Radiosensitizing Drugs Research Articles

Disulfiram Upgrades the Radiosensitivity of Osteosarcoma by Enhancing Apoptosis and P53-Induced Cell Cycle Arrest.

The prognosis of osteosarcoma has not been improved for decades. As radioresistance is one of the major reasons, effective radiotherapy sensitization drugs need to be discovered. HOS and K7M2 osteosarcoma cell lines were treated with disulfiram (DSF) and radiation to assess cell viability, proliferation, migration ability, apoptosis level, ROS and Ca2+ level, and cell cycle in vitro. A HOS-derived subcutaneous tumor mouse model was constructed to evaluate tumor growth after DSF combined with radiation, and the Tunel assay and immunohistochemistry of Ki67 were conducted. Western blot was used to evaluate the protein expression level. The IC50 and working concentration of DSF in osteosarcoma cell lines were ascertained. When combined with radiation, DSF effectively suppressed cell viability, proliferation, and migration, while enhancing apoptosis in osteosarcoma cells. The cell cycle postirradiation exhibited a downward shift in the G1 phase, but the addition of DSF counteracted this trend. The combination of DSF and radiation exhibited inhibitory effects on tumor growth invivo, which was corroborated by Ki67 staining and Tunel assay. Western blot analysis revealed that DSF upregulated the expression of P53, P21, CDKN2C, BAX, and cleaved Caspase-3 while downregulating BCL2, CDK4/6, and CyclinD1 after irradiation. Our results document that DSF exerts its radiosensitization effects invivo and in vitro, and is a valuable radiosensitizing drug option for osteosarcoma. The radiosensitization effect is mainly achieved by activating the apoptotic pathway and promoting cell cycle arrest induced by P53/P21 and CDKN2C after irradiation.

Abstract 1129: A multifaceted study of X-Ray radiation therapy across diverse mouse tumor models

Abstract In this study, we showcase the application of X-ray radiation therapy of tumor-bearing mice across 13 syngeneic tumors, one intracranial tumor, and in combination with radiosensitizing drugs, aiming to develop potent cancer treatment strategies. 1.We examined the effects of different radiation levels on multiple tumor models using a therapeutic device delivering targeted radiation to the tumor site. 2.We investigated the combined benefits of radiation with the radiosensitizer Gemcitabine on the H22 liver tumor model. 3.We assessed the impact of radiation sensitization on the HCC1975-luc intracranial tumor model in combination with AZD0156, a drug that cannot cross the blood-brain barrier. The integrity of the blood-brain barrier and the presence of the pharmacodynamic marker pRAD50 of AZD0156 were evaluated. The results show that X-ray radiation has anti-tumor effects across diverse models, with combination drug treatment with both Gemcitabine and AZD0156 showing enhanced therapeutic effects. Furthermore, pRAD50 showed decreasing trend in the single dose PD study in HCC1975-luc intracranial model. We conclude that our platform provides robust methods for evaluating the therapeutic effects of X-Ray radiation, offering invaluable insights for the creation of new cancer therapies. Citation Format: Jingqi Huang, Wentao Li, Xiaoyan He, Weiwei Cheng, Lingyun Zhang, Guannan Li, Qiuliang Li, Yongfei Wang, Xuesong Ren, Zhi Wei, Long Shi, Yiran Wei, Jing Jin, Linfeng Li, Wei Yun. A multifaceted study of X-Ray radiation therapy across diverse mouse tumor 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 1129.

Intraoperative radiotherapy in breast cancer: Alterations to the tumor microenvironment and subsequent biological outcomes (Review).

Intraoperative radiotherapy (IORT) is a precise, single high‑dose irradiation directly targeting the tumor bed during surgery. In comparison with traditional external beam RT, it minimizes damage to other normal tissues, ensures an adequate dose to the tumor bed and results in improved cosmetic outcomes and quality of life. Furthermore, IORT offers a shorter treatment duration, lower economic costs and therapeutic efficacy comparable with traditional RT. However, its relatively higher local recurrence rate limits its further clinical applications. Identifying effective radiosensitizing drugs and rational RT protocols will improve its advantages. Furthermore, IORT may not only damage DNA to directly kill breast tumor cells but also alter the tumor microenvironment (TME) to exert a sustained antitumor effect. Specific doses of IORT may exert anti‑angiogenic effects, and consequently antitumor effects, by impacting post‑radiation peripheral blood levels of vascular endothelial growth factor and delta‑like 4. IORT may also modify the postoperative wound fluid composition to continuously inhibit tumor growth, e.g. by reducing components such as microRNA (miR)‑21, miR‑221, miR‑115, oncostatin M, TNF‑β, IL‑6 and IL‑8, and by elevating levels of components such as miR‑223, to inhibit the ability of postoperative wound fluid to induce proliferation, invasion and migration of residual cancer cells. IORT can also modify cancer cell glucose metabolism to inhibit the proliferation of residual tumor cells. In addition, IORT can induce a bystander effect, eliminating the postoperative wound fluid‑induced epithelial‑mesenchymal transition and tumor stem cell phenotype. Insights gained at the molecular level may provide new directions for identifying novel therapeutic targets and approaches. A more comprehensive understanding of the effects of IORT on the breast cancer (BC) TME may further its clinical application. Hence, the present article reviews the primary effects of IORT on BC and its impact on the TME, aiming to offer fresh research perspectives for relevant professionals.

