IMMU-40. INVESTIGATING THE ROLE OF MUTATIONAL BURDEN IN TUMOR IMMUNOGENICITY USING A PRE-CLINICAL<i>MLH1</i>-DEFICIENT GLIOBLASTOMA MODEL

Abstract BACKGROUND Although there is an established correlation between tumor mutational burden (TMB) and response to immune checkpoint inhibition (ICI) in many cancers, such an association has not been definitively shown in high-grade gliomas. Most studies of ICI therapy in gliomas, including glioblastoma (GBM), have failed to demonstrate efficacy, with the exception of hypermutated cases resulting from rare inherited defects in mismatch repair (MMR) genes. We sought to investigate the association of TMB to immune response using a syngeneic murine model with a spectrum of mutational burdens. METHODS We used CRISPR-Cas9 to knockout the MMR gene Mlh1 in the murine glioma model SB28, an ICI-resistant cell line with a very low mutational burden (108 non-synonymous mutations). RESULTS Single-cell sorted Mlh1 knockout SB28 clones revealed a two- to six-fold increase in TMB. Importantly, loss of Mlh1 also brought about global changes in immune- and inflammatory-related gene expression in vitro, without correlation to increasing TMB. While all clones had the same rate of in vitro proliferation, flank injection of a high TMB vs a low TMB SB28 Mlh1 knockout clone resulted in similar survival benefits compared to Mlh1 wildtype SB28. CONCLUSIONS These preliminary results suggest that, although disruption of the MMR system may result in heightened tumor immunogenicity, elevated TMB may not be the sole significant determinant. We are currently investigating tumor proliferation after flank injection in immunocompromised mice to confirm that slowed growth is immune-related, as well as clone-specific response to ICI treatment. We are also exploring the role of specific cytokines/chemokines in conferring this survival advantage and potential immune response. By generating a GBM model of varied mutational burdens and immune profiles and comparing to the baseline SB28 cell line, we can interrogate the relative significance of TMB in tumor immunogenicity and ICI response.