Regulation Of DNA Damage Response Research Articles

FAM135B sustains the reservoir of Tip60-ATM assembly to promote DNA damage response.

BackgroundRecently, the mechanism by which cells adapt to intrinsic and extrinsic stresses has received considerable attention. Tat‐interactive protein 60‐kDa/ataxia–telangiectasia‐mutated (TIP60/ATM) axis‐mediated DNA damage response (DDR) is vital for maintaining genomic integrity.MethodsProtein levels were detected by western blot, protein colocalisation was examined by immunofluorescence (IF) and protein interactions were measured by co‐immunoprecipitation, proximity ligation assay and GST pull‐down assays. Flow cytometry, comet assay and IF assays were used to explore the biological functions of sequence similarity 135 family member B (FAM135B) in DDR. Xenograft tumour, FAM135B transgenic mouse models and immunohistochemistry were utilised to confirm in vitro observations.ResultsWe identified a novel DDR regulator FAM135B which could protect cancer cells from genotoxic stress in vitro and in vivo. The overexpression of FAM135B promoted the removal of γH2AX and 53BP1 foci, whereas the elimination of FAM135B attenuated these effects. Consistently, our findings revealed that FAM135B could promote homologous recombination and non‐homologous end‐joining repairs. Further study demonstrated that FAM135B physically bound to the chromodomain of TIP60 and improved its histone acetyltransferase activity. Moreover, FAM135B enhanced the interactions between TIP60 and ATM under resting conditions. Intriguingly, the protein levels of FAM135B dramatically decreased following DNA damage stress but gradually increased during the DNA repair period. Thus, we proposed a potential DDR mechanism where FAM135B sustains a reservoir of pre‐existing TIP60‐ATM assemblies under resting conditions. Once cancer cells suffer DNA damage, FAM135B is released from TIP60, and the functioning pre‐assembled TIP60‐ATM complex participates in DDR.Conclusions: We characterised FAM135B as a novel DDR regulator and further elucidated the role of the TIP60‐ATM axis in response to DNA damage, which suggests that targeting FAM135B in combination with radiation therapy or chemotherapy could be a potentially effective approach for cancer treatment.

The role of USP51 in attenuating chemosensitivity of lung cancer cells to cisplatin by regulating DNA damage response.

Lung cancer is the most frequent type of cancer affecting both men and women globally, and it is associated with a high mortality rate. It is clinically treated with cisplatin, a platinum-based drug that works by generating DNA lesions, which activates DNA damage response and induces cell death. However, chemoresistance by cancer cells limits the clinical usefulness of cisplatin as an anticancer drug. Here, we uncovered a role of ubiquitin-specific protease 51 (USP51) in the chemosensitivity of lung cancer cells to cisplatin by regulating DNA damage response. USP51 was more upregulated in lung cancer tissues of chemotherapy-resistant patients than those of chemotherapy-sensitive patients with adjacent, nontumor tissues. USP51 overexpression in lung cancer cells in vitro reduced γ-H2AX formation and promoted checkpoint kinase 1 (CHK1) phosphorylation, whereas USP51 knockdown showed opposite effects, indicating that USP51 played an important role in promoting DNA damage repair. Finally, USP51 knockdown weakened cisplatin resistance in A549/DDP cells and significantly suppressed tumor growth in vivo, suggesting that a USP51 inhibitor combined with cisplatin may be considered as an effective treatment strategy to eliminate drug-resistant lung cancer cells.

DNA Damage Response Regulation by Histone Ubiquitination.

Cells are constantly exposed to numerous genotoxic stresses that induce DNA damage. DNA double-strand breaks (DSBs) are among the most serious damages and should be systematically repaired to preserve genomic integrity. The efficiency of repair is closely associated with chromatin structure, which is regulated by posttranslational modifications of histones, including ubiquitination. Recent evidence shows crosstalk between histone ubiquitination and DNA damage responses, suggesting an integrated model for the systematic regulation of DNA repair. There are two major pathways for DSB repair, viz., nonhomologous end joining and homologous recombination, and the choice of the pathway is partially controlled by posttranslational modifications of histones, including ubiquitination. Histone ubiquitination changes chromatin structure in the vicinity of DSBs and serves as a platform to select and recruit repair proteins; the removal of these modifications by deubiquitinating enzymes suppresses the recruitment of repair proteins and promotes the convergence of repair reactions. This article provides a comprehensive overview of the DNA damage response regulated by histone ubiquitination in response to DSBs.

