A genome-wide CRISPR/Cas9 screen identifies DNA-PK as a sensitiser to 177Lutetium-DOTA-octreotate radionuclide therapy.

ABSTRACT

Peptide receptor radionuclide therapy (PRRT) using 177Lutetium-DOTA-octreotate (LuTate) for neuroendocrine tumours (NET) is now an approved treatment available in many countries, though primary or secondary resistance continue to limit its effectiveness or durability. We hypothesised that a genome-wide CRISPR/Cas9 screen would identify key mediators of response to LuTate and gene targets that might offer opportunities for novel combination therapies for NET patients. Methods: We utilised a genome-wide CRISPR-Cas9 screen in LuTate-treated cells to identify genes that impact on the sensitivity or resistance of cells to LuTate. Hits were validated through single-gene knockout. LuTate-resistant cells were assessed to confirm LuTate uptake and retention, and persistence of somatostatin receptor 2 (SSTR2) expression. Gene knockouts conferring LuTate sensitivity were further characterised by pharmacological sensitisation using specific inhibitors and in vivo analysis of the efficacy of these inhibitors in combination with LuTate. Results: The CRISPR-Cas9 screen identified several potential targets for both resistance and sensitivity to PRRT. Two gene knockouts which conferred LuTate resistance in vitro, ARRB2 and MVP, have potential mechanisms related to LuTate binding and retention, and modulation of DNA-damage repair (DDR) pathways, respectively. The screen showed that sensitivity to LuTate treatment in vitro can be conferred by the loss of a variety of genes involved in DDR pathways, with loss of genes involved in Non-Homologous End-Joining (NHEJ) being the most lethal. Loss of the key NHEJ gene, PRKDC (DNA-PK), either by gene loss or inhibition by two different inhibitors, resulted in significantly reduced cell survival upon exposure of cells to LuTate. In SSTR2-positive xenograft-bearing mice, the combination of nedisertib (a DNA-PK specific inhibitor) and LuTate produced a more robust control of tumour growth and increased survival compared to LuTate alone. Conclusions: DDR pathways are critical for sensing and repairing radiation-induced DNA damage, and our study shows that regulation of DDR pathways may be involved in both resistance and sensitivity to PRRT. Additionally, the use of a DNA-PK inhibitor in combination with LuTate PRRT significantly improves the efficacy of the treatment in pre-clinical models, providing further evidence for the clinical efficacy of this combination.