Induction of a mismatch repair deficient genotype by tailored chemical mutagenesis in experimental models of cancer

Abstract

Mismatch repair deficient (MMRd) tumors harbor thousands of somatic mutations enriched for insertion-deletion (indels) conferring high sensitivity to immunotherapy. We sought to reproduce this phenotype using mutagenic agents to engineer an MMRd genotype in immunoresistant cells. The combination of temozolomide (TMZ) and cisplatin led to a rapid accumulation of a high mutational load enriched for indels in murine cell lines resulting from the epigenetic loss of Msh2. Pretreated cells showed sensitivity to PD-1 blockade. Systemic treatment with TMZ, cisplatin, and anti-PD-1 bearing immunoresistant tumor cells led to increased survival, intratumoral T cell infiltration, and downregulation of Msh2 expression without affecting healthy tissues. In a clinical trial with 18 patients with refractory mismatch repair proficient colorectal cancer, no responses were seen, but MMRd signatures emerged in cell-free DNA. These findings show that recapitulating an MMRd genotype through chemical mutagenesis can generate an immunogenic phenotype.

Keywords: cancer; chemotherapy; immune checkpoints; immunogenicity; mismatch repair; mutagenesis; neoantigens.

Figure 1.. The combination of temozolomide and cisplatin induces a mismatch repair deficient (MMRd) genotype

(A) Strategy to identify mutagenic drugs that induce immunogenicity. 8W: 8 weeks; 1 W: 1 week. (B) Selection of compounds aimed at inducing an MMRd genotype, increasing TMB and frameshift mutations. 7,12-Dimethyl benz[a]anthracene (DMBA) was used as a mutagenic control. (C) Viability (MTS assay) of CT26 and B16-F10 cells treated with increasing doses of temozolomide, cisplatin, and DMBA. Data shown as mean viability (percentage) and standard error of the mean for each concentration of drug. (D) Whole exome sequencing (WES, ≥250X mean coverage) of CT26 cells after 8 weeks exposure to vehicle, mutagenic compounds, or combination: temozolomide (TMZ) 20 μM, cisplatin (CDDP) 0.5 μM, temozolomide 20 μM + cisplatin 0.5 μM (TMZ+CDDP), DMBA 25 μM. Mutations are classified as shared (blue) or gained (red) compared to vehicle. TMB changes assessed via non-synonymous single nucleotide variants (NSSNV, red) and frameshift mutations (FS, blue). The microsatellite instability (MSI) score corresponds to absolute value after treatment. (E) Single base substitution (SBS) by trinucleotide context and related COSMIC V3 SBS signatures of gained mutations assessed by WES after exposure of CT26 cells for 8 weeks to vehicle, TMZ 20 μM, CDDP 0.5 μM, TMZ 20 μM + CDDP 0.5 μM or DMBA 25 μM. Gained mutations were obtained by subtracting shared mutations with the CT26 parental cell line and deconvolved for mutational SBS. Total number of mutations and cosine similarity are displayed. Abbreviations: Ultraviolet (UV) light, Reactive Oxygen Species (ROS), Polymerase Epsilon proofreading defect (POLE), Chemotherapy (Chemo). See also Figures S1 and S2.

Figure 2.. The combination of temozolomide and cisplatin induces MSH2 loss through epigenetic silencing of Msh2

Treatments applied to CT26 cells include vehicle, TMZ 20 μM, CDDP 0.5 μM, and TMZ (20 μM) + CDDP (0.5 μM). (A) Protein expression of MLH1, MSH2 and MSH6 assessed by western blot for CT26 cell line pretreated for 8W and CT26 Mlh1^−/− and CT26 Msh2^−/−. (B) Relative protein expression of MLH1, MSH2 and MSH6 blot for CT26 cell line pretreated for 8W. One-way ANOVA. (C) Assessment of microsatellite instability induced by 72h treatment in CT26 cell line. CT26 cells transfected with reporter plasmid containing out of frame luciferase due to microsatellite (CA17 repeats). The luciferase expression is restored by Indels. The boxplot represents the median (middle line), upper quartile (top line), and lower quartile (bottow line). Confidence interval at 90% is reported as whiskers. Please note that there were no statistical differences between groups. (D) Assessment of Msh2 mRNA expression by RT-qPCR after 2, 4 or 8 weeks of treatment. One-way ANOVA. (E) Promoter methylation assessment by MSP-PCR of Mlh1, Msh2 in the CT26 cell line pretreated 8 weeks. (F) Quantification of Msh2 promoter methylation pretreated 4 weeks or 8 weeks. One-way ANOVA. (G) Assessment of microsatellite instability in CT26 parental, CT26 Msh2^−/− and CT26 pretreated 8 weeks by TMZ 20 μM + CDDP 0.5 μM using the same reporter plasmid as in Figure 2C. The boxplot represents the median (middle line), upper quartile (top line), and lower quartile (bottow line). Confidence interval at 90% is reported. One-way ANOVA. (H) Comparison of WES results for the CT26 cell line pretreated 2, 4 and 8 weeks. TMB gain assessment was assessed compared to the baseline TMB of parental CT26. Total frameshift mutations and MSI score per condition are displayed. ****: p ≤ 0.0001, ***: p ≤ 0.001, **: p ≤ 0.01; *: p > 0.01 and ≤0.05. Data are means +/− SEM. “ns” dictates not significant. See also Figure S3.