Model-based translation of DNA damage signaling dynamics across cell types

Interindividual variability in DNA damage response (DDR) dynamics may evoke differences in susceptibility to cancer. However, pathway dynamics are often studied in cell lines as alternative to primary cells, disregarding variability. To compare DDR dynamics in the cell line HepG2 with primary human hepatocytes (PHHs), we developed a HepG2-based computational model that describes the dynamics of DDR regulator p53 and targets MDM2, p21 and BTG2. We used this model to generate simulations of virtual PHHs and compared the results to those for PHH donor samples. Correlations between baseline p53 and p21 or BTG2 mRNA expression in the absence and presence of DNA damage for HepG2-derived virtual samples matched the moderately positive correlations observed for 50 PHH donor samples, but not the negative correlations between p53 and its inhibitor MDM2. Model parameter manipulation that affected p53 or MDM2 dynamics was not sufficient to accurately explain the negative correlation between these genes. Thus, extrapolation from HepG2 to PHH can be done for some DDR elements, yet our analysis also reveals a knowledge gap within p53 pathway regulation, which makes such extrapolation inaccurate for the regulator MDM2. This illustrates the relevance of studying pathway dynamics in addition to gene expression comparisons to allow reliable translation of cellular responses from cell lines to primary cells. Overall, with our approach we show that dynamical modeling can be used to improve our understanding of the sources of interindividual variability of pathway dynamics.

A Non-Cell-Autonomous Mode of DNA Damage Response in Soma of Caenorhabditis elegans.

Life has evolved a mechanism called DNA damage response (DDR) to sense, signal and remove/repair DNA damage, and its deficiency and dysfunction usually lead to genomic instability and development of cancer. The signaling mode of the DDR has been believed to be of cell-autonomy. However, the paradigm is being shifted with in-depth research into model organism Caenorhabditis elegans. Here, we mainly investigate the effect of DDR activation on the radiosensitivity of vulva of C. elegans, and first found that the vulval radiosensitivity is mainly regulated by somatic DDR, rather than the DDR of germline. Subsequently, the worm lines with pharynx-specific rescue of DDR were constructed, and it is shown that the 9-1-1-ATR and MRN-ATM cascades in pharynx restore approximately 90% and 70% of vulval radiosensitivity, respectively, through distantly regulating the NHEJ repair of vulval cells. The results suggest that the signaling cascade of DDR might also operate in a non-cell autonomous mode. To further explore the underlying regulatory mechanisms, the cpr-4 mutated gene is introduced into the DDR-rescued worms, and CPR-4, a cysteine protease cathepsin B, is confirmed to mediate the inter-tissue and inter-individual regulation of DDR as a signaling molecule downstream of 9-1-1-ATR. Our findings throw some light on the regulation of DNA repair in soma of C. elegans, and might also provide new cues for cancer prevention and treatment.

Abstract 5493: Activity of RP12146, a novel, selective, and potent small molecule inhibitor of PARP 1/2, in solid tumors

Abstract Introduction: The Poly (ADP-ribose) polymerase (PARP) family plays an important role in the regulation of transcription, apoptosis and DNA damage response. Several PARP inhibitors have been approved for the treatment of BRCA-mutated ovarian, breast and pancreatic cancer. Through ‘synthetic lethality’, the applicability PARP inhibitors can be expanded to cancers beyond the well-defined BRCA defects either as monotherapy or in combination with established therapy across various solid tumor types. Herein, we describe the efficacy of RP12146 as a single agent and in combination with approved therapies in preclinical models of solid tumors. Methods: Enzymatic potency was evaluated using a PARP Chemiluminescent Activity Assay Kit (BPS biosciences). Cell growth was determined following incubation with RP12146 as a single agent or in combination with approved agents in various solid tumor cell lines. Apoptosis was evaluated following incubation of cell lines with compound for 48 or 72 h, subsequent staining with Annexin-V-PE and 7-AAD, and analysis by flow cytometry. Anti-tumor potential of RP12146 as a single agent or in combination with chemotherapeutic agents was tested in OVCAR-3 and NCI-H69 Xenografts. Expression of downstream PAR, PARP-trapping, and cleaved PARP expression were determined in cell lines and xenograft tumor samples by Western blotting. Plasma and tumor concentration of RP12146 in NCI-H69 xenograft samples were also determined. Results: RP12146 demonstrated equipotent inhibition of PARP1 (0.6 nM) and PARP2 (0.5 nM) with several fold selectivity over the other members of the PARP family. Compound caused a dose-dependent growth inhibition of both BRCA mutant and non-mutant cancer cell lines with GI50 in the range of 0.04 µM to 9.6 µM. Combination of RP12146 with Temozolomide, Topotecan, Lurbinectedin, and Abemaciclib demonstrated either additive or synergistic effects manifested by an inhibition in growth of solid tumor cell lines. RP12146 exhibited anti-tumor potential with TGI of 28% and 21.8% as a single agent in OVCAR-3 and NCI-H69 Xenograft model respectively. In NCI-H69 xenograft model, RP12146 in combination with cisplatin exhibited TGI of 69.3 %. Conclusion: Data demonstrate the therapeutic potential of RP12146 as single agent and in combination in solid tumors. RP12146 is currently being evaluated in Phase 1 clinical trials in patients with locally advanced or metastatic solid tumors (NCT05002868). Citation Format: Srikant Viswanadha, Satyanarayana Eleswarapu, Kondababu Rasamsetti, Haritha Polimati, Sridhar Veeraraghavan, Swaroop Vakkalanka. Activity of RP12146, a novel, selective, and potent small molecule inhibitor of PARP 1/2, in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5493.

