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

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Engbers, Thom G.A. Reuvers, José María Heredia-Genestar, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7271457/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 21 Nov, 2025 Read the published version in EJNMMI Research → Version 1 posted 4 You are reading this latest preprint version Abstract Background Peptide receptor radionuclide therapy (PRRT) employing [ 177 Lu]Lu-[DOTA-Tyr 3 ]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. Peptide receptor radionuclide therapy 177Lu-DOTA-TATE HSP90 ganetespib radiosensitization neuroendocrine tumors Figures Figure 1 Figure 2 Figure 3 Figure 4 BACKGROUND Peptide receptor radionuclide therapy (PRRT) has been established as an effective treatment for patients with metastatic neuroendocrine tumors (NETs) that overexpress the somatostatin receptor 2 (SSTR2) [ 1 ]. This therapy employs [ 177 Lu]Lu-[DOTA-Tyr 3 ]octreotate ( 177 Lu-DOTA-TATE), in which somatostatin analogues are conjugated with radionuclides and administered systemically. Upon binding of 177 Lu-DOTA-TATE to SSTR2 expressed on the tumor cell membrane, the complex is internalized and induces DNA damage following radio-active decay, of which double-strand breaks (DSBs) are most cytotoxic [ 2 ]. While PRRT improves survival and quality of life, complete cures remain rare and its therapeutic potential is restricted by dose-limiting toxicity [ 3 , 4 ]. Thus, further refinement of this treatment is required to enhance its clinical outcome. A widely used approach for improving PRRT efficacy involves combination treatment with radiosensitizing agents that potentiate radiation-induced cytotoxicity. These radiosensitizers typically function by either amplifying radiation-induced DNA damage or impairing DNA damage repair [ 5 ]. An example of a broader-spectrum radiosensitizer is the inhibition of heat shock protein 90 (HSP90), a molecular chaperone protein of the HSP family that plays a central role in protein folding, stability and homeostasis. HSP90 regulates the stability of over a hundred client proteins, including oncogenic signaling molecules and key DNA damage response (DDR) components, thereby promoting cellular survival and stress adaptation [ 6 , 7 ]. Moreover, HSP90 is frequently overexpressed in various cancers, including NETs, highlighting its potential as a target for radiosensitization to PRRT [ 8 , 9 ]. Previous studies have demonstrated that HSP90 inhibitors enhance the efficacy of external beam radiotherapy (EBRT), primarily through inhibition of radiation-induced survival pathways such as the phosphatidylinositol 3-kinase (PI3K)/protein kinase B(Akt)- and microtubule associated protein kinase (MAPK)/extracellular signal-regulated kinase(ERK)-signaling pathways, as well as suppression of the DDR [ 10 ]. While HSP90 inhibition also has been shown to potentiate PRRT, the precise mechanisms underlying its sensitizing effects remain unclear [ 11 – 13 ]. Given the fundamental differences in radiation kinetics between EBRT and PRRT, including differential dose rates and exposure timings, the mechanism of radiosensitization by HSP90 inhibition possibly varies between these modalities. To better understand how these treatments synergize and use this information to potentially increase their efficacy, we investigated the mechanistic basis of HSP90 inhibition as a radiosensitizing strategy for PRRT. MATERIALS & METHODS Cell culture Human small intestine NET cell-line GOT1 was kindly gifted by Ola Nilsson (Sahlgrenska Cancer Center, University of Gothenburg, Sweden) [ 14 ] and cultured in RPMI 1640 (Gibco) supplemented with 10% fetal calf serum (FCS), 1% penicillin-streptomycin (PS), 5 mM L-glutamine (Stemcell Technologies), 5 µg/mL human insulin (Sigma Aldrich) and 5 µg/mL human transferrin (Sigma Aldrich, T8158). Human pancreatic NET cells BON1-SSTR2 were generated as described previously [ 15 ] and maintained in DMEM/F12 (Gibco), supplemented with 10% FCS and 1% PS in presence of 500 µg/mL G418 (Invitrogen). Cells were maintained at 37°C in a humidified incubator with 5% CO2. EBRT, PRRT and ganetespib EBRT was applied by X-ray irradiation with a RS320 cabinet irradiator (X-strahl) at a dose rate of 1.6 Gy/min. Lu-Mark (IDB Holland) was used for in-house production of 177 Lu-DOTA-TATE, which met standard characteristics for patient treatment (specific activity 53 MBq/nmol, radiometal incorporation > 95% and radiochemical purity > 90%). Ganetespib (STA-9090; Selleckchem, S1159) was stored as a 10 mM stock solution in DMSO at -80°C until further use. Western blotting Western blotting GOT1 and BON1-SSTR2 cells were treated with ganetespib (10 or 25 nM) or vehicle and harvested in Laemmli buffer (4% SDS, 20% glycerol, 120 mM Tris pH 6.8) at 0, 0.5, 2, 6, 24 and 72 h post-treatment. Protein concentrations were determined by Lowry method. A total of 10 µg (GOT1) or 5 µg (BON1-SSTR2) protein was loaded per lane on precast Mini-PROTEAN TGX 4–15% gels (BioRad, 4561084) and gel electrophoresis was run at 100V. Proteins were blotted onto a PVDF Transfer Membrane (Merck Millipore, IPVH00010) for 1 h at 100V in blotting buffer (1X Tris Glycine buffer with 20% methanol). Membranes were blocked in PBS + 0.05% Tween (Sigma-Aldrich) + 3% skimmed milk (Merck Millipore) and incubated with primary antibodies rat anti-HSP70 (Cell Signaling Technology, 4873S, 1:1000) and mouse anti-actin (Millipore, LV1547855, 1:500.000) at 4°C overnight. After washing, membranes were incubated for 1 h with secondary antibodies horseradish peroxidase-conjugated polyclonal rabbit-anti-rat (Dako, P0459, 1:2000) and horseradish peroxidase-conjugated AffiniPure ™ sheep-anti-mouse IgG (Jackson ImmunoResearch, 515-035-003, 1:2000) in blocking buffer at RT. Protein bands were detected by enhanced chemiluminescence (ECL) substrate and imaged on an Amersham Imager 600 (GE Healthcare Life Sciences). The experiment was performed as 3 independent biological replicates. Viability assay Cells were treated with 177 Lu-DOTA-TATE (1 MBq/mL, 1.9x10 -8 M) or vehicle in suspension in culture medium (2x10 5 cells/mL) in 15 mL tubes. After 4 h, radioactive medium was washed away and cells were seeded into white walled, clear flat bottom 96-well plates at a density of 3x10 4 (GOT1) or 2x10 3 (BON1-SSTR2) cells/well in 200 µL, in presence of ganetespib (1, 5, 10, 25 or 50 nM) or vehicle. In parallel, cells were first seeded into 96-well plates, pre-treated with ganetespib for 2 h and subsequently irradiated by EBRT (2 Gy). For BON1-SSTR2 cells, ganetespib was removed after 24 h and replaced with fresh medium. After 7 days, metabolic activity was measured by CellTiter-Glo® 2.0 Assay (Promega, G9241). In short, 100 µL of CellTiter-Glo® 2.0 Reagent was added per well and plates were incubated for 10 min at RT. Luminescence was measured on the Spectramax iD3 microplate reader (Molecular Devices). Experiments were performed as 3 independent biological replicates each with technical triplicates. Cell death assay Cells were treated with 177 Lu-DOTA-TATE (1 MBq/mL, 1.9x10 -8 M) or vehicle in suspension in culture medium (2x10 5 cells/mL). After 4 h, radioactive medium was washed away and cells were seeded into 6-well plates at a density of 1x10 6 (GOT1) or 3.5x10 5 (BON1-SSTR2) cells/well, in presence of ganetespib (10 or 25 nM) or vehicle. In parallel, cells were first seeded into 6-well plates, pre-treated with ganetespib for 2 h and subsequently irradiated by EBRT (2 Gy). For BON1-SSTR2 cells, medium was refreshed after 24 h to remove ganetespib. At day 3, live and dead cells were collected and incubated with SYTOX ™ AADvanced ™ Dead Cell Stain (1 µM) for 5 min at RT. Fluorescent signal was detected by flow cytometry on a LSRFortessa flow cytometer (BD Biosciences). Gating and analysis were performed using FlowJo ™ software (BD Biosciences). Experiments were performed as 3 independent biological replicates. RAD51 immunofluorescent staining and foci quantification GOT1 and BON1-SSTR2 were seeded on glass coverslips in 12-well plates and treated with vehicle, 10 or 25 nM ganetespib 2 h prior to irradiation (5 Gy). To label replicating cells, samples were incubated with 30 µM 5-ethynyl-2’-deoxyuridine (EdU) for 3 (GOT1) or 1 h (BON1-SSTR2) prior to fixation. At 2 and 6 h after EBRT, cells were pre-extracted on ice for 1 min in pre-extraction buffer (0.5% Triton X-100, 20 mM HEPES-KOH [pH7.9], 50 mM NaCl, 3 mM MgCl 2 , 300 mM sucrose), followed by fixation in 2% PFA for 15 min. Samples were blocked in PBS + 3% bovine serum albumin (BSA) and permeabilized in PBS + 0.1% Triton X-100. Then, cells were incubated for 30 min at RT with a reaction mix consisting of 40 mM Tris buffer, 4 mM CuSO 4 (Sigma), 30 µM Atto 488 azide (ATTO-TEC) and 4 mM ascorbic acid (Sigma), protected from light. For immunostaining, cells were incubated at RT in PBS + 0.5% BSA + 20 mM glycine with primary RAD51 antibody (homemade rabbit polyclonal clone #2307, 1:10.000) for 1.5 h. Cells were again washed in PBS + 0.1% Triton X-100 and incubated with secondary Goat-anti-rabbit IgG Alexa Fluor 594 (Thermofisher, A-11012, 1:1000) in PBS + 0.5% BSA + 20 mM glycine for 1 h in the dark. Coverslips were mounted on microscope slides using Vectashield (Vector Laboratories) containing DAPI. Imaging of RAD51 foci was performed on a TCS SP5 confocal microscope (Leica) using a 63x oil immersion objective and excitation lasers at 405 nm (DAPI), 488 (EdU) and 561 (RAD51). Images were acquired as Z-stacks from 4 different fields of view per condition. Image processing and quantification of RAD51 foci per EdU-positive nucleus were conducted using homemade scripts in FIJI [ 16 ]. In short, EdU-positive cells were segmented by thresholding the 488 nm channel to define the region of interest. Within each region of interest, RAD51 foci were segmented using the 561 nm channel, with predefined minimum and maximum foci sizes and intensities. At least 25 cells were analyzed per condition per replicate, and experiments were performed as 3 independent biological replicates. 