Mediator Of DNA Damage Checkpoint Research Articles

DNA Checkpoint Gene Mutation as a Biomarker for Immune Checkpoint Inhibitor Therapy in Advanced Biliary Tract Cancer.

The DNA checkpoint (DNACHK) pathway is engaged in signaling the need for cell cycle arrest. This pathway is being actively researched to assess its role in cancer immunotherapy. A total of 62 patients participated in this study. These patients were treated with immune checkpoint inhibitors (ICIs) for advanced biliary tract cancers (BTCs) from March 2020 to August 2022 at Samsung Medical Center. DNACHK mutated were defined as genomic alterations, such as single nucleotide variants, multi-nucleotide variants, and short insertion and deletions in seven genes; checkpoint kinase 1 (CHEK1), checkpoint kinase 2 (CHEK2), BRCA1, DNA repair-associated (BRCA1), the serine/threonine kinase ATM, the serine/threonine kinase ATR, mediator of DNA damage checkpoint 1 (MDC1) and tumor protein p53 binding protein 1 (TP53BP1). We analyzed the effect of DNACHK mutations on the efficacy of ICIs in advanced BTCs. Patient median age at diagnosis was 68.0 years. 10 patients (16.1%) had GB cancer; the remaining patients (n=52, 83.9%) were diagnosed with cholangiocarcinoma. Thirty-seven (59.7%) patients were categorized into the DNACHK wild-type (WT) group and 25 (40.3%) into the DNACHK mutated (MT) group. The most observed DNA checkpoint mutations were ATM mutations (n=14). Patients in the DNACHK MT group had better disease control rate (DCR) than patients in the DNACHK WT (60.0% vs. 48.6%, p=0.53). Median overall survival (OS) was 8.1 months (95% CI 5.1-22.8) in the MT group and 5.6 months (95%CI 3.1-11.0) in the WT group (p=0.33). The DNACHK pathway is expected to serve as a potential biomarker for ICI treatment.

Abstract 7624: DNA checkpoint gene mutation as a biomarker of immune checkpoint inhibitor in advanced biliary tract cancer

Abstract Introduction: The DNA checkpoint (DNACHK) pathway is engaged in signaling the need for cell cycle arrest. The G1 and S phase checkpoint allows for repair of damaged DNA, preventing damaged DNA from being copied. The G2 and M phase checkpoint prevents transmission of mutated DNA to the next generation. This pathway, along with mismatch repair (MMR), homologous recombination (HR), and base excision repair (BER), is being actively researched to assess its role in cancer immunotherapy. Methods: Sixty-two patients participated in this study. These patients were treated with immune checkpoint inhibitors (ICIs) for advanced biliary tract cancers (BTCs) from Mar. 2020 to Aug. 2022 at Samsung Medical Center. DNACHK mutations were defined as genomic alterations such as SNVs, MNVs, and short insertion and deletions in seven genes These genes include checkpoint kinase 1 (CHEK1), checkpoint kinase 2 (CHEK2), BRCA1, DNA repair associated (BRCA1), the serine/threonine kinase ATM, the serine/threonine kinase ATR, and mediator of DNA damage checkpoint 1 (MDC1). We analyzed the effect of DNACHK mutations on the efficacy of ICIs in advanced BTCs. Results: Patient median age at diagnosis was 68.0 years. Ten patients (16.1%) had gall bladder (GB) cancer; the remaining patients (n = 52, 83.9%) were diagnosed with cholangiocarcinoma. Among the 62 patients with advanced BTCs treated with ICIs, 37 (59.7%) were categorized into the DNACHK wild-type (WT) group and 25 (40.3%) into the DNACHK mutated (MT) group. The most observed DNA checkpoint mutations were ATM mutations (n=14). MDC1 mutations were found in seven patients, ATR mutations in three, BRCA1 mutations in four, and CHEK2 mutations in three. Patients in the DNACHK MT group had better disease control rates than patients in the DNACHK WT group (60.0% vs. 48.6%, p=0.53). Median overall survival (OS) was 8.1 months (95% CI, 5.1, 22.8) in the MT group and 5.6 months (95% CI, 3.1, 11.0) in the WT group (p=0.33). Conclusions: The results of this study suggest that the DNACHK pathway may be a valuable source of biomarkers for ICI and worthy of further study. Citation Format: Ji Eun Shin, Seung Tae Kim, Dae-Ho Choi, Junho Lee, Jiyeon Hyeon. DNA checkpoint gene mutation as a biomarker of immune checkpoint inhibitor in advanced biliary tract cancer [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 7624.

