Radiosensitivity Of Cells Research Articles

AM-18002, a derivative of natural anmindenol A, enhances radiosensitivity in mouse breast cancer cells.

Natural anmindenol A isolated from the marine-derived bacteria Streptomyces sp. caused potent inhibition of inducible nitric oxide synthase without any significant cytotoxicity. This compound consists of a structurally unique 3,10-dialkylbenzofulvene skeleton. We previously synthesized and screened the novel derivatives of anmindenol A and identified AM-18002, an anmindenol A derivative, as a promising anticancer agent. The combination of AM-18002 and ionizing radiation (IR) improved anticancer effects, which were exerted by promoting apoptosis and inhibiting the proliferation of FM3A mouse breast cancer cells. AM-18002 increased the production of reactive oxygen species (ROS) and was more effective in inducing DNA damage. AM-18002 treatment was found to inhibit the expansion of myeloid-derived suppressor cells (MDSC), cancer cell migration and invasion, and STAT3 phosphorylation. The AM-18002 and IR combination synergistically induced cancer cell death, and AM-18002 acted as a potent anticancer agent by increasing ROS generation and blocking MDSC-mediated STAT3 activation in breast cancer cells.

Radiosensitizing effect of a novel CTSS inhibitor by enhancing BRCA1 protein stability in triple-negative breast cancer cells.

Triple-negative breast cancer (TNBC) patients harboring wild-type breast cancer susceptibility gene 1 (BRCA1) account for most TNBC patients but lack adequate targeted therapeutic options. Although radiotherapy (RT) is the primary treatment modality for TNBC patients, radioresistance is one of the major challenges. RT-induced increase in cathepsin S (CTSS) causes radioresistance through suppressing BRCA1-mediated apoptosis of tumor cells, which was induced by CTSS-mediated degradation of BRCA1. Targeting CTSS may provide a novel therapeutic opportunity for TNBC patients. Publicly available data and human tissue microarray slides were analyzed to investigate the relationship between CTSS and BRCA1 in breast cancer patients. A CTSS enzyme assay and in silico docking analysis were conducted to identify a novel CTSS inhibitor. RO5461111 was used first to confirm the concept of targeting CTSS for radiosensitizing effects. The MDA-MB-231 TNBC cell line was used for invitro and invivo assays. Western blotting, promoter assay, cell death assay, clonogenic survival assay, and immunohistochemistry staining were conducted to evaluate novel CTSS inhibitors. CTSS inhibitors were further evaluated for their additional benefit of inhibiting cell migration. A novel CTSS inhibitor, TS-24, increased BRCA1 protein levels and showed radiosensitization in TNBC cells with wild-type BRCA1 and invivo in a TNBC xenograft mouse model. These effects were attributed by BRCA1-mediated apoptosis facilitated by TS-24. Furthermore, TS-24 demonstrated the additional effect of inhibiting cell migration. Our study suggests that employing CTSS inhibitors for the functional restoration of BRCA1 to enhance RT-induced apoptosis may provide a novel therapeutic opportunity for TNBC patients harboring wild-type BRCA1.

Purine salvage promotes treatment resistance in H3K27M-mutant diffuse midline glioma

BackgroundDiffuse midline gliomas (DMG), including diffuse intrinsic pontine gliomas (DIPGs), are a fatal form of brain cancer. These tumors often carry a driver mutation on histone H3 converting lysine 27 to methionine (H3K27M). DMG-H3K27M are characterized by altered metabolism and resistance to standard of care radiation (RT) but how the H3K27M mediates the metabolic response to radiation and consequent treatment resistance is uncertain.MethodsWe performed metabolomics on irradiated and untreated H3K27M isogenic DMG cell lines and observed an H3K27M-specific enrichment for purine synthesis pathways. We profiled the expression of purine synthesis enzymes in publicly available patient data and our models, quantified purine synthesis using stable isotope tracing, and characterized the in vitro and in vivo response to de novo and salvage purine synthesis inhibition in combination with RT.ResultsDMG-H3K27M cells activate purine metabolism in an H3K27M-specific fashion. In the absence of genotoxic treatment, H3K27M-expressing cells have higher relative activity of de novo synthesis and apparent lower activity of purine salvage demonstrated via stable isotope tracing of key metabolites in purine synthesis and by lower expression of hypoxanthine-guanine phosphoribosyltransferase (HGPRT), the rate-limiting enzyme of purine salvage into IMP and GMP. Inhibition of de novo guanylate synthesis radiosensitized DMG-H3K27M cells in vitro and in vivo. Irradiated H3K27M cells upregulated HGPRT expression and hypoxanthine-derived guanylate salvage but maintained high levels of guanine-derived salvage. Exogenous guanine supplementation decreased radiosensitization in cells treated with combination RT and de novo purine synthesis inhibition. Silencing HGPRT combined with RT markedly suppressed DMG-H3K27M tumor growth in vivo.ConclusionsOur results indicate that DMG-H3K27M cells rely on highly active purine synthesis, both from the de novo and salvage synthesis pathways. However, highly active salvage of free purine bases into mature guanylates can bypass inhibition of the de novo synthetic pathway. We conclude that inhibiting purine salvage may be a promising strategy to overcome treatment resistance in DMG-H3K27M tumors.

