Inhibition Of DNA Damage Repair Research Articles

Nano-enabled coordination platform of bismuth nitrate and cisplatin prodrug potentiates cancer chemoradiotherapy via DNA damage enhancement.

The combination of chemotherapy and radiotherapy (chemoradiotherapy) is a promising strategy, extensively studied and applied clinically. Meanwhile, radiosensitizers play an important role in improving clinical radiotherapy therapeutic efficacy. There are still some disadvantages in practical applications, because radiosensitizers and drugs are difficult to deliver spatio-temporally to tumor sites and work simultaneously with low efficiency for DNA damage and repair inhibition, leading to an inferior synergistic effect. Herein, a suitable radiosensitizer of nano-enabled coordination platform (NP@PVP) with bismuth nitrate and cisplatin prodrug is developed by a simple synthetic route to improve the effectiveness of chemo-radiation synergistic therapy. When NP@PVP is internalized by a tumor cell, the bismuth in NP@PVP can sensitize radiation therapy (RT) by increasing the amount of reactive oxygen species generation to enhance DNA damage after X-ray radiation; meanwhile, the cisplatin in NP@PVP can inhibit DNA damage repair with spatio-temporal synchronization. NP@PVP is demonstrated to exhibit higher sensitization enhancement ratio (SER) of 2.29 and excellent tumor ablation capability upon irradiation in vivo in comparison with cisplatin (SER of 1.78). Our strategy demonstrates that the RT sensitization effect of bismuth and cisplatin based NP@PVP has great anticancer potential in chemo-radiation synergistic therapy, which is promising for clinical application.

Ailanthus Altissima-derived Ailanthone enhances Gastric Cancer Cell Apoptosis by Inducing the Repression of Base Excision Repair by Downregulating p23 Expression.

Chemotherapy plays an irreplaceable role in the treatment of GC, but currently available chemotherapeutic drugs are not ideal. The application of medicinal plants is an important direction for new drug discovery. Through drug screening of GC organoids, we determined that ailanthone has an anticancer effect on GC cells in vitro and in vivo. We also found that AIL can induce DNA damage and apoptosis in GC cells. Further transcriptome sequencing of PDX tissue indicated that AIL inhibited the expression of XRCC1, which plays an important role in DNA damage repair, and the results were also confirmed by western blotting. In addition, we found that AIL inhibited the expression of P23 and that inhibition of P23 decreased the expression of XRCC1, indicating that AIL can regulate XRCC1 via P23. The results of coimmunoprecipitation showed that AIL can inhibit the binding of P23 and XRCC1 to HSP90. These findings indicate that AIL can induce DNA damage and apoptosis in GC cells. Meanwhile, AIL can decrease XRCC1 activity by downregulating P23 expression to inhibit DNA damage repair. The present study sheds light on the potential application of new drugs isolated from natural medicinal plants for GC therapy.

Palbociclib induces DNA damage and inhibits DNA repair to induce cellular senescence and apoptosis in oral squamous cell carcinoma

Palbociclib is an FDA-approved cyclin-dependent kinase (CDK) 4/6 inhibitor that has been clinically proven to be effective in breast cancer. However, its use in oral cancer is not well researched. In this study, we investigated the inhibitory activity of palbociclib against oral squamous cell carcinoma (OSCC) cells and explored the mechanism of inhibition. The effects of palbociclib on the cytotoxicity of OSCC cells were determined by MTT and colony formation assays. β-Galactosidase staining and cell-cycle analysis were used to determine palbociclib-induced cellular senescence and apoptosis of OSCC cells. Wound healing and transwell assays were performed to assess the effects of palbociclib treatment on migration and invasion ability of OSCC cells. Whole transcriptome sequencing was conducted to show the relationship between DNA damage repair of OSCC cells and palbociclib treatment. Palbociclib-induced DNA damage and repair capacity of OSCC cells were confirmed by comet assay and immunofluorescence confocal microscopy. Western blotting was used to verify the palbociclib-mediated changes in the CDK/pRB/c-Myc/CDC25A pathway. Finally, invitro findings were tested in a mouse xenograft model. Our results showed that palbociclib can significantly inhibit the growth, migration, and invasive ability of OSCC cells and can accelerate cellular senescence and apoptosis. We found that palbociclib induced DNA damage and p21 expression through the p53-independent pathway, thereby downregulating c-Myc and CDC25A expression to inhibit cell cycle progression. In addition, palbociclib downregulated RAD51 expression to inhibit DNA damage repair ability of OSCC cell. Palbociclib was found to have anti-oral squamous cell carcinoma activity and to simultaneously induce DNA damage and inhibit its repair, and to accelerated cellular senescence and apoptosis.

