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MO_4_2531 - Elucidation of an Exquisite Synergistic Interaction Between ATR Inhibitors and Alkylating Agents in MGMT-Methylated Glioma cells

Monday, October 22
10:45 AM - 12:15 PM
Location: Innovation Hub, Exhibit Hall 3

Elucidation of an Exquisite Synergistic Interaction Between ATR Inhibitors and Alkylating Agents in MGMT-Methylated Glioma cells
C. Jackson1, S. Noorbakhsh1, and R. S. Bindra2; 1Yale University, New Haven, CT, 2Department of Therapeutic Radiology, Yale School of Medicine, New Haven, CT

Purpose/Objective(s): Glioblastoma (GBM) is the most common primary brain tumor in adults. Current treatment of GBM includes maximal resection followed by concurrent temozolomide (TMZ) and radiation therapy (RT). TMZ is an alkylating agent that methylates guanine bases at the O6 position (O6meG). O6meG-DNA methyltransferase (MGMT) repairs TMZ-induced DNA damage by removing these alkyl adducts. The promoter region of MGMT contains CpG islands that are methylated in approximately 45% of glioblastoma cases. Patients with a methylated MGMT promoter respond more readily to TMZ and survive 9 months longer than patients with an unmethylated MGMT promoter. Despite this prognostic benefit, these patients still have a median survival of only 22 months. The purpose of this study is two-fold: 1) to better understand the DNA damage response pathways that are activated in MGMT-methylated glioma cells upon treatment with alkylating agents, and 2) to determine if inhibiting these activated pathways sensitizes the glioma cells to alkylation damage, with or without ionizing radiation (IR).

Materials/Methods: Our methods include comet assays, immunofluorescent imaging of DNA damage foci, western blotting, clonogenic survival assays, growth delay, flow cytometry, and mouse xenograft studies. Our assays were performed on a collection of human GBM cell lines engineered to express MGMT or not, including LN229 and U87 cells.

Results: Using comet assays and DNA damage foci, we showed that TMZ induces DNA double strand breaks (DSBs) in MGMT-methylated cells. These DSBs correspond temporally with cell cycle arrest in the S, G2, and M phases, as quantified by high-content microscopy and flow cytometry. We demonstrated that TMZ activates markers of replication stress in MGMT-methylated cells, specifically pChk1 S345 and pRPA32 S33. As these proteins are markers of ATR activation, we hypothesized that inhibition of ATR would sensitize MGMT-methylated cells to TMZ. This hypothesis was verified through clonogenic survival assays and growth delay assays; ATR inhibitors increase sensitivity to TMZ by over 100-fold in MGMT-methylated cells, but not in MGMT-expressing cells. Further exploration of the mechanism suggests that ATR inhibition with TMZ abrogates the normal cell cycle checkpoint that prevents replication with alkylated DNA, allowing cells to undergo replication catastrophe. Because we also showed that TMZ sensitizes MGMT-methylated cells to IR, our work suggests that the combination of ATR inhibition, TMZ and IR may further increase the therapeutic index over MGMT-expressing cells.

Conclusion: Here, we report exquisite synergistic interactions between TMZ and inhibitors of a key DNA damage response protein, ATR. These interactions depend on MGMT status. These data lay the foundation for future clinical trials testing this combination in MGMT-methylated glioma. Since ATR inhibitors are radiosensitizers, we believed that this “triple combination” will be highly synergistic in the treatment of GBM.

Author Disclosure: C. Jackson: None. S. Noorbakhsh: None. R.S. Bindra: None.

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