Radiation Biology

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SU_37_2377 - Combination of Ionizing Radiation and a Cell Cycle Checkpoint Kinase Inhibitor Leads to Increased Micronuclei Formation

Sunday, October 21
1:15 PM - 2:45 PM
Location: Innovation Hub, Exhibit Hall 3

Combination of Ionizing Radiation and a Cell Cycle Checkpoint Kinase Inhibitor Leads to Increased Micronuclei Formation
H. H. Chao1, I. Karagounis2, C. Thomas2, J. Karar2, C. Koumenis2, and A. Maity3; 1Department of Radiation Oncology, University of Pennsylvania, Philadelphia, PA, 2University of Pennsylvania, Philadelphia, PA, 3Department of Radiation Oncology, Perelman School of Medicine of the University of Pennsylvania, Philadelphia, PA

Purpose/Objective(s): Despite recent successes with the use of immune checkpoint blockade across disease sites, many patients have an inadequate response to immunotherapy. Therefore, it is imperative to identify regimens that increase the efficacy of immunomodulatory agents. Recently it has been shown that mitotic progression following DNA damage results in formation of micronuclei (MN). MN can trigger activation of the inflammatory pathway including interferon signaling, and activate cell-intrinsic immune surveillance mechanisms against neoplastic processes. We hypothesize that the combination of ionizing radiation (IR) with checkpoint kinase inhibitors abrogating the G2/M cell cycle checkpoint will result in increased formation of MN.

Materials/Methods: MCF10A (mammary epithelial) and H460 (lung carcinoma) cells were treated using IR +/- AZD7762, a CHK1/2 inhibitor. MCF10A cells were treated with 25nM of AZD7762 1 hour prior to receiving 16 Gy IR. H460 cells received 4 Gy IR following AZD7762 treatment. mRNA was collected at 24h and 48h following IR and assessed for IFN-b levels using the Taqman Gene Expression Assay. All cells were also cultured on chamber slides and fixed using 4% paraformaldehyde at the 24h and 48h timepoints followed by Hoechst nuclear staining in order to identify MN. Images were obtained using Immunofluorescence microscopy using a UV filter at 40X magnification. The percentage of cells containing MN were quantified per imaged fields containing > 50 cells. T-tests were performed to assess for differences in gene expression levels and MN percentage.

Results: 5% of MCF10A cells treated with radiation alone developed MN at 24 hours, as compared to 16% with the combination of IR + AZD7762 (p = <.001). Similarly, at 48 hours following IR, 8% of MCF10A cells developed MN as compared to 16% with the combination of IR + AZD7762 (p =0.04). A similar effect was seen in H460 cells, with 19% of cells with MN following IR alone vs. 35% of cells with IR + AZD7762 at 24 hours (p = 0.01). At 48 hours post treatment, 18% of H460 cells developed MN with IR alone vs. 35% with IR + AZD7762 (p = 0.03). IFN-b expression levels exhibited a similar trend as MN development. MCF10A cells treated with IR + AZD7762 had a 4.3-fold increase in IFN-b levels as compared to IR alone at 24 hours (p = 0.029) and a 6.1-fold increase at 48 hours (p = 0.006). H460 cells treated with IR + AZD7762 had a 5.2-fold increase in IFN-b levels as compared to IR alone at 24 hours (p = 0.012) and a 2.0-fold increase at 48 hours (p = 0.07)

Conclusion: The combination of IR with agents targeting the G2/M checkpoint to drive mitotic progression, such as CHK1/2 inhibitors, is able to induce increased MN formation and interferon signaling. This presents a promising means of stimulating immune surveillance, and we are pursuing further investigations to determine whether this combination will increase the efficacy of immune checkpoint blockade.

Author Disclosure: H. Chao: None. I. Karagounis: None. C. Thomas: None. J. Karar: None. C. Koumenis: None.

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