CHD4 acts as a prognostic factor and drives radioresistance in HPV negative HNSCC

Despite great efforts in improving existing therapies, the outcome of patients with advanced radioresistant HPV-negative head and neck squamous cell carcinoma (HNSCC) remains poor. The chromatin remodeler Chromodomain helicase DNA binding protein 4 (CHD4) is involved in different DNA-repair mechanisms, but the role and potential in HNSCC has not been explored yet. In the present study, we evaluated the prognostic significance of CHD4 expression using in silico analysis of the pan-cancer dataset. Furthermore, we established a monoclonal HNSCC CHD4 knockdown cell clone utilizing the CRISPR/Cas9 system. Effects of lower CHD4 expression on radiosensitivity after increasing doses of ionizing radiation were characterized using clonogenic assays and cell numbers. The in silico analysis revealed that high CHD4 expression is associated with significant poorer overall survival of HPV-negative HNSCC patients. Additionally, the knockdown of CHD4 significantly increased the radiosensitivity of HNSCC cells. Therefore, CHD4 might be involved in promoting radioresistance in hard-to-treat HPV-negative HNSCC entities. We conclude that CHD4 could serve as a prognostic factor in HPV-negative HNSCC tumors and is a potential target protein overcoming radioresistance in HNSCC. Our results and the newly established cell clone laid the foundation to further characterize the underlying mechanisms and ultimately use CHD4 in HNSCC therapies.

Abstract LB015: Differential expression of deubiquitinating enzymes and its related DNA damage response genes in neuroblastoma heterogeneity

Abstract SK-N-AS and SK-N-DZ are two cell lines of pediatric solid tumors neuroblastomas well characterized to evaluate heterogeneity between different neuroblastomas and are presumed as models for differences between different neuroblastoma. Using this model system, we recently evaluated if radiation could improve efficacy of radiation therapy and to follow these studies later with animal models to determine decrease of tumor burden. Differences between the N-type and S-type neuroblastoma cell lines SK-N-DZ and SK-N-AS, respectively were observed to be high and their responses to radiation stress were equally heterogeneous. Ubiquitin signaling plays a key role during the DNA damage response in protein recruitment and protein interactions. Deubiquitinating enzymes (DUBs) deconjugate ubiquitin from ubiquitylated substrate proteins to control their activity and stability and are shown to regulate various biological processes. DUBs may play a role in regulating the radiosensitivity of cancer cells by modulating DNA damage response and repair pathways. Determination of specific DUBs that are involved in regulating radiosensitivity in cancer cells will provide opportunity for drug targeting and visa-vis enhance the efficacy of radiation therapy in neuroblastoma cancer treatment. Here, using bioinformatic tools, the differentially regulated DUBs were identified. The relationship between DEGs-DUBs with the genes involved in various DNA repair pathways (NHEJ, HR and Fanconi anemia pathway; KEGG pathway database) are described. We identified 32 DEGs (14 upregulated and 18 downregulated) with these neuroblastoma cell lines. USP22 and USP41 DUBs and its differential expression in these cell lines denotes the importance of such analysis. Especially considering USP22 a potentially biomarker for predicting the outcome of patients with neuroblastoma. These DEG-DUBs were likewise correlated with 91 DNA damage response (DDR) genes belonging to three different pathways (NHEJ, HR and Fanconi anemia pathway). Out of which 44 of the DDR genes showed significant correlation with DEG-DUBs. Further studies are needed to determine the mechanisms underlying the complex relationship of these DUBs and to determine the critical DUBs for drug targeting and to enhance the efficacy of radiation therapy in cancer treatment. Citation Format: Vaibhav Vaibhav, Fahika Nazeerulla, Tatjana Paunesku, Ekta Tripathi, Gayle Woloschak, Prashanthi Karyala, Rao V. Papineni. Differential expression of deubiquitinating enzymes and its related DNA damage response genes in neuroblastoma heterogeneity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB015.

Evaluation of core-shell Fe3O4@Au nanoparticles as radioenhancer in A549 cell lung cancer model