53BP1 immunofluorescent staining and foci quantification GOT1 and BON1-SSTR2 cells were plated in 12-well plates on glass coverslips. For EBRT, cells were incubated with vehicle, 10 or 25 nM ganetespib 2 h prior to irradiation (2 Gy). For PRRT, adherent cells were incubated for 4 h with 177 Lu-DOTA-TATE (1 MBq/mL, 1.9x10 -8 M), after which the radioactive medium was replaced with medium containing ganetespib (10 or 25 nM) or vehicle. At 30 min, 6 and 24 h after radiation treatment, cells were fixed in 2% paraformaldehyde (PFA) for 15 min. For immunostaining, cells were permeabilized in PBS + 0.1% Triton X-100. Samples were blocked in PBS + 0.5% BSA + 20 mM glycine and incubated with primary antibody for 53BP1 (Novus Biologicals, NB100-904, 1:1000) in blocking buffer for 1 h at RT. Cells were then washed with PBS + 0.1% Triton X-100 and incubated in secondary Goat-anti-rabbit IgG Alexa Fluor 594 (Thermofisher, A-11012, 1:1000) in blocking buffer for 1 h at RT in the dark. Samples were mounted using Vectahield (Vector Laboratories) containing DAPI. Imaging of 53BP1 foci was done on a TCS SP5 confocal microscope (Leica) using a 63x oil immersion objective and 405 nm (DAPI) and 561 nm (53BP1) lasers. Imaging and analysis were done in a similar manner to the RAD51 foci quantification. For analysis, nuclei were segmented in the DAPI channel, after which in each nuclear mask foci were segmented in the 53BP1 channel by thresholding, using a defined minimum and maximum foci size and intensity. At least 40 cells were analyzed per condition per replicate and experiments were performed as 3 independent biological replicates. End-joining assay GOT1 or BON1-SSTR2 cells were plated in 6-well plates. As a positive control, 1 µM DNA-PKcs inhibitor AZD7648 (Medchemexpress) was added at the moment of seeding. After 24 h, treatment with 10 or 25 nM ganetespib was started. Assessment of the relative amount of classical non-homologous end joining (c-NHEJ) and microhomology-mediated end joining (MMEJ) used by cells was done as described before [ 17 ]. In short, 2 h after starting ganetespib treatment, a linear DNA substrate (pDVG94 plasmid) was transfected into cells. Extrachromosomal DNA was isolated 2 days after transfection. Plasmid DNA was PCR-amplified and digested by using the restriction enzyme BstXI, DNA fragments were separated and visualized using gel electrophoresis in a 6% polyacrylamide gel in Tris/borate/ethylenediaminetetraacetic acid (TBE) buffer using ethidium bromide. The experiment was performed as 2 independent biological replicates. RNA sequencing GOT1 cells were treated in suspension 177 Lu-DOTA-TATE (1 MBq/mL, 1.9x10 -8 M) or vehicle for 4 h and seeded in 6-well plates in presence or absence of 10 nM ganetespib. Total RNA was isolated at 24 and 72 h post-PRRT using the RNeasy Mini Kit (QIAGEN) according to the manufacturer’s protocol and stored at -80°C before further processing. RNA concentration, purity and quality were assessed on a Nanodrop 2000 Spectrophotometer (Thermo Scientific) and Bioanalyzer 2100 (Agilent). Library preparation and RNA sequencing were performed at Genomescan B.V. (Leiden, The Netherlands). In short, libraries were prepared using the NEBNext Ultra II Directional RNA Library Prep Kit (New England Biolabs). Clustering and DNA sequencing on the NovaSeq6000 (Illumina) was performed at an average sequencing depth of 28 million reads and 150 base pair read length. Samples sent for RNA sequencing comprised 3 biological replicates per condition. RNA-seq processing, differential gene expression, and gene set enrichment analysis The raw fastq files were pre-processed with fastp v0.23.2 [ 18 ]. Samples were then aligned using STAR v2.7.10a [ 19 ] against the human reference genome hs38 with ENSEMBL v107 annotation. Quality control was assessed using fastqc v0.11.9 ( https://github.com/s-andrews/FastQC/ ) and RSeQC v5.0.1 [ 20 , 21 ]. MultiQC v1.15.dev0 [ 22 ] was used to summarize the quality control of these two programs and the different steps of the data processing. Read counts overlapping the entire gene body were generated using htseq-count v2.0.2 [ 23 ]. The resulting gene-count matrices were analyzed using R (version 4.4). One sample (001, Control 24h) showed a diverging expression profile and it was excluded from subsequent analyses. Differential gene expression between groups and conditions was analyzed using DESeq2 v1.44.0 [ 24 ] by two different models, with an adjusted p-value threshold of 0.05 and |Fold Change| ≥ 1.2. The first model used was “design = ~ group”, where group represents the combination of treatment and time point. In addition, an interaction model (“design = ~ radiation*inhibitor”) was used (only for samples at 24 h) to decouple the effects of PRRT and ganetespib monotherapies and evaluate the synergistic effects of both therapies in the combination treatment samples. Gene Set Enrichment Analysis (GSEA) was performed using the R package fGSEA v1.30.0 [ 25 ] and MSigDB (v2023.2) human GO: Biological Process and GO: Molecular Function were used as a reference [ 26 , 27 ]. Each comparison between groups and conditions was analyzed separately using the log2 Fold Change of all detected genes. Genes not present in the data were excluded from the background. Statistical analysis Statistical analyses for all experiments, except for RNA sequencing, were performed in Graphpad Prism (version 9.4.0). Results are presented as mean ± standard error of the mean (SEM). For viability and cell death assays, statistical significance between treatments was determined by an unpaired t-test. For foci analysis experiments (53BP1, RAD51), a one-way ANOVA followed by Tukey’s multiple comparisons test was performed. P-values were considered significant if p < 0.05. RESULTS Ganetespib activates the heat shock response and sensitizes NET cells to EBRT and PRRT To sensitize NET cell lines GOT1 and BON1-SSTR2 to ionizing radiation, we treated the cells with small molecule inhibitor ganetespib, which competitively binds to the N-terminal ATP-binding pocket of HSP90 [ 28 ]. To confirm effective HSP90 inhibition and assess its temporal dynamics, we characterized the cellular response to ganetespib according to HSP70 protein levels, which is an established biomarker for detection of HSP90 inhibition and the subsequent activation of the heat shock response (HSR) [ 29 ]. In both GOT1 and BON1-SSTR2 cells, HSP70 levels were increased following ganetespib treatment within 6 h, and remained elevated for 72 h (Fig. 1 A), suggesting successful inhibition of HSP90 for (at least) 72 h. To evaluate the radiosensitizing potential of ganetespib in our setup, we indirectly measured cell viability by evaluating metabolic activity seven days after starting the treatment. In GOT1 cells, PRRT and EBRT monotherapy resulted in a relative cell viability of 0.60 and 0.48 compared to control, respectively. In contrast, BON1-SSTR2 cells exhibited markedly lower sensitivity to both radiation treatments, with relative viabilities of 1.02 for PRRT and 0.87 for EBRT (Fig. 1 B). In GOT1 cells, combination treatment with ganetespib and either PRRT or EBRT significantly reduced cell viability compared to ganetespib monotherapy (Fig. 1 B). However, in BON1-SSTR2 cells, continuous ganetespib treatment was excessively toxic by itself (Supplementary Fig. 1A). Therefore, we modified the treatment protocol by washing out ganetespib after 24 h of incubation and replacing it with drug-free medium. This resulted in a small but significant reduction in cell viability after seven days of combination treatment compared to ganetespib monotherapy treated cells (Fig. 1 B). To further validate whether the observed decrease in cell viability was attributable to cell death induction, we quantified cell death three days after treatment initiation by flow cytometry (Supplementary Fig. 1B). In GOT1 cells, a dose-dependent decrease in living cells was observed, which was significantly stronger in EBRT and PRRT treated cells compared to the ganetespib monotherapy (Fig. 1 C). In contrast, in BON1-SSTR2 cells no such effect was observed. Additionally, consistent with the cell viability assay, these cells showed negligible sensitivity to ionizing irradiation itself (Fig. 1 C). Treatment with ganetespib has limited impact on the persistence of radiation-induced DSBs The radiosensitizing effect of HSP90 inhibition has previously been associated with inhibitory effects on the DDR, more specifically through inhibition of DSB repair [ 30 – 33 ]. To determine whether the observed increase in cell death following combined ganetespib and EBRT or PRRT was associated with elevated DNA damage as a result of perturbed DNA repair, we quantified p53 binding protein 1 (53BP1) foci as marker of DSB levels [ 34 ]. Following EBRT, both GOT1 and BON1-SSTR2 cells exhibited a significant increase in 53BP1 foci at 0.5 h post-irradiation, which gradually declined to baseline level within 24 h. In contrast, PRRT induced a milder but still significant increase in 53BP1 foci at 0.5 h post-incubation (Fig. 2 B, D). Ganetespib monotherapy did not increase the number of 53BP1 foci in GOT1 cells, nor did it enhance DSB levels when combined with EBRT or PRRT at the analyzed time-points (Fig. 2 A, B). At the 24 h time-point, even a modest reduction in the number of 53BP1 foci was observed following ganetespib treatment in combination with EBRT and PRRT, though this difference was relatively small and therefore might lack biological relevance. In BON1-SSTR2 cells, a trend toward a dose-dependent increase in 53BP1 foci following ganetespib treatment was observed when combined with EBRT or PRRT; however, the differences were again relatively minor and not statistically significant across all conditions and time points (Fig. 2 C, D). Together, these findings suggest that despite its radiosensitizing effect, ganetespib has a limited impact on overall DSB levels and persistence of DSBs after EBRT and PRRT under the tested conditions. HSP90 inhibition impairs homologous recombination through decreasing irradiation-induced RAD51 foci formation Since the effect of ganetespib on DSB persistence was not clearly visible, we further investigated the role of specific DSB repair pathways in the sensitization. Therefore, we analyzed the effect of ganetespib on homologous recombination (HR) and classical non-homologous end-joining (c-NHEJ), the two major DSB pathways. To determine the effect on HR, we analyzed the effect of ganetespib on RAD51 foci formation after irradiation, specifically in S-phase cells by selecting for EdU-positive nuclei (Fig. 3 A,C). RAD51 specifically accumulates on DSBs being repaired by HR. Since the previously used radiation conditions (2 Gy EBRT; 1 MBq/mL PRRT) did not induce a detectable number of RAD51 foci (data not shown), we focused on a higher dose of EBRT (5 Gy) as a general indicator for HR functioning. Irradiated GOT1 and BON1-SSTR2 cells showed a clear presence of RAD51 foci both at 2 h and 6 h post-irradiation in proliferating cells. Treatment with ganetespib resulted in a significant and dose-dependent decrease in the number of RAD51 foci, with the most pronounced effect observed at 6h (Fig. 3 B, D), indicating that HR is (partially) blocked by ganetespib. Subsequently, effects on DSB repair by c-NHEJ were assessed by an end-joining assay. This assay measures the balance between c-NHEJ and microhomology-mediated end joining (MMEJ) using an exogenous repair product. Positive control, DNA-PKcs inhibitor AZD7648 induced a profound shift from c-NHEJ to MMEJ products, indicative of a strong inhibitory effect on c-NHEJ. However, 2 h treatment with ganetespib pre-transfection did not induce a visible shift in repair products for both cell lines (Fig. 3 E). Increasing the duration of ganetespib treatment before transfection to 24 h as a control for treatment scheme-dependent effects, also did not induce a shift to MMEJ. These results, together with the 53BP1 foci quantification, indicate that ganetespib impairs HR in GOT1 and BON1-SSTR2 cells, but this has a limited effect on the overall repair of DSBs induced by EBRT and PRRT. Transcriptomic analysis shows activation of the heat shock response upon HSP90 inhibition Given the significant effect on overall survival, but limited impact of HSP90 inhibition on DSB repair following both EBRT and PRRT, we hypothesized that additional cell mechanisms may contribute to the observed radiosensitizing effects. To gain deeper insight into the cellular response to HSP90 inhibition in the context of PRRT, we performed RNA sequencing in GOT1 cells, which showed the strongest degree in radiosensitization (Fig. 1 B). The effect of ganetespib, PRRT and combination treatment was analyzed after 24 and 72 h of treatment by differential gene expression analysis. To evaluate