Landscape of the Radiosensitizing Properties of Targeted Agents from the NCI CTEP Portfolio across Genomically Diverse 3D Tumor Models

Clinical translation of molecular targeted radiosensitizers remains challenging. To improve the robustness of preclinical data, we sought to broadly examine across a wide spectrum of genomic cancer features the radiosensitizing properties of targeted drugs from the NCI Cancer Therapy Evaluation Program (CTEP). As part of a U01 Consortium, we adapted a robotic high-throughput platform for testing ionizing radiation (IR) plus drug combinations in 3D cultures derived from non-small cell lung carcinoma (NSCLC), head/neck (HNSCC), and pancreatic cancers. All cell lines were annotated, mycoplasma free, and re-authenticated. Signal windows were established for all 3D growth formats. CTEP drugs were added in four concentrations 24 hours prior to IR exposure of 384-well plates. Five days after IR, cell viability was assessed, an endpoint previously benchmarked using colony formation. Each data point was based on 4-6 technical replicates with 2 biological repeats. A multistep statistical method combining Wilcoxon signed-rank and Spearman rank-order correlation tests together with adjusted Benjamini-Hochberg corrections for multiple comparisons was used to identify radiosensitization hits. Select validation experiments of hits were carried out. We screened 73 3D tumor models with 34-42 CTEP drugs each plus IR (n = 10,744 cell line/drug concentration combinations). IR/drug patterns were comparable for the three cancer types. DNA damage response (DDR) inhibitors constituted the most active class of radiosensitizers with DNA-PKcs, PARP, and ATR being the top targets. In contrast, the majority of the other agents radiosensitized only a minority of models. Highlighting NSCLC, out of 1,120 cell line/drug combinations, 328 (29.3%) were associated with statistically significant radiosensitization. PARP inhibitors, of particular interest for clinical translation in this space, demonstrated relatively broad radiosensitization for talazoparib (62%) and olaparib (44%) but less so for veliparib (28%), which correlated with differential PARP trapping potentials. Radiosensitization was evident even for tumor genotypes associated with radioresistance such as mutant KRAS, KEAP1, and STK11. Interestingly, single-agent activity of olaparib was more pronounced in 11/30 3D cultures compared to 2D growth conditions but with only 2/11 having known homologous recombination defects. Lastly, for HPV negative HNSCC (735 combinations), there was radiosensitization in subsets of models for IAP, ATR, and AKT targets, which are under clinical investigation. We present a unique resource for examining radiosensitizing drug properties in 3D cultures. The observed radiosensitization patterns strongly argue for the use of larger panels of clinically relevant tumor models rather than a few non-representative cell lines. Our data highlight the urgent need for predictive biomarkers to guide IR/drug combinations, even for some DDR inhibitors.

GSH-activatable camptothecin prodrug-loaded gold nanostars coated with hyaluronic acid for targeted breast cancer therapy via multiple radiosensitization strategies.

Breast cancer has overtaken lung cancer to rank as the top malignant tumor in terms of incidence. Herein, a gold nanostar (denoted as AuNS) is used for loading disulfide-coupled camptothecin-fluorophore prodrugs (denoted as CPT-SS-FL) to form a nanocomposite of AuNS@CPT-SS-FL (denoted as AS), which, in turn, is further encapsulated with hyaluronic acid (HA) to give the final nanoplatform of AuNS@CPT-SS-FL@HA (denoted as ASH). ASH effectively carries the prodrug and targets the CD44 receptor on the surface of tumor cells. The endogenously overexpressed glutathione (GSH) in tumor cells breaks the disulfide bond to activate the prodrug and release the radiosensitizer drug camptothecin (CPT) and the fluorescence imaging reagent rhodamine derivative as a fluorophore (FL). The released FL can track the precise release position of the radiosensitizer camptothecin in tumor cells in real time. The AuNS has strong X-ray absorption and deposition ability due to the high atomic coefficient of elemental Au (Z = 79). At the same time, the AuNS can alleviate the tumor microenvironment (TME) hypoxia through its mild photothermal therapy (PTT). Therefore, through the multiple radiosensitizing effects of GSH depletion, the high atomic coefficient of Au, and hypoxia alleviation, accompanied by the radiosensitizer camptothecin, the designed ASH nanoplatform can effectively induce strong immunogenic cell death (ICD) at the tumor site via radiosensitizing therapy combined with PTT. This work provides a new way of constructing a structurally compact and highly functionalized hierarchical system toward efficient breast cancer treatment through ameliorating the TME with multiple modalities.

Supramolecular Radiosensitizer Based on Hypoxia-Responsive Macrocycle.