Abstract 1951: Circulating tumor cell-derived explant models reveal DNA damage response-based therapeutic opportunities in non-small cell lung cancer

Abstract Background: DNA damage and genomic instability contribute to non-small cell lung cancer (NSCLC) etiology and progression. However, their therapeutic exploitation is disappointing. CTC-derived eXplants (CDX) offer systems for mechanistic investigation of CTC metastatic potency and biology-driven therapeutic testings. We perform in-depth molecular and functional characterization of CDX models and demonstrate that targeting defects in the DNA damage response (DDR) and genome integrity regulators impedes CTC-driven metastasis in NSCLC. Methods: CTCs were enriched from 30 mL blood samples of 56 advanced NSCLC patients and implanted subcutaneously into Nod/Scid-IL2Rγ-/- (NSG) mice. Tumors were palpable within a median of 108 days. Among the four CDX models established, three CDX-derived cell lines (GR-CDXL1, GR-CDXL3, GR-CDXL4) were obtained. CDX and cell lines were characterized by immunofluorescence (IF), immunohistochemistry and whole-exome sequencing (WES). Chromosomal instability (CIN) and DDR activity were evaluated by IF and western blot. Gene expression was quantified by qRT-PCR. Tumorigenic potential of CDX-derived cell lines was assessed in the chick embryo chorioallantoic membrane and NSG mice engrafted intravenously. IC50 was assessed using CellTiter-Glo®. Results: Four CDX models and three CDX-derived cell lines were established from NSCLC CTCs and recapitulated patient tumor histology (available for three patients) and response to platinum-based chemotherapy. WES analysis showed considerable mutational landscape similarity between the CDX (GR-CDXL1, GR-CDXL2, GR-CDXL3, GR-CDXL4), corresponding patient tumor biopsy and/or single CTCs. Truncal alterations in key DDR and genome integrity-related genes were prevalent across models and assessed as therapeutic targets in vitro, in ovo and in vivo. GR-CDXL1 presented homologous recombination deficiency linked to bi-allelic BRCA2 mutation, FANCA deletion and unrepaired DNA lesions post-mitosis. GR-CDXL1 cells were sensitive to PARP inhibitor (PARPi) olaparib, despite chemoresistance, which challenges the current clinical hypothesis claiming that chemosensitive NSCLC patients should respond to PARPi. Targeting CIN through centrosome clustering inhibition in GR-CDXL3 impeded tumor growth in ovo and in vivo. In GR-CDXL4, olaparib sensitivity was dictated by SLFN11 overexpression, which also correlated with increased neuroendocrine marker expression at patient disease progression, suggesting a predictive value of SLFN11 in histological transformation of NSCLC into SCLC. Conclusion: This study unravels distinct DDR profiles as a central mechanism underpinning CTC metastatic potency. Our CDX models provide a robust platform for ex vivo drug testing of DDR-targeted strategies to expand patient categories that may benefit from precision medicine in metastatic NSCLC. Citation Format: Tala Tayoun, Vincent Faugeroux, Marianne Oulhen, Olivier Deas, Judith Michels, Laur Brulle-Soumare, Stefano Cairo, Jean-Yves Scoazec, Virginie Marty, Agathe Aberlenc, David Planchard, Jordi Remon, Santiago Ponce, Benjamin Besse, Patricia Kannouche, Jean-Gabriel Judde, Patrycja Pawlikowska, Françoise Farace. Circulating tumor cell-derived explant models reveal DNA damage response-based therapeutic opportunities in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1951.

Abstract 1918: A novel computational framework predicts synthetic lethal interactions between key regulators of the DNA damage response and chromatin modifiers