In radiotherapy, metallic nanoparticles are of high interest in the fight against cancer for their radiosensitizing effects. This study aimed to evaluate the ability of core-shell Fe3O4@Au nanoparticles to potentiate the irradiation effects on redox-, pro-inflammatory markers, and cell death of A549 human pulmonary cancer cells. The hybrid Fe3O4@Au nanoparticles were synthesized using green chemistry principles by the sonochemistry method. Their characterization by transmission electron microscopy demonstrated an average size of 8 nm and a homogeneous distribution of gold. The decreased hydrodynamic size of these hybrid nanoparticles compared to magnetite (Fe3O4) nanoparticles showed that gold coating significantly reduced the aggregation of Fe3O4 particles. The internalization and accumulation of the Fe3O4@Au nanoparticles within the cells were demonstrated by Prussian Blue staining. The reactive oxygen species (ROS) levels measured by the fluorescent probe DCFH-DA were up-regulated, as well as mRNA expression of SOD, catalase, GPx antioxidant enzymes, redox-dependent transcription factor Nrf2, and ROS-producing enzymes (Nox2 and Nox4), quantified by RT-qPCR. Furthermore, irradiation coupled with Fe3O4@Au nanoparticles increased the expression of canonical pro-inflammatory cytokines and chemokines (TNF-α, IL-1β, IL-6, CXCL8, and CCL5) assessed by RT-qPCR and ELISA. Hybrid nanoparticles did not potentiate the increased DNA damage detected by immunofluorescence following the irradiation. Nevertheless, Fe3O4@Au caused cellular damage, leading to apoptosis through activation of caspase 3/7, secondary necrosis quantified by LDH release, and cell growth arrest evaluated by clonogenic-like assay. This study demonstrated the potential of Fe3O4@Au nanoparticles to potentiate the radiosensitivity of cancerous cells.

Circ-POSTN promotes the progression and reduces radiosensitivity in esophageal cancer by regulating the miR-876-5p/FYN axis.

Circular RNAs (circRNAs) play critical roles in the tumorigenesis and radiosensitivity of multiple cancers. Nevertheless, the biological functions of circRNA periostin (circ-POSTN) in esophageal cancer (EC) progression and radiosensitivity have not been well elucidated. The expression of circ-POSTN, microRNA-876-5p (miR-876-5p), and proto-oncogene tyrosine-protein kinase (FYN) was analyzed by quantitative reverse transcription PCR (RT-qPCR). Cell proliferation was assessed by MTT, colony formation, and 5-ethynyl-2'-deoxyuridine (EDU) assays. All protein levels were detected by western blot assay. Cell apoptosis and invasion were assessed by flow cytometry analysis and transwell assay, respectively. Dual-luciferase reporter and RNA immunoprecipitation (RIP) assays were used to validate the interaction between miR-876-5p and circ-POSTN or FYN. The role of circ-POSTN in vivo was explored by establishing mice xenograft model. Circ-POSTN was overexpressed in EC tissues and cells. Knockdown of circ-POSTN inhibited cell proliferation and invasion and elevated apoptosis and radiosensitivity in EC cells. MiR-876-5p was a direct target of circ-POSTN, and its knockdown reversed the role of sh-circ-POSTN in EC cells. FYN was a direct target of miR-876-5p, and FYN elevation weakened the effects of miR-876-5p overexpression on the progression and radiosensitivity of EC cells. Moreover, circ-POSTN acted as a miR-876-5p sponge to regulate FYN expression. Circ-POSTN interference also suppressed tumor growth and enhanced radiosensitivity in vivo. Circ-POSTN knockdown inhibited proliferation and invasion, but increased apoptosis and enhanced radiosensitivity in EC cells via modulating miR-876-5p/FYN axis, which might be a potential diagnostic and therapeutic target for EC.

Abstract 1126: Extracellular vesicle mediated radioresistance in H3K27M-diffuse midline glioma

Abstract H3K27M-Diffuse Midline Gliomas (DMGs) are a subset of uncurable malignant pediatric gliomas and the deadliest form of brain tumors in children worldwide. Radiation therapy, the current standard of care, is initially effective but all tumors become resistant within 4-6 months. H3K27M-DMGs are heterogenous, comprised of an average of 6 genetically distinct subclones. Some subclones harbor mutations in genes previously linked to radiation resistance, such as TP53. We have found subclones are also functionally distinct, with some clones harboring intrinsic radiation resistance. Extracellular signaling between clones is linked with therapy resistance and tumor progression in other tumors, and we hypothesized that radioresistant H3K27M-DMG release small extracellular vesicles (sEVs) that impact the radiosensitivity of neighboring tumor cells. We found that all patient-derived H3K27M-DMG samples examined (n=6) produced sEVs (< 220nm), indicating this is a common feature in this tumor type. We isolated sEVs from a radioresistant (RR) patient-derived H3K27M-DMG cell line and used them to treat radiosensitive (RS) DMG cells. Using flow cytometry to profile sEV uptake dynamics, we found that RR-sEVs are internalized by RS cells within 2 hours of exposure and are retained at 18 hours (p<0.001, compared to control). This uptake can be significantly abrogated by proteinase K treatment of the sEVs, (p<0.001), indicating that uptake involves sEV surface proteins. We then applied a biosensor approach to assess single-cell phenotypic changes in RS DMG after RR-sEV treatment on radiation-induced cell death, DNA damage, and cell senescence, three dynamic processes. Using a genetically encoded death indicator (GEDI), we found that RR-sEVs significantly enhanced the survival of RS cells after acute radiation exposure (p<0.001). This was due to enhanced DNA damage repair as measured by 53BP1 foci (p<0.01). Small RNA sequencing of sEV cargo demonstrated that RR-sEVs express multiple miRNAs that are involved in oncogenic signaling such as the miR-7 family, miR-1246, and miR-1290 at several log fold higher than RS-sEVs. Overall, this data reveals the first functional role for sEVs in the context of radiation induced tumor transformation in DMG. We plan to further investigate the primary route of sEV uptake and define the mechanism through which sEV cargo alters radiosensitivity. This study provides new insight into the impact of heterogeneity and sEVs in mediating radioresistance in H3K27M-DMG and may lead to the development of novel radiosensitizers, which are critically needed for all patients. Citation Format: Viral Oza, Shilpa Sampathi, Yelena Chernyavskava, Kenan Flores, Jessica Blackburn. Extracellular vesicle mediated radioresistance in H3K27M-diffuse midline glioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1126.

