Background
Glioblastoma (GBM) carries a dismal prognosis with a median survival under 15 months. Temozolomide (TMZ) is the standard frontline chemotherapeutic treatment in glioblastoma patients and works by depositing a methyl group to purine bases of DNA (O6-guanine; N7-guanine and N3-adenine. The O6-methylguanine (O6-MeG) lesion is directly repaired by O6-Methylguanine Methyltransferase (MGMT). In tumor cells with loss of MGMT, the unrepaired methyl adducts result in a mismatched nucleotide base pairing. The mismatch repair (MMR) pathway recognizes the damage and induces cell death via futile cycles of MMR. Therefore, loss-of-function mutations in MMR proteins confer resistance to TMZ. KL-50 is a newly developed, novel alkylating agent that deposits a 2-fluoroethyl lesion at O6-guanine.
Methods
Here, we used CRISPR/Cas9 to generate isogenic SB28 murine glioma cell lines with knockout (KO) of MMR proteins MSH6 and MLH1. We confirmed KO by western blot and performed in vitro cell viability assays. For in vivostudies, we injected cells subcutaneously or intracranially and treated with TMZ and KL-50 both at 25mg/kg/dose via oral gavage for 5 days/week x 2 cycles. Anti-PD1 (RMP1-14, Bioxcell) was given at 10mg/kg via i.p. injection for 2 doses/week x 6 doses.
Results
We performed in vitro short-term viability and clonogenic survival assays demonstrating that cells with loss of MMR displayed resistance to TMZ while retaining sensitivity to KL-50. The MMR-independent efficacy of KL-50 was also demonstrated in vivo in both flank and intracranial tumor models. Notably, there was no difference in the in vivo anti-tumor activity of immune checkpoint blockade (ICB) with anti-PD-1 monotherapy between WT and MLH1 KO tumors, consistent with clinical findings in patients with MMR deficient GBM. However, we did find modest in vivo synergy of ant-PD-1 combined with KL-50.
Conclusions
These findings suggest that KL-50 may provide a new treatment approach for TMZ resistant glioma while mediating immunologic effects that improve tumor immunogenicity, which will be explored in future studies. Moreover, the development of a new syngeneic glioma model with somatic loss of MMR will facilitate studies on the immunomodulatory effects of alkylating agents and testing of chemoimmunotherapy combinations.
Acknowledgements
JCV is supported in part by the NIH/NCI K08 Career Development award # 1-K08 CA258796-01, the Robert Wood Johnson Harold Amos Medical Faculty Development Program, the Fund to Retain Clinical Scientists at Yale, sponsored by the Doris Duke Charitable Foundation award #2015216 and the Yale Center for Clinical Investigation, and by an American Cancer Society Institutional Research Grant, #IRG-21-132-60-IRG.