Abstract The concept of synthetic lethality (SL) has made a pivotal impact for the development of the anti-cancer drug olaparib, the first approved agent targeting the DNA Damage Response (DDR). Typically, SL is described as the interaction of two genes, whereby simultaneous inactivation of both genes results in cell death whereas loss of one gene can be tolerated. Given the importance of SL to develop highly selective anti-cancer therapeutics, large efforts have been undertaken to identify these interactions, both experimentally and computationally. Here, we present a novel computational framework harnessing large-scale cell line gene inactivation screens (DepMap, Project Score), as well as patient data (TCGA), to discover known and novel SL gene pairs. Overall, we implemented six statistical tests considering gene dependency scores, genomic profiles, gene expression and patient survival as parameters. We further utilized data from public drug screening consortia to validate our top-ranking pairs. We applied our framework to a defined target space covering genes relating to DDR, chromatin binding, cell cycle and druggable genes (overall > 2.5 M. pairs were tested). When focusing on SL partners for three DDR genes with promising inhibitors in early clinical development: ATM, ATR and DNA-PK; we noticed that chromatin modifiers were enriched in the top ranking pairs. In particular, SL interactions were predicted with histone (de)methylases, histone (de)acetyltransferases and members of SWI/SNF family. For instance, we observed that loss of function mutations in several members of the KMT2 family, also known as MLL family, would render cancers cells more dependent on ATR or ATM. Similarly, drug sensitivity was increased for selected DDR inhibitors in cell lines with KMT2 mutations. Members of the KMT2 family play essential roles in transcription, but have recently also been shown to be recruited to DNA damage sites and may be mediators for PARP inhibitor sensitivity. The KMT2 family is frequently mutated in several cancers types such as endometrial and bladder, thus underpinning their suitability as potential selection biomarkers as well as relevance to cancer. Furthermore, we observed that patients with mutations in EP300, a histone lysine acetyl transferase, exhibited an increase in ATR expression which may comprise a compensatory mechanism. In addition, patients with ATR and EP300 double mutation tend to have a better probability of survival, suggesting decreased tumor fitness. In summary, we present a modular framework to predict SL gene pairs to a defined target space. Here, we focused on discovering novel SL relationships for the key DDR regulators ATM, ATR and DNA-PK. Our results provide not only new biomarker hypotheses for further validation, but also suggest that cancers with a high mutation rate in chromatin modifying genes may be efficiently targeted by DDRi. Citation Format: Anna M. Coenen-Stass, Magda Markowska, Magdalena Budzinska-Zaniewska, Krzysztof Kolmus, Ewa Szczurek, Eike Staub. A novel computational framework predicts synthetic lethal interactions between key regulators of the DNA damage response and chromatin modifiers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1918.

Abstract 5467: NTX301 targets platinum resistant ovarian cancer

Abstract Background: The majority of patients with ovarian cancer (OC) experience tumor relapse and develop chemoresistant tumors, which are fatal. Epigenetic alterations, especially DNA methylation, result in silencing of tumor suppressor and differentiation-associated genes, confer chemoresistance and are linked to stemness. Although our and others’ previous studies demonstrated that hypomethylating agents (HMA) could re-sensitize OC cells to chemotherapy, translation to the clinic has been slow due the poor stability and bioavailability of existing HMAs. Novel agents are critically needed. Methods: NTX 301 is a novel, highly potent and orally bioavailable HMA, in early clinical development, provided by PinotBio. Here, we assessed its anti-tumor effects in OC models by using cell proliferation, stemness assays, and RNA sequencing and validation. Results: OC cells (SKOV3, IC50=5.089nM; OVCAR4, IC50=29.68nM; and OVCAR5 IC50=3.664nM) were highly sensitive to NTX301 (p<0.05) compared to immortalized fallopian tube epithelial cells (FT-190) (IC50=103.3nM). Treatment with NTX301 induced more significant downregulation of the DNA methyltransferases (DNMTs) 1-3 expression in SKOV3 and OVCAR5 cells compared with decitabine (p<0.05). Treatment with low dose NTX301 (100nM) reset the transcriptome of OC cells, inducing about 15,000 differentially expressed genes (DEGs) compared to DMSO (P<0.05). Gene Ontology Enrichment analysis classified upregulated DEGs enriched in biological processes related to cellular response to DNA damage stimulus, DNA repair, positive regulation of cell cycle process; down-regulated DEGs enriched in processes of regulation of alcohol, cholesterol, and fatty acid biosynthesis and regulation of cell migration. GO analysis indicated NTX301 down-regulated molecular functions related to aldehyde dehydrogenase (ALDH) and oxidoreductase activity, known features of cancer stem cells (CSCs). Indeed, treatment with low dose NTX301 (100nM, 4 days) reduced ALDH(+) cell population, inhibited their self-renewal ability, and repressed the stemness-related transcription factor, Sox2 (P<0.05). We next tested the effects NTX301 in cisplatin (CDDP) resistant OC cells, developed by repeated treatments with CDDP. Priming with NTX301 reduced the IC50 (~two-fold) to CDDP in OVCAR5_CDDP (5.43μM vs. 9.79μM) and OVCAR4_CDDP (2.19μM vs. 9.29μM) resistant cells (p<0.05). NTX301 with CDDP induced phospho-γH2AX and cleaved caspase 3. To discover the mechanisms by which NTX301 restored chemosensitivity, we measured its effects on Schlafen-11 (SLFN11), a DNA damage response regulator silenced through methylation. SLFN11 expression was repressed in CDDP resistant compared to parental cells (P<0.05) and treatment with NTX301 induced its re-expression (P<0.05). Conclusions: Our data indicate that NTX301 is a potent HMA which targets ovarian CSCs and re-sensitizes OC cells to chemotherapy, supporting its further development in OC. Citation Format: Yinu Wang, Xiaolei Situ, Horacio Cardenas, Daniela Matei. NTX301 targets platinum resistant ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5467.

