Radiosensitization of NET cells by HSP90 inhibitor ganetespib is mediated through pleiotropic stress responses

Background: Peptide receptor radionuclide therapy (PRRT) employing [¹⁷⁷Lu]Lu-[DOTA-Tyr³]octreotate has been established as treatment for patients with metastatic neuroendocrine tumors (NETs) that overexpress the somatostatin receptor (SSTR). While PRRT improves survival and quality of life, curative responses remain rare. One way to enhance PRRT efficacy is to combine it with radiosensitizing agents such as heat shock protein 90 (HSP90) inhibitors. HSP90 is a highly conserved molecular chaperone essential for the maturation and stabilization of over a hundred proteins, including proteins involved in the DNA damage response and oncogenic signaling. HSP90-inhibition has been shown to potentiate PRRT, however the mechanism behind this radiosensitizing effect remains unknown. This study aimed to elucidate mechanisms involved in the radiosensitizing effect of HSP90 inhibition. Results: The radiosensitizing effect of HSP90 inhibitor ganetespib in the context of PRRT and external beam radiotherapy (EBRT) was tested using viability assays for NET cell models GOT1 and BON1-SSTR2. Ganetespib significantly enhanced radiation-induced cytotoxicity in both models. To explore underlying mechanisms, we assessed DNA double-strand break (DSB) repair by quantifying 53BP1 foci numbers, and functionally evaluated DSB repair pathways by RAD51 foci quantification and end-joining assay. Although HSP90 inhibition reduced RAD51 foci numbers, its effect on non-homologous end joining and overall DSB persistence was limited. Finally, potential DSB repair-independent mechanisms of radiosensitization were assessed for GOT1 cells using RNA sequencing. Transcriptomic analysis revealed enrichment of pathways related to loss of HSP90 function, such as protein folding and response to heat stress, following combination treatment. This was consistent with effects observed after HSP90 inhibitor monotherapy. Conclusions: Given the lack of significant effects on direct DNA repair or transcriptomic responses, our findings suggest that HSP90 inhibition radiosensitizes NET cells by inducing a pleiotropic effect on multiple stress-related pathways at the protein level, rather than solely through disruption of DNA damage response mechanisms. This effect is likely driven by loss of HSP90 function and subsequent cumulated unfolded protein and proteotoxic stress.