Abstract 688: Radium-223 in combination with darolutamide exhibits synergistic antitumor efficacy in LNCaP prostate cancer models

Abstract Radium-223 dichloride (radium-223) is a targeted alpha therapy that binds to newly formed abnormal bone in bone metastases and induces DNA double-strand breaks (DSBs) in cancer cells, osteoblasts and osteoclasts. Radium-223 is used for the treatment of patients with metastatic castration-resistant prostate cancer (mCRPC). Darolutamide is an androgen receptor inhibitor indicated for patients with metastatic hormone-sensitive prostate cancer or non-metastatic CRPC. We studied the antitumor effects of radium-223 in combination with darolutamide using LNCaP human prostate cancer cells in vitro and an intratibial LNCaP model mimicking prostate cancer metastasized to bone. The in vitro antiproliferative effects of radium-223 and darolutamide were determined using LNCaP cells. Gene set enrichment analysis (GSEA) was conducted on RNA sequencing data from LNCaP cells treated with the synthetic androgen R1881 alone or in combination with darolutamide. LNCaP cells were inoculated into the right tibia of male NOD.scid mice. The mice (n=7-9/group) were randomized based on serum prostate-specific antigen (PSA) and treated with vehicle, radium-223 (330 kBq/kg, Q4Wx2, i.v.), darolutamide (100 mg/kg, BID, p.o.), or with a combination of radium-223 and darolutamide, for 41 days. PSA and the bone turnover markers PINP and CTX-I were measured every second week in serum. Tumor-induced abnormal bone area, bone formation rate and radium-223 uptake in tumor-bearing tibiae were determined by X-ray, histomorphometry and gamma counter, respectively. The combination of radium-223 and darolutamide showed synergistic antiproliferative effects with combination indexes between 0.69-0.75 in vitro. GSEA demonstrated a prominent darolutamide-induced downregulation of DNA damage response (DDR) signaling pathways. In vivo, the combination treatment showed synergistic antitumor efficacy (p=0.04) as demonstrated by lower PSA concentrations when compared with the vehicle, radium-223 or darolutamide monotherapies (p=0.004, p=0.011 and p=0.002, respectively). In the vehicle and combination treatment groups, the mean serum PSA was 541% and 95.9% of the pre-treatment level, respectively. Furthermore, radium-223 alone or in combination with darolutamide inhibited increased bone turnover, tumor-induced abnormal bone growth and trabecular bone formation when compared to vehicle. Concurrent administration of darolutamide did not affect radium-223 uptake in tibiae. Radium-223 in combination with darolutamide exhibited synergistic antitumor efficacy both in vitro and in vivo. The synergistic effects could be due to the radiosensitization of cancer cells by darolutamide-induced downregulation of DDR pathways. Our results suggest that the combination of targeted alpha therapy with an androgen receptor inhibitor is a promising treatment strategy for mCRPC. Citation Format: Christoph A. Schatz, Mari I. Suominen, Andreas Schlicker, Matias Knuuttila, Esa Alhoniemi, Sanna-Maria Käkönen, Bernard Haendler, Urs B. Hagemann, Arne Scholz. Radium-223 in combination with darolutamide exhibits synergistic antitumor efficacy in LNCaP prostate cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 688.

Abstract 567: Fisetin sensitizes triple negative breast cancer cells to radiation