Kif4A mediates resistance to neoadjuvant chemoradiotherapy in patients with advanced colorectal cancer via regulating DNA damage response.

More and more patients with advanced colorectal cancer (CRC) have benefited from surgical resection or ablation following neoadjuvant chemoradiotherapy (nCRT), but nCRT may be ineffective and have potential risks to some patients. Therefore, it is necessary to discover effective biomarkers for predicting the nCRT efficacy in CRC patients. Chromokinesin Kif4A plays a critical role in mitosis, DNA damage repair and tumorigenesis, but its relationship with nCRT efficacy in advanced CRC remains unclear. Here, we find that Kif4A expression in pretreated tumor tissue is positively correlated with poorer tumor regression after receiving nCRT ( P=0.005). Knockdown of endogenous Kif4A causes an increased sensitivity of CRC cells to chemotherapeutic drugs 5-fluorouracil (5-FU) and Cisplatin (DDP), while overexpression of Kif4A enhances resistance of CRC cells to the chemotherapeutic drugs. Furthermore, depending on its motor domain and tail domain, Kif4A regulates DNA damage response (DDR) induced by 5-FU or DDP treatment in CRC cells. In conclusion, we demonstrate that Kif4A may be a potential independent biomarker for predicting the nCRT efficacy in advanced CRC patients, and Kif4A regulates chemosensitivity of CRC cells through controlling DDR.

The impact of genetic aberrations on response to radium-223 treatment for castration-resistant prostate cancer with bone metastases.

Radium (Ra)-223 is an established treatment option for patients with metastatic castrate-resistant prostate cancer (mCRPC) who have symptomatic bone metastases without soft tissue disease. Studies have indicated genetic aberrations that regulate DNA damage response (DDR) in prostate cancer can increase susceptibility to treatments such as poly ADP-ribose polymerase inhibitors and platinum-based therapies. This study aims to evaluate mCRPC response to Ra-223 stratified by tumor genomics. This is a retrospective study of mCRPC patients who received Ra-223 and genetic testing within the Mayo Clinic database (Arizona, Florida, and Minnesota) and Tulane Cancer Center. Patient demographics, genetic aberrations, treatment responses in terms of alkaline phosphatase (ALP) and prostate-specific antigen (PSA), and survival were assessed. Primary end points were ALP and PSA response. Secondary end points were progression-free survival (PFS) and overall survival (OS) from time of first radium treatment. One hundred and twenty-seven mCRPC patients treated with Ra-223 had germline and/or somatic genetic sequencing. The median age at time of diagnosis and Ra-223 treatment was 61.0 and 68.6 years, respectively. Seventy-nine (62.2%) had Gleason score ≥ 8 at time of diagnosis. 50.4% received prior docetaxel, and 12.6% received prior cabazitaxel. Notable alterations include TP53 (51.7%), BRCA 1/2 (15.0%), PTEN (13.4%), ATM (11.7%), TMPRSS2-ERG (8.2%), RB deletion (3.4%), and CDK12 (1.9%). There was no significant difference in ALP or PSA response among the different genetic aberrations. Patients with a TMPRSS2-ERG mutation exhibited a trend toward lower OS 15.4 months (95% confidence interval [CI] 10.0-NR) versus 26.8 months (95% CI 20.9-35.1). Patients with an RB deletion had a lower PFS 6.0 months (95% CI 1.28-NR) versus 9.0 months (95% CI 7.3-11.1) and a lower OS 13.9 months (95% CI 5.2-NR) versus 26.5 months (95% CI 19.8-33.8). Among mCRPC patients treated with Ra-223 at Mayo Clinic and Tulane Cancer Center, we did not find any clear negative predictors of biochemical response or survival to treatment. TMPRSS2-ERG and RB mutations were associated with a worse OS. Prospective studies and larger sample sizes are needed to determine the impact of genetic aberrations in response to Ra-223.

A phase I trial of elimusertib in combination with cisplatin or with cisplatin plus gemcitabine in advanced solid tumors with an emphasis on urothelial carcinoma.

