Two main mutational processes operate in the absence of DNA mismatch repair

The analysis of tumour genome sequences has demonstrated high rates of base substitution mutagenesis upon the inactivation of DNA mismatch repair (MMR), and the resulting somatic mutations in MMR deficient tumours appear to significantly enhance the response to immune therapy. A handful of different algorithmically derived base substitution mutation signatures have been attributed to MMR deficiency in tumour somatic mutation datasets. In contrast, mutation data obtained from whole genome sequences of isogenic wild type and MMR deficient cell lines in this study, as well as from published sources, show a more uniform experimental mutation spectrum of MMR deficiency. In order to resolve this discrepancy, we reanalysed mutation data from MMR deficient tumour whole exome and whole genome sequences. We derived two base substitution signatures using non-negative matrix factorisation, which together adequately describe mutagenesis in all tumour and cell line samples. The two new signatures broadly resemble COSMIC signatures 6 and 20, but perform better than existing COSMIC signatures at identifying MMR deficient tumours in mutation signature deconstruction. We show that the contribution of the two identified signatures, one of which is dominated by C to T mutations at CpG sites, is biased by the different sequence composition of the exome and the whole genome. We further show that the identity of the inactivated MMR gene, the tissue type, the mutational burden or the patient's age does not influence the mutation spectrum, but that a tendency for a greater contribution by the CpG mutational process is observed in tumours as compared to cultured cells. Our analysis suggest that two separable mutational processes operate in the genomes of MMR deficient cells. © 2020 The Author(s

PO-332 Genomic landscapes, neoantigen profiles and biological impact of MLH1 inactivation in cancer cells

Introduction Alterations in DNA repair pathways are thought to fuel tumour progression. Mismatch Repair (MMR) deficient cancers show peculiar biological features such as an indolent progression and a resolute therapeutic response to checkpoint inhibitors. The genomic and biological bases of the peculiar clinical features are poorly understood. Further progress in this area is limited by the paucity of models to study the impact of MMR genes inactivation at the genomic and biological levels. To address this issue we developed a bioinformatic workflow to monitor the neoantigen repertoire induced by inactivation of the Mlh1 gene (a key player of the MMR machinery), in murine cell lines. Material and methods We inactivated Mlh1 throughout the CRISPR-Cas9 technology in CT26 (colon cancer), PDAC (pancreatic cancer) and TSA (breast cancer) murine cell lines. We performed whole exome sequencing (WES) at different time points and then we quantified the amount of mutations (SNVs and indels). We generated a pipeline that characterises the neoantigen repertoire, starting from annotated alterations and the HLA of specific murine strain. In parallel, we inoculated MMR-proficient and -deficient cells in immuno-compromised and -competent mice and monitored their growth. Results and discussions In all pre-clinical models analysed we found a massive increment in the number of non-synonymous alterations (up to 100% increase respect to basal population) after Mlh1 inactivation. Notably, analysis of MMR deficient mouse cells at different time points showed a renewal of mutational profile and consequently an accumulation of predicted neoantigens. We further characterised the SNVs and frameshifts acquired by Mlh1-knockout cells. In agreement with data in human tumours, the fraction of predicted neoantigens derived from frameshifts was higher than the SNV-derived neoantigens. When injected in immuno-compromised mice the Mlh1-knockout cells and their wild type counterpart showed comparable growth. On the contrary, MMR-deficient cells but not their control counterpart grew poorly in immuno-competent mice and responded promptly to treatment with checkpoint inhibitors. Conclusion We find that Mlh1 gene inactivation drives dynamic neoantigen profiles, which can be monitored with an ad hoc bioinformatic pipeline. These analyses provide mechanistic support to understand why MMR deficient cells engage the immune system of the host, foster immune surveillance and tumour control

Additional file 1 of Non-canonical antigens are the largest fraction of peptides presented by MHC class I in mismatch repair deficient murine colorectal cancer

Additional file 1. Supplementary figures S1-S9

Genetic and pharmacological modulation of DNA mismatch repair heterogeneous tumors promotes immune surveillance

Patients affected by colorectal cancer (CRC) with DNA mismatch repair deficiency (MMRd), often respond to immune checkpoint blockade therapies, while those with mismatch repair-proficient (MMRp) tumors generally do not. Interestingly, a subset of MMRp CRCs contains variable fractions of MMRd cells, but it is unknown how their presence impacts immune surveillance. We asked whether modulation of the MMRd fraction in MMR heterogeneous tumors acts as an endogenous cancer vaccine by promoting immune surveillance. To test this hypothesis, we use isogenic MMRp (Mlh1+/+) and MMRd (Mlh1-/-) mouse CRC cells. MMRp/MMRd cells mixed at different ratios are injected in immunocompetent mice and tumor rejection is observed when at least 50% of cells are MMRd. To enrich the MMRd fraction, MMRp/MMRd tumors are treated with 6-thioguanine, which leads to tumor rejection. These results suggest that genetic and pharmacological modulation of the DNA mismatch repair machinery potentiate the immunogenicity of MMR heterogeneous tumors

Dendritic cell-targeted therapy expands CD8 T cell responses to bona-fide neoantigens in lung tumors

International audienceCross-presentation by type 1 DCs (cDC1) is critical to induce and sustain antitumoral CD8 T cell responses to model antigens, in various tumor settings. However, the impact of cross-presenting cDC1 and the potential of DC-based therapies in tumors carrying varied levels of bona-fide neoantigens (neoAgs) remain unclear. Here we develop a hypermutated model of non-small cell lung cancer in female mice, encoding genuine MHC-I neoepitopes to study neoAgs-specific CD8 T cell responses in spontaneous settings and upon Flt3L + αCD40 (DC-therapy). We find that cDC1 are required to generate broad CD8 responses against a range of diverse neoAgs. DC-therapy promotes immunogenicity of weaker neoAgs and strongly inhibits the growth of high tumor-mutational burden (TMB) tumors. In contrast, low TMB tumors respond poorly to DC-therapy, generating mild CD8 T cell responses that are not sufficient to block progression. scRNA transcriptional analysis, immune profiling and functional assays unveil the changes induced by DC-therapy in lung tissues, which comprise accumulation of cDC1 with increased immunostimulatory properties and less exhausted effector CD8 T cells. We conclude that boosting cDC1 activity is critical to broaden the diversity of anti-tumoral CD8 T cell responses and to leverage neoAgs content for therapeutic advantage