the transcriptional effect of ganetespib monotherapy, differentially expressed genes (DEGs) were identified by comparing ganetespib treatment to control. Similarly, the effect of ganetespib in the context of PRRT was assessed by comparing combination treatment to PRRT monotherapy. After 24 h of treatment, there was an overlap of 80 (62%) upregulated and 21 (15%) downregulated DEGs being shared between the two comparisons (Fig. 4 A). To focus on DEGs specific to ganetespib treatment in presence of PRRT, non-overlapping DEGs were further analyzed by an interaction model, that uses the effects of PRRT and ganetespib monotherapies as covariates. The interaction effect revealed no significant DEGs, suggesting that at 24 h, the expression changes observed in the combination treatment were the additive effects of PRRT and HSP90 inhibition, with no synergistic effects observed (Supplementary Fig. 2A). Comparing the effects of ganetespib in the context of PRRT at both 24 and 72 h time points revealed a similar response that became much more attenuated after 72 h, as indicated by the number of significant DEGs identified (237 DEGs at 24 h vs. 16 DEGs at 72 h) and the generally lower fold change observed at 72 h (Fig. 4 B). Due to the lack of a control sample at 72 h, interaction model analysis could not be performed at this time point. Instead, the effect of HSP90 inhibition in the context of PRRT (combination vs. PRRT) relative to both monotherapies (ganetespib vs. PRRT) was analyzed for overlapping DEGs. This comparison showed a high level of concordance, with 15 out of 16 (94%) genes being shared between conditions (Fig. 4 A). For all these comparisons the top significant DEGs were mainly upregulated canonical chaperone genes involved in the compensatory HSR upon HSP90 inhibition (e.g. HSP90AA2P, HSP90AA3p, HSP90AA1 ), again validating the biological activity of ganetespib at the analyzed time points. GSEA of DEGs between combination and PRRT monotherapy revealed exclusive enrichment of pathways related to HSP90 function, including response to heat and protein folding, which was the strongest at 24 h (Fig. 4 C). At this time point, similar affected pathways were found after ganetespib mono-treatment (Supplementary Fig. 2B). Taken together, these results demonstrate that HSP90 inhibition induces a detectable transcriptomic response at 24 h which diminishes by 72 h, likely reflecting its predominant mode of action at the protein level. The transcriptomic profile of the combination treatment suggests an orthogonal and functionally distinct transcriptomic response. This likely results in an additive, rather than synergistic, effect of PRRT and HSP90 inhibition on cell survival. DISCUSSION HSP90 inhibition has been established as an effective radiosensitization strategy in the context of EBRT across various cancer types [ 10 ]. More recently, HSP90 inhibition has shown promising results in enhancing sensitivity of NETs to PRRT employing 177 Lu-DOTA-TATE, although the underlying mechanisms remain incompletely understood [ 11 – 13 ]. In this study, we aimed to elucidate the pathways contributing to the radiosensitizing effect of HSP90 inhibition to PRRT. Consistent with previous studies, we showed that the HSP90 inhibitor ganetespib enhances the efficacy of both EBRT and PRRT in NET cell lines GOT1 and BON1-SSTR2. Notably, the degree of radiosensitization varied between the two models, with BON1-SSTR2 cells exhibiting pronounced toxicity to ganetespib monotherapy, likely reflecting a stronger dependency on HSP90 function. To mitigate this toxicity, we modified the treatment regimen by removing the inhibitor after 24 h, which indeed reduced monotherapy toxicity and resulted in a small but significant radiosensitizing effect. Further optimization of ganetespib treatment strategies, e.g. by adjusting exposure duration or using a lower concentration range of the inhibitor, may help expand the therapeutic window of HSP90 inhibition. Next, we validated whether the observed decrease in cell viability was attributable to enhanced radiation-induced cell death. In GOT1 cells, a dose-dependent increase in cell death was detected, whereas no such effect was observed in BON1-SSTR2 cells. This aligns with the differences in radiosensitization between cell lines as observed in the viability assays, and may also reflect variations in the timing of cell death induction between the two cell lines. Indeed, BON1-SSTR2 cells proliferate rapidly (doubling time ~ 34 h [ 35 ]), while GOT1 cells much more slowly (> 18 days [ 14 ]). As consequence, it is possible that by 72 h post-treatment, the surviving fraction BON1-SSTR2 cell population may have undergone sufficient proliferation to mask the effects of initial cell death, whereas in the slower-proliferating GOT1 cells, these effects remain detectable. Alternatively, the reduced viability in BON1-SSTR2 cells following combination treatment may primarily reflect decreased proliferation or metabolic activity rather than increased radiation-induced cell death. Finally, differences in radiosensitization could correlate with intrinsic radiosensitivity of cell lines, as both EBRT and PRRT monotherapy were less toxic for BON1-SSTR2 cells than for GOT1 cells. Since DSBs are the primary determinant of radiation-induced effects, targeting DDR components, likely involved in radiosensitization mechanisms, is a widely studied approach to enhance radiation sensitivity [ 36 ]. The two primary DSB repair pathways are HR and c-NHEJ. HR is a high-fidelity repair mechanism that utilizes the homologous sister chromatid as a template for repair and is therefore restricted to cells in the S and G2 phases of the cell cycle. In contrast, c-NHEJ mediates repair by directly ligating the broken DNA ends without the need of a homologous template, making it a more error-prone but widely utilized mechanism throughout all cell cycle phases [ 37 ]. Both disruption of c-NHEJ and HR have demonstrated radiosensitizing potential [ 15 , 38 , 39 ]. As HSP90 stabilizes proteins at various stages of DNA damage detection, repair and cell cycle check points, it is likely that HSP90 inhibition affects the DDR in a multi-target manner. Previous studies have shown that HSP90 inhibition leads to reduction of the overall cellular levels of key HR proteins such as BRCA2 and RAD51 [ 31 , 32 , 40 ]. However, since these proteins are predominantly expressed during S and G2 phases of the cell cycle, their downregulation at the population level may largely reflect reduced proliferation and a consequent decrease in the proportion of HR-competent cells, rather than a direct impairment of HR itself. To address this, we specifically assessed the effect of ganetespib on HR in EdU-positive (S/G2 phase) nuclei and confirmed that HSP90 inhibition indirectly impairs HR as indicated by a reduced number of RAD51 foci. In contrast, limited data is available showing effects of HSP90 inhibition on c-NHEJ, or more indirectly by showing a decrease in levels of c-NHEJ mediators such as DNA-PKcs [ 40 – 42 ]. Our end-joining assay did not show a shift from c-NHEJ to MMEJ as an alternative pathway after treatment, suggesting that c-NHEJ is not affected by treatment with radiosensitizing doses of ganetespib. Despite HR inhibition, ganetespib had little impact on overall DSB persistence following EBRT and PRRT as reflected by the minimal changes in 53BP1 foci numbers. One possible explanation is that the inhibitory effect was insufficient to substantially impair overall DSB repair. Alternatively, HR may contribute too little to DSB repair under the examined conditions to produce a detectable effect, which partly aligns with the established predominance of c-NHEJ as the primary DSB repair pathway in mammalian cells [ 43 ]. This may account for the modest, dose-dependent increase in 53BP1 foci observed in BON1-SSTR2 cells, as their higher proliferation rate likely makes them more reliant on HR compared to the slower dividing GOT1 cells. While our analysis confirmed that ganetespib impairs HR following EBRT, it remained unclear whether similar mechanisms contribute to radiosensitization in the context of PRRT, which differs in radiation delivery and kinetics. To address this, we performed RNA sequencing in GOT1 cells, which we selected for their pronounced response to PRRT combined with HSP90 inhibition. To specifically isolate the impact of HSP90 inhibition on cellular pathways during PRRT, our analysis focused primarily on transcriptomic differences between PRRT monotherapy and the PRRT-HSP90 inhibitor combination. It is well established that HSP90 inhibition activates a compensatory HSR, which is transcriptionally regulated by heat shock factor 1 (HSF1). In response to HPS90 inhibition, HSF1 upregulates a set of HSR-related genes and induces molecular chaperones such as HSP70, to restore proteostasis [ 44 ]. Our transcriptomic analysis indeed confirmed a robust induction of this compensatory HSR at 24 h post-treatment. However, by 72 h, the transcriptional response was markedly reduced, with only 16 DEGs remaining between PRRT and combination treatment. Moreover, the overall transcriptomic response to the combination treatment was relatively modest, characterized by low-magnitude fold changes across most genes. This limited transcriptional signature is likely a reflection of HSP90’s primary mode of action at the post-translational level. Considering the temporal difference between transcriptomic and proteomic responses, comprehensive proteomic analysis following HSP90 inhibition may offer deeper insight into the dynamic regulation of affected pathways. GSEA between PRRT and combination treatment solely showed enrichment of pathways associated with HSP90 function, such as protein folding and response to heat. As GSEA between control and HSP90 monotherapy showed enrichment of similar pathways, it is likely that the increased efficacy after combination treatment is an additive result of HSP90 inhibition and PRRT. These findings suggest that the radiosensitizing effect of HSP90 inhibition is not attributed to synergistic disruption of specific signaling pathways, but instead is caused by a pleiotropic effect resulting from loss of HSP90 function. The pleiotropic nature of HSP90 inhibition is supported by its broad list of client proteins and central role in managing cellular stress, particularly in tumor cells [ 10 ]. For example, HSP90 is essential for the stability and function of proteins regulating cell cycle progression and checkpoint control, such as cyclin-dependent kinases CDK4 and CDK1 [ 33 ], and upstream Serine/threonine-protein kinase (ATR) [ 45 ]. Degradation of these proteins upon HSP90 inhibition results in cell cycle arrest at the G2/M phase, the stage at which cells are most sensitive to ionizing irradiation [ 46 ]. In addition, HSP90 supports multiple radioresistance pathways. In the PI3K/Akt pathway, it maintains key proteins like Akt [ 47 – 49 ], PDK1 and mTOR [ 49 , 50 ], resulting in downregulation of this signaling axis upon inhibition of HSP90. Similarly, the MAPK/ERK pathway is disrupted upon HSP90 inhibition through depletion of Raf and ERK proteins [ 51 , 52 ]. Importantly, in certain cancer types, inhibition of the PI3K/Akt pathway has been shown to induce compensatory activation of the MAPK/ERK pathway, thereby sustaining cell survival despite inhibition of PI3K [ 53 ]. This compensatory mechanism highlights the advantage of HSP90 inhibition