeneic mice. This workflow can help accelerate the discovery and development of alternative therapies and a deeper understanding of the immune consequences of tumor mutations, with potential clinical implications.

Summary: Sequencing of genes, such as BRCA1 and BRCA2, is recommended for individuals with a personal or family history of early onset and/or bilateral breast and/or ovarian cancer or a history of male breast cancer. Such sequencing efforts have resulted in the identification of more than 17,000 BRCA2 variants. The functional significance of most variants remains unknown; consequently, they are called variants of uncertain clinical significance (VUSs). We have previously developed mouse embryonic stem cell (mESC)-based assays for functional classification of BRCA2 variants. We now developed a next-generation sequencing (NGS)-based approach for functional evaluation of BRCA2 variants using pools of mESCs expressing 10–25 BRCA2 variants from a given exon. We use this approach for functional evaluation of 223 variants listed in ClinVar. Our functional classification of BRCA2 variants is concordant with the classification reported in ClinVar or those reported by other orthogonal assays. Motivation: We have previously used mouse ESCs for functional classification of BRCA2 variants using XTT-based cell proliferation assays. While the results are reliable, the approach is time consuming because each variant is analyzed individually. This has impeded the number of variants that can be examined at a time. To overcome this, we developed an NGS-based medium-throughput approach for functional evaluation of multiple BRCA2 variants at a time.

Characterizing the somatic mutation landscape of a cohort of patients has become a routine task in cancer research in recent years. Such studies are often highly interdisciplinary, requiring iterative analysis that must be evaluated at each step by many researchers. Therefore, there is a growing need for reporting tools that can easily generate interactive reports for sharing data and results with collaborators. Here we present an R package, MAFDash, that tries to simplify summarization and visualization of mutation data from Mutation Annotation Format (MAF) files. The output HTML dashboard is a self-contained report that can be used for downstream analysis and sharing results. MAFDash is freely available on Github (https://github.com/CCBR/MAFDash).