Radiotherapy (RT) has been viewed as one of the most effective and extensively applied curatives in clinical cancer therapy. However, the radioresistance of tumor severely discounts the radiotherapy outcomes. Here, an innovative supramolecular radiotherapy strategy, based on the complexation of a hypoxia‐responsive macrocycle with small‐molecule radiosensitizer, is reported. To exemplify this tactic, a carboxylated azocalix[4]arene (CAC4A) is devised as molecular container to quantitatively package tumor sensitizer banoxantrone dihydrochloride (AQ4N) through reversible host–guest interaction. Benefited from the selective reduction of azo functional groups under hypoxic microenvironment, the supramolecular prodrug CAC4A•AQ4N exhibits high tumor accumulation and efficient cellular internalization, thereby significantly amplifying radiation‐mediated tumor destruction without appreciable systemic toxicity. More importantly, this supramolecular radiotherapy strategy achieves an ultrahigh sensitizer enhancement ratio (SER) value of 2.349, which is the supreme among currently reported noncovalent‐based radiosensitization approach. Further development by applying different radiosensitizing drugs can make this supramolecular strategy become a general platform for boosting therapeutic effect in cancer radiotherapies, tremendously promising for clinical translation.

Advances of Nanomedicine in Radiotherapy.

Radiotherapy (RT) remains one of the current main treatment strategies for many types of cancer. However, how to improve RT efficiency while reducing its side effects is still a large challenge to be overcome. Advancements in nanomedicine have provided many effective approaches for radiosensitization. Metal nanoparticles (NPs) such as platinum-based or hafnium-based NPs are proved to be ideal radiosensitizers because of their unique physicochemical properties and high X-ray absorption efficiency. With nanoparticles, such as liposomes, bovine serum albumin, and polymers, the radiosensitizing drugs can be promoted to reach the tumor sites, thereby enhancing anti-tumor responses. Nowadays, the combination of some NPs and RT have been applied to clinical treatment for many types of cancer, including breast cancer. Here, as well as reviewing recent studies on radiotherapy combined with inorganic, organic, and biomimetic nanomaterials for oncology, we analyzed the underlying mechanisms of NPs radiosensitization, which may contribute to exploring new directions for the clinical translation of nanoparticle-based radiosensitizers.

Effectiveness of Concomitant Chemoradiotherapy with Gemcitabine in Locally Advanced Cervical Cancer Patients with Comorbidities.

PurposeThe standard treatment for locally advanced cervical cancer (LACC) is concomitant chemoradiotherapy with cisplatin (CDDP) followed by brachytherapy. The presence of comorbidities are risk factors for nephrotoxicity and are associated with lower survival. Gemcitabine is a radiosensitizing drug that has shown efficacy and safety in this context. The effectiveness of concomitant chemoradiotherapy with gemcitabine was evaluated versus cisplatin in LACC patients with comorbidities and preserved renal function.Materials and MethodsAn observational, longitudinal and paired study was carried out that included patients treated between February 2003 and December 2015. The primary objectives were to evaluate response rates, progression-free survival, and overall survival; the secondary objectives were to evaluate toxicity and renal function.ResultsSixty-three patients treated with gemcitabine at 300 mg/m2 weekly and 126 patients treated with CDDP 40 mg/m2 weekly were included. There were no significant differences in response rates and survival rates. Treatment with cisplatin presented a higher frequency of hematological toxicities, while gemcitabine presented a higher frequency of gastrointestinal toxicities. A decrease in glomerular filtration rate (GFR; baseline vs. 1-year post-treatment) was observed in the cisplatin group (p=0.002), while not in the gemcitabine group (p=0.667). In a multivariate analysis, it is observed that only CDDP correlates with the decrease in GFR (hazard ratio, 2.42; p=0.012).ConclusionIn LACC patients with comorbidities, gemcitabine and CDDP show the same efficacy, with different toxicity profiles. Treatment with cisplatin is associated with a significant decrease in GFR during follow-up, compared to treatment with gemcitabine that does not decrease it.

Targeting a Radiosensitizing Antibody-Drug Conjugate to a Radiation-Inducible Antigen.

We recently discovered that anti-TIP1 antibody activates endocytosis in cancer cells, which facilitates retention of antibody and dissociation of a conjugated drug. To improve the pharmacokinetics and cancer specificity of radiosensitizing drugs, we utilized antibody-drug conjugates (ADCs) that bind specifically to radiation-inducible antigen, TIP1, on non-small cell lung cancer (NSCLC). This approach exploits the long circulation time of antibodies to deliver a radiosensitizing drug to cancer each day during radiotherapy. Antibodies to TIP1 were prioritized based on affinity, cancer-specific binding, and internalization. The lead antibody, 7H5, was conjugated with a cytotoxic drug MMAE because of its ability to radiosensitize cancer. Cytotoxicity, colony formation, and tumor growth studies were performed with 7H5-VcMMAE in combination with radiation. 7H5 showed a high affinity to recombinant TIP1 protein and radiation-inducible TIP1 on the cancer cell surface. 7H5 undergoes endocytosis in NSCLC cells in vitro. We obtained an average drug-to-antibody ratio (DAR) of 4.25 for 7H5-VcMMAE. A 70% reduction in viable cells was observed following 7H5-VcMMAE treatment compared with 7H5 alone in both A549 and H1299 cells. 7H5-VcMMAE sensitized NSCLC cells to radiation, thereby significantly decreasing the surviving fraction. The ADC combined with radiation showed a prolonged delay in tumor growth and improved survival in A549 and H1299 tumor models. Targeting radiation-inducible TIP1 with a radiosensitizing ADC is a promising strategy to enhance the therapeutic efficacy of NSCLC. This novel approach of targeting with ADCs to radiation-inducible antigens will lead to clinical trials in lung cancer patients treated with radiotherapy.