TPS3174 Background: Cisplatin, a well-established backbone of combination therapy of various advanced solid tumors, inhibits DNA synthesis by forming DNA cross-links and adducts. Despite the activity of cisplatin, tumor cells can either be refractory or develop resistance to treatment. Cisplatin has been demonstrated to cause cell cycle G2/M arrest, which may allow for DNA damage response (DDR) and repair. Ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad 3-related (ATR) protein kinases are key regulators of DDR, and contribute to maintaining genomic integrity in response to various exogenous and endogenous genotoxic insults like cytotoxic chemotherapy. In fact, cisplatin has been shown to transiently increase ATR expression. Inhibitors of ATR have been studied in combination with cisplatin both in vitro and in vivo, demonstrating enhanced activity. The oral small molecule ATR inhibitor elimusertib, which has been studied as a single agent in a Phase I study, has also demonstrated enhanced activity with cisplatin in vitro in lung cancer and bladder cancer cell lines. We sought to conduct a Phase I study evaluating the combination of elimusertib with cisplatin or with cisplatin and gemcitabine. Methods: In the first cohort, patients with histologically confirmed advanced solid tumors for which cisplatin-based therapy would be considered appropriate, who exhibit adequate organ function, and have received < 300 mg/m2 of cisplatin previously are treated with cisplatin on Day (D) 1 and with elimusertib on D2 & 9 of each 21-day cycle. The study follows a phase I queue (IQ) 3+3 dose escalation design, following standard practices for dose-limiting toxicity (DLT) impact on escalation, and when the maximum tolerated dose (MTD) is established with cisplatin alone, this will inform the first dose level of the next cohort, in which patients will be treated with cisplatin on D1, gemcitabine on D1 & 8, and elimusertib on D2 & 9 of each 21-day cycle. An expansion cohort will enroll urothelial carcinoma patients when the second MTD is established. The primary objective of the study is to evaluate the safety and MTD of elimusertib in combination with cisplatin, as well as in combination with cisplatin and gemcitabine. Secondary study objectives include evaluation of pharmacokinetics of elimusertib in these combinations, preliminary efficacy, and evaluating the association between ATM expression and responses to therapy. Currently, 6 patients have been enrolled to the first cohort of the study. Clinical trial information: NCT04491942.

Investigation of CREBBP mutation and correlation with immunotherapy biomarker in Chinese bladder cancer patients.

e16537 Background: The CREBBP gene provides instructions for making CREB binding protein, is ubiquitously expressed and is involved in the transcriptional coactivation of many different transcription factors. Previous research on CREBBP gene revealed that CREBBP is correlated with tumor occurrence and the efficacy of immune checkpoint inhibitors (ICIs) through directly regulate DNA damage response (DDR) pathway. However, the CREBBP characteristics, its correlation with immunogenic marker, and the predictive value of immunotherapy in bladder cancer was unknown. Methods: An independent cohorts (the MSKCC study cohort) with next generation sequencing (NGS) data from 211 patients with bladder cancer of pan-cancer, which were used to analyze the prognostic effect of CREBBP on immunotherapy. Tumor tissue samples from Chinese bladder cancer were analyzed using NGS (panel on 381/733-gene). TMB was defined as total number of somatic non-synonymous mutations in coding region. MSI was evaluated by NGS of 500 known MSI loci. PD-L1 expression was evaluated using immunohistochemistry (Dako 22C3). Estimation of immune filtration cells scores were conducted via TIMER2.0. Results: In MSKCC cohort, there were 30 (14.2%) patients harbored CREBBP mutation ( CREBBPmut), and it (median, 15.71 Muts/Mb) was associated with higher TMB (P ＜0.0001) than the wild-type ( CREBBPwt) (median, 7.81 Muts/Mb). In terms of prognostic effect with ICIs therapy, there was significant difference on overall survival (OS) (HR = 0.43; 95% CI, 0.21–0.90; P = 0.02) between CREBBPmut (median, 12 months) and CREBBPwt (median, 8 months). In Chinese patients, genetic mutation of 184 bladder cancer patients were analyzed with NGS and 24 (13.0%) harbored CREBBPmut, similar to western patients of the MSKCC cohort. In bladder cancer patients with CREBBPmut, the most frequently co-mutated genes were TP53 (71%), followed by ATM (46%), BLM (42%), and BRCA1 (29%). CREBBPmut (median, 15.10 Muts/Mb) was associated with higher TMB (P = 0.0008) than CREBBPwt (median, 9.50 Muts/Mb), which was significant difference. Similarly, there was significant difference in MSI between CREBBPmut and CREBBPwt (P = 0.0095). However, there was no difference in PD-L1 expression between the two groups (P = 0.53). In the immune microenvironment of TCGA-SKCM cohort, only CD4+ T cell (P = 0.022) and activated NK cell (P = 0.042) were significantly higher in CREBBPmut patients. Conclusions: The results showed that the CREBBP gene had a high correlation with TMB and MSI in bladder cancer, and might a potential biomarker for ICIs therapy.