as a multitarget approach, capable of synchronously impairing multiple survival and radioresistance pathways. CONCLUSIONS Taken together, our data suggests that the radiosensitizing effect of HSP90 inhibition is unlikely to be mediated by the suppression of a single signaling pathway. Instead, it likely results from the simultaneous, moderate disruption of several interconnected pathways – including those regulating cell survival, proliferation, radioresistance, and cell cycle progression – ultimately leading to a synergistic reduction in tumor cell viability when combined with PRRT. Abbreviations Akt: protein kinase B ATR : Serine/threonine-protein kinase CDK : cyclin-dependent kinase c-NHEJ : classical non-homologous end joining DEG: differentially expressed gene DDR : DNA damage response DSB : double-stranded break EBRT : external beam radiotherapy EDU: 5-ethynyl-2’-deoxyuridine ERK : extracellular signal-regulated kinase GSEA : gene set enrichment analysis HR : homologous recombination HSF1 : heat shock factor 1 HSP90 : heat shock protein 90 HSR: heat shock response MAPK : microtubule associated protein kinase MMEJ : microhomology-mediated end joining mTOR : mammalian target of rapamycin NETs: neuroendocrine tumors PDK1 : 3-phosphoinositide-dependent protein kinase-1 PI3K : phosphatidylinositol 3-kinase PRRT : peptide receptor radionuclide therapy SSTR2 : somatostatin receptor 2 53BP1 : p53 binding protein 1 177 Lu-DOTA-TATE : [ 177 Lu]Lu-[DOTA-Tyr 3 ]octreotate Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Availability of data and material Data is available upon reasonable request to the corresponding author. RNA-sequencing dataset generated in the current study is available in the XXX repository. Competing interests The authors declare that they have no competing interests. Funding This work was supported by the Dutch Cancer Society (grant number 11840) and ERC grant (101042537 RADIOBIO). Author’s contributions PE, TR, MS and JN contributed to conceptualization of the study. PE, TR, JH, JC and NS contributed to acquisition, visualization and analysis of the data. MS and JN supervised the project. All authors helped draft, review or edit the manuscript and approved the submission. Acknowledgements We would like to thank Prof. Ola Nilsson (Sahlgrenska Cancer center, University of Gothenburg, Sweden) for kindly providing the GOT1 cell line, and Dr. Joris Pothof, Dr. Roland Kanaar and Danilo Remmers for their contributions to this study. 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Biochem Soc Trans. 2012;40:139-46. Supplementary Files 202508025HSP90ipapersupplementaldata.docx Cite Share Download PDF Status: Published Journal Publication published 21 Nov, 2025 Read the published version in EJNMMI Research → Version 1 posted Reviewers agreed at journal 07 Sep, 2025 Reviewers invited by journal 04 Sep, 2025 Editor assigned by journal 04 Sep, 2025 First submitted to journal 03 Sep, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7271457","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":510430705,"identity":"716bdd40-ae23-4b71-8a90-56ad726aa1d2","order_by":0,"name":"Pleun A.M. 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A) \u003c/strong\u003eTime-dependent induction of HSP70 protein expression after start of ganetespib treatment (10 or 25 nM) for GOT1 (left) and BON1-SSTR2 (right) cells. β-actin was used as loading control. \u003cstrong\u003eB) \u003c/strong\u003eViability assay for GOT1 (left) and BON1-SSTR2 (right) cells of a concentration range of ganetespib as monotherapy, or combined with 2 Gy EBRT or 1 MBq/mL PRRT 7 days post treatment initiation. All curves are normalized to their respective viability without ganetespib (0 nM). Data points represent the mean of 3 independent biological replicates with technical triplicates. Below the graphs, the viability of PRRT or EBRT monotherapy is depicted. \u003cstrong\u003eC) \u003c/strong\u003eAssessment of cell death for GOT1 (left) and BON1-SSTR2 (right) cells after ganetespib (0, 10 or 25 nM) as monotherapy, or combined with 2 Gy EBRT or 1 MBq/mL PRRT 3 days post treatment initiation. Values are expressed as the percentage of living cells and normalized to their respective control condition (without ganetespib, 0 nM) for each curve. Data points represent the mean of 3 independent biological replicates. Below the graphs, the relative percentage of live cells of PRRT or EBRT monotherapy is depicted. All error bars represent the SEM. Asterisks indicate the level of significance of the difference for each corresponding point with control. *p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7271457/v1/d221497cdffe497239483567.png"},{"id":91086686,"identity":"484a0767-c0bc-435e-b1d9-e1fc87752579","added_by":"auto","created_at":"2025-09-11 12:29:42","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":707612,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of HSP90 inhibition on EBRT- and PRRT-induced 53BP1 foci. \u003c/strong\u003eCells were treated with 2 Gy EBRT or 1 MBq/mL PRRT and 10 or 25 nM ganetespib or vehicle. \u003cstrong\u003eA,C)\u003c/strong\u003eRepresentative images of 53BP1 foci (red) and cell nucleus (blue) 6 h post treatment initiation in GOT1 (A) and BON1-SSTR2 (C) cells. Scale bars indicate 10 µm. \u003cstrong\u003eB,D)\u003c/strong\u003e Quantification of 53BP1 foci per nucleus in GOT1 (B) and BON1-SSTR2 (D) cells at 0.5, 6 and 24 h after start of treatment. Figures represent data from 3 independent biological replicates pooled together. Black horizontal lines indicate the mean. Ns = not significant, \u0026nbsp;** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7271457/v1/85e4734f171d8b6f1f444f35.png"},{"id":91086684,"identity":"3101abb4-8753-4a0e-afe6-aff3bca9d824","added_by":"auto","created_at":"2025-09-11 12:29:42","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":927492,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFunctional analysis of the effect of ganetespib on DSB repair pathways HR and c-NHEJ. A-D) \u003c/strong\u003eAssessment of HR in GOT1 and BON1-SSTR2 cells after EBRT (5 Gy) and ganetespib (0, 10, 25 or 50 nM). \u003cstrong\u003eA,C)\u003c/strong\u003eRepresentative images of RAD51 foci (red), EdU+ cells (green) and cell nucleus (blue) 2 h post-irradiation in GOT1 (A) and BON1-SSTR2 (B) cells. Scale bars indicate 10 µm. \u003cstrong\u003eB,D)\u003c/strong\u003e Quantification of the number of RAD51 foci per EdU+ nucleus in GOT1 (B) and BON1-SSTR2 (D) cells 2 and 6 h post-irradiation. Figures represent data from 3 independent biological replicates pooled together. Red horizontal lines indicate the mean and error bars indicate SEM. Ns = not significant, * p ≤ 0.05, *** p ≤ 0.001, **** p ≤ 0.0001. \u003cstrong\u003eE) \u003c/strong\u003eAssessment of the balance between c-NHEJ and MMEJ by end-joining assay, non-treated, after 2 h of ganetespib treatment (10 or 25 nM), or after 24 h of ganetespib (25 nM) or AZD648 (1 µM) treatment in GOT1 and BON1-SSTR2 cells.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7271457/v1/f28d66c1575323499117e1d7.png"},{"id":91085468,"identity":"9baceb36-a3fc-44e7-819d-d832bb4ef247","added_by":"auto","created_at":"2025-09-11 12:21:42","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":913889,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTranscriptomic analysis of the response of GOT1 cells to PRRT and ganetespib at 24 and 72 h. A) \u003c/strong\u003eVenn diagrams showing the number and overlap of DEGs after ganetespib monotherapy (ganetespib vs. control) and in the context of PRRT (combination vs. PRRT) after 24 h of treatment, and the effect of HSP90 inhibition in the context of PRRT (combination vs. PRRT) relative to both monotherapies (ganetespib vs. PRRT) after 72 h of treatment. \u003cstrong\u003eB)\u003c/strong\u003e Scatter plot showing non-significant (black) and significant DEGs between combination and PRRT at 24 h (blue) and 72 h (red) or both time points (green). Genes that were also differentially expressed between both monotherapies (ganetespib vs. PRRT) at 72 h are indicated with a diamond. The horizontal and vertical dashed lines denote the minimum log2FoldChange threshold set for significance (log2(1.2)). The diagonal dashed lines denote a slope of 1. \u003cstrong\u003eC) \u003c/strong\u003eGSEA results for the comparison of combination and PRRT using GO database at 24 and 72 h after treatment. Circle size indicates the number of genes represented in the pathway. Circle color indicates upregulation (red) or downregulation (blue) of the pathway.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7271457/v1/2f097bfd4aaff862035a66a4.png"},{"id":96650395,"identity":"b05d0f07-969c-4589-9f3a-4bc886906b7a","added_by":"auto","created_at":"2025-11-24 16:11:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4380492,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7271457/v1/b2020fc5-5939-4398-aaf0-771db361d0cf.pdf"},{"id":91085470,"identity":"fe234cfb-2aff-4043-858c-33940234a507","added_by":"auto","created_at":"2025-09-11 12:21:42","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":948935,"visible":true,"origin":"","legend":"","description":"","filename":"202508025HSP90ipapersupplementaldata.docx","url":"https://assets-eu.researchsquare.com/files/rs-7271457/v1/9428f39cb0ef902f2c34108c.docx"}],"financialInterests":"","formattedTitle":"Radiosensitization of NET cells by HSP90 inhibitor ganetespib is mediated through pleiotropic stress responses","fulltext":[{"header":"BACKGROUND","content":"\u003cp\u003ePeptide receptor radionuclide therapy (PRRT) has been established as an effective treatment for patients with metastatic neuroendocrine tumors (NETs) that overexpress the somatostatin receptor 2 (SSTR2) [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. This therapy employs [\u003csup\u003e177\u003c/sup\u003eLu]Lu-[DOTA-Tyr\u003csup\u003e3\u003c/sup\u003e]octreotate (\u003csup\u003e177\u003c/sup\u003eLu-DOTA-TATE), in which somatostatin analogues are conjugated with radionuclides and administered systemically. Upon binding of \u003csup\u003e177\u003c/sup\u003eLu-DOTA-TATE to SSTR2 expressed on the tumor cell membrane, the complex is internalized and induces DNA damage following radio-active decay, of which double-strand breaks (DSBs) are most cytotoxic [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. While PRRT improves survival and quality of life, complete cures remain rare and its therapeutic potential is restricted by dose-limiting toxicity [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Thus, further refinement of this treatment is required to enhance its clinical outcome.