SHLD2 loss is a synthetic vulnerability to Polθ inhibition combined with radiotherapy

DNA polymerase theta (Polθ) plays a crucial role in the repair of DNA double-strand breaks (DSBs) by microhomology-mediated end joining (MMEJ). We previously demonstrated that Polθ inhibition (Polθi) is an effective and well-tolerated approach to sensitise tumours to radiotherapy (RT). Here, we profiled 54 cancer cell lines and found that Polθi induces significant radiosensitisation in most models, though with marked variability not explained by indicators of Polθ activity. To pinpoint molecular determinants of radiosensitisation by Polθi, we performed a CRISPR knockout screen which revealed loss of the TP53BP1/Shieldin pathway component SHLD2 (FAM35A) as a vulnerability to Polθi combined with RT. We demonstrated that SHLD2 loss not only increases sensitivity to RT alone, but also enhances the radiosensitising effect of Polθi, both in vitro and in vivo. Importantly, we found that SHLD2 is deleted in a subset of human prostate cancers, often co-occurring with PTEN loss, an adverse prognostic factor. Furthermore, we show that SHLD2-deficient cancer cells are more reliant on Polθ to prevent DSB accumulation and chromosomal instability. In summary, we discovered SHLD2 loss as a novel collateral vulnerability that can be exploited through combined treatment with Polθi and RT.