Modeling response to alkylating chemotherapy in a syngeneic model of MGMT overexpression and MMR-deficient glioma

Glioblastoma (GBM) is associated with dismal prognosis, having a median survival of less than 15 months. The frontline chemotherapeutic agent used in the treatment of GBM patients, temozolomide (TMZ), is limited by predictable resistance mechanisms. TMZ-induced O 6 -methylguanine (O 6 -MeG) lesion is repaired directly by O 6 -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 promotes cell death through futile cycles of MMR. Loss of function mutations in MMR proteins are now recognized as a key mechanism of acquired TMZ resistance in gliomas, specifically in tumors with MGMT promoter methylation. MGMT serves as a prognostic marker in GBM and is found to be related to improved response to TMZ. Although alkylator therapy has shown relative efficacy in treating MGMT-methylated tumors, standard treatments are largely ineffective for GBM patients with MGMT-unmethylated tumors. For this, it is crucial to overcome intrinsic resistance to TMZ, which can be possible with the use of MGMT inhibitors such as O 6 -benzylguanine (O 6 -BG) and Lomeguatrib. Fluoroethylating agents, KL-50, is a recently developed agent which acts through a unique DNA-modifying mechanism involving evolution of a primary alkyl lesion to a DNA interstrand crosslink particularly in the absence of MGMT.Using CRISPR/Cas9, isogenic SB28 murine glioma cell lines with knockout of MSH6 and MLH1 were generated and MGMT overexpression models created by overexpressing FLAG-tagged MGMT. 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. MGMT overexpression showed resistance to TMZ and KL-50 and this resistance was reversed when cells were treated with O 6 -BG or Lomeguatrib as MGMT inhibitors. We assessed the TMZ and KL-50 efficacy in flank and intracranially implanted tumor models and observed that KL-50 extended the median survival in comparison to TMZ or vehicle control in the MLH1 KO setting. Therefore, this suggested that KL-50 forms DNA interstrand crosslinks selectively in MGMT-silenced tumor models and maintains its efficacy even in the loss of functional MMR, emphasizing its potential to fill a critical therapeutic gap in recurrent, treatment-refractory tumors.Furthermore, in MGMT+ models, Lomeguatrib given concurrently with KL-50 demonstrated a significantly increased survival benefit, indicating that KL-50 in combination with the MGMT inhibitor can be used as a potential clinical approach for GBM patients with MGMT-unmethylated tumors. Our findings suggest that KL-50 may provide a new treatment approach for TMZ-resistant gliomas and in the MGMT+ setting, combination with Lomeguatrib could be beneficial.