Figure 3.. In vivo immunogenicity of cell lines exposed to temozolomide and cisplatin

Treatments applied to CT26 cells include vehicle, TMZ 20 μM, CDDP 0.5 μM, and TMZ (20 μM) + CDDP (0.5 μM) or DMBA (25 μM). (A) Tumor growth assessment in immunodeficient NSG mice engrafted with 1 million CT26 cells pretreated for 8W or CT26 Msh2^−/− (n = 5 mice per group). One-way ANOVA at last time point. (B) Follow up of tumor volume (mm³) of CT26 cells pretreated for 8W engrafted in syngeneic BALB/c mice (n = 6 per group). CT26 Msh2^−/− is included as a control. Comparison is reported at 20 days (D20). (C) Tumor growth assessment in syngeneic mice engrafted with 1 million CT26 B2m^−/− cells pretreated 8W or CT26 B2m^WT (wild type) pre-treated 8W by Vehicle or TMZ+CDDP (n = 5 mice per group). One-way ANOVA at last time point. (D) Randomized preclinical sensitivity assessment to anti PD-1 (aPD-1) versus isotype control (IgG) of tumors derived from CT26 cell line pre-treated for 8W. engrafted with 1 million cells in syngeneic BALB/c mice (N = 5 per group). CT26 Msh2^−/− cell line is used as a positive control. No statistical comparison was performed due to high immunorejection rate in the IgG group for the TMZ+CDDP condition. (E and F) Exome sequencing comparison of missense (E) and frameshift (F) mutations in tumor compared to baseline cells. One-way ANOVA. Only significant comparisons are displayed. (G) Absolute loss of TMB in mice tumors samples derived from CT26 cell lines pretreated 8W and engrafted in syngeneic mice compared to cell line from matched condition before engraftment. TMB was assessed by WES. One-way ANOVA. (H) SBS signatures of lost mutations assessed by WES in CT26 derived tumors pretreated 8W before engraftment. Lost mutations were determined by comparing tumoral mutations with the baseline cell line. The total number of lost mutations and cosine similarity are reported. Abbreviations: Ultra Violet (UV) light, Mismatch Repair Deficiency (MMRd), Reactive Oxygen Species (ROS), Polymerase Epsilon proofreading defect (POLE), Chemotherapy (Chemo). (I) MLH1, MSH2, and MSH6 protein expression assessment by immunohistochemistry of syngeneic orthotopic mice tumors derived from CT26 pretreated 8W or CT26 Msh2^−/− at time of sacrifice. Representative staining based on two tumor samples and multiple fields assessment are displayed. *: p ≤ 0.05; **: p ≤ 0.01; ***: p ≤ 0.001; ****: p ≤ 0.0001, Data are means +/− SEM. “NS” dictates not significant. See also Figures S4 and S5.

Figure 4.. Reprogramming of the tumor immune microenvironment after cancer cells exposure to temozolomide and cisplatin

(A and B) Uniform Manifold Approximation and Projection (UMAP) representation of single cell clusters from tumor derived from CT26 pretreated by vehicle for 8W (A) and CT26 pretreated by TMZ+CDDP for 8W (B). ISG+: Interferon-Stimulated Gene positive cells; CAFs: Cancer-Associated Fibroblasts. NK: Natural Killers. (C) Gene ontology analysis of bulk scRNA-seq and in specific clusters of cells displaying enriched pathways compared to vehicle. *: p ≤ 0.05; **: p ≤ 0.01; ***: p ≤ 0.001; ****: p ≤ 0.0001, ANOVA corrected for multiplicity. Only significant comparisons are displayed. (D and E) VDJ TCR sequencing from T cells from tumors derived from CT26 cells pretreated 8 weeks by vehicle, TMZ 20 μM, CDDP 0.5 μM or combination of TMZ 20 μM and CDDP 0.5 μM (TMZ+CDDP) engrafted in syngeneic BALB/c mice (D) and TCR diversity score (E). See also Figure S6.