Artesunate Impairs Growth in Cisplatin-Resistant Bladder Cancer Cells by Cell Cycle Arrest, Apoptosis and Autophagy Induction.

Cisplatin, which induces DNA damage, is standard chemotherapy for advanced bladder cancer (BCa). However, efficacy is limited due to resistance development. Since artesunate (ART), a derivative of artemisinin originating from Traditional Chinese Medicine, has been shown to exhibit anti-tumor activity, and to inhibit DNA damage repair, the impact of artesunate on cisplatin-resistant BCa was evaluated. Cisplatin-sensitive (parental) and cisplatin-resistant BCa cells, RT4, RT112, T24, and TCCSup, were treated with ART (1–100 µM). Cell growth, proliferation, and cell cycle phases were investigated, as were apoptosis, necrosis, ferroptosis, autophagy, metabolic activity, and protein expression. Exposure to ART induced a time- and dose-dependent significant inhibition of tumor cell growth and proliferation of parental and cisplatin-resistant BCa cells. This inhibition was accompanied by a G0/G1 phase arrest and modulation of cell cycle regulating proteins. ART induced apoptos is by enhancing DNA damage, especially in the resistant cells. ART did not induce ferroptosis, but led to a disturbance of mitochondrial respiration and ATP generation. This impairment correlated with autophagy accompanied by a decrease in LC3B-I and an increase in LC3B-II. Since ART significantly inhibits proliferative and metabolic aspects of cisplatin-sensitive and cisplatin-resistant BCa cells, it may hold potential in treating advanced and therapy-resistant BCa.

Alterations of DNA damage repair in cancer: from mechanisms to applications.

DNA damage repair (DDR) pathways are essential to ensure the accurate transmission of genetic material. However, different endogenous and exogenous factors challenge genomic integrity. Mechanisms involved in the alterations of DDR pathways mainly include genetic inactivation and epigenetic mechanisms. The development and progression of carcinomas are closely associated with DDR pathway aberrations, including the epigenetic silencing of gene O6-alkylguanine-DNA methyltransferase (MGMT); deficiencies of mismatch repair (MMR) genes, including MutL homolog 1 (MLH1), MutS protein homologue (MSH)-2 (MSH2), MSH6, and PMS1 homolog 2; the mismatch repair system component (PMS2); and mutations of homologous recombination repair (HRR) genes, such as the breast cancer susceptibility gene 1/2 (BRCA1/2). Understanding the underlying mechanisms and the correlations between alterations to DDR pathways and cancer could improve the efficacy of antitumor therapies. Emerging evidence suggests that survival is higher in patients with DDR-deficient tumors than in those with DDR-proficient tumors. Thus, DDR alterations play a predictive and prognostic role in anticancer therapies. Theoretical studies on the co-administration of DDR inhibitors and other anticancer therapies, including chemotherapy, radiotherapy, immunotherapy, endocrine therapy, and epigenetic drugs, hold promise for cancer treatments. In this review, we focus on the basic mechanisms, characteristics, current applications, and combination strategies of DDR pathways in the anticancer field.

Fucoidan-coated nanoparticles target radiation-induced P-selectin to enhance chemoradiotherapy in murine colorectal cancer.

