Polθ Inhibition Enhances Proton and Photon Radiotherapy By Increasing DNA Damage and Tumor Immunogenic Remodeling
Abstract
Purpose
To investigate the effects of combining proton and photon radiotherapy (RT) with a DNA polymerase theta inhibitor (Polθi) on DNA damage and promotion of tumor immunogenic remodeling.
Methods
RT±Polθi effects were evaluated in MDA-MB-436 (BRCA1-mutated), its BRCA1-restored isogenic line (MDA-MB-436BRCA1+/+), 4T1 and HUVEC (normal cell). Except MDA-MB-436, other cell lines were proficient in homologous recombination (HR) and non-homologous end-joining (NHEJ). Cells were irradiated with photons (6 MV x-rays) or protons (3.9 keV/mm) to assess clonogenic survival, cell cycle, and DNA damage foci (γH2AX and 53BP1). Additionally, genomic instability and innate immune activation (micronuclei and cGAS colocalization), immune marker surface expression (CD80, CD86, PD-L1, CTLA-4, Tim-3, Lag-3, CD47, MHC-I, FasL, and calreticulin) and cytokines secretion (GM-CSF, IFN-β, IFN-γ, IL-1α, IL-1β, IL-6, IL-10, IL-12p70, IL-17A, IL-23, IL-27, MCP-1 and TNF-α) were assessed to determine immunogenic remodeling in vitro. RT±Polθi was tested in vivo using a fractionated regimen (3x8 Gy of protons or photons), with Polθi administered daily for 12 consecutive days starting one day prior to RT. Tumor growth and survival were assessed.
Results
RT+Polθi enhanced radiosensitivity in all cancer cell lines—but not in normal cells—to both radiation types. Polθi increased RT-induced DNA damage, G2/M-phase arrest and promoted micronuclei with cGAS colocalization. RT alone upregulated all immune surface markers and increased release of pro-inflammatory cytokines, which were further modulated by Polθi. Tumor immunogenic remodeling varied depending on radiation type and presence of Polθi, with protons inducing more pronounced effects. In-vivo, RT+Polθi significantly delayed tumor growth, with improved survival observed only in protons+Polθi.
Conclusion
These findings indicate that Polθ inhibition enhances RT-induced DNA damage, promotes innate immune activation, and modulates immune signaling in a radiation type-dependent manner. Interestingly, these effects were observed regardless of HR status. Moreover, Polθ inhibition improves radiosensitivity, with protons providing the greatest therapeutic benefit in vivo.