MSAI-Path: Predicting Microsatellite Instability From Routine Histology Slides Without Reinventing the Wheel.

Single-cell transcriptome profiling of post-treatment and treatment-naïve colorectal cancer: Insights into putative mechanisms of chemoresistance.

Benchmarking pathology foundation models for predicting microsatellite instability in colorectal cancer histopathology.

Proteogenomic profiling predicts outcomes of adjuvant chemotherapy in extrahepatic cholangiocarcinoma.

Colorectal cancer (CRC) becomes highly lethal upon progression to advanced or metastatic stages. Treatment options are particularly limited for refractory metastatic CRC (mCRC) harboring KRAS mutations. In this study, we established a series of patient-derived organoids (PDOs) and patient-derived xenografts (PDXs) from mCRC patients to identify effective therapeutic compounds. We employed RNA sequencing to characterize the transcriptomic profiles of KRAS-mutant microsatellite stable (MSS) PDOs and analyzed single-cell RNA sequencing data to examine features of KRAS-mutant CRC epithelial cells. Transcriptomic analysis revealed that KRAS mutations induce elevated global transcription activity in both PDOs and epithelial cells. A large-scale drug screen of 786 Food and Drug Administration (FDA)-approved anticancer agents identified the acridine compound amsacrine hydrochloride as a potent inhibitor of PDOs and cell lines. We subsequently synthesized a series of acridine derivatives for further screening. Finally, LS-1-2 was discovered to overcome chemotherapy resistance and suppress liver metastasis in KRAS-mutated CRC. Mechanistically, LS-1-2 binds to non-muscle myosin heavy chain IIA (NMHC IIA), blocking its phosphorylation. This inhibition disrupts the PI3K/ERK/FOXO/PLK1 signaling pathway and attenuates KRAS-driven hypertranscription. In conclusion, the acridine derivative LS-1-2 emerges as a promising candidate from this preclinical investigation, providing a rationale for future clinical trials in KRAS-mutant CRC.

Immune escape in colorectal cancer: Mechanisms and challenges of immune checkpoint inhibitor resistance.

ABSTRACT The recent introduction of immune checkpoint inhibitor therapy has significantly improved outcomes for patients with colorectal cancer (CRC). The most pronounced clinical benefits were observed in patients with immunogenic microsatellite instable (MSI)/deficient MMR (dMMR) tumors. However, emerging evidence indicates that a subset of patients with microsatellite stable tumors may also respond to therapy. Finding predictive markers to identify these patients is critical. In this study, we analyzed the immunohistochemical expression of immune checkpoints CTLA-4, PD-1, and PD-L1 using multispectral imaging in 151 CRC patients with defined molecular characteristics. Consistent with prior reports, MSI tumors had higher levels of all immune checkpoints analyzed than microsatellite stable tumors. Notably, distinct patterns of immune checkpoint expression were associated with KRAS and BRAF mutation status. KRAS-mutated tumors showed lower, and BRAF-mutated tumors higher, expression of immune checkpoints compared to wild-type/wild-type tumors. The strongest association with KRAS and BRAF mutations was observed for PD-L1 expression. The relationship between PD-L1 and KRAS/BRAF-mutational status was validated in a second cohort of 527 CRC patients, finding the association for PD-L1 expression in both stroma and in tumor cells. Furthermore, the role of BRAF mutation on immunity in CRC was found to be partly independent of MSI status. The strongest prognostic role was found for PD-L1 in stroma, underscoring the clinical significance of this marker. In conclusion, our findings suggest that KRAS and BRAF mutations, alongside MSI, may serve as valuable biomarkers for identifying CRC patient subgroups likely to benefit from immune checkpoint blockade in CRC.

To investigate the expression characteristics of Microsatellite Instability (MSI) and Mismatch Repair (MMR) protein in gastric cancer, analyze their correlation with clinicopathological parameters, prognosis, and response to immunotherapy, and provide a basis for individualized treatment of gastric cancer.