Colorectal cancer (CRC) is a leading cause of cancer-related death for both men and women, highlighting the need for new treatment strategies. Advanced disease is often treated with a combination of radiation and cytotoxic agents, such as DNA damage repair inhibitors and DNA damaging agents. To optimize the therapeutic window of these multimodal therapies, advanced nanomaterials have been investigated to deliver sensitizing agents or enhance local radiation dose deposition. In this study, we demonstrate the feasibility of employing an inflammation targeting nanoscale metal-organic framework (nMOF) platform to enhance CRC treatment. This novel formulation incorporates a fucoidan surface coating to preferentially target P-selectin, which is over-expressed or translocated in irradiated tumors. Using this radiation stimulated delivery strategy, a combination PARP inhibitor (talazoparib) and chemotherapeutic (temozolomide) drug-loaded hafnium and 1,4-dicarboxybenzene (Hf-BDC) nMOF was evaluated both in vitro and in vivo. Significantly, these drug-loaded P-selectin targeted nMOFs (TT@Hf-BDC-Fuco) show improved tumoral accumulation over multiple controls and subsequently enhanced therapeutic effects. The integrated radiation and nanoformulation treatment demonstrated improved tumor control (reduced volume, density, and growth rate) and increased survival in a syngeneic CRC mouse model. Overall, the data from this study support the continued investigation of radiation-priming for targeted drug delivery and further consideration of nanomedicine strategies in the clinical management of advanced CRC.

Deltex E3 Ubiquitin Ligase 3L confers radioresistance in prostate cancer via Akt pathway

Purpose: To determine the effect of Deltex E3 Ubiquitin Ligase 3L (DTX3L) on the radioresistance of prostate cancer (PCa).Methods: A PCa cell model of radioresistance was established via exposure of cancer cell lines to fractionated radiation. The MTT {(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)} assay and western blotting were performed to evaluate the impact of DTX3L on cell survival and DNA damage repair. The molecular mechanism of action was evaluated by western blotting.Results: DTX3L was elevated in PCa cell lines compared with normal primary prostate epithelial cells (p < 0.01). The survival of PCa cells exposed to radiation was promoted by overexpression of DTX3L, while knockdown of DTX3L abrogated the radioresistance. Moreover, overexpression of DTX3Ldecreased phosphorylation of histone H2AX (γH2AX) and increased Rad51 levels (p < 0.01). However, knockdown of DTX3L reversed the accumulation of γH2AX and Rad51. Phosphorylation of AKT was promoted by DTX3L overexpression, but was reduced by DTX3L knockdown (p < 0.01). Inhibition of AKT (protein kinase B) counteracted the promotion ability of DTX3L on the radioresistance of PCa cells via decreased cell survival ratio, and also inhibited DNA damage repair via accumulation of γ-H2AX and depletion of Rad51 (p < 0.01).Conclusion: DTX3L increases the resistance of prostate cancer to radiotherapy and DNA damage repair in PCa via AKT pathway, indicating a potential therapeutic strategy to overcome radioresistance in PCa.
 Keywords: DTX3L (Deltex E3 Ubiquitin Ligase 3L), DNA damage, Phosphorylation, Radioresistance, AKT, Protein kinase B, Prostate cancer

Factors Associated with the Improvement of Outcomes of High-Risk Relapsed Hodgkin Lymphoma (HL) Patients Receiving High-Dose Chemotherapy (HDC) and Autologous Stem-Cell Transplantation (ASCT): The MD Anderson Cancer Center Experience

Factors Associated with the Improvement of Outcomes of High-Risk Relapsed Hodgkin Lymphoma (HL) Patients Receiving High-Dose Chemotherapy (HDC) and Autologous Stem-Cell Transplantation (ASCT): The MD Anderson Cancer Center Experience

Improving Radiation Response in Glioblastoma Using ECO/siRNA Nanoparticles Targeting DNA Damage Repair.