Abstract PO-086: Selective radiosensitization in preclinical models of HPV-negative squamous cell carcinoma

Abstract Background: Radiotherapy is a mainstay of curative treatment for squamous cell carcinomas (SCC) and is frequently combined with radiosensitizing drugs to improve efficacy. Outcomes for HPV-negative SCCs remain heterogeneous, in part due to variable resistance to ionizing radiation (IR) and modest benefit of existing radiosensitizing drugs. Hypothesis driven testing of novel radiosensitizing drugs has previously been limited due to a lack of robust, efficient, and reproducible methods to assess drug radiation combinations. Aims: To improve the throughput of preclinical studies on radiosensitizers, we have developed an in vitro system for assessing drug/radiation combinations, validated using the known radiosensitizing chemotherapeutic drug cisplatin. We extended the assay to assess the effect of ML-385, a NRF2 inhibitor that increases cellular reactive oxygen species (ROS), and abemaciclib and palbociclib, two CDK4/6 inhibitors predicted to radiosensitize cancer cells by inhibiting entry into more resistant phases of the cell cycle. Experimental Methods: We adapted the 9-day viability assay of Abazeed et al. (Cancer Res., 2013) to enable drug/radiation efficacy assessments. We first validated the assay’s ability to recapitulate clonogenic survival among 19 HPV-negative SCC cell lines. Drug/radiation experiments were performed in the 6 most radioresistant SCC cell lines. ROS levels after 4Gy IR was used to confirm NRF2 suppression. Flow cytometry was used to evaluate cell cycle phase. AKT inhibitor GSK690693 was used in combination with a NRF2 shRNA knockdown cell line to investigate the mechanism of sensitivity. Western blot was used to detect p16. Cellular response to multiple IR doses was summarized using an area-under-the-curve (AUC) metric. The delta AUC across drug doses was used to evaluate radiosensitization. Results: Among the 19 cell lines, AUCs with the clonogenic and 9-day viability assays were strongly correlated (Pearson r=0.74, p=3.00 × 10–4). Six (32%) of the cell lines were reproducibly radioresistant (AUC >3.5) using both assays. All of these cell lines saw sensitization with cisplatin. None of these 6 cell lines harboured mutations in the canonical NRF2 pathway, whereas all 6 harboured either CCND1 amplification, CDKN2A mutation, or both. Only one cell line showed radiosensitization with the NRF2 inhibitor ML-385, an effect that was abrogated by PIK3CA or AKT pathway inhibition. In contrast, 5 of the 6 cell lines showed reproducible radiosensitization following CDK4/6 inhibition; with robust p16 expression detected in the lone unaffected cell line. Conclusion: The 9-day viability assay allows efficient evaluation of putative radiosensitizers. Our tests of putative radiosensitizing drugs in a cohort of radioresistant cell lines have identified CDK4/6 inhibitors to be of interest for further investigation in biomarker-selected populations. Citation Format: Meghan Lambie, Rita Gill, Laurie Ailles, Scott V. Bratman. Selective radiosensitization in preclinical models of HPV-negative squamous cell carcinoma [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-086.

Review of Experimental Studies to Improve Radiotherapy Response in Bladder Cancer: Comments and Perspectives.

Simple SummaryBladder cancer is a major global health problem. Bladder removal surgery is the standard treatment for muscle-invasive bladder cancer (25% of all bladder cancer), but this treatment negatively affects the quality of life, especially for elderly and frail patients. Tumour resection followed by combination of radiotherapy and chemotherapy has emerged as a promising bladder preserving strategy. However, this strategy is unable to avoid radiation-related bladder side effects. Therefore, it is of great interest to discover novel strategies radiosensitising tumours while sparing normal bladder tissue. In this review, we analysed the experimental studies of radiosensitising strategies in bladder cancer and provided suggestions to improve forthcoming studies.Bladder cancer is among the top ten most common cancer types in the world. Around 25% of all cases are muscle-invasive bladder cancer, for which the gold standard treatment in the absence of metastasis is the cystectomy. In recent years, trimodality treatment associating maximal transurethral resection and radiotherapy combined with concurrent chemotherapy is increasingly used as an organ-preserving alternative. However, the use of this treatment is still limited by the lack of biomarkers predicting tumour response and by a lack of targeted radiosensitising drugs that can improve the therapeutic index, especially by limiting side effects such as bladder fibrosis. In order to improve the bladder-preserving treatment, experimental studies addressing these main issues ought to be considered (both in vitro and in vivo studies). Following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines for systematic reviews, we conducted a literature search in PubMed on experimental studies investigating how to improve bladder cancer radiotherapy with different radiosensitising agents using a comprehensive search string. We made comments on experimental model selection, experimental design and results, formulating the gaps of knowledge still existing: such as the lack of reliable predictive biomarkers of tumour response to chemoradiation according to the molecular tumour subtype and lack of efficient radiosensitising agents specifically targeting bladder tumour cells. We provided guidance to improve forthcoming studies, such as taking into account molecular characteristics of the preclinical models and highlighted the value of using patient-derived xenografts as well as syngeneic models. Finally, this review could be a useful tool to set up new radiation-based combined treatments with an improved therapeutic index that is needed for bladder preservation.

