Increased immunogenicity of hypermutated SB28 syngeneic glioblastoma is partially mediated by alterations in tumor chemokine expression

Abstract Background Glioblastoma (GBM) prognosis remains poor, and although immune checkpoint inhibitors (ICI) have transformed the treatment of many tumors; they are ineffective in GBM. However, response to ICIs occurs in high-tumor-mutational-burden (TMB) GBMs. To address the immunological impact of high TMB in GBM, we created a high TMB syngeneic mouse model from the low TMB SB28 GBM cell line. Methods We used CRISPR-Cas9 to target murine Msh2, Mlh1, CXCL10, and CCL5. Single cell-sorted clones were characterized by whole exome, bulk RNA-sequencing, and neoantigen prediction. Clones were injected subcutaneously or intracranially with or without anti-PD-1/anti-CTLA4 and dexamethasone. Results Loss of mismatch repair (MMR) proteins Msh2 or Mlh1 increased nonsynonymous mutations. A fraction of mice with intracranial Msh2KO but not Mlh1KO SB28 showed long-term survival with anti-PD-1/anti-CTLA4 treatment plus dexamethasone. Long-term surviving mice from Msh2 KO SB28 rejected rechallenged subcutaneous tumors. Subcutaneous tumors from clones with increased TMB grew more slowly. This was fully abrogated in Rag1 null mice for Msh2KO but only partially for Mlh1KO SB28. Hypermutant Msh2KO clones spontaneously secreted CXCL10, CCL5, and increased pro-inflammatory chemokines after IFN-γ stimulation. Knockout of CXCL10 or CCL5 in the highest TMB Msh2KO clone restored flank tumor growth, indicating loss of immune response despite elevated TMB. Conclusion Mismatch repair-deficient SB28 tumors were more immunogenic, but this was not completely correlated with TMB. Rather, rejection depended on increased secretion of pro-inflammatory chemokines. Msh2 and Mlh1 loss was not equivalent, suggesting that additional studies are needed to elucidate germline and somatic mismatch repair gene-specific immune alterations.