Abstract Triple negative breast cancer (TNBC) represents a highly aggressive subtype, constituting approximately 20% of all breast cancer cases. Y-box binding protein-1 (YB-1) is often overexpressed in various tumor types and has been implicated in conferring resistance to cell death induced by radiotherapy. Fisetin, a flavonoid compound with demonstrated anti-cancer properties, operates in part through inhibiting phosphorylation of YB-1 by p90 ribosomal S6 kinase (RSK). In this study, we investigated the potential synergistic effects of combining fisetin with radiotherapy in TNBC. The activation status of the RSK signaling pathway was assessed in both total cell lysate and subcellular fractions using Western blotting. A standard clonogenic assay was employed to evaluate post-irradiation cell survival. To investigate the frequency of double-strand breaks (DSBs) and chromosomal aberrations, γH2AX foci assay and 3-color fluorescence in situ hybridization analyses were conducted, respectively. The DSB repair pathways targeted by fisetin were examined in cells expressing genomically integrated reporter constructs by quantifying green fluorescence protein expression through flow cytometry. Apoptosis and autophagy were measured using flow cytometric quantification of sub-G1 cells and the assessment of LC3-II protein expression, respectively. Kinase array and phosphoproteomics analyses were carried out to study the potential impact of fisetin on the signaling involved in the DNA damage response (DDR) after irradiation. We demonstrated that fisetin mimics the effects of RSK pharmacological inhibitors in terms of effect on YB-1 phosphorylation. Treatment with fisetin increased frequency of DSB in non-irradiated cells and impaired repair of DSB after irradiation. Fisetin attenuated DSB repair by suppressing the classical non-homologous end-joining and homologous recombination repair pathways. The frequency of chromosomal aberration was enhanced by fisetin treatment. The effect of fisetin on inhibiting DSB repair was partially YB-1 dependent. Phosphoproteomic analysis revealed that fisetin inhibits DDR signaling, which leads to radiosensitization in TNBC cells, as shown in combination with single dose or fractionated doses irradiation. Fisetin neither inhibited DSB repair nor radiosensitized normal human fibroblast HSF7 cells. The radiosensitizing effect of fisetin was not attributed to enhancing apoptosis or modulating autophagy. In summary, combining fisetin with radiotherapy may enhance TNBC radiotherapy outcomes. -This research was supported by a grant from the German Research Council (DFG,TO 685/2-3). Citation Format: Shayan Khozooei, Soundaram Veerappan, Mahmoud Toulany. Fisetin sensitizes triple negative breast cancer cells to radiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 567.

Abstract 694: Evaluation of a synergistic drug combination with 177Lu-rhPSMA-10.1 for prostate cancer: Results of an in vitro screen and in vivo proof of concept study

Abstract Purpose: Prostate-specific membrane antigen (PSMA)-targeted radioligand therapy (RLT) has been shown to extend survival in men with advanced prostate cancer (PCa). Novel radiohybrid (rh) PSMA-targeted 177Lu-rhPSMA-10.1 has shown promising preclinical efficacy and advantageous radiation dosimetry in humans. We conducted an in vitro screen to identify known anticancer drugs with potential for synergistic interaction with 177Lu-rhPSMA-10.1. Here we present key screening data and a subsequent in vivo efficacy analysis of the lead novel drug combination in PSMA-expressing 22Rv1 PCa xenografts. Methods: Over 150 FDA-approved anticancer drugs were screened in a clonogenic survival assay of 22Rv1 cells using the test drug alone, at a range of concentrations <20 µM to determine the IC50, and results compared to incubations of the drug + 15 MBq/mL 177Lu-rhPSMA-10.1 after 10 d. A focused screen of 5 lead candidates was then conducted to determine the impact of 177Lu-rhPSMA-10.1 (0-25 MBq/mL) on the drug IC50. A synergy score was determined using the zero interaction potency (ZIP) reference model and the multi-dimensional synergy of combinations (MuSyc) platform. Subsequently, to evaluate the efficacy of the lead combination, 177Lu-rhPSMA-10.1 (single 30 MBq iv dose) and Cobimetinib (0.25 mg orally per day for 21 d) alone and in combination were administered to 22Rv1 tumor-bearing NMRI nude mice (n = 8 per group plus untreated controls). Tumor volume was measured 2x week for 69 d. Two-way ANOVA and Tukey’s multiple comparisons test (data analyzed until n = 3 remained per group) and Kaplan-Meier Log-rank survival analyses were performed. Results: The in vitro screen identified MEK inhibitor Cobimetinib as a lead candidate for synergistic combination with 177Lu-rhPSMA-10.1 across a wide concentration range, with a ZIP synergy score of 13.25% (95% CI ± 2.17) and promising results on MuSyc analysis. The 177Lu-rhPSMA-10.1 + Cobimetinib combination significantly suppressed tumor growth in vivo vs untreated controls (from day 13-30; p<0.01) and 177Lu-rhPSMA-10.1 alone (from day 17-30; p<0.001). The median survival in the combination group (49 d) was significantly longer vs the untreated group (23 d; p=0.001) and the group treated with 177Lu-rhPSMA-10.1 alone (36 d; p=0.002). Conclusions: Through an extensive in vitro screen, we identified Cobimetinib to have potential for a synergistic anti-tumor effect in combination with 177Lu-rhPSMA-10.1. This may be due to inhibition of the MEK-MAPK pathway by Cobimetinib during DNA damage response, resulting in radiosensitization of cancer cells to 177Lu-labeled RLT agents such as 177Lu-rhPSMA-10.1. This novel combination showed an enhanced therapeutic efficacy vs the single agents in 22Rv1 xenografts and the lack of overlapping monotherapy toxicity reported in the clinic supports clinical investigation in men with PCa. Citation Format: Caroline Foxton, Bart Cornelissen, Edward O'Neill, Bradley Waldron, Freja Pretzmann, Rikke Veggerby Grønlund, Mathias Wikke Hallund, Daniel J. Stevens. Evaluation of a synergistic drug combination with 177Lu-rhPSMA-10.1 for prostate cancer: Results of an in vitro screen and in vivo proof of concept study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 694.