\u003c/p\u003e\u003cp\u003eA widely used approach for improving PRRT efficacy involves combination treatment with radiosensitizing agents that potentiate radiation-induced cytotoxicity. These radiosensitizers typically function by either amplifying radiation-induced DNA damage or impairing DNA damage repair [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. An example of a broader-spectrum radiosensitizer is the inhibition of heat shock protein 90 (HSP90), a molecular chaperone protein of the HSP family that plays a central role in protein folding, stability and homeostasis. HSP90 regulates the stability of over a hundred client proteins, including oncogenic signaling molecules and key DNA damage response (DDR) components, thereby promoting cellular survival and stress adaptation [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Moreover, HSP90 is frequently overexpressed in various cancers, including NETs, highlighting its potential as a target for radiosensitization to PRRT [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e\u003cp\u003ePrevious studies have demonstrated that HSP90 inhibitors enhance the efficacy of external beam radiotherapy (EBRT), primarily through inhibition of radiation-induced survival pathways such as the phosphatidylinositol 3-kinase (PI3K)/protein kinase B(Akt)- and microtubule associated protein kinase (MAPK)/extracellular signal-regulated kinase(ERK)-signaling pathways, as well as suppression of the DDR [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. While HSP90 inhibition also has been shown to potentiate PRRT, the precise mechanisms underlying its sensitizing effects remain unclear [\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Given the fundamental differences in radiation kinetics between EBRT and PRRT, including differential dose rates and exposure timings, the mechanism of radiosensitization by HSP90 inhibition possibly varies between these modalities.\u003c/p\u003e\u003cp\u003eTo better understand how these treatments synergize and use this information to potentially increase their efficacy, we investigated the mechanistic basis of HSP90 inhibition as a radiosensitizing strategy for PRRT.\u003c/p\u003e"},{"header":"MATERIALS \u0026 METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eCell culture\u003c/h2\u003e\u003cp\u003eHuman small intestine NET cell-line GOT1 was kindly gifted by Ola Nilsson (Sahlgrenska Cancer Center, University of Gothenburg, Sweden) [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] and cultured in RPMI 1640 (Gibco) supplemented with 10% fetal calf serum (FCS), 1% penicillin-streptomycin (PS), 5 mM L-glutamine (Stemcell Technologies), 5 \u0026micro;g/mL human insulin (Sigma Aldrich) and 5 \u0026micro;g/mL human transferrin (Sigma Aldrich, T8158). Human pancreatic NET cells BON1-SSTR2 were generated as described previously [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] and maintained in DMEM/F12 (Gibco), supplemented with 10% FCS and 1% PS in presence of 500 \u0026micro;g/mL G418 (Invitrogen). Cells were maintained at 37\u0026deg;C in a humidified incubator with 5% CO2.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eEBRT, PRRT and ganetespib\u003c/h3\u003e\n\u003cp\u003eEBRT was applied by X-ray irradiation with a RS320 cabinet irradiator (X-strahl) at a dose rate of 1.6 Gy/min. Lu-Mark (IDB Holland) was used for in-house production of \u003csup\u003e177\u003c/sup\u003eLu-DOTA-TATE, which met standard characteristics for patient treatment (specific activity 53 MBq/nmol, radiometal incorporation\u0026thinsp;\u0026gt;\u0026thinsp;95% and radiochemical purity\u0026thinsp;\u0026gt;\u0026thinsp;90%). Ganetespib (STA-9090; Selleckchem, S1159) was stored as a 10 mM stock solution in DMSO at -80\u0026deg;C until further use.\u003c/p\u003e\n\u003ch3\u003eWestern blotting\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003eWestern blotting\u003c/div\u003e\u003cp\u003eGOT1 and BON1-SSTR2 cells were treated with ganetespib (10 or 25 nM) or vehicle and harvested in Laemmli buffer (4% SDS, 20% glycerol, 120 mM Tris pH 6.8) at 0, 0.5, 2, 6, 24 and 72 h post-treatment. Protein concentrations were determined by Lowry method. A total of 10 \u0026micro;g (GOT1) or 5 \u0026micro;g (BON1-SSTR2) protein was loaded per lane on precast Mini-PROTEAN TGX 4\u0026ndash;15% gels (BioRad, 4561084) and gel electrophoresis was run at 100V. Proteins were blotted onto a PVDF Transfer Membrane (Merck Millipore, IPVH00010) for 1 h at 100V in blotting buffer (1X Tris Glycine buffer with 20% methanol). Membranes were blocked in PBS\u0026thinsp;+\u0026thinsp;0.05% Tween (Sigma-Aldrich)\u0026thinsp;+\u0026thinsp;3% skimmed milk (Merck Millipore) and incubated with primary antibodies rat anti-HSP70 (Cell Signaling Technology, 4873S, 1:1000) and mouse anti-actin (Millipore, LV1547855, 1:500.000) at 4\u0026deg;C overnight. After washing, membranes were incubated for 1 h with secondary antibodies horseradish peroxidase-conjugated polyclonal rabbit-anti-rat (Dako, P0459, 1:2000) and horseradish peroxidase-conjugated AffiniPure\u003csup\u003e\u0026trade;\u003c/sup\u003e sheep-anti-mouse IgG (Jackson ImmunoResearch, 515-035-003, 1:2000) in blocking buffer at RT. Protein bands were detected by enhanced chemiluminescence (ECL) substrate and imaged on an Amersham Imager 600 (GE Healthcare Life Sciences). The experiment was performed as 3 independent biological replicates.\u003c/p\u003e\n\u003ch3\u003eViability assay\u003c/h3\u003e\n\u003cp\u003eCells were treated with \u003csup\u003e177\u003c/sup\u003eLu-DOTA-TATE (1 MBq/mL, 1.9x10\u003csup\u003e-8\u003c/sup\u003eM) or vehicle in suspension in culture medium (2x10\u003csup\u003e5\u003c/sup\u003e cells/mL) in 15 mL tubes. After 4 h, radioactive medium was washed away and cells were seeded into white walled, clear flat bottom 96-well plates at a density of 3x10\u003csup\u003e4\u003c/sup\u003e (GOT1) or 2x10\u003csup\u003e3\u003c/sup\u003e (BON1-SSTR2) cells/well in 200 \u0026micro;L, in presence of ganetespib (1, 5, 10, 25 or 50 nM) or vehicle. In parallel, cells were first seeded into 96-well plates, pre-treated with ganetespib for 2 h and subsequently irradiated by EBRT (2 Gy). For BON1-SSTR2 cells, ganetespib was removed after 24 h and replaced with fresh medium. After 7 days, metabolic activity was measured by CellTiter-Glo\u0026reg; 2.0 Assay (Promega, G9241). In short, 100 \u0026micro;L of CellTiter-Glo\u0026reg; 2.0 Reagent was added per well and plates were incubated for 10 min at RT. Luminescence was measured on the Spectramax iD3 microplate reader (Molecular Devices). Experiments were performed as 3 independent biological replicates each with technical triplicates.\u003c/p\u003e\n\u003ch3\u003eCell death assay\u003c/h3\u003e\n\u003cp\u003eCells were treated with \u003csup\u003e177\u003c/sup\u003eLu-DOTA-TATE (1 MBq/mL, 1.9x10\u003csup\u003e-8\u003c/sup\u003eM) or vehicle in suspension in culture medium (2x10\u003csup\u003e5\u003c/sup\u003e cells/mL). After 4 h, radioactive medium was washed away and cells were seeded into 6-well plates at a density of 1x10\u003csup\u003e6\u003c/sup\u003e (GOT1) or 3.5x10\u003csup\u003e5\u003c/sup\u003e (BON1-SSTR2) cells/well, in presence of ganetespib (10 or 25 nM) or vehicle. In parallel, cells were first seeded into 6-well plates, pre-treated with ganetespib for 2 h and subsequently irradiated by EBRT (2 Gy). For BON1-SSTR2 cells, medium was refreshed after 24 h to remove ganetespib. At day 3, live and dead cells were collected and incubated with SYTOX\u003csup\u003e\u0026trade;\u003c/sup\u003e AADvanced\u003csup\u003e\u0026trade;\u003c/sup\u003e Dead Cell Stain (1 \u0026micro;M) for 5 min at RT. Fluorescent signal was detected by flow cytometry on a LSRFortessa flow cytometer (BD Biosciences). Gating and analysis were performed using FlowJo\u003csup\u003e\u0026trade;\u003c/sup\u003e software (BD Biosciences). Experiments were performed as 3 independent biological replicates.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eRAD51 immunofluorescent staining and foci quantification\u003c/h2\u003e\u003cp\u003eGOT1 and BON1-SSTR2 were seeded on glass coverslips in 12-well plates and treated with vehicle, 10 or 25 nM ganetespib 2 h prior to irradiation (5 Gy). To label replicating cells, samples were incubated with 30 \u0026micro;M 5-ethynyl-2\u0026rsquo;-deoxyuridine (EdU) for 3 (GOT1) or 1 h (BON1-SSTR2) prior to fixation. At 2 and 6 h after EBRT, cells were pre-extracted on ice for 1 min in pre-extraction buffer (0.5% Triton X-100, 20 mM HEPES-KOH [pH7.9], 50 mM NaCl, 3 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 300 mM sucrose), followed by fixation in 2% PFA for 15 min. Samples were blocked in PBS\u0026thinsp;+\u0026thinsp;3% bovine serum albumin (BSA) and permeabilized in PBS\u0026thinsp;+\u0026thinsp;0.1% Triton X-100. Then, cells were incubated for 30 min at RT with a reaction mix consisting of 40 mM Tris buffer, 4 mM CuSO\u003csub\u003e4\u003c/sub\u003e (Sigma), 30 \u0026micro;M Atto 488 azide (ATTO-TEC) and 4 mM ascorbic acid (Sigma), protected from light. For immunostaining, cells were incubated at RT in PBS\u0026thinsp;+\u0026thinsp;0.5% BSA\u0026thinsp;+\u0026thinsp;20 mM glycine with primary RAD51 antibody (homemade rabbit polyclonal clone #2307, 1:10.000) for 1.5 h. Cells were again washed in PBS\u0026thinsp;+\u0026thinsp;0.1% Triton X-100 and incubated with secondary Goat-anti-rabbit IgG Alexa Fluor 594 (Thermofisher, A-11012, 1:1000) in PBS\u0026thinsp;+\u0026thinsp;0.5% BSA\u0026thinsp;+\u0026thinsp;20 mM glycine for 1 h in the dark. Coverslips were mounted on microscope slides using Vectashield (Vector Laboratories) containing DAPI. Imaging of RAD51 foci was performed on a TCS SP5 confocal microscope (Leica) using a 63x oil immersion objective and excitation lasers at 405 nm (DAPI), 488 (EdU) and 561 (RAD51). Images were acquired as Z-stacks from 4 different fields of view per condition.\u003c/p\u003e\u003cp\u003eImage processing and quantification of RAD51 foci per EdU-positive nucleus were conducted using homemade scripts in FIJI [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In short, EdU-positive cells were segmented by thresholding the 488 nm channel to define the region of interest. Within each region of interest, RAD51 foci were segmented using the 561 nm channel, with predefined minimum and maximum foci sizes and intensities. At least 25 cells were analyzed per condition per replicate, and experiments were performed as 3 independent biological replicates.\u003c/p\u003e\u003cp\u003e\u003cb\u003e53BP1 immunofluorescent staining and foci quantification\u003c/b\u003e\u003c/p\u003e\u003cp\u003eGOT1 and BON1-SSTR2 cells were plated in 12-well plates on glass coverslips. For EBRT, cells were incubated with vehicle, 10 or 25 nM ganetespib 2 h prior to irradiation (2 Gy). For PRRT, adherent cells were incubated for 4 h with \u003csup\u003e177\u003c/sup\u003eLu-DOTA-TATE (1 MBq/mL, 1.9x10\u003csup\u003e-8\u003c/sup\u003eM), after which the radioactive medium was replaced with medium containing ganetespib (10 or 25 nM) or vehicle. At 30 min, 6 and 24 h after radiation treatment, cells were fixed in 2% paraformaldehyde (PFA) for 15 min. For immunostaining, cells were permeabilized in PBS\u0026thinsp;+\u0026thinsp;0.1% Triton X-100. Samples were blocked in PBS\u0026thinsp;+\u0026thinsp;0.5% BSA\u0026thinsp;+\u0026thinsp;20 mM glycine and incubated with primary antibody for 53BP1 (Novus Biologicals, NB100-904, 1:1000) in blocking buffer for 1 h at RT. Cells were then washed with PBS\u0026thinsp;+\u0026thinsp;0.1% Triton X-100 and incubated in secondary Goat-anti-rabbit IgG Alexa Fluor 594 (Thermofisher, A-11012, 1:1000) in blocking buffer for 1 h at RT in the dark. Samples were mounted using Vectahield (Vector Laboratories) containing DAPI. Imaging of 53BP1 foci was done on a TCS SP5 confocal microscope (Leica) using a 63x oil immersion objective and 405 nm (DAPI) and 561 nm (53BP1) lasers.\u003c/p\u003e\u003cp\u003eImaging and analysis were done in a similar manner to the RAD51 foci quantification. For analysis, nuclei were segmented in the DAPI channel, after which in each nuclear mask foci were segmented in the 53BP1 channel by thresholding, using a defined minimum and maximum foci size and intensity. At least 40 cells were analyzed per condition per replicate and experiments were performed as 3 independent biological replicates.