DDB1 and CUL4-associated factor 7 (DCAF7) is a WD-repeat adaptor that recruits substrates to the CUL4DDB1 ubiquitinligase complex, but its pan-cancer relevance and mechanistic contribution to tumor progression remain unclear. Multi-omics datasets (genomic, transcriptomic, epigenomic, proteomic and single-cell) from 33 tumor types were integrated to define DCAF7 expression, regulation, and clinical significance. Somatic alterations and copy-number variation were analysed across cohorts, and promoter methylation and RNA modification signatures were interrogated. Immune associations were assessed by computational deconvolution and checkpoint-gene profiling. Pathway and network analyses were performed to infer DCAF7-linked programmes. Mechanistic and functional validation was conducted in hepatocellular carcinoma (LIHC) cell lines (HepG2, Huh7) using DCAF7 perturbation and pharmacologic Wnt inhibition. DCAF7 was overexpressed in most cancers, consistent with copy-number gain, focal promoter hypomethylation and putative m6A-linked post-transcriptional regulation, whereas hypermethylation at two CpG loci predicted poor prognosis in LIHC. DCAF7 alterations, predominantly amplifications, were associated with shorter overall survival in LIHC and positively correlated with DCAF7 mRNA abundance across cohorts. Immunogenomic analyses linked high DCAF7 to CD4+ T-cell enrichment, broad upregulation of checkpoint genes (PD-1/PD-L1, CTLA-4, TIGIT), and increased tumour mutational burden, microsatellite instability and neoantigen load, suggesting an immune-evasive phenotype. Network and enrichment analyses converged on canonical Wnt/β-catenin, Hippo and cell-cycle programs. In vitro, DCAF7 promoted LIHC cell proliferation and migration by stabilising β-catenin via increased inhibitory Ser9 phosphorylation of GSK-3β, thereby inducing c-Myc and cyclin D1; DCAF7 knockdown or the Wnt inhibitor XAV939 attenuated these effects. Drug-response modelling further predicted increased sensitivity of DCAF7-high tumours to 17-AAG, docetaxel and alsterpaullone. DCAF7 is frequently activated by genetic and epigenetic mechanisms across cancers, associates with an immunotherapy-relevant tumour immune milieu, and drives Wnt/β-catenindependent malignant phenotypes in LIHC. These findings support DCAF7 as a prognostic biomarker and a candidate therapeutic target, particularly for stratified intervention in LIHC. DCAF7 is up-regulated in various tumours and correlates with poor prognosis, particularly in LIHC. High DCAF7 expression is linked to CD4+ T cell infiltration, up-regulation of immune checkpoint genes and increased tumour mutational burden, suggesting a role in tumour immune escape. DCAF7 stabilises β-catenin by enhancing GSK-3β Ser9 phosphorylation, thereby driving c-Myc/cyclin D1 expression and contributing to proliferation and migration in LIHC. DCAF7-high tumours demonstrate therapeutic vulnerability to 17-AAG, docetaxel and CDK/GSK-3 inhibitor, revealing potential targeted treatment strategies.

ABSTRACT Colorectal cancer (CRC) remains a formidable clinical challenge, many patients exhibit limited responses to conventional chemotherapy and targeted therapies. Although immunotherapy has demonstrated potential, its efficacy is largely restricted to a subset of patients with high microsatellite instability (MSI-H), highlighting the critical need to identify key molecular drivers of immune evasion in CRC. Through comprehensive bioinformatic analysis, we identified the deubiquitinating enzyme JOSD2 as a key player in CRC progression, with elevated expression correlating with poor prognosis in MSI-H patients (HR = 4.79, 95% CI: 3.6–7.96) and dysregulation of multiple immune-related pathways. Mechanistically, we discovered that JOSD2 suppresses cGAS enzymatic activity by removing K27-linked ubiquitination, thereby promoting M2 polarization of macrophages, a process critical for immunosuppression in the tumor microenvironment. Furthermore, using the JOSD2 catalytic inhibitor HY041004, we demonstrated both in vitro and in vivo that the inhibition of JOSD2 activated the cGAS-STING signaling pathway, leading to robust anti-tumor effects in CRC. These findings not only uncover a novel immunomodulatory mechanism in colorectal cancer but also provide a therapeutic rationale for the development of JOSD2-targeted anticancer strategies.