Abstract 705: miR-21-5p induces radioresistance through reinforced DNA damage response in oral squamous cell carcinoma

Abstract Cancers arising from oral cavity and oropharynx are commonly treated with radiotherapy, whereas tumor radioresistance results in poor outcome. The prerequisite for developing more effective interventions for these patients is that the molecular mechanisms leading to a radioresistant phenotype in these tumors are clear to researchers. Unfortunately, the radioresistant mechanisms remain elusive to date. We recently identified that a highly conserved and cancer-related microRNA, i.e. miR-21-5p, plays an important role in radioresistance of oral squamous cell carcinoma (OSCC). We analyzed the RNA-sequencing data of 290 tissue samples from TCGA database, and found the expression of miR-21-5p was significantly upregulated in many OSCC samples (N=271) when comparing to adjacent normal tissues (N=19). The difference was further confirmed on our freshly collected OSCC tissues from patients in operation room. We performed RNA-sequencing analysis in 11 OSCC samples and their adjacent normal tissues. Among the 2106 differentially expressed genes between OSCC and normal control groups, we found that several important DNA repair genes (RAD50 double strand break repair protein gene, RAD50; DNA Polymerase Theta, POLQ; Ataxia Telangiectasia and Rad3-related, ATR) were significantly overexpressed in OSCC samples (P<0.05), while positive correlations between miR-21-5p expression and DNA repair genes RAD51 and XRCC4 were identified in the OSCC cohort. Notably, ATR is an important DNA damage response gene, while XRCC4, RAD51, and POLQ are involved in DNA double strand break (DSB) repair through non-homologous end joining, homologous recombination, and microhomology-mediated end joining, respectively. OSCC cell lines were further recruited to study the relations of miR-21-5p with radiation-induced DSB repair. By analyzing kinetics of 53BP1 foci, a biomarker for detecting DSBs, we found that anti-miR-21-5p transfected cells showed more residual foci, indicating of unfinished repair of DSBs, at 5 hours and 18 hours post irradiation. The deficient DSB repair resulted from anti-miR-21-5p transfection were further confirmed by single cell gel electrophoresis with comet assay. Consistently, the same treatment resulted in fewer clonogenic survivors in colony formation assay, demonstrating increased radiosensitivity of OSCC cells when miR-21-5p was antagonized. This work provided novel insights about the mechanism of miR-21-5p-induced radioresistance in OSCC, which may give rise to novel therapeutic strategy for improved treatment of OSCC patients. Citation Format: Qi Liu, Weitao Hu, Xinghan Li, Yongqiang Deng, Lin Ma. miR-21-5p induces radioresistance through reinforced DNA damage response in oral squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 705.

Abstract 1113: USP18 impairs the sensitivity of radiotherapy for nasopharyngeal carcinoma by promoting the ubiquitination and nuclear translocation of TRIM29

Abstract Radiotherapy is the main and preferred treatment for nasopharyngeal carcinoma (NPC), owing to its high sensitivity to radiation. However, there are ~20% patients suffering from tumor recurrence. Accumulating evidences show that protein ubiquitination plays a vital role in radiation caused DNA damage response. Here, we identified that the deubiquitinase USP18 is highly expressed in NPC tissues, and inversely associated with radiosensitivity of NPC cells. USP18 interacts with TRIM29 and promotes its K27-linked ubiquitination independent of its deubiquitinase activity. Further investigation reveals that USP18 acts as a scaffold to recruit the E3 ubiquitin ligase TRIM21 to directly ubiquitinate TRIM29 at Lys307, thus promoting its oligomerization and nuclear translocation, and thereby facilitates the DNA damage repair of NPC cells after irradiation. Clinically, higher expression of USP18 indicates poor response to radiotherapy and worse prognosis in NPC patients. Our findings reveal the critical role of USP18 in regulating radiosensitivity, and targeting USP18-TRIM21-TRIM29 axis maybe a novel strategy to synergize with radiotherapy for NPC patients. Citation Format: Jia-Yi Lin, Wei-Wei Zhang, Qing-Jie Li, Xue-Liang Fang, Jun-Yan Li, Shi-Wei He, Ying-Qin Li, Jun Ma, Na Liu, Yin Zhao, Rui Guo. USP18 impairs the sensitivity of radiotherapy for nasopharyngeal carcinoma by promoting the ubiquitination and nuclear translocation of TRIM29 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1113.