Simple SummaryGlioblastoma (GBM) is the most common form of brain cancer and among the most lethal of human cancers. Radiation therapy is a mainstay in the standard of care for GBM, killing tumor cells by creating DNA damage. Inhibiting DNA damage repair (DDR) proteins enhances radiation therapy by not allowing tumor cells to repair the DNA damage caused by radiation. The aim of our study was to investigate whether the novel nanoparticle material, ECO, could be used to deliver small interfering RNA (siRNA) to GBM tumor cells and temporarily reduce the production of DDR proteins to improve radiation therapy outcomes. SiRNAs can be designed to target an innumerable number of genes and with the right delivery vehicle can be used in a variety of disease settings. Our work provides support for the use of the novel ECO material for delivery of siRNA in GBM.Radiation therapy is a mainstay in the standard of care for glioblastoma (GBM), thus inhibiting the DNA damage response (DDR) is a major strategy to improve radiation response and therapeutic outcomes. Small interfering RNA (siRNA) therapy holds immeasurable potential for the treatment of GBM, however delivery of the siRNA payload remains the largest obstacle for clinical implementation. Here we demonstrate the effectiveness of the novel nanomaterial, ECO (1-aminoethylimino[bis(N-oleoylcysteinylaminoethyl) propionamide]), to deliver siRNA targeting DDR proteins ataxia telangiectasia mutated and DNA-dependent protein kinase (DNApk-cs) for the radiosensitzation of GBM in vitro and in vivo. ECO nanoparticles (NPs) were shown to efficiently deliver siRNA and silence target protein expression in glioma (U251) and glioma stem cell lines (NSC11, GBMJ1). Importantly, ECO NPs displayed no cytotoxicity and minimal silencing of genes in normal astrocytes. Treatment with ECO/siRNA NPs and radiation resulted in the prolonged presence of γH2AX foci, indicators of DNA damage, and increased radiosensitivity in all tumor cell lines. In vivo, intratumoral injection of ECO/siDNApk-cs NPs with radiation resulted in a significant increase in survival compared with injection of NPs alone. These data suggest the ECO nanomaterial can effectively deliver siRNA to more selectively target and radiosensitize tumor cells to improve therapeutic outcomes in GBM.

EGFR overexpression increases radiotherapy response in HPV-positive head and neck cancer through inhibition of DNA damage repair and HPV E6 downregulation.

High-risk Human Papillomavirus (HPV) infections have recently emerged as an independent risk factor in head and neck squamous cell carcinoma (HNSCC). There has been a marked increase in the incidence of HPV-induced HNSCC subtype, which demonstrates different genetics with better treatment outcome. Despite the favourable prognosis of HPV-HNSCC, the treatment modality, consisting of high dose radiotherapy (RT) in combination with chemotherapy (CT), remains similar to HPV-negative tumours, associated with toxic side effects. Epidermal growth factor receptor (EGFR) is overexpressed in over 80% of HNSCC and correlates with RT resistance. EGFR inhibitor Cetuximab is the only FDA approved targeted therapy for both HNSCC subtypes, however the response varies between HNSCC subtypes. In HPV-negative HNSCC, Cetuximab sensitises HNSCC to RT improving survival rates. To reduce adverse cytotoxicity of CT, Cetuximab has been approved for treatment de-escalation of HPV-positive HNSCC. The results of several recent clinical trials have concluded differing outcome to HPV-negative HNSCC. Here we investigated the role of EGFR in HPV-positive HNSCC response to RT. Remarkably, in HPV-positive HNSCC cell lines and in vivo tumour models, EGFR activation was strongly indicative of increased RT response. In response to RT, EGFR activation induced impairment of DNA damage repair and increased RT response. Furthermore, EGFR was found to downregulate HPV oncoproteinE6 expression and induced p53 activity in response to RT. Collectively, our data uncovers a novel role for EGFR in virally induced HNSCC and highlights the importance of using EGFR-targeted therapies in the context of the genetic makeup of cancer.

Chemoproteomic Profiling of an Ibrutinib Analogue Reveals its Unexpected Role in DNA Damage Repair.

Ibrutinib is an FDA-approved drug to treat B-lymphoid malignancies, which functions mechanistically as a covalent inhibitor for Bruton's tyrosine kinase (BTK). During the course of screening more potent and selective BTK inhibitors, we discovered that MM2-48, an ibrutinib analogue that contains the alkynyl amide functional group in place of the acrylamide warhead, exhibits a much stronger cytotoxicity. Comparative chemoproteomic profiling of the targets of ibrutinib and MM2-48 revealed that the alkynyl amide warhead exhibits much higher reactivity in proteomes. Unexpectedly, MM2-48 covalently targets a functional cysteine in a BRCA2 and CDKN1A-interacting protein, BCCIP, and significantly inhibits DNA damage repair. Our findings suggest that simultaneous inhibition of BTK activity and DNA damage repair might be a more effective therapeutic strategy for combating B-cell malignancies.

