Pancreatic ductal adenocarcinoma (PDAC) is characterized by strong resistance against radio(chemo )therapy and potent immunosuppression. Improving the therapeutic outcome might be achieved by a combined modality approach of radiotherapy plus immunostimulation. Intratumoral type I interferons (IFNs) are known to be of essential importance for the stimulation of effective anti-tumor immunity, and they have been described to contribute to successful tumor control upon radiotherapy by orchestrating adaptive immune responses. In turn, radiotherapeutic failure might be overcome by combining radiotherapy with agents that favor intratumoral type I IFN production.

We propose targeting crucial regulators of the DNA damage response (DDR) by pharmacological inhibitors as well as bi-functional 5’ triphosphate siRNAs (ppp siRNAs) which combine silencing of crucial DDR regulators and activation of retinoic acid inducible gene I (RIG I), a cytosolic sensor for viral RNA, as a strategy to (i) sensitize PDAC to irradiation-induced cell death, (ii) activate intratumoral type I IFN production, and (iii) stimulate adaptive anti-tumor immune responses in order to increase the efficacy of PDAC radiotherapy.

Potential DDR targets have already been identified by dimensionality reduction of clonogenic survival data and correlation analyses with transcriptomic data in our previous work, and this screening approach will be expanded to cell culture models and tissue samples provided by S01. Target validation will be carried out by pharmacological inhibition and RNA interference as well as CRISPR/Cas9-mediated deletion followed by analyses of DDR kinetics, short-term viability, long-term clonogenic survival, cell death, and senescence induction as well as global gene expression and (phospho)proteomic changes (with S02, P15, P18). Bi-functional ppp siRNAs will be designed, and their impact on the cellular response upon irradiation will be examined. Therapeutic efficacy of our approach in vivo will be evaluated in orthotopic syngeneic tumor transplantation models as well as GEMMs of PDAC. CT-based 3D-conformal radiotherapy will be employed alone or in combination with the DDR-targeting, immunostimulatory approach, and tumor control and metastasis formation will be monitored by contrast-enhanced CT scans. Local and systemic immune monitoring will be performed, and the role of different immune cell subsets will be assessed by antibody-mediated depletion and genetic deletion (with P06). Anti-PDAC immunity will be evaluated by analyzing tumor-specific T cell responses and tumor rechallenge models (with P17). Immunological memory will be tested in adoptive transfer experiments and bone marrow chimeras. Optimizing tumor-specific delivery of the ppp siRNAs using superparamagnetic iron oxide nanoparticles (SPIONs) will be performed with P14 and the influence of obesity versus fasting on efficacy and toxicity of radioimmunotherapy will be studied together with P08.

Overall, our project will contribute to elucidate the performance of DDR-targeting, ppp siRNA-driven, combinatorial radioimmunotherapy approaches for PDAC treatment and will evaluate their translational potential in elaborate in vitro and in vivo model systems.