Abstract 704: Targeting hypoxic survival pathways to overcome radiotherapy-related treatment resistance

Abstract Tumor cells undergo biological adaptations in order to survive stress conditions, such as hypoxia and nutrient deprivation. These biological adaptations include the activation of hypoxic survival pathways that play an important role in acquiring radioresistance which becomes most challenging with treatment regimens using high doses of radiation/fraction (e.g., hypofractionated radiotherapy). Therefore, inhibiting key regulators within these pathways and thereby exploiting these survival mechanisms, gives rise to new potential drug targets for combined treatment modalities. This novel strategy investigated in our laboratory relies on the concept of biological cooperation: while ionizing radiation kills primarily well-oxygenated cells, such inhibitors of survival kinases will drive quiescent tumor cells under hypoxia and/or nutrient deprivation either directly into cell death or into re-entry into a proliferative and thereby more ionizing radiation-sensitive state. One of the survival kinases we are investigating in combination with ionizing radiation (IR) is DYRK1B (dual-specificity tyrosine regulated kinase 1B). We demonstrated that the expression of DYRK1B was upregulated under serum-starvation and hypoxia, but not in response to IR. The small molecule DYRK1B inhibitor AZ191 and shRNA-mediated DYRK1B knockdown significantly reduced proliferative activity and clonogenicity of SW620 tumor cells alone and in combination with IR under serum-starved conditions, which correlated with increased ROS levels and DNA damage. Furthermore, AZ191 successfully targeted the hypoxic core of tumor spheroids while IR preferentially targeted normoxic cells in the rim of the spheroids. A combined treatment effect was also observed in patient-derived colorectal carcinoma organoids but not in healthy tissue organoids. This data shows that inhibition of DYRK1B in quiescent tumor cells could drive tumor cells on their own into crisis or into a more radiosensitive cell cycle phase and thus supports our strategy of biological cooperation. In the continuation of this project, we have performed a drug-screen with approx. 3’000 clinically relevant compounds in colorectal tumor cells lines under normoxic and hypoxic conditions in order to explore additional hypoxia-mediated survival pathways contributing to radioresistance. Thereby we aim to identify novel combined treatment modalities to overcome radiotherapy-induced hypoxia and hypoxia-related resistance mechanisms. Citation Format: Claire Beckers, Lazaros Vasilikos, Alba Sanchez-Fernandez, Lorena Moor, Martin Pruschy. Targeting hypoxic survival pathways to overcome radiotherapy-related treatment resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 704.

Clobetasol propionate, a Nrf-2 inhibitor, sensitizes human lung cancer cells to radiation-induced killing via mitochondrial ROS-dependent ferroptosis.

Combining radiotherapy with Nrf-2 inhibitor holds promise as a potential therapeutic strategy for radioresistant lung cancer. Here, the radiosensitizing efficacy of a synthetic glucocorticoid clobetasol propionate (CP) in A549 human lung cancer cellswas evaluated. CP exhibited potent radiosensitization in lung cancer cells via inhibition of Nrf-2 pathway, leading to elevation of oxidative stress. Transcriptomic studies revealed significant modulation of pathways related to ferroptosis, fatty acid and glutathione metabolism. Consistent with these findings, CP treatment followed by radiation exposure showed characteristic features of ferroptosis in terms of mitochondrial swelling, rupture and loss of cristae. Ferroptosis is a form of regulated cell death triggered by iron-dependent ROS accumulation and lipid peroxidation. In combination with radiation, CP showed enhanced iron release, mitochondrial ROS, and lipid peroxidation, indicating ferroptosis induction. Further, iron chelation, inhibition of lipid peroxidation or scavenging mitochondrial ROS prevented CP-mediated radiosensitization. Nrf-2 negatively regulates ferroptosis through upregulation of antioxidant defense and iron homeostasis. Interestingly, Nrf-2 overexpressing A549 cells were refractory to CP-mediated ferroptosis induction and radiosensitization. Thus, this study identifiedanti-psoriatic drug clobetasol propionate can be repurposed as a promising radiosensitizer for Keap-1 mutant lung cancers.

Enhancing radiosensitivity in triple-negative breast cancer through targeting ELOB.

Enhancing radiotherapy sensitivity is crucial for improving treatment outcomes in triple-negative breast cancer (TNBC) patients. In this study, we investigated the potential of targeting Elongin B (ELOB) to enhance radiotherapy efficacy in TNBC. Analysis of TNBC patient cohorts revealed a significant association between high ELOB expression and poor prognosis in patients who received radiation therapy. Mechanistically, we found that ELOB plays a pivotal role in regulating mitochondrial function via modulating mitochondrial DNA expression and activities of respiratory chain complexes. Targeting ELOB effectively modulated mitochondrial function, leading to enhanced radiosensitivity in TNBC cells. Our findings highlight the importance of ELOB as a potential therapeutic target for improving radiotherapy outcomes in TNBC. Further exploration of ELOB's role in enhancing radiotherapy efficacy may provide valuable insights for developing novel treatment strategies for TNBC patients.

Nicaraven attenuates the acquired radioresistance of established tumors in mouse models via PARP inhibition.