Abstract 6272: Redirecting tumor extracellular proteases to target radiosensitizer delivery

Abstract Patients with locally advanced cancers are treated with combined radiotherapy and chemotherapy, however curability is poor and side effects severe. Drugs that sensitize tumors to radiotherapy have been tried to improve cure, but tumor specificity remains a challenge. To achieve tumor selectivity of small molecule radiosensitizers, we tested as a strategy active tumor targeting utilizing peptide-based drug conjugates that split the responsibilities for targeting and radiosensitization into distinct molecular tasks. The tumor milieu possesses localizing beacons that can guide drug delivery using tumor cell surface receptors and microenvironment extracellular matrix metalloproteinases (MMPs). Here we applied peptide-based linkers to test for tumor selective drug delivery and radiosensitization by chemically conjugating the DNA damage checkpoint kinase 1/2 inhibitor AZD7762 to antibody to or cell penetrating peptides. AZD7762 governs DNA damage response and irradiated cell fate. We synthesized drug conjugates of AZD7762 and compared their anti-tumor efficacy in combination with ionizing radiation (IR). An antibody drug conjugate (ADC) was built by attaching AZD7762 to the anti-EGFR antibody cetuximab. Polycationic cell penetrating peptide drug conjugates were constructed by linking AZD7762 to a 9 amino acid repeat of D-arginine and keeping it bare (r9-AZD7762) or cloaking r9 within a MMP-2/9 triggered activatable cell penetrating peptide scaffold (ACPP-AZD7762). In contrast to receptor directed ADCs, ACPPs redirect extracellular tumor microenvironment proteases to guide drug delivery. Intact, ACPPs are charge neutral which blocks the conjugated drug from cell entry. Upon cleavage by tumor overexpressed MMPs, the ACPP scaffold releases drug conjugated polycationic peptide which is then internalized by cells. We found that while cetuximab-AZD7762 ADC had high affinity for EGFR overexpressing tumor cells and prolonged blood circulation in murine models, cetuximab-AZD7762 lacked anti-tumor efficacy. On the other extreme, naked r9-AZD7762 drug conjugate was indiscriminately cytotoxic and radiosensitized a panel of tumor cell lines in cell culture: CAL27, A549, PANC1 and HCT116. However, r9 cell penetrating peptides lacked tumor specificity when intravenously injected into mice. In contrast, MMP-2/9 sensitive ACPPs balanced tumor selectivity with drug efficacy. Intact, ACPPs held conjugated AZD7762 in a pro-drug inactive state that upon tumor microenvironment ACPP cleavage resulted in tumor selective drug accumulation and anti-tumor efficacy in combination with IR. Importantly, a control non-cleavable ACPP-AZD7762 did not improve tumor control with IR, demonstrating the necessity of ACPP cleavage to release active drug. These results highlight the utility of the ACPP technology platform to target amine containing radiosensitizing drugs to tumor tissues based on extracellular protease activity that overcome the limitations of ADC conjugated radiosensitizers. Citation Format: Dina V. Hingorani, Maria F. Camargo, Maryam A. Quraishi, Joseph Aguilera, Stephen R. Adams, Sunil J. Advani. Redirecting tumor extracellular proteases to target radiosensitizer delivery [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6272.

Redirecting extracellular proteases to molecularly guide radiosensitizing drugs to tumors

Patients with advanced cancers are treated with combined radiotherapy and chemotherapy, however curability is poor and treatment side effects severe. Drugs sensitizing tumors to radiotherapy have been developed to improve cell kill, but tumor specificity remains challenging. To achieve tumor selectivity of small molecule radiosensitizers, we tested as a strategy active tumor targeting using peptide-based drug conjugates. We attached an inhibitor of the DNA damage response to antibody or cell penetrating peptides. Antibody drug conjugates honed in on tumor overexpressed cell surface receptors with high specificity but lacked efficacy when conjugated to the DNA damage checkpoint kinase inhibitor AZD7762. As an alternative approach, we synthesized activatable cell penetrating peptide scaffolds that accumulated within tumors based on matrix metalloproteinase cleavage. While matrix metalloproteinases are integral to tumor progression, they have proven therapeutically elusive. We harnessed these pro-tumorigenic extracellular proteases to spatially guide radiosensitizer drug delivery using cleavable activatable cell penetrating peptides. Here, we tested the potential of these two drug delivery platforms targeting distinct tumor compartments in combination with radiotherapy and demonstrate the advantages of protease triggered cell penetrating peptide scaffolds over antibody drug conjugates to deliver small molecule amine radiosensitizers.