Inhibition of intracellular ATP synthesis impairs the recruitment of homologous recombination factors after ionizing radiation.

Ionizing radiation (IR)-induced double-strand breaks (DSBs) are primarily repaired by non-homologous end joining or homologous recombination (HR) in human cells. DSB repair requires adenosine-5'-triphosphate (ATP) for protein kinase activities in the multiple steps of DSB repair, such as DNA ligation, chromatin remodeling, and DNA damage signaling via protein kinase and ATPase activities. To investigate whether low ATP culture conditions affect the recruitment of repair proteins at DSB sites, IR-induced foci were examined in the presence of ATP synthesis inhibitors. We found that p53 binding protein 1 foci formation was modestly reduced under low ATP conditions after IR, although phosphorylated histone H2AX and mediator of DNA damage checkpoint 1 foci formation were not impaired. Next, we examined the foci formation of breast cancer susceptibility gene I (BRCA1), replication protein A (RPA) and radiation 51 (RAD51), which are HR factors, in G2 phase cells following IR. Interestingly, BRCA1 and RPA foci in the G2 phase were significantly reduced under low ATP conditions compared to that under normal culture conditions. Notably, RAD51 foci were drastically impaired under low ATP conditions. These results suggest that HR does not effectively progress under low ATP conditions; in particular, ATP shortages impair downstream steps in HR, such as RAD51 loading. Taken together, these results suggest that the maintenance of cellular ATP levels is critical for DNA damage response and HR progression after IR.

Identification of plants’ functional counterpart of the metazoan mediator of DNA Damage checkpoint 1

Induction of DNA damage triggers rapid phosphorylation of the histone H2A.X (γH2A.X). In animals, mediator of DNA damage checkpoint 1 (MDC1) binds γH2A.X through a tandem BRCA1 carboxyl-terminal (tBRCT) domain and mediates recruitment of downstream effectors of DNA damage response (DDR). However, readers of this modification in plants have remained elusive. We show that from the Arabidopsis BRCT domain proteome, BCP1-4 proteins with tBRCT domains are involved in DDR. Through its tBRCT domain BCP4 binds γH2A.X in vitro and localizes to DNA damage-induced foci in an H2A.X-dependent manner. BCP4 also contains a domain that interacts directly with NBS1 and thus acts as a functional counterpart of MDC1. We also show that BCP1, that contains two tBRCT domains, co-localizes with γH2A.X but it does not bind γH2A.X suggesting functional similarity with human PAXIP1. A phylogenetic analysis supports that PAXIP1 and MDC1 in metazoa and their plant counterparts evolved independently from common ancestors with tBRCT domains. Collectively, our study reveals missing components and provides mechanistic and evolutionary insights into plant DDR.

Mitotic tethering enables inheritance of shattered micronuclear chromosomes.

Chromothripsis, the shattering and imperfect reassembly of one (or a few) chromosome(s)1, is an ubiquitous2 mutational process generating localized and complex chromosomal rearrangements that drive genome evolution in cancer. Chromothripsis can be initiated by mis-segregation errors in mitosis3,4 or DNA metabolism5-7 that lead to entrapment of chromosomes within micronuclei and their subsequent fragmentation in the next interphase or following mitotic entry6,8-10. Here we use inducible degrons to demonstrate that chromothriptically produced pieces of a micronucleated chromosome are tethered together in mitosis by a protein complex consisting of mediator of DNA damage checkpoint 1(MDC1), DNA topoisomerase II-binding protein 1(TOPBP1) and cellular inhibitor of PP2A (CIP2A), thereby enabling en masse segregation to the same daughter cell. Such tethering is shown to be crucial for the viability of cells undergoing chromosome mis-segregation and shattering after transient inactivation of the spindle assembly checkpoint. Transient, degron-induced reduction in CIP2A following chromosome micronucleation-dependent chromosome shattering is shown to drive acquisition of segmental deletions and inversions. Analyses of pancancer tumour genomes showed that expression of CIP2A and TOPBP1 was increased overallin cancers withgenomic rearrangements, including copy number-neutralchromothripsiswith minimal deletions, but comparatively reduced in cancers with canonical chromothripsis in which deletions were frequent. Thus, chromatin-bound tethers maintain the proximity of fragments of a shattered chromosome enabling their re-encapsulation into, and religation within, a daughter cell nucleus to form heritable, chromothriptically rearranged chromosomes found in the majority of human cancers.

