Exploiting the DNA damage response to improve peptide receptor radionuclide therapy outcome

Objectives Targeted molecular imaging & treatment (PRRT) of neuroendocrine tumors (NET) with radiolabeled somatostatin analogues is successfully applied in clinical trials. PRRT of metastasized NETs results in improved patient outcome, but cure is reached in low percentages only. To further improve treatment outcome, we investigated DNA damaging effects of PRRT with the aim to enhance these effects through modulation of the DNA damage response. We aimed specifically to sensitize tumor cells without further damage to healthy tissues using PARP inhibitors (PARPi). Although PRRT induces cytotoxic double strand breaks (DSBs), the majority of the induced lesions are single strand breaks (SSBs) that require PARP activity for repair. When these breaks are not repaired efficiently (by using PARPi), they cause replication fork arrest and more difficult to repair DSB formation in replicating cells.

Methods Somatostatin receptor-positive tumor cells were incubated with increasing doses of 177Lu-[DOTA,Tyr3]octreotate (DOTA-TATE), followed by non-radionuclide chases with and without PARPi. Cells were fixed and fluorescently stained with antibodies to visualize accumulation of DNA repair proteins to the DSBs and apoptosis. Proliferation was measured using fluorescent Click-iT labeling and sulforhodamine B. Cellular survival was measured using colony assay.

Results Using several molecular biological and fluorescent microscopy techniques, we have characterized the induced DNA damage and DNA damage response after PRRT. Our results show that after PRRT tumor cells showed accumulation of the DSB repair protein 53BP1 in so-called foci, indicating the induction and ongoing repair of DSBs. These foci were detectable for up to 3 days after treatment. In contrast, treatment with 177Lu-DTPA caused transient DSBs that were no longer detectable after 1 day. Interestingly, addition of PARPi led to increased and prolonged DNA damage (up to 6 days), specifically in replicating cells. This was also confirmed by the higher percentage of cells with chromosomal damage, shown by micronuclei. PRRT eradicated cells in a dose dependent manner and cell killing (via apoptosis) was strongly enhanced by PARPi.

Conclusions In conclusion, we show that PRRT triggers the DNA damage response by producing DSBs in preclinical models. Through modulation of the DNA damage response with PARPi, we were able to enhance the DNA-damaging effects of PRRT, specifically in replicating cells. We expect that our results will eventually improve the current PRRT outcome, leading to increased patient survival rates.