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eEnd-joining assay\u003c/h3\u003e\n\u003cp\u003eGOT1 or BON1-SSTR2 cells were plated in 6-well plates. As a positive control, 1 \u0026micro;M DNA-PKcs inhibitor AZD7648 (Medchemexpress) was added at the moment of seeding. After 24 h, treatment with 10 or 25 nM ganetespib was started. Assessment of the relative amount of classical non-homologous end joining (c-NHEJ) and microhomology-mediated end joining (MMEJ) used by cells was done as described before [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In short, 2 h after starting ganetespib treatment, a linear DNA substrate (pDVG94 plasmid) was transfected into cells. Extrachromosomal DNA was isolated 2 days after transfection. Plasmid DNA was PCR-amplified and digested by using the restriction enzyme BstXI, DNA fragments were separated and visualized using gel electrophoresis in a 6% polyacrylamide gel in Tris/borate/ethylenediaminetetraacetic acid (TBE) buffer using ethidium bromide. The experiment was performed as 2 independent biological replicates.\u003c/p\u003e\n\u003ch3\u003eRNA sequencing\u003c/h3\u003e\n\u003cp\u003eGOT1 cells were treated in suspension \u003csup\u003e177\u003c/sup\u003eLu-DOTA-TATE (1 MBq/mL, 1.9x10\u003csup\u003e-8\u003c/sup\u003eM) or vehicle for 4 h and seeded in 6-well plates in presence or absence of 10 nM ganetespib. Total RNA was isolated at 24 and 72 h post-PRRT using the RNeasy Mini Kit (QIAGEN) according to the manufacturer\u0026rsquo;s protocol and stored at -80\u0026deg;C before further processing. RNA concentration, purity and quality were assessed on a Nanodrop 2000 Spectrophotometer (Thermo Scientific) and Bioanalyzer 2100 (Agilent). Library preparation and RNA sequencing were performed at Genomescan B.V. (Leiden, The Netherlands). In short, libraries were prepared using the NEBNext Ultra II Directional RNA Library Prep Kit (New England Biolabs). Clustering and DNA sequencing on the NovaSeq6000 (Illumina) was performed at an average sequencing depth of 28\u0026nbsp;million reads and 150 base pair read length. Samples sent for RNA sequencing comprised 3 biological replicates per condition.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eRNA-seq processing, differential gene expression, and gene set enrichment analysis\u003c/h2\u003e\u003cp\u003eThe raw fastq files were pre-processed with fastp v0.23.2 [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Samples were then aligned using STAR v2.7.10a [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] against the human reference genome hs38 with ENSEMBL v107 annotation. Quality control was assessed using fastqc v0.11.9 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/s-andrews/FastQC/\u003c/span\u003e\u003cspan address=\"https://github.com/s-andrews/FastQC/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and RSeQC v5.0.1 [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. MultiQC v1.15.dev0 [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] was used to summarize the quality control of these two programs and the different steps of the data processing. Read counts overlapping the entire gene body were generated using htseq-count v2.0.2 [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe resulting gene-count matrices were analyzed using R (version 4.4). One sample (001, Control 24h) showed a diverging expression profile and it was excluded from subsequent analyses. Differential gene expression between groups and conditions was analyzed using DESeq2 v1.44.0 [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] by two different models, with an adjusted p-value threshold of 0.05 and |Fold Change| \u0026ge; 1.2. The first model used was \u0026ldquo;design\u0026thinsp;=\u0026thinsp;~\u0026thinsp;group\u0026rdquo;, where group represents the combination of treatment and time point. In addition, an interaction model (\u0026ldquo;design\u0026thinsp;=\u0026thinsp;~\u0026thinsp;radiation*inhibitor\u0026rdquo;) was used (only for samples at 24 h) to decouple the effects of PRRT and ganetespib monotherapies and evaluate the synergistic effects of both therapies in the combination treatment samples.\u003c/p\u003e\u003cp\u003eGene Set Enrichment Analysis (GSEA) was performed using the R package fGSEA v1.30.0 [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] and MSigDB (v2023.2) human GO: Biological Process and GO: Molecular Function were used as a reference [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Each comparison between groups and conditions was analyzed separately using the log2 Fold Change of all detected genes. Genes not present in the data were excluded from the background.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eStatistical analyses for all experiments, except for RNA sequencing, were performed in Graphpad Prism (version 9.4.0). Results are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). For viability and cell death assays, statistical significance between treatments was determined by an unpaired t-test. For foci analysis experiments (53BP1, RAD51), a one-way ANOVA followed by Tukey\u0026rsquo;s multiple comparisons test was performed. P-values were considered significant if p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eGanetespib activates the heat shock response and sensitizes NET cells to EBRT and PRRT\u003c/h2\u003e\u003cp\u003eTo sensitize NET cell lines GOT1 and BON1-SSTR2 to ionizing radiation, we treated the cells with small molecule inhibitor ganetespib, which competitively binds to the N-terminal ATP-binding pocket of HSP90 [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. To confirm effective HSP90 inhibition and assess its temporal dynamics, we characterized the cellular response to ganetespib according to HSP70 protein levels, which is an established biomarker for detection of HSP90 inhibition and the subsequent activation of the heat shock response (HSR) [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. In both GOT1 and BON1-SSTR2 cells, HSP70 levels were increased following ganetespib treatment within 6 h, and remained elevated for 72 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), suggesting successful inhibition of HSP90 for (at least) 72 h.\u003c/p\u003e\u003cp\u003eTo evaluate the radiosensitizing potential of ganetespib in our setup, we indirectly measured cell viability by evaluating metabolic activity seven days after starting the treatment. In GOT1 cells, PRRT and EBRT monotherapy resulted in a relative cell viability of 0.60 and 0.48 compared to control, respectively. In contrast, BON1-SSTR2 cells exhibited markedly lower sensitivity to both radiation treatments, with relative viabilities of 1.02 for PRRT and 0.87 for EBRT (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). In GOT1 cells, combination treatment with ganetespib and either PRRT or EBRT significantly reduced cell viability compared to ganetespib monotherapy (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). However, in BON1-SSTR2 cells, continuous ganetespib treatment was excessively toxic by itself (Supplementary Fig.\u0026nbsp;1A). Therefore, we modified the treatment protocol by washing out ganetespib after 24 h of incubation and replacing it with drug-free medium. This resulted in a small but significant reduction in cell viability after seven days of combination treatment compared to ganetespib monotherapy treated cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003eTo further validate whether the observed decrease in cell viability was attributable to cell death induction, we quantified cell death three days after treatment initiation by flow cytometry (Supplementary Fig.\u0026nbsp;1B). In GOT1 cells, a dose-dependent decrease in living cells was observed, which was significantly stronger in EBRT and PRRT treated cells compared to the ganetespib monotherapy (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). In contrast, in BON1-SSTR2 cells no such effect was observed. Additionally, consistent with the cell viability assay, these cells showed negligible sensitivity to ionizing irradiation itself (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eTreatment with ganetespib has limited impact on the persistence of radiation-induced DSBs\u003c/h2\u003e\u003cp\u003eThe radiosensitizing effect of HSP90 inhibition has previously been associated with inhibitory effects on the DDR, more specifically through inhibition of DSB repair [\u003cspan additionalcitationids=\"CR31 CR32\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. To determine whether the observed increase in cell death following combined ganetespib and EBRT or PRRT was associated with elevated DNA damage as a result of perturbed DNA repair, we quantified p53 binding protein 1 (53BP1) foci as marker of DSB levels [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Following EBRT, both GOT1 and BON1-SSTR2 cells exhibited a significant increase in 53BP1 foci at 0.5 h post-irradiation, which gradually declined to baseline level within 24 h. In contrast, PRRT induced a milder but still significant increase in 53BP1 foci at 0.5 h post-incubation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, D).\u003c/p\u003e\u003cp\u003eGanetespib monotherapy did not increase the number of 53BP1 foci in GOT1 cells, nor did it enhance DSB levels when combined with EBRT or PRRT at the analyzed time-points (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B). At the 24 h time-point, even a modest reduction in the number of 53BP1 foci was observed following ganetespib treatment in combination with EBRT and PRRT, though this difference was relatively small and therefore might lack biological relevance. In BON1-SSTR2 cells, a trend toward a dose-dependent increase in 53BP1 foci following ganetespib treatment was observed when combined with EBRT or PRRT; however, the differences were again relatively minor and not statistically significant across all conditions and time points (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, D). Together, these findings suggest that despite its radiosensitizing effect, ganetespib has a limited impact on overall DSB levels and persistence of DSBs after EBRT and PRRT under the tested conditions.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eHSP90 inhibition impairs homologous recombination through decreasing irradiation-induced RAD51 foci formation\u003c/h2\u003e\u003cp\u003eSince the effect of ganetespib on DSB persistence was not clearly visible, we further investigated the role of specific DSB repair pathways in the sensitization. Therefore, we analyzed the effect of ganetespib on homologous recombination (HR) and classical non-homologous end-joining (c-NHEJ), the two major DSB pathways.\u003c/p\u003e\u003cp\u003eTo determine the effect on HR, we analyzed the effect of ganetespib on RAD51 foci formation after irradiation, specifically in S-phase cells by selecting for EdU-positive nuclei (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA,C). RAD51 specifically accumulates on DSBs being repaired by HR. Since the previously used radiation conditions (2 Gy EBRT; 1 MBq/mL PRRT) did not induce a detectable number of RAD51 foci (data not shown), we focused on a higher dose of EBRT (5 Gy) as a general indicator for HR functioning. Irradiated GOT1 and BON1-SSTR2 cells showed a clear presence of RAD51 foci both at 2 h and 6 h post-irradiation in proliferating cells. Treatment with ganetespib resulted in a significant and dose-dependent decrease in the number of RAD51 foci, with the most pronounced effect observed at 6h (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, D), indicating that HR is (partially) blocked by ganetespib.