Inflammation plays a critical role in cancer initiation and progression by modulating the tumor microenvironment and immune responses. Interleukin-1 receptor-associated kinase 2 (IRAK2) is a key mediator of the Toll-like receptor and interleukin-1 receptor signaling pathways, its pan-cancer expression patterns, genomic and epigenetic features, immune-related roles, and clinical relevance remain unclear. The expression patterns of IRAK2 across multiple cancer types, transcript variants, single-cell distribution, prognostic significance, and biological functions were comprehensively evaluated through analyses of multiple databases and multi-dimensional datasets. Furthermore, the correlations of IRAK2 with the immune microenvironment, epigenetic modifications, and drug sensitivity were investigated. The potential role of IRAK2 in hepatocellular carcinoma was further explored through both in vitro and in vivo experiments. Aberrant expression of IRAK2 was observed in the majority of cancer types, with a relatively high proportion of expression detected in macrophages, and was found to be associated with the prognosis of certain cancers. In most cancer types, IRAK2 expression showed significant correlations with immune cell infiltration, the cancer-immunity cycle, major histocompatibility complex molecules, immune checkpoints, tumor mutational burden, microsatellite instability, RNA modifications (including m1A, m5C, and m6A), and DNA methylation sites. Both in vitro and in vivo experiments demonstrated that knockdown of IRAK2 markedly reduced the proliferative capacity of hepatocellular carcinoma cells. The present study highlights the potential of IRAK2 expression as a novel biomarker for predicting the prognosis and immunotherapeutic response across various human cancers.

Background/Objectives: PHF6 is a chromatin-binding protein located in the nucleus, and it is involved in transcriptional regulation. However, limited research exists on the specific roles and mechanisms of PHF6 across various tumors. Methods: Based on The Cancer Genome Atlas (TCGA) database, we analyzed PHF6 expression in pan-cancer. We first evaluated the relevance between PHF6 and prognosis; then, the relevance between PHF6 and immune cell infiltration in pan-cancer were analyzed. Subsequently, we explored the correlation between PHF6 and cancer heterogeneity, such as tumor mutation burden (TMB) and microsatellite instability (MSI), as well as cancer stemness. Finally, the role of PHF6 was validated in liver cancer and pancreatic cancer cell lines by cell proliferation assays. Results: PHF6 expression was higher in the vast majority of cancers than their normal counterparts. PHF6 was substantially correlated with prognosis and immune cell infiltration in various cancers. Moreover, PHF6 expression showed a strong correlation with cancer heterogeneity and stemness in certain cancer types. Additionally, the depletion of PHF6 inhibited cell proliferation in both liver and pancreatic cancer cells. Conclusions: PHF6 expression was closely associated with the occurrence and development of many types of cancer, and it might promote cancer progression by inhibiting the function of the immune microenvironment, while knockout of PHF6 significantly inhibited the tumor cells proliferation.