Dopamine receptor D2 activation suppresses the radiosensitizing effect of aripiprazole via activation of AMPK.

Drug repositioning has garnered attention as an alternative strategy to the discovery and development of novel anticancer drug candidates. In this study, we screened 321 FDA‐approved drugs against nonirradiated and irradiated MCF‐7 cells, revealing that aripiprazole, a dopamine receptor D2 (D2R) partial agonist, enhances the radiosensitivity of MCF‐7 cells. Unexpectedly, D2R‐selective antagonist treatment significantly enhanced the radiosensitizing effects of aripiprazole and prevented aripiprazole‐induced 5' adenosine monophosphate‐activated protein kinase (AMPK) phosphorylation. Direct AMPK activation with A769662 treatment blunted the radiosensitizing effects of aripiprazole. These results indicate that aripiprazole has potential as a radiosensitizing drug. Furthermore, prevention of D2R/AMPK activation might enhance these anticancer effects of aripiprazole in breast cancer cells.

Abstract 3925: A screen for epigenetic radiosensitizers in small cell lung cancer

Abstract Objective: Small cell lung cancer (SCLC) is a highly aggressive neuroendocrine malignancy. Post-platinum-based chemotherapy, rapid relapse of chemoresistant tumor is commonly seen. Unidentifiable causative mutations and from reversible nature of acquired chemoresistance, involvement of epigenetic switch regulating SCLC progression is evident. Aim of this screen is to identify epigenetic modifiers that could sensitize SCLC cells to ionizing radiation (IR). Methods: To recapitulate molecular diversity of patients, a range of SCLC cell lines (n=10) from classic or variant and ASCL1 or NeuroD1 sub-groups, driven by varying MYC family amplifications were picked. Short term viability experiment was setup in 3 groups: (a) Epigenetic monotherapy: cells were screened with 10 epigenetic modifiers (0 to 10 µM). (b) IR monotherapy: cells were irradiated from 0 to 8 gy. (c) Combination therapy: cells were treated with different doses of epigenetic probes and IR. Radiosensitization was measured as dose modifying factor at SF63 for each epigenetic modulator. Results: (a) Epigenetic monotherapy: All 10 cell lines were responsive to GSK-J4 (KDMi, IC50 3.1-0.9 µM), SAHA (HDACi, IC50 2.9-0.79 µM) and JIB04 (Jumonji KDMi, IC50 74-2.4 nM). Other interesting epi-probe to which 9/10 cell lines responded was JQ1 (BET-BRDi, IC50 5.7 to 0.15 µM, except SBC-5). Compounds with lesser universal potency were MS023 (PRMTi), UNC0642 (G9a/GLPi) and UNC1999 (EZH2i). All cell lines were irresponsive to PFI3 (SMARCA2/4 BRDi), BAY598 (SMYD2 MTi) and OICR-9429 (WDR5i). (b) IR monotherapy: All 10 cell lines demonstrated different sensitivities to 4 days of IR. They’re ranked in descending order of radiosensitivity as H446, H82, SHP77, LX22, H889, H1092, H196, H69, SBC5, H526. (c) Epi-probes + IR combination therapy: Epi-probes ranked in descending order of number of cell lines they radiosensitized are JQ1, JIB-04, GSK-J4, SAHA, UNC0642, BAY598, MS023, UNC1999, OICR9429 and PFI3. Interestingly, drugs that demonstrated potency as a single agent did not necessarily radiosensitize the cell lines. On the contrary, certain epi-probes that were ineffective as monotherapy radiosensitized a few of the cell lines. H82 is a sensitive cell line which was radiosensitized by all epi-probes used. Conclusions: This screen shows that treating cells with IR in conjunction with epigenetic modifiers may potentially improve therapeutic efficacy of radiotherapy in SCLC. Further validation is being done with long term colonogenic assays followed by in vivo studies for the potent radiosensitizing candidate drugs. It would be therapeutically relevant to correlate the probability of radiosensitization by an epi-probe with the molecular profile of the patients for more predictive targeted therapeutics. Citation Format: Mansi K. Aparnathi, Lifang Song, Ratheesh Subramaniam, Richard Marcellus, Rima Al-awar, Benjamin H. Lok. A screen for epigenetic radiosensitizers in small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3925.

National Cancer Institute (NCI) state of the science: Targeted radiosensitizers in colorectal cancer.

