Abstract 2923: DNA-PK inhibitor, M3814, is a potent inducer of inflammatory micronucleation in irradiated p53-deficient cancer cells: Implications for combination radio-immunotherapy
Authors: Carr Michael , Zimmermann Astrid , Guo Yige , Liu Xiaohong , Steiner Patrick , Hahn Susanne , Zenke Frank , Blaukat Andree , Vassilev Lyubomir T. 
Published: August 2019

A novel, clinical-stage DNA-PK inhibitor, M3814, potently and selectively blocks the non-homologous end-joining pathway for repair of DNA double strand breaks (DSB) and synergizes with ionizing radiation (IR) and DSB-inducing chemotherapy. We have previously shown that M3814 boosts ATM/p53 signaling and reinforces cell cycle checkpoint controls in irradiated p53 wild-type cancer cells, inducing irreversible premature senescence. However, in the absence of functional p53 this protective mechanism is disrupted, cancer cells progress through the cell cycle with unrepaired DSBs, leading to aberrant mitotic division and ultimately cell death. Here, we investigated the mechanistic consequences of DNA-PK inhibition in p53-dysfunctional cancer cells exposed to IR. We found that cell cycle progression with persistent DSB damage, promoted by M3814, causes severe chromosome aberrations, misalignment, and mis-segregation. This leads to the formation of micronuclei after each abnormal mitotic division. Live cell imaging and immunofluorescence analysis revealed high numbers of γH2AX foci, marking unrepaired DSBs inside the micronuclei, and lamin-B1-deficient micronuclei lamina. Cyclic GMP-AMP synthase (cGAS) was found to colocalize with micronuclei, suggesting that compromised laminar integrity exposes damaged DNA to the cytosol and activates the STING pathway. Gene expression analyses revealed activation of over 60 immune-related genes in cells exposed to IR+M3814, consistent with a STING-driven inflammatory response. Indeed, many cyto-/chemokines with immunomodulatory properties were detected in culture media from IR+M3814 treated cells. In addition, elevated expression of PD-L1 protein was found in irradiated p53-deficient cancer cells exposed to M3814. Together, these results provided a clear rationale for combination with PD-L1 inhibitory therapy. Murine p53-mutant cancer cells, MC38, were grown subcutaneously bilaterally in syngeneic mice. One of the tumors was exposed to 5 x 3.6 Gy daily IR fractions given locally and daily oral M3814 at previously established efficacious doses, followed by three applications (every 3-4 days) of the PD-L1 inhibitory antibody, avelumab. The other tumors were left unirradiated to monitor for abscopal effect. Study results demonstrated a superior benefit of adding M3814 to the IR/avelumab combination, including enhancement of the abscopal effect of IR/avelumab combination. Altogether, our results revealed that selective DNA-PK inhibition by M3814 provides a powerful new mechanism for induction of micronucleation and STING inflammatory signaling in p53-disfunctional cancer cells and offers a new approach to combination radio-immunotherapy of cancer.

Citation Format: Michael Carr, Astrid Zimmermann, Yige Guo, Xiaohong Liu, Patrick Steiner, Susanne Hahn, Frank Zenke, Andree Blaukat, Lyubomir T. Vassilev. DNA-PK inhibitor, M3814, is a potent inducer of inflammatory micronucleation in irradiated p53-deficient cancer cells: Implications for combination radio-immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2923.