This study aimed to identify and predict bioactive small molecules and immunogenic antigen epitopes targeting proteins with point mutations in colitis-associated colorectal cancer (CAC), using integrative bioinformatics approaches. Genes associated with colorectal cancer (CRC), ulcerative colitis (UC), diabetes mellitus (DM), hyperlipidemia (HL), gout, and functional dyspepsia (FD) were obtained from disease databases and GEO. Overlapping genes were identified using jvenn. CIBERSORT was used for immune infiltration analysis. Prognostic significance was evaluated using KM Plotter, and gene co-expression correlations in CRC were assessed via TIMER. cBioPortal was employed to analyze mutation profiles, tumor mutational burden (TMB), and microsatellite instability (MSI). Allosteric sites were predicted using AlloDriver. Molecular docking (AutoDock Vina v1.2.3) and 100-ns molecular dynamics simulations (GROMACS v2022.03) assessed protein-ligand interactions. Neoantigen and B-cell epitope immunogenicity were predicted using NetMHCpan-4.1 and IEDB, respectively. SERPINE1, SELE, SPP1, PLAU, COL1A1, PTGES, IL6, KDR, and ICAM1 were significantly upregulated in CAC. Lower expression correlated with improved relapse-free and overall survival (log-rank P < 0.05) and was associated with CRC molecular subtypes and biomarkers (P < 0.05). Mutations PLAUY151H, SERPINE1V166A, and KDRP359H were located in allosteric sites. Anacetrapib and LY2090314-metabolism-related compounds-showed high affinity for wild-type KDR, PLAU, and SERPINE1.The KDRP359H and PLAUY151H significantly weakened LY2090314 binding, and SERPINE1V166A mutation significantly reduced Anacetrapib binding, whereas SERPINE1V166A significantly enhanced LY2090314 binding. Although HLA-I affinity of mutation-derived antigens was low, four mutated B-cell epitopes (COL1A1R493H, KDRP551S, ICAM1T1259M, KDRT1260M) exhibited higher immunogenicity than wild-type, suggesting potential for antibody-based therapy. Based on bioinformatics analysis, this study identified nine genes significantly upregulated in CAC and associated with prognosis. Allosteric-site mutations in PLAU, SERPINE1, and KDR affect the binding stability of small-molecule drugs (Anacetrapib, LY2090314), suggesting their role in drug response. Four mutation-specific B-cell epitopes (COL1A1R493H, KDRP551S, ICAM1T1259M, KDRT1260M) exhibit enhanced immunogenicity, indicating potential for antibody-based therapy.

Background: Mismatch repair (MMR) and microsatellite instability (MSI) testing have become essential biomarkers in the molecular classification of endometrial cancer (EC), guiding adjuvant treatment decisions and eligibility for immune checkpoint inhibition. Although international guidelines recommend universal testing, real-world implementation remains heterogeneous. This study aimed to evaluate trends in MMR and MSI testing and associated molecular diagnostics in Germany between 2018 and 2022. Methods: A retrospective multicenter analysis was conducted across German tertiary care centers. Data from patients with histologically confirmed EC between 2018 and 2022 were extracted from standardized electronic pathology records. Annual testing rates for MSI, MMR, POLE, TP53, and L1CAM were analyzed using descriptive statistics and trend analysis (Chi-square test for trend, p < 0.05). Therapeutic data were collected to assess the use of immune checkpoint inhibitors. Results: There was a significant increase in the annual rates of molecular testing for MSI, POLE, TP53, and L1CAM over the five-year observation period (all p < 0.05). TP53 testing showed the highest increase (13.1% → 78.6%), while MSI testing rose from 82.9% to 97.4%. Both POLE and L1CAM testing were virtually absent in 2018 (0% and 1.6%) but reached 15.7% by 2022. Conclusions: This study demonstrates a rapid and substantial implementation of MMR and MSI testing in German clinical practice, reflecting successful translation of trial results into routine care. However, implementation of testing in guidelines appeared time-shifted. For bridging this gap, annual guideline updates seem to be necessary.