Inhibition of PLK‐1 by using a Small Molecule Inhibitor HMN‐214 is a Novel Therapeutic Approach for High‐risk Neuroblastoma

Polo‐like kinase1 (PLK‐1), a member of the polo‐like kinase family of serine/threonine protein kinases, plays an essential role in regulating the cell cycle. PLK‐1 has a strong relationship with numerous regulatory events progressing during G2/M transition, chromosomal segregation, spindle assembly maturation, and mitotic exit. In addition, PLK‐1 also regulates DNA damage response, DNA replication, transcription, translation, chromosomes dynamics, and checkpoint adoption. Elevated levels of PLK‐1 have been observed in a plethora of cancers, including neuroblastoma (NB). NB is the most prevalent solid tumor that develops during the early embryonic stage and accounts for 15% of pediatric cancer‐related deaths. Current cytotoxic treatment strategies for NB are limiting and failed to prevent relapse and metastasis. In the present study, we hypothesized that inhibition of cell cycle regulator PLK‐1 by a small molecule inhibitor HMN‐214 will inhibit NB growth. To determine the role of PLK‐1 in NB, we analyzed multiple NB patient datasets and observed that PLK‐1 expression is inversely correlated with overall patient survival. PLK‐1 also strongly correlates with MYCN amplification and overall NB disease progression in our patient dataset analysis. Further, we used six NB cell lines, including MYCN‐ amplified and ‐non‐amplified cell lines, to evaluate the anti‐proliferative effects of HMN‐214 in NB. Results showed that HMN‐214 significantly and in a dose‐dependent manner inhibits NB proliferation and colony formation capacity. Additionally, we found that HMN‐214 significantly induces apoptosis and blocks cell cycle progression at the G2‐M phase in treatment groups compared to controls. Our molecular analysis further revealed that HMN‐214 significantly inhibits the mRNA and protein levels of PLK‐1 and CDK1 in contrast to control treatments. Further, we developed a 3D spheroid tumor model for NB to mimic in vivo tumor growth and utilized it to determine the effects of HMN‐214 on NB tumor growth. Results showed that HMN‐214 significantly and in a dose‐dependent manner inhibits NB 3D spheroidal tumor growth by specifically inducing tumor cell death. Overall, our data highlights the role of PLK‐1 in the oncogenic progression of NB and showed the efficacy of HMN‐214 in inhibiting NB growth. Our future efforts will be directed towards elucidating the role of PLK‐1 in NB and developing effective therapeutic strategies incorporating PLK‐1 inhibitor.

Integrative analysis of therapy resistance and transcriptomic profiling data in glioblastoma cells identifies sensitization vulnerabilities for combined modality radiochemotherapy

BackgroundInherent resistance to radio/chemotherapy is one of the major reasons for early recurrence, treatment failure, and dismal prognosis of glioblastoma. Thus, the identification of resistance driving regulators as prognostic and/or predictive markers as well as potential vulnerabilities for combined modality treatment approaches is of pivotal importance.MethodsWe performed an integrative analysis of treatment resistance and DNA damage response regulator expression in a panel of human glioblastoma cell lines. mRNA expression levels of 38 DNA damage response regulators were analyzed by qRT-PCR. Inherent resistance to radiotherapy (single-shot and fractionated mode) and/or temozolomide treatment was assessed by clonogenic survival assays. Resistance scores were extracted by dimensionality reduction and subjected to correlation analyses with the mRNA expression data. Top-hit candidates with positive correlation coefficients were validated by pharmacological inhibition in clonogenic survival assays and DNA repair analyses via residual γH2AX/53BP1-foci staining.ResultsInherent resistance to single-shot and similarly also to fractionated radiotherapy showed strong positive correlations with mRNA expression levels of known vulnerabilities of GBM, including PARP1, NBN, and BLM, as well as ATR and LIG4—two so far underestimated targets. Inhibition of ATR by AZD-6738 resulted in robust and dose-dependent radiosensitization of glioblastoma cells, whereas LIG4 inhibition by L189 had no noticeable impact. Resistance against temozolomide showed strong positive correlation with mRNA expression levels of MGMT as to be expected. Interestingly, it also correlated with mRNA expression levels of ATM, suggesting a potential role of ATM in the context of temozolomide resistance in glioblastoma cells. ATM inhibition exhibited slight sensitization effects towards temozolomide treatment in MGMT low expressing glioblastoma cells, thus encouraging further characterization.ConclusionsHere, we describe a systematic approach integrating clonogenic survival data with mRNA expression data of DNA damage response regulators in human glioblastoma cell lines to identify markers of inherent therapy resistance and potential vulnerabilities for targeted sensitization. Our results provide proof-of-concept for the feasibility of this approach, including its limitations. We consider this strategy to be adaptable to other cancer entities as well as other molecular data qualities, and its upscaling potential in terms of model systems and observational data levels deserves further investigation.

