Olaparib and radiotherapy induce type I interferon and CD8+ T cell-dependent sensitization to immunotherapy in pancreatic cancer

PARP inhibitors sensitize pancreatic ductal adenocarcinoma (PDAC) to radiation by inducing DNA damage and replication stress. These mechanisms also have the potential to enhance radiation-induced type I interferon (T1IFN) mediated anti-tumoral immune responses. We hypothesized that the PARP inhibitor olaparib would also potentiate radiation-induced T1IFN to promote anti-tumor immune responses and sensitization of otherwise resistant PDAC to immunotherapy. To test this hypothesis, we assessed the effects of olaparib and radiation on T1IFN production and sensitivity to aPD-L1 immunotherapy, as well as consequent effects on the tumor microenvironment by single-cell RNA sequencing (scRNA-seq). We found that olaparib enhanced T1IFN production following radiation and had superior therapeutic efficacy in immune competent models. Treatment with olaparib and radiation sensitized PDAC tumors to aPD-L1 resulting in decreased tumor burden and a 33% complete response rate. Combination treatment provided durable immune responses as shown by tumor rejection upon tumor rechallenge of previously cured mice. Furthermore, scRNA-seq analysis revealed that combination treatment induced an immunogenic tumor microenvironment, characterized by interferon responses in both PDAC and myeloid cell populations, macrophage polarization, and increased CD8+ terminal effector T cell frequency and activity. Furthermore, CD8+ T cells and T1IFN signaling were required for therapeutic efficacy as host depletion of CD8+ T cells or the T1IFN receptor diminished treatment responses. Taken together, our results indicate that olaparib enhances radiation-induced T1IFN-mediated immune signaling and subsequently an adaptive immune response thus sensitizing pancreatic cancer to aPD-L1 therapy, supporting an ongoing clinical trial of this therapy in patients with PDAC. Overall design: 6-8 week old C57/Bl6 mice with subcutaneous mT4 tumors were treated with AZD1390 (20mg/kg) for 5 days starting on day 0, 1 dose of radiation (8Gy) on day 0, and 3 doses of anti-PD-L1 blocking antibody (100µg) once every three days starting on day -1. 6-8 tumors were harvested for each of the following treatment groups: IgG/control, AZD1390, RT+anti-PD-L1, and AZD1390+RT+anti-PD-L1. Tumors were harvested on day 5 and digested in a collagenase digest buffer, and filtered through 40µm mesh filter. Samples were depleted of dying cells twice using Dead Cell Removal Kit from Miltenyi. Cells were then seperated based on CD45 status, and mixed back in equal parts CD45+ and CD45- to enrich immune cells present in the samples. Samples were diluted to a final concentraiotn of 700-1000 cells/ul. Single cell sequencing was performed by the University of Michigan Advanced Genomics Core Research Facility. Library generation was performed on the 10x Genomica Chromium Controller following manufacturers protocol for the 3'v3.1 chemistry with NextGEM Chip G reagents. Library quality was assess by LabChip GXII HT and quantified by Qubit. Pooled libraries were subjected to 150 base pair, paired-end sequencing according to manufacturer protocol (Illumina NovaSeq 6000). Bcl2fastq2 Conversion Software was used to de-multiplex fastq files and CellRanger v7 Pipeline was used to align reads and generate count matrices.