Low-Dose Triptolide Enhanced Activity of Idarubicin Against Acute Myeloid Leukemia Stem-like Cells Via Inhibiting DNA Damage Repair Response.

Leukemia stem cells (LSCs) are considered to be the root of relapse for acute myeloid leukemia (AML). Conventional chemotherapeutic drugs fail to eliminate LSCs. Therefore, new therapeutic strategies eliminating LSCs are urgently needed. Our results showed that low-dose Triptolide (TPL) enhanced the anti-AML activity of Idarubicin (IDA) in vitro against LSC-like cells (CD34 + CD38- KG1αand CD34 + CD38- kasumi-1 cells) and CD34+ primary AML cells, while sparing normal cells. Inspiringly, the combination treatment with low-dose TPL and IDA was also effective against CD34 + blasts from AML patients with FLT3-ITD mutation, which is an unfavorable risk factor for AML patients. Moreover, the combination of TPL and IDA induced a remarkable suppression of human leukemia growth in a xenograft mouse model. Mechanistically, the enhanced effect of low dose TPL on IDA against LSCs was attributed to inhibiting DNA damage repair response. Thus, our study may provide a theoretical basis to facilitate the development of a novel LSCs-targeting strategy for AML.Graphical abstract.

Erlotinib enhanced chemoradiotherapy sensitivity via inhibiting DNA damage repair in nasopharyngeal carcinoma CNE2 cells.

Nasopharyngeal carcinoma (NPC), arising from nasopharynx epithelium, is a rare type of malignant carcinoma that has a specific geographical distribution and a high risk of distant metastases. For most of the diagnosed NPC patients, the total survival rate decreased significantly due to the high local recurrence rate and metastasis rate. Concurrent chemoradiotherapy (CCRT), as routine therapy strategy of NPC, usually accompanies with high-dosage cytotoxic agents and serious toxic side reaction. Therefore, there is an urgent need for update the existing therapy strategies. In this study, we sought to investigate the effects of a combined therapy strategy, erlotinib combined with cisplatin and radiotherapy, on biological characteristics of NPC CNE2 cells and the potential reasons. CNE2 cells in logarithmic phase seeding in 96-well plates received concentration gradients of erlotinib (at 0, 10, 20, 40, 80, 160, 320 mmol/L) or cisplatin (at 0, 0.25, 0.5, 1, 2, 4, 8 mg/L), in order to obtain the optimal working concentration of erlotinib and cisplatin via 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Then, cells were divided into control group and four treatment groups (Group I-IV). All treatment groups received irradiation of 4 Gy, moreover, Group II-IV respectively received erlotinib, cisplatin and erlotinib plus cisplatin at optimal working concentration. After 24 and 48 h of irradiation, growth inhibition rate was determined; invasion ability and migration ability was respectively detected by Boyden's chamber assay and cell scratch test; flow cytometry was performed for determining apoptosis rate and cell cycle distribution; the expression levels of epidermal growth factor receptor (EGFR) signal pathway proteins were semi-quantitatively analyzed by Western-blot. Compared with Group I, all the other treatment groups showed better inhibition effect on cell viability, invasion and migration ability and higher apoptosis rate, while Group IV showed the strongest growth inhibition effect and highest apoptosis rate. In addition, EGFR signal pathway proteins of Group IV showed the lowest expression level. In combined therapy with radiotherapy/chemotherapy, erlotinib could enhance radiotherapy/chemotherapy sensitivity, probably because it could suppress DNA damage repair after radiotherapy/chemotherapy, thus weakening radiotherapy/chemotherapy resistance of tumor cells.

Low dose novel PARP-PI3K inhibition via nanoformulation improves colorectal cancer immunoradiotherapy.