A plant-specific module for homologous recombination repair

Homologous recombination repair (HR) is an error-free DNA damage repair pathway to maintain genome stability and a basis of gene targeting using genome-editing tools. However, the mechanisms of HR in plants are still poorly understood. Through genetic screens for DNA damage response mutants (DDRM) in Arabidopsis, we find that a plant-specific ubiquitin E3 ligase DDRM1 is required for HR. DDRM1 contains an N-terminal BRCT (BRCA1 C-terminal) domain and a C-terminal RING (really interesting new gene) domain and is highly conserved in plants including mosses. The ddrm1 mutant is defective in HR and thus is hypersensitive to DNA-damaging reagents. Biochemical studies reveal that DDRM1 interacts with and ubiquitinates the transcription factor SOG1, a plant-specific master regulator of DNA damage responses. Interestingly, DDRM1-mediated ubiquitination promotes the stability of SOG1. Consistently, genetic data support that SOG1 functions downstream of DDRM1. Our study reveals that DDRM1-SOG1 is a plant-specific module for HR and highlights the importance of ubiquitination in HR.

Arabidopsis mediator subunit 17 connects transcription with DNA repair after UV-B exposure.

Mediator 17 (MED17) is a subunit of the Mediator complex that regulates transcription initiation in eukaryotic organisms. In yeast and humans, MED17 also participates in DNA repair, physically interacting with proteins of the nucleotide excision DNA repair system, but this function in plants has not been investigated. We studied the role of MED17 in Arabidopsis plants exposed to UV-B radiation. Our results demonstrate that med17 and OE MED17 plants have altered responses to UV-B, and that MED17 participates in various aspects of the DNA damage response (DDR). Comparison of the med17 transcriptome with that of wild-type (WT) plants showed that almost one-third of transcripts with altered expression in med17 plants were also changed by UV-B exposure in WT plants. Increased sensitivity to DNA damage after UV-B in med17 plants could result from the altered regulation of UV-B responsive transcripts but MED17 also physically interacts with DNA repair proteins, suggesting a direct role of this Mediator subunit during repair. Finally, we show that MED17 is necessary to regulate the DDR activated by ataxia telangiectasia and Rad3 related (ATR), and that programmed cell death 5 (PDCD5) overexpression reverts the deficiencies in DDR shown in med17 mutants. Our data demonstrate that MED17 is an important regulator of DDR after UV-B irradiation in Arabidopsis.

SETD2 mediates immunotherapy and radiotherapy efficacy via regulating DNA damage responses and genomic stability in lung adenocarcinoma

SETD2 mediates immunotherapy and radiotherapy efficacy via regulating DNA damage responses and genomic stability in lung adenocarcinoma

Development of novel urea-based ATM kinase inhibitors with subnanomolar cellular potency and high kinome selectivity

The ATM kinase is a key molecule regulating DNA damage response and can be targeted resulting in efficient radio- or chemosensitization. Due to the enormous size of this protein and the associated difficulties in obtaining high-quality crystal structures, we sought to develop an accurate in silico model to identify new targeting possibilities. We identified a urea group as the most beneficial chemical anchor point, which could undergo multiple interactions in the aspartate-rich hydrophobic region I of the atypical ATM kinase domain.Based on in silico data, we designed and synthesized a comprehensive set of novel urea-based inhibitors and characterized them in diverse biochemical assays. Using this strategy, we identified inhibitors with subnanomolar potency, which were further evaluated in cellular models, selectivity and early DMPK properties. Finally, the two lead compounds 34 and 39 exhibited subnanomolar cellular activity along with an excellent selectivity profile and favorable metabolic stability.

The Small DdrR Protein Directly Interacts with the UmuDAb Regulator of the Mutagenic DNA Damage Response in Acinetobacter baumannii.

Acinetobacter baumannii poses a great threat in health care settings worldwide, with clinical isolates displaying an ever-evolving multidrug resistance. In strains of A. baumannii, expression of multiple error-prone polymerase genes are corepressed by UmuDAb, a member of the LexA superfamily, and a small protein, DdrR. It is currently unknown how DdrR establishes this repression. Here, we used surface plasmon resonance spectrometry to show that DdrR formed a stable complex with the UmuDAb regulator. Our results indicated that the carboxy-terminal dimerization domain of UmuDAb formed the interaction interface with DdrR. Our in vitro data also showed that RecA-mediated inactivation of UmuDAb was inhibited when this transcription factor was bound to its target DNA. In addition, we showed that DdrR interacted with a putative prophage repressor, homologous to LexA superfamily proteins. These data suggested that DdrR modulated DNA damage response and prophage induction in A. baumannii by binding to LexA-like regulators. IMPORTANCE We previously identified a 50-residue bacteriophage protein, gp7, which interacts with and modulates the function of the LexA transcription factor from Bacillus thuringiensis. Here, we present data that indicates that the small DdrR protein from A. baumannii likely coordinates the SOS response and prophage processes by also interacting with LexA superfamily members. We suggest that similar small proteins that interact with LexA-like proteins to coordinate DNA repair and bacteriophage functions may be common to many bacteria that mount the SOS response.