\u003c/p\u003e\u003cp\u003eSubsequently, effects on DSB repair by c-NHEJ were assessed by an end-joining assay. This assay measures the balance between c-NHEJ and microhomology-mediated end joining (MMEJ) using an exogenous repair product. Positive control, DNA-PKcs inhibitor AZD7648 induced a profound shift from c-NHEJ to MMEJ products, indicative of a strong inhibitory effect on c-NHEJ. However, 2 h treatment with ganetespib pre-transfection did not induce a visible shift in repair products for both cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Increasing the duration of ganetespib treatment before transfection to 24 h as a control for treatment scheme-dependent effects, also did not induce a shift to MMEJ. These results, together with the 53BP1 foci quantification, indicate that ganetespib impairs HR in GOT1 and BON1-SSTR2 cells, but this has a limited effect on the overall repair of DSBs induced by EBRT and PRRT.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eTranscriptomic analysis shows activation of the heat shock response upon HSP90 inhibition\u003c/h2\u003e\u003cp\u003eGiven the significant effect on overall survival, but limited impact of HSP90 inhibition on DSB repair following both EBRT and PRRT, we hypothesized that additional cell mechanisms may contribute to the observed radiosensitizing effects. To gain deeper insight into the cellular response to HSP90 inhibition in the context of PRRT, we performed RNA sequencing in GOT1 cells, which showed the strongest degree in radiosensitization (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). The effect of ganetespib, PRRT and combination treatment was analyzed after 24 and 72 h of treatment by differential gene expression analysis.\u003c/p\u003e\u003cp\u003eTo evaluate the transcriptional effect of ganetespib monotherapy, differentially expressed genes (DEGs) were identified by comparing ganetespib treatment to control. Similarly, the effect of ganetespib in the context of PRRT was assessed by comparing combination treatment to PRRT monotherapy. After 24 h of treatment, there was an overlap of 80 (62%) upregulated and 21 (15%) downregulated DEGs being shared between the two comparisons (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). To focus on DEGs specific to ganetespib treatment in presence of PRRT, non-overlapping DEGs were further analyzed by an interaction model, that uses the effects of PRRT and ganetespib monotherapies as covariates. The interaction effect revealed no significant DEGs, suggesting that at 24 h, the expression changes observed in the combination treatment were the additive effects of PRRT and HSP90 inhibition, with no synergistic effects observed (Supplementary Fig.\u0026nbsp;2A).\u003c/p\u003e\u003cp\u003eComparing the effects of ganetespib in the context of PRRT at both 24 and 72 h time points revealed a similar response that became much more attenuated after 72 h, as indicated by the number of significant DEGs identified (237 DEGs at 24 h vs. 16 DEGs at 72 h) and the generally lower fold change observed at 72 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Due to the lack of a control sample at 72 h, interaction model analysis could not be performed at this time point. Instead, the effect of HSP90 inhibition in the context of PRRT (combination vs. PRRT) relative to both monotherapies (ganetespib vs. PRRT) was analyzed for overlapping DEGs. This comparison showed a high level of concordance, with 15 out of 16 (94%) genes being shared between conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). For all these comparisons the top significant DEGs were mainly upregulated canonical chaperone genes involved in the compensatory HSR upon HSP90 inhibition (e.g. \u003cem\u003eHSP90AA2P, HSP90AA3p, HSP90AA1\u003c/em\u003e), again validating the biological activity of ganetespib at the analyzed time points.\u003c/p\u003e\u003cp\u003eGSEA of DEGs between combination and PRRT monotherapy revealed exclusive enrichment of pathways related to HSP90 function, including response to heat and protein folding, which was the strongest at 24 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). At this time point, similar affected pathways were found after ganetespib mono-treatment (Supplementary Fig.\u0026nbsp;2B). Taken together, these results demonstrate that HSP90 inhibition induces a detectable transcriptomic response at 24 h which diminishes by 72 h, likely reflecting its predominant mode of action at the protein level. The transcriptomic profile of the combination treatment suggests an orthogonal and functionally distinct transcriptomic response. This likely results in an additive, rather than synergistic, effect of PRRT and HSP90 inhibition on cell survival.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eHSP90 inhibition has been established as an effective radiosensitization strategy in the context of EBRT across various cancer types [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. More recently, HSP90 inhibition has shown promising results in enhancing sensitivity of NETs to PRRT employing \u003csup\u003e177\u003c/sup\u003eLu-DOTA-TATE, although the underlying mechanisms remain incompletely understood [\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In this study, we aimed to elucidate the pathways contributing to the radiosensitizing effect of HSP90 inhibition to PRRT.\u003c/p\u003e\u003cp\u003eConsistent with previous studies, we showed that the HSP90 inhibitor ganetespib enhances the efficacy of both EBRT and PRRT in NET cell lines GOT1 and BON1-SSTR2. Notably, the degree of radiosensitization varied between the two models, with BON1-SSTR2 cells exhibiting pronounced toxicity to ganetespib monotherapy, likely reflecting a stronger dependency on HSP90 function. To mitigate this toxicity, we modified the treatment regimen by removing the inhibitor after 24 h, which indeed reduced monotherapy toxicity and resulted in a small but significant radiosensitizing effect. Further optimization of ganetespib treatment strategies, e.g. by adjusting exposure duration or using a lower concentration range of the inhibitor, may help expand the therapeutic window of HSP90 inhibition.\u003c/p\u003e\u003cp\u003eNext, we validated whether the observed decrease in cell viability was attributable to enhanced radiation-induced cell death. In GOT1 cells, a dose-dependent increase in cell death was detected, whereas no such effect was observed in BON1-SSTR2 cells. This aligns with the differences in radiosensitization between cell lines as observed in the viability assays, and may also reflect variations in the timing of cell death induction between the two cell lines. Indeed, BON1-SSTR2 cells proliferate rapidly (doubling time\u0026thinsp;~\u0026thinsp;34 h [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]), while GOT1 cells much more slowly (\u0026gt;\u0026thinsp;18 days [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]). As consequence, it is possible that by 72 h post-treatment, the surviving fraction BON1-SSTR2 cell population may have undergone sufficient proliferation to mask the effects of initial cell death, whereas in the slower-proliferating GOT1 cells, these effects remain detectable. Alternatively, the reduced viability in BON1-SSTR2 cells following combination treatment may primarily reflect decreased proliferation or metabolic activity rather than increased radiation-induced cell death. Finally, differences in radiosensitization could correlate with intrinsic radiosensitivity of cell lines, as both EBRT and PRRT monotherapy were less toxic for BON1-SSTR2 cells than for GOT1 cells.\u003c/p\u003e\u003cp\u003eSince DSBs are the primary determinant of radiation-induced effects, targeting DDR components, likely involved in radiosensitization mechanisms, is a widely studied approach to enhance radiation sensitivity [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The two primary DSB repair pathways are HR and c-NHEJ. HR is a high-fidelity repair mechanism that utilizes the homologous sister chromatid as a template for repair and is therefore restricted to cells in the S and G2 phases of the cell cycle. In contrast, c-NHEJ mediates repair by directly ligating the broken DNA ends without the need of a homologous template, making it a more error-prone but widely utilized mechanism throughout all cell cycle phases [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Both disruption of c-NHEJ and HR have demonstrated radiosensitizing potential [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. As HSP90 stabilizes proteins at various stages of DNA damage detection, repair and cell cycle check points, it is likely that HSP90 inhibition affects the DDR in a multi-target manner. Previous studies have shown that HSP90 inhibition leads to reduction of the overall cellular levels of key HR proteins such as BRCA2 and RAD51 [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. However, since these proteins are predominantly expressed during S and G2 phases of the cell cycle, their downregulation at the population level may largely reflect reduced proliferation and a consequent decrease in the proportion of HR-competent cells, rather than a direct impairment of HR itself. To address this, we specifically assessed the effect of ganetespib on HR in EdU-positive (S/G2 phase) nuclei and confirmed that HSP90 inhibition indirectly impairs HR as indicated by a reduced number of RAD51 foci. In contrast, limited data is available showing effects of HSP90 inhibition on c-NHEJ, or more indirectly by showing a decrease in levels of c-NHEJ mediators such as DNA-PKcs [\u003cspan additionalcitationids=\"CR41\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Our end-joining assay did not show a shift from c-NHEJ to MMEJ as an alternative pathway after treatment, suggesting that c-NHEJ is not affected by treatment with radiosensitizing doses of ganetespib.\u003c/p\u003e\u003cp\u003eDespite HR inhibition, ganetespib had little impact on overall DSB persistence following EBRT and PRRT as reflected by the minimal changes in 53BP1 foci numbers. One possible explanation is that the inhibitory effect was insufficient to substantially impair overall DSB repair. Alternatively, HR may contribute too little to DSB repair under the examined conditions to produce a detectable effect, which partly aligns with the established predominance of c-NHEJ as the primary DSB repair pathway in mammalian cells [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. This may account for the modest, dose-dependent increase in 53BP1 foci observed in BON1-SSTR2 cells, as their higher proliferation rate likely makes them more reliant on HR compared to the slower dividing GOT1 cells. While our analysis confirmed that ganetespib impairs HR following EBRT, it remained unclear whether similar mechanisms contribute to radiosensitization in the context of PRRT, which differs in radiation delivery and kinetics. To address this, we performed RNA sequencing in GOT1 cells, which we selected for their pronounced response to PRRT combined with HSP90 inhibition. To specifically isolate the impact of HSP90 inhibition on cellular pathways during PRRT, our analysis focused primarily on transcriptomic differences between PRRT monotherapy and the PRRT-HSP90 inhibitor combination.