Figure 5.. Systemic administration of temozolomide and cisplatin with anti-PD-1 to mice bearing immunoresistant tumors

(A) BALB/c mice were engrafted intraperitoneally (IP) with CT26 parental cells and treated with vehicle TMZ + CDDP ± anti-PD-1 or IgG starting 4 days post-engraftment. Mice received daily IP injections of DMSO (0.08%) or TMZ (40 μg, 5 days on, 2 days off), weekly CDDP (1 μg IP), and anti-PD-1 or IgG (200 μg IP, three times weekly) for up to 4 weeks or until sacrifice. The table displays final tumor rejection rates. (B) Kaplan-Meier cancer-free survival analysis with relevant group comparisons (log rank test). (C) Immunofluorescence assessment of CD3⁺ and CD8⁺ intratumoral cells. Quantification was performed on 10 independent fields of 0.2 mm² for each tumor. One-way ANOVA. Data are represented as means +/− SEM. (D) Normalized mRNA expression in tumors originating from CT26 cell line and challenged intraperitoneally by vehicle, TMZ+CDDP and TMZ+CDDP + anti-PD-1. RT-qPCR was performed on 10 tumors samples of 1 mm³ per condition. The violin plot represents the median (middle dashed line), upper quartile (top dashed line), and lower quartile (bottom dashed line). The violin plot extends to minimum and maximum observed values. One-way ANOVA. (E) Exome sequencing of healthy tissues (brain, liver, bone marrow, GI tract, lung) from mice after 8W exposure to vehicle control (mouse 1: M1) or TMZ+CDDP +anti PD-1 (mouse 4: M4, mouse 5: M5). Number of mutations are displayed as mutation per megabase by class. BM: Bone Marrow. One-way ANOVA. Data are means +/− SEM. (F) Comparison of TMB and MSI scores in healthy tissues of mice treated by vehicle (5 organs from 1 mouse) or intraperitoneal combination of TMZ+CDDP + anti PD-1 (9 organs from 2 mice). Mice received 8 weeks treatment. The violin plot represents the median (middle dashed line), upper quartile (top dashed line), and lower quartile (bottow dashed line). The violin plot extends to minimum and maximum observed values. Student’s t test. (G) SBS signatures identified by WES in healthy tissues of mice treated by vehicle (5 organs/mouse) or intraperitoneal combination of TMZ+CDDP+ anti PD-1 (9 organs from 2 mice). Lost mutations were determined by comparing tumoral mutations with the baseline cell line. The total number of lost mutations and cosine similarities are reported. Abbreviations: cytosine deaminases (APOBEC). (H) Immunohistochemistry assessment of MLH1, MSH2 and MSH6 expression in tumors derived from mice challenged intraperitoneally by TMZ+CDDP. Tumors were collected 8W after engraftment at time of sacrifice. Regions with focal losses are highlighted and not observed with vehicle treatment. T: Tumor deposit; GI: gastrointestinal region. ****: p ≤ 0.0001; ***: p ≤ 0.001, **: p ≤ 0.01, *: p ≤ 0.05, ns: p > 0.05. See also Figure S6 and Data S1.

Figure 6.. Treatment of patients with refractory metastatic colorectal cancers with temozolomide, cisplatin and PD-1 blockade and resulting genomic profiles assessed by ctDNA

(A) Study design. Patients with refractory, mismatch repair proficient colorectal cancer were treated with temozolomide, cisplatin and nivolumab. Baseline status for low mutational burden and microsatellite score was confirmed by tumoral sequencing using the MSK-IMPACT panel and blood TMB and blood MSI score dynamics were studied using cell-free DNA analyses. (B and C) Cell-free DNA measurements of (B) blood tumor mutational burden (bTMB), (C) blood microsatellite instability (bMSI) score at baseline and at last available sample. One-way ANOVA mixed-effects analysis. (D) Number of mutations from baseline, to cycle 3 to cycle 5. Lines indicate samples from the same patients. Paired t-test. (E) Microsatellite score from baseline, to cycle 3 to cycle 5. Lines indicate samples coming from the same patients. Paired t-test. (F) Tumor fraction from baseline, to cycle 3 to cycle 5. Lines represent samples coming from the same patients. Paired t-test. (G) Relative contribution of SBS signatures between mutations in baseline samples vs. gained in subsequent samples. (H) Relative contribution of SBS signatures between potentially immunoedited and non-immunoedited mutations. HRD, Homologous recombination. The violin plots (B–F) represent the median (middle dashed line), upper quartile (top dashed line), and lower quartile (bottow dashed line). Each violin plot extends to minimum and maximum observed values. ***: p ≤ 0.001; **: p ≤ 0.01; *: p ≤ 0.05; ns: p > 0.05. See also Figure S7.

Figure 7.. Survival outcomes of patients with refractory metastatic colorectal cancers treated with temozolomide and cisplatin and PD-1 blockade based on acquired genotypes

(A) Swimmer plot summarizing the genotype and clinical outcomes of each patient. Gain in bTMB and bMSI score are defined based on the median increases observed in the clinical trial. (B) Overall survival (OS) according to acquired bTMB and bMSI score on treatment. Gain in bTMB and in bMSI score are defined based on the median increases observed in the clinical trial as explained above. We report the median OS per group. Log rank test. (C) Assessment of OS according to acquired bTMB and bMSI score from Cycle 3 (C3). Log rank test. (D) Oncoprint displaying genomic features of patients at baseline and at the end of treatment with temozolomide, cisplatin, and nivolumab. Copy number gains in mismatch repair (MMR) genes are reported at baseline and at the end of treatment. See also Figure S7 and Table S1.