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Supplementary Figure from DNA-PK Inhibitor Peposertib Amplifies Radiation-Induced Inflammatory Micronucleation and Enhances TGFβ/PD-L1 Targeted Cancer Immunotherapy

Radiotherapy is the most widely used cancer treatment and improvements in its efficacy and safety are highly sought-after. Peposertib (also known as M3814), a potent and selective DNA-dependent protein kinase (DNA-PK) inhibitor, effectively suppresses the repair of radiation-induced DNA double-strand breaks (DSB) and regresses human xenograft tumors in preclinical models. Irradiated cancer cells devoid of p53 activity are especially sensitive to the DNA-PK inhibitor, as they lose a key cell-cycle checkpoint circuit and enter mitosis with unrepaired DSBs, leading to catastrophic consequences. Here, we show that inhibiting the repair of DSBs induced by ionizing radiation with peposertib offers a powerful new way for improving radiotherapy by simultaneously enhancing cancer cell killing and response to a bifunctional TGFβ “trap”/anti-PD-L1 cancer immunotherapy. By promoting chromosome misalignment and missegregation in p53-deficient cancer cells with unrepaired DSBs, DNA-PK inhibitor accelerated micronuclei formation, a key generator of cytosolic DNA and activator of cGAS/STING-dependent inflammatory signaling as it elevated PD-L1 expression in irradiated cancer cells. Triple combination of radiation, peposertib, and bintrafusp alfa, a fusion protein simultaneously inhibiting the profibrotic TGFβ and immunosuppressive PD-L1 pathways was superior to dual combinations and suggested a novel approach to more efficacious radioimmunotherapy of cancer.Implications:Selective inhibition of DNA-PK in irradiated cancer cells enhances inflammatory signaling and activity of dual TGFβ/PD-L1 targeted therapy and may offer a more efficacious combination option for the treatment of locally advanced solid tumors.

Radiotherapy is the most widely used cancer treatment and improvements in its efficacy and safety are highly sought-after. Peposertib (also known as M3814), a potent and selective DNA-dependent protein kinase (DNA-PK) inhibitor, effectively suppresses the repair of radiation-induced DNA double-strand breaks (DSB) and regresses human xenograft tumors in preclinical models. Irradiated cancer cells devoid of p53 activity are especially sensitive to the DNA-PK inhibitor, as they lose a key cell-cycle checkpoint circuit and enter mitosis with unrepaired DSBs, leading to catastrophic consequences. Here, we show that inhibiting the repair of DSBs induced by ionizing radiation with peposertib offers a powerful new way for improving radiotherapy by simultaneously enhancing cancer cell killing and response to a bifunctional TGFβ “trap”/anti-PD-L1 cancer immunotherapy. By promoting chromosome misalignment and missegregation in p53-deficient cancer cells with unrepaired DSBs, DNA-PK inhibitor accelerated micronuclei formation, a key generator of cytosolic DNA and activator of cGAS/STING-dependent inflammatory signaling as it elevated PD-L1 expression in irradiated cancer cells. Triple combination of radiation, peposertib, and bintrafusp alfa, a fusion protein simultaneously inhibiting the profibrotic TGFβ and immunosuppressive PD-L1 pathways was superior to dual combinations and suggested a novel approach to more efficacious radioimmunotherapy of cancer. Implications: Selective inhibition of DNA-PK in irradiated cancer cells enhances inflammatory signaling and activity of dual TGFβ/PD-L1 targeted therapy and may offer a more efficacious combination option for the treatment of locally advanced solid tumors.

The clinical success of antibody-mediated immune checkpoint blockade therapies has transformed the cancer therapy paradigm by demonstrating that durable antitumor immune responses and long-term remissions may be achieved in a subset of patients across a diverse range of cancers. However, the majority of patients fail to respond to antibody therapies targeting single immune checkpoint pathways and antibodies exhibit a long in vivo half-life (>15-20 days with >70% target occupancy for months) which may contribute to the emergence of immune-related adverse events. Additionally, antibody therapies must be administered by intravenous infusion in a hospital or clinic which places an additional burden on patients who may have mobility challenges. Thus, there is a significant opportunity for a novel immune checkpoint therapy that can address the shortcomings associated with the current antibody therapies. CA-170 is a small molecule, orally bioavailable antagonist of the PD-L1, PD-L2 and VISTA/PD-1H immune checkpoint pathways which is currently undergoing Phase I clinical testing. In preclinical safety studies conducted in rodents and non-human primates, orally administered CA-170 shows no signs of toxicity when dosed up to 1000 mg/kg for 28 consecutive days. CA-170 exhibits an oral bioavailability of approximately 40% and Citation Format: Adam S. Lazorchak, Troy Patterson, Yueyun Ding, Pottayil G. Sasikumar, Naremaddepalli S. Sudarshan, Nagaraj M. Gowda, Raghuveer K. Ramachandra, Dodheri S. Samiulla, Sanjeev Giri, Rajesh Eswarappa, Murali Ramachandra, David Tuck, Timothy Wyant. CA-170, an oral small molecule PD-L1 and VISTA immune checkpoint antagonist, promotes T cell immune activation and inhibits tumor growth in pre-clinical models of cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A36.