Multimodal therapy is often used in oncology to overcome dosing limitations and chemoresistance. Recently, combination immunoradiotherapy has shown great promise in a select subset of patients with colorectal cancer (CRC). Furthermore, molecularly targeted agents delivered in tandem with immunotherapy regimens have been suggested to improve treatment outcomes and expand the population of responding patients. In this study, radiation-sensitizing small molecules niraparib (PARP inhibitor) and HS-173 (PI3K inhibitor) are identified as a novel combination that synergistically enhance toxicity and induce immunogenic cell death both in vitro and in vivo in a CRC model. These inhibitors were co-encapsulated in a polymer micelle to overcome solubility limitations while minimizing off-target toxicity. Mice bearing syngeneic colorectal tumors (CT26) were administered these therapeutic micelles in combination with X-ray irradiation and anti-CTLA-4 immunotherapy. This combination led to enhanced efficacy demonstrated by improved tumor control and increased tumor infiltrating lymphocytes. This report represents the first investigation of DNA damage repair inhibition combined with radiation to potentiate anti-CTLA-4 immunotherapy in a CRC model.

Biomimetic carrier-free nanoparticle delivers digoxin and doxorubicin to exhibit synergetic antitumor activity in vitro and in vivo

We previously reported that digoxin could enhance the antitumor effect of the DNA damaging agent doxorubicin by inhibiting DNA damage repair (DDR). Digoxin has a narrow therapeutic window, and doxorubicin often causes toxicity in organs such as the liver and heart. Therefore, we recently designed a biomimetic carrier-free nanoparticle system containing digoxin and doxorubicin to increase the antitumor efficacy further while reducing side effects, via specifically targeting tumor cells. In this system, the DDR inhibitor digoxin and the DNA damage agent doxorubicin were combined to formulate carrier-free nanoparticles (DDNPs), which were further embedded into the NSCLC cell line A549 cell membrane camouflaged framework (termed as DDMNPs). The DDMNPs were specifically internalized into A549 cells but not to other cells such as prostate cancer DU145 and macrophage RAW264.7 cells. Compared with the free drug combination of digoxin and doxorubicin (DIG + DOX) or simple DDNPs, the biomimetic DDMNP system displayed amplified synergistic therapeutic effects by causing cell apoptosis and enhancing DNA damage, probably due to the increased expression of DNA damage markers γH2AX and Replication protein A (RPA), and simultaneously suppressed mRNA and protein expression of DDR proteins. Consistently, more potent in vivo antitumor efficacy was demonstrated in zebrafish and nude mouse A549 xenograft models. Additionally, while the free drug combination (DIG + DOX) caused liver toxicity, as shown by histological analysis, DDNPs and biomimetic DDMNPs did not, suggesting improved safety. In conclusion, the codelivery of a DDR inhibitor and a DNA damaging agent via biomimetic carrier-free nanoparticles may become an innovative strategy for cancer chemotherapy, while more preclinical and clinical evidence is required.

CircRNA inhibits DNA damage repair by interacting with host gene

BackgroundDeregulated circular RNAs (circRNAs) are associated with the development of cancer and therapy resistance. However, functional research of circRNAs mostly focus on potential miRNA or protein binding and more potential regulation of circRNA on host gene DNA in cancers are yet to be inspected.MethodWe performed total RNA sequencing on clinical breast cancer samples and identified the expression patterns of circRNAs and corresponding host genes in patient blood, tumor and adjacent normal tissues. qPCR, northern blot and in situ hybridization were used to validate the dysregulation of circRNA circSMARCA5. A series of procedures including R-loop dot-blotting, DNA-RNA immunoprecipitation and mass spectrum, etc. were conducted to explore the regulation of circSMARCA5 on the transcription of exon 15 of SMARCA5. Moreover, immunofluorescence and in vivo experiments were executed to investigate the overexpression of circSMARCA5 with drug sensitivities.ResultsWe found that circRNAs has average higher expression over its host linear genes in peripheral blood. Compared to adjacent normal tissues, circSMARCA5 is decreased in breast cancer tissues, contrary to host gene SMARCA5. The enforced expression of circSMARCA5 induced drug sensitivity of breast cancer cell lines in vitro and in vivo. Furthermore, we demonstrated that circSMARCA5 can bind to its parent gene locus, forming an R-loop, which results in transcriptional pausing at exon 15 of SMARCA5. CircSMARCA5 expression resulted in the downregulation of SMARCA5 and the production of a truncated nonfunctional protein, and the overexpression of circSMARCA5 was sufficient to improve sensitivity to cytotoxic drugs.ConclusionOur results revealed a new regulatory mechanism for circRNA on its host gene and provided evidence that circSMARCA5 may serve as a therapeutic target for drug-resistant breast cancer patients.