OR07-2 Endocrine Response And Resistance Is Driven By Novel Transcriptional Co-Regulator Activity In Invasive Lobular Carcinoma Of The Breast

Abstract Invasive lobular carcinoma of the breast (ILC) affects ∼40,000 US women annually and is a top ten most diagnosed cancer in women. ∼95% of ILC express estrogen receptor alpha (ER), but ILC is associated with poor long-term outcomes and anti-estrogen resistance, suggesting ER function is distinct in ILC cells. We reported that MDC1 (mediator of DNA damage checkpoint 1) is an ILC-specific ER co-regulator required for ER-driven proliferation, and that MDC1 knockdown dysregulates the ER transcriptome in ILC. Mechanisms of MDC1 co-regulator activity are unknown, but must be defined to understand endocrine response and resistance in ILC. To understand MDC1 co-regulator functions, we profiled ER/MDC1 genomic binding (ie. "cistrome") using CUT&RUN in ER+ ILC cell line MDA MB 134VI (MM134). We identified ∼2500 binding sites each for ER and MDC1, but only ∼8% of sites were bound by both factors. However, using BETA to integrate cistrome and transcriptome data predicted that both ER and MDC1 activate estrogen-regulated genes with over half of direct ER target genes also direct MDC1 targets (p=9.8×10-375). Among shared ER: MDC1 direct target genes (n=760), 58% had MDC1 binding in the promoter region with ER bound at nearby enhancers, while only 15% were linked to shared ER: MDC1 binding sites, suggesting MDC1 primarily facilitates promoter access for ER-bound enhancers. Further, MDC1 knockdown reduced ER binding at enhancer sites at ER: MDC1 target genes, implicating MDC1 in regulating chromatin access. Importantly, MDC1 does not have known enzymatic activity to directly regulate chromatin structure. We profiled MDC1-interacting proteins in ILC cells by RIME (Rapid immunoprecipitation mass spectrometry of endogenous proteins), and determined whether knockdown of putative ER: MDC1 partners disrupted ER function. This identified the SWI/SNF complex protein BRG1 (SMARCA4) as a critical ER: MDC1 partner. BRG1 was required for ILC cell growth, and BRG1 knockdown specifically suppressed ER regulation of MDC1-dependent ER target genes. Taken together, our data suggest that in ILC cells, MDC1 facilitates ER access to and activity at target gene promoters. The BRG1 SWI/SNF complex is a putative ER: MDC1 partner and mediator of MDC1 co-regulator functions. Ongoing research is focused on understanding how MDC1 regulates ILC-specific ER activity by licensing access to ILC-specific target genes, and the role of the SWI/SNF complex in mediating genomic ER: MDC1 activity. Presentation: Saturday, June 11, 2022 11:45 a.m. - 12:00 p.m.

Cytotoxic, genotoxic, and toxicogenomic effects of heated tobacco products and cigarette smoke in human primary keratinocytes.

INTRODUCTIONHeated tobacco products (HTPs) appear to be less harmful to health than conventional cigarettes (CCs). However, limited analytical data are available to support this claim. This study aimed to compare the cytotoxic, genotoxic, and toxicogenomic effects of HTPs and CCs in carcinogenesis via multistep gene mutations in the oral mucosal cells.METHODSCigarette smoke extract (CSE) was obtained from HTPs and CCs. Primary human oral keratinocytes (HOKs) were treated with 5% and 20% CSE from HTPs and CCs. Cell survival rate assays were performed after 6, 12, and 24 h. After 6 h, DNA double-strand breaks (DSBs) were evaluated using anti-γH2AX antibodies with immunohistochemistry. mRNAs expressions of mediator of DNA damage checkpoint 1 (MDC1) and ataxia telangiectasia and Rad3-related protein (ATR), were analyzed. Expressions of miR-22 and miR-185 were analyzed because miR-22 targets MDC1 and miR-185, ATR.RESULTSThe HOKs had equivalent survival rates after exposure to the same concentrations of CSE from CCs and HTPs. HTPs increased foci formation of γH2AX in HOKs, as did CCs (without CSE vs 20% HTP, p<0.05; without CSE vs 20% CC, p<0.05). Expressions of MDC1 and ATR decreased in cells exposed to CSE from CCs and HTPs (MDC1: without CSE vs 20% HTP, p<0.05; without CSE vs 20% CC, p<0.05; ATR: without CSE vs 20% HTP, p<0.05; without CSE vs 20% CC, p<0.05). Expressions of miR-22 and miR-185 were not significantly increased when exposed to CSE from CCs or HTPs.CONCLUSIONSHTPs and CCs had similar cytotoxic effects. HTPs are genotoxic, can cause DSBs, and have toxicogenomic damage because they inhibit the MDC1 and ATR-CHK1 DNA repair pathways in the oral mucosa. The miRNA-mRNA axis was not related to these inhibitions.

