Trimodal Therapy Consisting of DNA-PK Inhibition, PD-L1 Immune Checkpoint Blockade and Radiotherapy with Carbon Ions

Preferential radiosensitizing effects of peposertib (M3814), a novel small molecule serine-threonine kinase inhibitor of DNA-dependent protein kinase (DNAPKi), in hypoxic tumors was recently reported. This effect was augmented by high-linear energy transfer (LET) carbon ion irradiation (CIR). We aimed to investigate the impact of PD-L1/PD1 immune-checkpoint blockade (ICB), DNAPKi and radiotherapy in a trimodal setting. PD-L1 expression, DSB repair kinetic, micronucleus formation and stimulator of interferon genes (STING)-Interferon type I pathway was assessed by functional studies. To counteract immune evasive mechanisms mediated by therapy-induced PD-L1 expression, Anti-PD-L1-based ICB was investigated in syngeneic B16 melanoma orthotopically growing in C57BL/6 mice. Immune profiling was performed by, transcriptome analysis, TCR-Sequencing and immune cytometry (CyToF). For CIR, tumor region was irradiated at HIT within a spread-out Bragg peak of 20 mm (147-266 MeV/u, μLET 75KeV/μm) at a water equivalent depth of 10 cm. Stimulation of PD-L1 was discovered as a tumor immune evasive mechanism in response to mono- as well as dual therapy with DNAPKi and RT. Consequently, anti-PDL1-based ICB reversed the immune evasive effect of therapy-induced PD-L1 expression. Moreover, the efficacy of combined treatment with ICB and DNAPKi was further improved by high-LET CIR. Mechanistic studies revealed activation of STING pathway to be centrally involved in radiation and DNAPKi-mediated PD-L1 expression. In line with this observation, beneficial effects of trimodal therapy were in part abrogated by tumor-specific STING knockdown. This study provides a biological rationale for application of combined radiotherapy and DNA-PK inhibition to improve eradication of radioresistant tumors. Moreover, addition of ICB to this regimen can switch the immune balance towards enhanced anti-tumor immunity. This trimodal concept is currently investigated in NCT03724890 clinical trial.