Abstract 5355: MSLN-targeted thorium-227 conjugate demonstrates increased antitumor activity in combination with bevacizumab and regorafenib

Abstract Mesothelin-targeted thorium-227 conjugate (MSLN-TTC) is a targeted alpha therapeutic consisting of a MSLN-targeting antibody anetumab covalently attached to a 3,2-HOPO chelator complexed with the alpha emitter thorium-227. It has potent in vivo activity in cell and patient-derived xenograft (PDX) models in monotherapy and in combination with DNA damage repair inhibitors. We evaluated the antitumor efficacy of MSLN-TTC in a mesothelin overexpressing ovarian PDX model (ST206B) in monotherapy and in combination bevacizumab or the multikinase inhibitor regorafenib which both have anti-angiogenic activity. The biodistribution of MSLN-TTC was studied in parallel. Athymic mice were transplanted s.c. with ST206B-derived tumor fragments and treated with MSLN-TTC (250 kBq/kg, Q7Dx2, 0.43 mg/kg) alone or in combination with bevacizumab (1.25 or 5 mg/kg, i.p., Q5D; or 5 mg/kg, single dose) or regorafenib (7.5, 15, or 30 mg/kg, QD, p.o. for 28 days). The biodistribution of the respective treatment groups was analyzed using a germanium detector. For molecular analyses, tumors were stained for γ-H2AX, Ki67, CD31 and α-SMA. MSLN-TTC monotherapy showed high in vivo potency at 2 × 250 kBq/kg with a Treated/Control (T/C) ratio of 0.28 (n=5) on day 29. Monotherapy treatment with bevacizumab and regorafenib showed dose-dependent antitumor activity with T/C ratios ranging from 0.31 (regorafenib 30 mg/kg) to 0.33 (bevacizumab 5 mg/kg, single dose). The in vivo efficacy of MSLN-TTC was further enhanced by combination therapy with bevacizumab (T/C 0.21; n=5) or regorafenib (T/C 0.15; n=5). Combination of MSLN-TTC and regorafenib (30 mg/kg) resulted in more complete and partial responses (3/4 CRs; 1/4 PR) as compared to MSLN-TTC (2/3 PRs; 1/3 PD) at day 141. All treatments were well-tolerated based on body weight assessment; a reversible myelosuppression of white blood cells was observed for MSLN-TTC monotherapy and combination with bevacizumab or regorafenib did not worsen it. In the biodistribution study, tumor-specific accumulation of MSLN-TTC was observed over 336 h (14 days) with lower accumulation in the MSLN-TTC + bevacizumab group (1.25 mg/kg, Q5D, p<0.05; 5 mg/kg, single dose, p<0.05). On the molecular level, MSLN-TTC- and MSLN-TTC/ regorafenib (15 and 30 mg/kg) -treated tumors showed an increase in the DNA damage marker γ-H2AX compared to vehicle. The endothelial cell marker CD31 was decreased in tumors treated with MSLN-TTC and bevacizumab (1.25 mg/kg, Q5D; 5 mg/kg, single dose), but not in MSLN-TTC monotherapy (p<0.05). In summary, MSLN-TTC effectively reduced the growth of primary tumors in the ST206B ovarian cancer model which was further enhanced by addition of bevacizumab and regorafenib. Further studies are needed to investigate the underlying molecular mechanisms. Citation Format: Urs B. Hagemann, Christine Ellingsen, Claudia Kamfenkel, Dieter Zopf, Jesper Fonslet, Carsten Nielsen, Hartwig Hennekes, Alan S. Cuthbertson, Dominik Mumberg. MSLN-targeted thorium-227 conjugate demonstrates increased antitumor activity in combination with bevacizumab and regorafenib [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 5355.