\u003c/p\u003e\u003cp\u003eIt is well established that HSP90 inhibition activates a compensatory HSR, which is transcriptionally regulated by heat shock factor 1 (HSF1). In response to HPS90 inhibition, HSF1 upregulates a set of HSR-related genes and induces molecular chaperones such as HSP70, to restore proteostasis [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Our transcriptomic analysis indeed confirmed a robust induction of this compensatory HSR at 24 h post-treatment. However, by 72 h, the transcriptional response was markedly reduced, with only 16 DEGs remaining between PRRT and combination treatment. Moreover, the overall transcriptomic response to the combination treatment was relatively modest, characterized by low-magnitude fold changes across most genes. This limited transcriptional signature is likely a reflection of HSP90\u0026rsquo;s primary mode of action at the post-translational level. Considering the temporal difference between transcriptomic and proteomic responses, comprehensive proteomic analysis following HSP90 inhibition may offer deeper insight into the dynamic regulation of affected pathways.\u003c/p\u003e\u003cp\u003eGSEA between PRRT and combination treatment solely showed enrichment of pathways associated with HSP90 function, such as protein folding and response to heat. As GSEA between control and HSP90 monotherapy showed enrichment of similar pathways, it is likely that the increased efficacy after combination treatment is an additive result of HSP90 inhibition and PRRT. These findings suggest that the radiosensitizing effect of HSP90 inhibition is not attributed to synergistic disruption of specific signaling pathways, but instead is caused by a pleiotropic effect resulting from loss of HSP90 function.\u003c/p\u003e\u003cp\u003eThe pleiotropic nature of HSP90 inhibition is supported by its broad list of client proteins and central role in managing cellular stress, particularly in tumor cells [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. For example, HSP90 is essential for the stability and function of proteins regulating cell cycle progression and checkpoint control, such as cyclin-dependent kinases CDK4 and CDK1 [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], and upstream Serine/threonine-protein kinase (ATR) [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Degradation of these proteins upon HSP90 inhibition results in cell cycle arrest at the G2/M phase, the stage at which cells are most sensitive to ionizing irradiation [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. In addition, HSP90 supports multiple radioresistance pathways. In the PI3K/Akt pathway, it maintains key proteins like Akt [\u003cspan additionalcitationids=\"CR48\" citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e], PDK1 and mTOR [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e], resulting in downregulation of this signaling axis upon inhibition of HSP90. Similarly, the MAPK/ERK pathway is disrupted upon HSP90 inhibition through depletion of Raf and ERK proteins [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Importantly, in certain cancer types, inhibition of the PI3K/Akt pathway has been shown to induce compensatory activation of the MAPK/ERK pathway, thereby sustaining cell survival despite inhibition of PI3K [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. This compensatory mechanism highlights the advantage of HSP90 inhibition as a multitarget approach, capable of synchronously impairing multiple survival and radioresistance pathways.\u003c/p\u003e"},{"header":"CONCLUSIONS","content":"\u003cp\u003eTaken together, our data suggests that the radiosensitizing effect of HSP90 inhibition is unlikely to be mediated by the suppression of a single signaling pathway. Instead, it likely results from the simultaneous, moderate disruption of several interconnected pathways \u0026ndash; including those regulating cell survival, proliferation, radioresistance, and cell cycle progression \u0026ndash; ultimately leading to a synergistic reduction in tumor cell viability when combined with PRRT.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e\u003cstrong\u003eAkt:\u003c/strong\u003e protein kinase B\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eATR\u003c/strong\u003e: Serine/threonine-protein kinase\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCDK\u003c/strong\u003e: cyclin-dependent kinase\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ec-NHEJ\u003c/strong\u003e: classical non-homologous end joining\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDEG:\u003c/strong\u003e differentially expressed gene\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDDR\u003c/strong\u003e: DNA damage response\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDSB\u003c/strong\u003e: double-stranded break\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEBRT\u003c/strong\u003e: external beam radiotherapy\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEDU:\u0026nbsp;\u003c/strong\u003e5-ethynyl-2\u0026rsquo;-deoxyuridine\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eERK\u003c/strong\u003e: extracellular signal-regulated kinase\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGSEA\u003c/strong\u003e: gene set enrichment analysis\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHR\u003c/strong\u003e: homologous recombination\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHSF1\u003c/strong\u003e:\u0026nbsp;heat shock factor 1\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHSP90\u003c/strong\u003e: heat shock protein 90\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHSR:\u003c/strong\u003e heat shock response\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMAPK\u003c/strong\u003e: microtubule associated protein kinase\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMMEJ\u003c/strong\u003e: microhomology-mediated end joining\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003emTOR\u003c/strong\u003e: mammalian target of rapamycin\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNETs:\u003c/strong\u003e neuroendocrine tumors\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePDK1\u003c/strong\u003e: 3-phosphoinositide-dependent protein kinase-1\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePI3K\u003c/strong\u003e: phosphatidylinositol 3-kinase\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePRRT\u003c/strong\u003e: peptide receptor radionuclide therapy\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSSTR2\u003c/strong\u003e: somatostatin receptor 2\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e53BP1\u003c/strong\u003e: p53 binding protein 1\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003csup\u003e177\u003c/sup\u003e\u003c/strong\u003e\u003cstrong\u003eLu-DOTA-TATE\u003c/strong\u003e: [\u003csup\u003e177\u003c/sup\u003eLu]Lu-[DOTA-Tyr\u003csup\u003e3\u003c/sup\u003e]octreotate\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData is available upon reasonable request to the corresponding author. RNA-sequencing dataset generated in the current study is available in the XXX repository.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Dutch Cancer Society (grant number 11840) and ERC grant (101042537 RADIOBIO).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor’s contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePE, TR, MS and JN contributed to conceptualization of the study. PE, TR, JH, JC and NS contributed to acquisition, visualization and analysis of the data. MS and JN supervised the project. All authors helped draft, review or edit the manuscript and approved the submission.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank Prof. Ola Nilsson (Sahlgrenska Cancer center, University of Gothenburg, Sweden) for kindly providing the GOT1 cell line, and Dr. Joris Pothof, Dr. Roland Kanaar and Danilo Remmers for their contributions to this study. All confocal imaging experiments were performed in collaboration with the Erasmus MC Optical Imaging Center core facility. Additionally, we would like to thank colleagues from the Department of Radiology \u0026amp; Nuclear Medicine for the preparation of \u003csup\u003e177\u003c/sup\u003eLu-DOTA-TATE. The graphical abstract figure was generated in BioRender.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eStrosberg JR, Caplin ME, Kunz PL, Ruszniewski PB, Bodei L, Hendifar A, et al. 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Biochem Soc Trans. 2012;40:139-46.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"ejnmmi-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ejre","sideBox":"Learn more about [EJNMMI Research](http://ejnmmires.springeropen.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/ejre/default.aspx","title":"EJNMMI Research","twitterHandle":"@officialEANM","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Peptide receptor radionuclide therapy, 177Lu-DOTA-TATE, HSP90, ganetespib, radiosensitization, neuroendocrine tumors","lastPublishedDoi":"10.21203/rs.3.rs-7271457/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7271457/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003ePeptide receptor radionuclide therapy (PRRT) employing [\u003csup\u003e177\u003c/sup\u003eLu]Lu-[DOTA-Tyr\u003csup\u003e3\u003c/sup\u003e]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.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eThe 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.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eGiven 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.\u003c/p\u003e","manuscriptTitle":"Radiosensitization of NET cells by HSP90 inhibitor ganetespib is mediated through pleiotropic stress responses","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-11 12:21:37","doi":"10.21203/rs.3.rs-7271457/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-09-08T00:25:57+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-04T20:09:13+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-04T08:53:31+00:00","index":"","fulltext":""},{"type":"submitted","content":"EJNMMI Research","date":"2025-09-04T02:09:54+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"ejnmmi-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ejre","sideBox":"Learn more about [EJNMMI Research](http://ejnmmires.springeropen.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/ejre/default.aspx","title":"EJNMMI Research","twitterHandle":"@officialEANM","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b78e93e8-db02-4b89-bb92-1403984d2f20","owner":[],"postedDate":"September 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-11-24T16:07:43+00:00","versionOfRecord":{"articleIdentity":"rs-7271457","link":"https://doi.org/10.1186/s13550-025-01346-z","journal":{"identity":"ejnmmi-research","isVorOnly":false,"title":"EJNMMI Research"},"publishedOn":"2025-11-21 15:58:57","publishedOnDateReadable":"November 21st, 2025"},"versionCreatedAt":"2025-09-11 12:21:37","video":"","vorDoi":"10.1186/s13550-025-01346-z","vorDoiUrl":"https://doi.org/10.1186/s13550-025-01346-z","workflowStages":[]},"version":"v1","identity":"rs-7271457","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7271457","identity":"rs-7271457","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}