Colorectal cancer (CRC) represents a major public health problem as the second leading cause of cancer-related mortality in the United States. Of an estimated 140,000 newly diagnosed CRC cases in 2018, roughly one-third of these patients will have a primary tumor located in the distal large bowel or rectum. The current standard-of-care approach includes curative-intent surgery, often after preoperative (neoadjuvant) radiotherapy (RT), to increase rates of tumor down-staging, clinical and pathologic response, as well as improving surgical resection quality. However, despite advancements in surgical techniques, as well as sharpened precision of dosimetry offered by contemporary RT delivery platforms, the oncology community continues to face challenges related to disease relapse. Ongoing investigations are aimed at testing novel radiosensitizing agents and treatments that might exploit the systemic antitumor effects of RT using immunotherapies. If successful, these treatments may usher in a new curative paradigm for rectal cancers, such that surgical interventions may be avoided. Importantly, this disease offers an opportunity to correlate matched paired biopsies, radiographic response, and molecular mechanisms of treatment sensitivity and resistance with clinical outcomes. Herein, the authors highlight the available evidence from preclinical models and early-phase studies, with an emphasis on promising developmental therapeutics undergoing prospective validation in larger scale clinical trials. This review by the National Cancer Institute's Radiation Research Program Colorectal Cancer Working Group provides an updated, comprehensive examination of the continuously evolving state of the science regarding radiosensitizer drug development in the curative treatment of CRC.

Identification and validation of non-histone protein acetylation regulated by the DNA damage response

Event Abstract Back to Event Identification and validation of non-histone protein acetylation regulated by the DNA damage response Judith Nicholson1, Junetha Syed Jabarulla1, Iolanda Vendrell1, 2, Xin Ying Yeo1, Roman Fischer2, Benedikt M. Kessler2 and Anne Kiltie1* 1 Department of Oncology, Medical Sciences Division, University of Oxford, United Kingdom 2 Nuffield Department of Medicine, Medical Sciences Division, University of Oxford, United Kingdom Background: The DNA Damage Response (DDR) is an attractive target for developing radiosensitising drugs, which can downregulate the efficiency of DNA repair in cancer cells induced by ionising radiation. Post-translational modifications of DNA repair proteins tightly coordinate the DDR events, among which lysine acetylation plays an important role in regulating histone and non-histone proteins in the DDR process. Our previous work on modulating acetylation with histone deacetylase inhibitor (HDACi) panobinostat in bladder cancer cell lines has shown to increase radiosensitivity by targeting DDR without any side effects to the normal tissue. In this study, we identified acetylation of DDR-associated non-histone proteins after treatment with panobinostat with the long-term goal of finding effective radiosensitisers with less systemic toxicity. Methods: Tandem mass spectrometry was used to identify potential DDR candidates that become acetylated following irradiation +/- panobinostat treatment. Acetylated protein enrichment was achieved by Immunoprecipitation. Radiosensitisation was determined by clonogenic assay, gamma H2AX levels by western blotting and interacting partners of the protein of interest by APEX-mediated proximity labelling. Results: In total, 228 proteins harbouring 657 unique acetylation sites with a subset related to DNA repair functions were identified. The acetylation of two hit DDR candidates sMEK1 and THRAP3 was validated. The clonogenic survival assay and the pattern of H2AX phosphorylation with acetyl-mimic and acetyl-dead constructs indicated the important role of the identified acetylation site in THRAP3 for radiosensitivity. Conclusion: Several non-histone proteins are regulated on a post-translational level by acetylation during the DNA damage response. The acetylated proteins identified in this dataset represent a complex sample of non-histone proteins, THRAP3 is one of which we validated as having a role in radiosensitisation via a specific acetyl site. Identifying the interacting partners of acetylated THRAP3 would help in understanding the biological significance of identified acetylation site with respect to DDR. Keywords: DNA repair protein, Proteomics, Acetylation, HDACi, THRAP3 Conference: Bladder Cancer Translational Research Meeting, London, United Kingdom, 29 Mar - 29 Mar, 2019. Presentation Type: Poster Topic: Optimisation of diagnostic pathways Citation: Nicholson J, Syed Jabarulla J, Vendrell I, Yeo X, Fischer R, Kessler BM and Kiltie A (2019). Identification and validation of non-histone protein acetylation regulated by the DNA damage response. Front. Oncol. Conference Abstract: Bladder Cancer Translational Research Meeting. doi: 10.3389/conf.fonc.2019.01.00017 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 01 Mar 2019; Published Online: 27 Sep 2019. * Correspondence: Prof. Anne Kiltie, Department of Oncology, Medical Sciences Division, University of Oxford, Oxford, England, OX3 7DQ, United Kingdom, anne.kiltie@oncology.ox.ac.uk Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Judith Nicholson Junetha Syed Jabarulla Iolanda Vendrell Xin Ying Yeo Roman Fischer Benedikt M Kessler Anne Kiltie Google Judith Nicholson Junetha Syed Jabarulla Iolanda Vendrell Xin Ying Yeo Roman Fischer Benedikt M Kessler Anne Kiltie Google Scholar Judith Nicholson Junetha Syed Jabarulla Iolanda Vendrell Xin Ying Yeo Roman Fischer Benedikt M Kessler Anne Kiltie PubMed Judith Nicholson Junetha Syed Jabarulla Iolanda Vendrell Xin Ying Yeo Roman Fischer Benedikt M Kessler Anne Kiltie Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Abstract 854: Validation of a viability assay for assessing radiation response and investigating drug/radiation combinations