Abstract 4198: Role of receptor tyrosine kinase AXL in mediating DNA damage response

Abstract AXL is a receptor tyrosine kinase that is often overexpressed in many cancers. It contributes to tumor progression, metastasis and drug resistance through activating downstream signaling cascades, making it an emerging therapeutic target. Moreover, the first-in-class AXL inhibitor R428 (BGB321) was lately approved by the FDA for the treatment of relapsed or refractory acute myeloid leukemia. Previously, R428 (BGB321) was also reported to show selective sensitivity towards ovarian cancers with a Mesenchymal molecular subtype. Recently, a novel role of AXL in the regulation of DNA damage responses has been brought to light. Here, we further explored AXL's role in mediating DNA damage responses by using ovarian cancer as a disease model. AXL inhibition using R428 resulted in the increase DNA damage foci in ovarian cancer cells SKOV3 and HeyA8. This occurred concurrently with the upregulation of classic DNA damage response signaling molecules such as γH2Ax, ATM and ATR. RNA sequencing results further validated this observation, that R428-treated cells showed downregulation of genes involved in cell cycle control and DNA replication, suggesting the decrease in proliferation. Through SILAC co-immunoprecipitation mass spectrometry, we uncovered a novel binding partner of AXL that could potentially regulate the DNA damage repair upon the inhibition of AXL, giving us insights to how AXL inhibition might contribute to DNA damage. Furthermore, our results showed that AXL inhibition rendered cells more sensitive to the inhibition of ATR, a crucial mediator for replication stress, paving ways to the rationale for potential combinatory use of AXL and DNA damage repair inhibitors. Citation Format: Xun Hui Yeo, Wai Leong Tam, Ruby Yun-Ju Huang. Role of receptor tyrosine kinase AXL in mediating DNA damage response [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 4198.

Abstract 6171: High throughput screening identified new radiosensitizers which inhibit DNA damage repair pathway

Abstract Radiotherapy (RT) plays a crucial role in the treatment of cervical cancer. However, the efficacy of existing radiosensitizers in clinical applications is limited. The aim of this study was to established an efficient method of identifying new radiosensitizers and to investigated the specific mechanisms when used in combination with RT. An effective platform for radiosensitizers discovery was created based on high-throughput screening (HTS) technology. We established an automated and powerful HTS approach to identify potent radiosensitizers from the LOPAC1280 chemical library. We used the Geldanamycin for validation of our HTS method, which had been known as a radiosensitizer for cervical cancer cells. Z'-score of our HTS platform was 0.6. From 1280 compounds, we identified four hit compounds. Among them, we found Dihydroouabain (DHO) and Ro90-7501 which have not been previously known as radiosensitizers. Clonogenic survival assay showed that DHO and Ro90-7501 significantly enhanced radiosensitivity in cervical cancer cells. Western blot analysis showed that DHO inhibited the phosphorylation of Chk1, and Ro90-7501 inhibited the phosphorylation of ATM. These results indicated that DHO and Ro90-7501 inhibited DNA damage response pathway. In conclusion, we established the HTS method for radiosensitizer screening, and found DHO and Ro90-7501 as new radiosensitizers targeting DNA damage repair pathway. Citation Format: Keisuke Tamari, Zhihao Li, Hideki Matsutani, Yuji Seo, Yutaka Takahashi, Kazumasa Minami, Fumiaki Isohashi, Kazuhiko Ogawa. High throughput screening identified new radiosensitizers which inhibit DNA damage repair pathway [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 6171.

Synthetic lethality strategies: Beyond BRCA1/2 mutations in pancreatic cancer.

Cancer cells are often characterized by abnormalities in DNA damage response including defects in cell cycle checkpoints and/or DNA repair. Synthetic lethality between DNA damage repair (DDR) pathways has provided a paradigm for cancer therapy by targeting DDR. The successful example is that cancer cells with BRCA1/2 mutations are sensitized to poly(adenosine diphosphate [ADP]‐ribose)polymerase (PARP) inhibitors. Beyond the narrow scope of defects in the BRCA pathway, “BRCAness” provides more opportunities for synthetic lethality strategy. In human pancreatic cancer, frequent mutations were found in cell cycle and DDR genes, including P16, P73, APC, MLH1, ATM, PALB2, and MGMT. Combined DDR inhibitors and chemotherapeutic agents are under preclinical or clinical trials. Promoter region methylation was found frequently in cell cycle and DDR genes. Epigenetics joins the Knudson's “hit” theory and “BRCAness.” Aberrant epigenetic changes in cell cycle or DDR regulators may serve as a new avenue for synthetic lethality strategy in pancreatic cancer.