Targeting MDC1 promotes apoptosis and sensitizes Imatinib resistance in CML cells by mainly disrupting non-homologous end-joining repair.

The first-line drug Imatinib (IM) has achieved a curative effect in most chronic myeloid leukemia (CML) patients, but drug resistance remains a problem. More alternative therapeutic strategies need to explore. In recent years, targeting dysregulated DNA repair mechanisms provided promising options for cancer treatment. Here, we discovered the versatile Mediator of DNA Damage Checkpoint 1 (MDC1) interacted with γ-H2AX and 53BP1 in the early stage of the DNA damage response of cells. MDC1 overexpressed in CML cell lines and patients' bone marrow mononuclear cells. By knocking down MDC1, non-homologous end-joining pathways were mainly inhibited, leading to an intense accumulation of unrepaired intracellular DNA damage and an apparent cell apoptosis promotion. Notably, targeting MDC1 further enhanced drug sensitivity in IM-resistant CML cells. Our work revealed that MDC1 is a prospective target for CML treatment through regulating DNA damage repair mechanism, and also an alternative option for IM resistance dilemma. This study extends the understanding of regulating dysfunctional DNA repair mechanisms for cancer treatment.

AP4 suppresses DNA damage, chromosomal instability and senescence via inducing MDC1/Mediator of DNA damage Checkpoint 1 and repressing MIR22HG/miR-22-3p

BackgroundAP4 (TFAP4) encodes a basic helix-loop-helix leucine zipper (bHLH-LZ) transcription factor and is a direct target gene of the oncogenic transcription factor c-MYC. Here, we set out to determine the relevance of AP4 in human colorectal cancer (CRC) cells.MethodsA CRISPR/Cas9 approach was employed to generate AP4-deficient CRC cell lines with inducible expression of c-MYC. Colony formation, β-gal staining, immunofluorescence, comet and homologous recombination (HR) assays and RNA-Seq analysis were used to determine the effects of AP4 inactivation. qPCR and qChIP analyses was performed to validate differentially expressed AP4 targets. Expression data from CRC cohorts was subjected to bioinformatics analyses. Immunohistochemistry was used to evaluate AP4 targets in vivo. Ap4-deficient APCmin/+ mice were analyzed to determine conservation. Immunofluorescence, chromosome and micronuclei enumeration, MTT and colony formation assays were used to determine the effects of AP4 inactivation and target gene regulation on chromosomal instability (CIN) and drug sensitivity.ResultsInactivation of AP4 in CRC cell lines resulted in increased spontaneous and c-MYC-induced DNA damage, chromosomal instability (CIN) and cellular senescence. AP4-deficient cells displayed increased expression of the long non-coding RNA MIR22HG, which encodes miR-22-3p and was directly repressed by AP4. Furthermore, Mediator of DNA damage Checkpoint 1 (MDC1), a central component of the DNA damage response and a known target of miR-22-3p, displayed decreased expression in AP4-deficient cells. Accordingly, MDC1 was directly induced by AP4 and indirectly by AP4-mediated repression of miR-22-3p. Adenomas and organoids from Ap4-deficient APCmin/+ mice displayed conservation of these regulations. Inhibition of miR-22-3p or ectopic MDC1 expression reversed the increased senescence, DNA damage, CIN and defective HR observed in AP4-deficient CRC cells. AP4-deficiency also sensitized CRC cells to 5-FU treatment, whereas ectopic AP4 conferred resistance to 5-FU in a miR-22-3p and MDC1-dependent manner.ConclusionsIn summary, AP4, miR-22-3p and MDC1 form a conserved and coherent, regulatory feed-forward loop to promote DNA repair, which suppresses DNA damage, senescence and CIN, and contributes to 5-FU resistance. These findings explain how elevated AP4 expression contributes to development and chemo-resistance of colorectal cancer after c-MYC activation.Graphical abstract

Mediator of DNA Damage Checkpoint 1 (MDC1) Is a Novel Estrogen Receptor Coregulator in Invasive Lobular Carcinoma of the Breast.