Abstract Purpose: Radiotherapy (RT) is frequently used either alone or in combination with chemotherapy for curative treatment of squamous cell carcinomas (SCC) originating from the head and neck, lung, esophagus, and cervix. Treatment outcomes for SCCs are heterogeneous, in part due to variable degrees of resistance to ionizing radiation and modest benefit of existing radiosensitizing drugs. Preclinical research into intrinsic cancer cell radiosensitivity biomarkers and novel radiosensitizing drugs has been hampered by a lack of robust high throughput assays of radiation response. We sought to validate a recently published high throughput viability assay as a surrogate of clonogenic survival and extend its use to drug-radiation combinations. Experimental Methods: Clonogenic assays—the gold standard for assessing radiation survival in vitro—and viability assays were performed on 19 SCC cell lines (16 head and neck, 3 esophageal). The clonogenic assay was performed according to published procedures (Franken et al. Nat. Protoc, 2006) with colonies counted using ImageJ. The viability assay was adapted from Abazeed et al. (Cancer Res., 2013). Cells were seeded with a range of densities in 96 well plates then treated with a range of radiation doses (0, 2, 4, and 8 Gy); viability was measured using CellTitre Glo reagent after 9 days. Agreement between clonogenic and viability assays was measured using modified concordance index (C-index) and Pearson correlation. For the addition of drug, cells were allowed to adhere to plates for 2 hours before drug administration. Cells were irradiated 16 hours later again at a range of doses (0, 2, 4, and 6 Gy for drug analysis), and then quantified as above. All assays were performed with technical and biological triplicates. Results: There was a high degree of agreement between the area under the curve with the clonogenic assay and the viability assay (C-index = 0.80, p = 3.03x10-7; Pearson r = 0.70, p = 1.10x10–3). Significant concordance was also seen at 2 Gy and 4 Gy dose points (Pearson r = 0.62 and 0.73 respectively), however not at 8 Gy (Pearson r<0.5). The viability assay decreases the overall time for the experiment, as it has a 9-day endpoint rather than the 14 days typical for the clonogenic assay. The viability assay also eliminates cell counting time, as results can be analyzed in minutes. Survival curves with and without radiation with clinically utilized chemotherapeutic drugs cisplatin and paclitaxel were developed which showed an additive effect for drug combinations, demonstrating the ability of the assay to be combined with chemotherapeutic agents and allows for the investigation into dose-response effects. Conclusion: The proliferative assay recapitulates the clonogenic assay within our cohort of SCC cell lines. Novel higher throughput methods to analyze radiation response will allow for a more efficient measure of drug and radiation combinations. Citation Format: Meghan Lambie, Venkata S. Manem, Benjamin Haibe-Kains, Scott V. Bratman. Validation of a viability assay for assessing radiation response and investigating drug/radiation combinations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 854.

An evaluation in vitro of the efficacy of nutlin-3 and topotecan in combination with 177Lu-DOTATATE for the treatment of neuroblastoma.

Targeted radiotherapy of metastatic neuroblastoma using the somatostatin receptor (SSTR)-targeted octreotide analogue DOTATATE radiolabelled with lutetium-177 (177Lu-DOTATATE) is a promising strategy. This study evaluates whether its effectiveness may be enhanced by combination with radiosensitising drugs. The growth rate of multicellular tumour spheroids, derived from the neuroblastoma cell lines SK-N-BE(2c), CHLA-15 and CHLA-20, was evaluated following treatment with 177Lu-DOTATATE, nutlin-3 and topotecan alone or in combination. Immunoblotting, immunostaining and flow cytometric analyses were used to determine activation of p53 signalling and cell death. Exposure to 177Lu-DOTATATE resulted in a significant growth delay in CHLA-15 and CHLA-20 spheroids, but not in SK-N-BE(2c) spheroids. Nutlin-3 enhanced the spheroid growth delay induced by topotecan in CHLA-15 and CHLA-20 spheroids, but not in SK-N-BE(2c) spheroids. Importantly, the combination of nutlin-3 with topotecan enhanced the spheroid growth delay induced by X-irradiation or by exposure to 177Lu-DOTATATE. The efficacy of the combination treatments was p53-dependent. These results indicate that targeted radiotherapy of high risk neuroblastoma with 177Lu-DOTATATE may be improved by combination with the radiosensitising drugs nutlin-3 and topotecan.

Fragmentation patterns of 4(5)-nitroimidazole and 1-methyl-5-nitroimidazole-The effect of the methylation.

We present here the photofragmentation patterns of doubly ionized 4(5)-nitroimidazole and 1-methyl-5-nitroimidazole. The doubly ionized state was created by core ionizing the C 1s orbitals of the samples, rapidly followed by Auger decay. Due to the recent development of nitroimidazole-based radiosensitizing drugs, core ionization was selected as it represents the very same processes taking place under the irradiation with medical X-rays. In addition to the fragmentation patterns of the sample, we study the effects of methylation on the fragmentation patterns of nitroimidazoles. We found that methylation alters the fragmentation significantly, especially the charge distribution between the final fragments. The most characteristic feature of the methylation is that it effectively quenches the production of NO and NO+ , widely regarded as key radicals in the chemistry of radiosensitization by the nitroimidazoles.