Nicaraven has been reported to inhibit the activity of poly (ADP-ribose) polymerase (PARP). In this study, we investigated the probable ability of nicaraven to attenuate cancer radioresistance during fractionated radiotherapy. Tumor models were established in C57BL/6 mice and BALB/c nude mice by subcutaneous injection of Lewis mouse lung carcinoma cancer cells and A549 human lung cancer cells, respectively. When the tumors had grown to approximately 100 mm3, we initiated fractionated radiotherapy. Nicaraven or saline was administered immediately after each irradiation exposure. Compared to saline treatment, nicaraven administration significantly induced gamma-H2AX foci formation and cell apoptosis in tumors at 1 or 3days after an additional challenge exposure to 10Gy and inhibited tumor growth during the short-term follow-up period, suggesting increased radiosensitivity of cancer cells. Moreover, the expression of PARP in tumor tissue was decreased by nicaraven administration. Our data suggest that nicaraven likely attenuates the acquired radioresistance of cancers through PARP inhibition.

Theoretical evaluation of the impact of diverse treatment conditions by calculation of the tumor control probability (TCP) of simulated cervical cancer Hyperthermia-Radiotherapy (HT-RT) treatments in-silico

Introduction Hyperthermia (HT) induces various cellular biological processes, such as repair impairment and direct HT cell killing. In this context, in-silico biophysical models that translate deviations in the treatment conditions into clinical outcome variations may be used to study the extent of such processes and their influence on combined hyperthermia plus radiotherapy (HT + RT) treatments under varying conditions. Methods An extended linear-quadratic model calibrated for SiHa and HeLa cell lines (cervical cancer) was used to theoretically study the impact of varying HT treatment conditions on radiosensitization and direct HT cell killing effect. Simulated patients were generated to compute the Tumor Control Probability (TCP) under different HT conditions (number of HT sessions, temperature and time interval), which were randomly selected within margins based on reported patient data. Results Under the studied conditions, model-based simulations suggested a treatment improvement with a total CEM43 thermal dose of approximately 10 min. Additionally, for a given thermal dose, TCP increased with the number of HT sessions. Furthermore, in the simulations, we showed that the TCP dependence on the temperature/time interval is more correlated with the mean value than with the minimum/maximum value and that comparing the treatment outcome with the mean temperature can be an excellent strategy for studying the time interval effect. Conclusion The use of thermoradiobiological models allows us to theoretically study the impact of varying thermal conditions on HT + RT treatment outcomes. This approach can be used to optimize HT treatments, design clinical trials, and interpret patient data.

Assessing Radiation Effects on Chemo-Treated BT20 and 4T1 Breast Cancer, and Neuroblastoma Cell Lines: A Study of Single and Multiple-Cell Ionization via Infrared Laser Trapping

Background: Our study aimed to assess the radiation sensitivity of BT20, a human breast tumor cell line, using the laser-trapping technique and compare it with N2a and 4T1 cells. Additionally, we investigated the impact of the antitumor compound 2-Dodecyl-6-methoxycyclohexa-2,5-diene-1,4-dione (DMDD) on radiation sensitivity. Methods and Materials: We employed laser trapping to calculate both the threshold ionization energy (TIE) and threshold radiation dose (TRD) for BT20, N2a, and 4T1 cells. We assessed the effect of DMDD on BT20 cells’ radiosensitivity and conducted comparisons across these cell lines. Results: Our findings reveal that DMDD significantly enhances the radiosensitivity of BT20 breast carcinoma cells. Moreover, we observed distinct trends in TIE and TRD across the three cell lines, with differences attributed to variations in cell size and composition. When multiple cell ionizations were considered, a notable reduction in TRD was observed, implicating factors such as the chain effect of ionizing radiation and the influence of DMDD. The study found that TIE increased with the number of cells in the trap while TRD consistently decreased across all three cell lines, suggesting comparable radiation sensitivity, and oligostilbene treatment further reduced TRD, presenting the potential for enhancing therapeutic ratios in cancer treatment. Conclusion: The antitumor compound DMDD enhances the radiosensitivity of BT20 breast carcinoma cells, highlighting its potential in cancer treatment. Furthermore, our study underscores the impact of cell size and multiple-cell ionizations on TRD. Leveraging laser trapping techniques, biocompatible nanoparticles, and advanced optical tweezers opens promising avenues for personalized and effective cancer therapy approaches.

CircNOP14 increases the radiosensitivity of hepatocellular carcinoma via inhibition of Ku70-dependent DNA damage repair

Circular RNAs (circRNAs) are profoundly affected in hepatocellular carcinoma (HCC) through various pathways. However, the role of circRNAs in the radiosensitivity of HCC cells is yet to be explored. In this study, we identified a circRNA—hsa_circ_0006737 (circNOP14) involved in the radiosensitivity of HCC. We found that circNOP14 increased the radiosensitivity of HCC cells both in vitro and in vivo. Notably, using a circRNA pulldown assay and RNA-binding protein immunoprecipitation, we identified Ku70 as a novel and robust interacting protein of circNOP14. Mechanistically, circNOP14 interacts with Ku70 and prevents its nuclear translocation, thereby increasing irradiation-induced DNA damage. Therefore, our findings may provide a predictive indicator and intervention option for 125I brachytherapy or external radiotherapy in HCC.