The mismatch repair (MMR) system plays a crucial role in correcting replication errors; when the MMR system is deficient (dMMR), replication errors (particularly within microsatellites) accumulate throughout the genome, leading to microsatellite instability (MSI). The key MMR genes include MLH1, MSH2, MSH6, and PMS2. Germline mutations in these genes are associated with Lynch syndrome, whereas in sporadic solid tumors, dMMR is often caused by hypermethylation of the MLH1 promoter. As the clinical use of dMMR/MSI expands, the importance of reliable testing for dMMR or MSI in companion diagnostics continues to increase. dMMR/MSI is diagnosed using immunohistochemistry (IHC) for MMR proteins, polymerase chain reaction with fragmentation analysis, or next-generation sequencing. Although IHC has technical limitations, it requires less tissue, has a short processing time, and is cost-effective. Experienced or specialized pathologists and educational efforts are helpful for reliable diagnosis, in addition to the technical aspects. Solid tumors with dMMR/MSI exhibit distinct clinicopathological features, including prognostic significance and a predictive role in adjuvant cytotoxic chemotherapy. Solid tumors with dMMR/MSI are also characterized by a higher tumor mutational burden and abundant immune cell infiltration, making them promising candidates for immune checkpoint inhibitor therapy. However, the oncogenic processes and immune microenvironment are not identical across the organs of origin, between patients, and even within the same patient, which should be considered in future studies. This review provides an overview of the practical aspects of dMMR/MSI testing, along with the molecular mechanisms and immune microenvironments associated with dMMR/MSI solid tumors.

Approximately 24 000 people die of colorectal cancer (CRC) in Germany each year. New developments in prevention, diagnosis, and treatment are improving long-term outcomes. These measures have been incorporated in the updated guideline and are presented here along with the findings of other new randomized controlled trials (RCTs). A systematic search (2022-2023) was conducted for guidelines, reviews/meta-analyses, and primary studies (search term "colorectal cancer"; databases: international guideline registries, PubMed, Cochrane). 123 reviews were identified, evaluated, and used in the creation of the guideline. Early detection is performed with colonoscopy every 10 years, sigmoidoscopy every 5 years, or an immunological stool test for occult blood every 1-2 years; it can lower cancer-related mortality by up to 30%. Hereditary forms (Lynch syndrome, polyposis) require more intensive monitoring. All diagnosed cases must be presented to a tumor board. In the case of low-risk pT1 carcinoma, extended resection is not indicated after complete endoscopic removal. As for surgery, robotic and laparoscopic procedures are equivalent (3-year recurrence rate 1.6% and 4.0% respectively, in a recent RCT; adjusted hazard ratio, 0.39; 95% confidence interval: [0.19; 0.80]). Treatment of patients with mismatch repair deficiency (dMMR) or microsatellite instability (MSI) with checkpoint inhibitors yields response above 90%. Further systemic therapies are based on the molecular tumor profile (dMMR/MSI, RAS, and BRAF). Organ-preserving strategies after a complete response require intensive follow-up care. The efficacy of early detection is undisputed. Less evidence is available concerning the optimal level of intensity of follow-up care. The clinical and molecular stratification of tumors enables differential treatment. Organ-preserving strategies after a complete response to therapy are an important area of current research.

Complement Factor H (CFH) has been reported as an indispensable role in innate and adaptive immunity. Despite increasing interest in CFH, its clinicopathological implications and immunological mechanisms across different cancers remain unclear and are currently under debate. Investigating the function of CFH in 23 tumor types using The Cancer Genome Atlas (TCGA), Genotype-Tissue Expression (GTEx), The Tumor Immune Estimation Resource 2.0 (TIMER2.0) database, cBioportal, Human Protein Atlas (HPA). Then using Cell Counting Kit-8 (CCK8) assay, plate colony formation assay, and wound healing assay to detect the biological function of CFH in vitro. CFH exhibited dysregulated expression across multiple cancers, with associations observed between its expression levels, immune cell infiltration, clinical stage, and immune subtype. Additionally, CFH was critically involved in shaping the tumor microenvironment and regulating immune responses. The upregulated expression level of CFH was significantly related to tumor-infiltrating immune cells (TICs), tumor mutational burden (TMB), and microsatellite instability (MSI). Functionally, we found that knockdown of CFH could inhibit the tumor cell proliferation and invasion. This study implies the potential prognostic and immunological role of CFH in various cancers, and CFH could act as a biomarker for tumor immunotherapy.

Background and Objectives: BRCA1-interacting protein C-terminal helicase 1 (BRIP1) encodes a member of the RecQ DEAH helicase family and interacts with the BRCT repeats of breast cancer type 1 (BRCA1). It also participates in DNA damage repair and tumor suppression; thus, its mutations may be associated with an increased risk of several cancers, including fallopian tube and ovarian cancer. Recent research has explored whether BRIP1 dysregulation also contributes to the development and progression of other malignancies. This study investigated the clinical and prognostic value of BRIP1 in colorectal cancer (CRC). Materials and Methods: We first analyzed The Cancer Genome Atlas (TCGA) dataset to evaluate the prognostic significance of BRIP1 mRNA expression in CRC. BRIP1 expression was subsequently examined in tumor tissues from 60 CRC patients, and its associations with clinicopathological characteristics and clinical outcomes were assessed. Results: In rectal cancer, a higher BRIP1 expression was associated with younger age. In colon cancer, BRIP1 expression was correlated with gender, lymphatic invasion, carcinoembryonic antigen (CEA) level, pathological stage, M stage, N stage, microsatellite instability (MSI) status, and anatomical tumor location. Survival analysis showed that low BRIP1 expression was associated with poorer overall survival in both rectal and colon cancers significantly. In CRC patient tissues, lower BRIP1 expression was further related to elevated CEA levels and unfavorable clinical outcomes. Lower BRIP1 mRNA expression is significantly associated with aggressive clinicopathological features and poor prognosis in CRC. Conclusions: BRIP1 may serve as a promising biomarker for risk stratification and a potential therapeutic target in the management of CRC.

Immunotherapy has significantly improved survival outcomes for various cancer types, despite suboptimal efficacy in over 60% of patients. However, identifying reliable predictive biomarkers for patient response is still a focus of ongoing research, as current indicators often lack consistency. This network meta-analysis (NMA) systematically compared the predictive performance of 13 biomarkers across multiple cancer types to identify optimal predictors of immunotherapy efficacy. We systematically searched PubMed, OVID, Embase, Cochrane Trials, Web of Science, and trial registries (ClinicalTrials.gov, WHO ICTRP) from inception to September 1st, 2025, and conducted a comprehensive NMA evaluating sensitivity, specificity, diagnostic odds ratio (DOR), superiority, and area under the curve (AUC) for 13 biomarkers. These included circulating tumor DNA (ctDNA), programmed cell death ligand 1 (PD-L1; at varying thresholds), tumor mutational burden (TMB), CD8+tumor-infiltrating lymphocytes (CD8+TILs), microsatellite instability (MSI), and inflammatory markers such as neutrophil-lymphocyte ratio (NLR), platelet-lymphocyte ratio (PLR), lactate dehydrogenase (LDH), Lung Immune Prognostic Index (LIPI), and immune-related adverse effects (irAEs). Subgroup analyses were performed for non-small cell lung cancer (NSCLC), melanoma, gastrointestinal (GI) cancer, urothelial carcinoma, and head and neck squamous cell carcinoma (HNSCC). Heterogeneity and publication bias were assessed using I2 statistics and Deeks' funnel plots. This analysis included 54,634 patients from 194 clinical studies worldwide, evaluating various predictive biomarkers. PD-L1 expression was the most frequently assessed (n=212), with different cutoffs (≥1% [n=110],≥5% [n=45],≥10% [n=32],≥50% [n=25]), followed by TMB (n=68). Other markers included NLR (n=20), irAEs (n=17), MSI (n=14), CD8+TILs (n=14), ctDNA (n=14), PLR (n=12), LIPI (n=11), and LDH (n=10). Among these, ctDNA demonstrated the highest sensitivity (0.82, 95% CI: 0.72-0.89) and overall discriminative power (DOR=9.75, 95% CI: 5.20-16.73; AUC=0.769). PD-L1 demonstrated threshold-dependent performance:≥50% cutoff demonstrated the highest specificity among PD-L1 thresholds (0.78, 95% CI: 0.73-0.81) and diagnostic accuracy (DOR=2.60, 95% CI: 1.86-3.52; AUC=0.661) but the lowest sensitivity (0.42, 95% CI: 0.36-0.49).≥10% threshold showed sensitivity of 0.44 (95% CI: 0.38-0.51) with specificity of 0.74 (95% CI: 0.70-0.78; AUC=0.656).≥5% cutoff demonstrated sensitivity of 0.54 (95% CI: 0.48-0.60) and specificity of 0.66 (95% CI: 0.62-0.70; AUC=0.631). Conversely,≥1% cutoff achieved the highest sensitivity among PD-L1 thresholds (0.68, 95% CI: 0.65-0.71) at the cost of the lowest specificity (0.48, 95% CI: 0.45-0.51; AUC=0.601). TMB balanced sensitivity (0.56, 95% CI: 0.50-0.60) and specificity (0.69, 95% CI: 0.65-0.73; AUC=0.637). MSI status had the highest specificity (0.89, 95% CI: 0.85-0.93; AUC=0.727) but low sensitivity (0.36, 95% CI: 0.27-0.46), supporting its role in confirmatory testing. CD8+TILs showed good sensitivity (0.69, 95% CI: 0.58-0.79) but lower specificity (0.59, 95% CI: 0.49-0.67; AUC=0.632). irAEs displayed relatively higher sensitivity (0.69, 95% CI: 0.60-0.77) with moderate specificity (0.59, 95% CI: 0.50-0.67; AUC=0.674). Among inflammatory markers, PLR (AUC=0.623) showed slightly better predictive power than NLR (AUC=0.613), while LIPI and LDH exhibited the least overall effectiveness (AUC=0.585 and 0.544, respectively). Biomarker performance varies by cancer type and clinical context, underscoring the potential for individualized immunotherapy strategies. ctDNA, PD-L1 (high thresholds, as ≥50%), and TMB emerge as leading predictors, while combinations may optimize sensitivity and specificity. Future research should focus on overcoming heterogeneity and standardization challenges to further refine and individualize immunotherapy approaches and target patients who may benefit from immunotherapy.

Mutations in DNA mismatch repair (MMR) pathway genes (MSH2, MSH6, MLH1, and PMS2) are linked to acquired resistance to temozolomide (TMZ) and high tumor mutation burden (TMB) in high-grade gliomas (HGG), including glioblastoma (GBM). However, the specific roles of individual MMR genes in the initiation, progression, TMB, microsatellite instability (MSI), and resistance to TMZ in glioma remain unclear. Here, we developed de novo mouse models of germline and somatic MMR-deficient (MMRd) HGG. Surprisingly, loss of Msh2 or Msh6 does not lead to high TMB, MSI, nor confer response to anti-PD-1 in GBM. Similarly, human GBM shows discordance between MMR gene mutations and TMB/MSI.Germline MMRd leads to promoted progression from low-grade to HGG and reduced survival compared to MMR-proficient (MMRp) tumor-bearing mice. This effect is not tumor cell intrinsic but is associated with MMRd in the tumor immune microenvironment, driving immunosuppressive myeloid programs, reduced lymphoid infiltration, and CD8+ T cell exhaustion. Both MMR-reduced (MMRr) and MMRd GBM are resistant to temozolomide (TMZ), unlike MMRp tumors. Our study shows that KL-50, a imidazotetrazine-based DNA targeting agent inducing MMR-independent cross-link-mediated cytotoxicity, was effective against germline and somatic MMRr/MMRd GBM, offering a potential therapy for TMZ-resistant HGG with MMR alterations.