Invasive lobular carcinoma (ILC) is the most common special histologic subtype of breast cancer, and nearly all ILC tumors express estrogen receptor alpha (ER). However, clinical and laboratory data suggest ILC are strongly estrogen-driven but not equally antiestrogen-sensitive. We hypothesized ILC-specific ER coregulators mediate ER functions and antiestrogen resistance in ILC, and profiled ER-associated proteins by mass spectrometry. Three ER+ ILC cell lines (MDA MB 134VI, SUM44PE, and BCK4) were compared with ER+ invasive ductal carcinoma (IDC) line data, and we examined whether siRNA of identified proteins suppressed ER-driven proliferation in ILC cells. This identified mediator of DNA damage checkpoint 1 (MDC1), a tumor suppressor in DNA damage response (DDR), as a novel ER coregulator in ILC. We confirmed ER:MDC1 interaction was specific to ILC versus IDC cells, and found MDC1 knockdown suppressed ILC cell proliferation and tamoxifen resistance. Using RNA-sequencing, we found in ILC cells MDC1 knockdown broadly dysregulates the ER transcriptome, with ER:MDC1 target genes enriched for promoter hormone response elements. Importantly, our data are inconsistent with MDC1 tumor suppressor functions in DDR, but suggest a novel oncogenic role for MDC1 as an ER coregulator. Supporting this, in breast tumor tissue microarrays, MDC1 protein was frequently low or absent in IDC, but MDC1 loss was rare in ER+ ILC. ER:MDC1 interaction and MDC1 coregulator functions may underlie ER function in ILC and serve as targets to overcome antiestrogen resistance in ILC. IMPLICATIONS: MDC1 has novel ER coregulator activity in ILC, which may underlie ILC-specific ER functions, estrogen response, and antiestrogen resistance.

Genome-wide CRISPR screen reveals SLFN11 as a potent mediator of cisplatin sensitivity in muscle-invasive bladder cancer

Bladder cancer is among the top ten most common cancer types in the world, with approximately 550,000 new cases annually. The highest burden of bladder cancer is currently on most developed communities across the world and an estimated 9,000 Canadians are diagnosed with bladder cancer each year. Cisplatin-based Neoadjuvant chemotherapy (NAC) followed by radical cystectomy in patients with muscle-invasive bladder cancer (MIBC) has been shown to improve five-year survival, and is therefore currently the first-line standard of care in patients. However, 60% of patients are inherently resistant to NAC at the time of cystectomy. While several mechanisms of cellular resistance to cisplatin have been proposed, the mechanisms presented thus far still do not offer and effective patient response prediction in the context of MIBC. There is therefore, an urgent and unmet need to determine clinically actionable mechanisms of cisplatin resistance. In order to elucidate mechanisms of resistant to cisplatin, the study presented in this thesis takes advantage of a pooled genome‐wide CRISPR knock‐out library targeting 19,114 protein coding genes with 76,441 synthetic guide RNAs (sgRNAs) which allows for an unbiased screen. Upon completion of the screen the top hit was validated in vitro (CRISPR knockout cell lines), in vivo (mouse models) and in patient tumour tissue samples by immunohistochemistry. A full-scale screen revealed that several genes involved in the pro-apoptotic pathway (such as CASP8, BAX, and TNFSFR10A) and cell cycle regulation have the potential to confer resistance to cisplatin when knocked out. For this study however, we validated the top hit from our screen - Schlafen 11 (SLFN11) - and established that the complete loss of SLFN11 confers a cisplatin resistant phenotype in MIBC. We further established that SLFN11 is involved in the regulation of cell cycle progression upon cisplatin challenge and does so via interactions with Mediator of DNA Damage Checkpoint 1 (MDC1) protein. Overall, the study presented here offers SLFN11 as a potential biomarker to aid in clinical decision making and to anticipate resistance to cisplatin-based NAC in MIBC. Furthermore, targeting SLFN11 associated pathways could allow for the development of combination therapies to be used in conjunction with cisplatin in the future.

OR12-05 MDC1 Is a Novel Estrogen Receptor Co-Regulator in Invasive Lobular Carcinoma of the Breast

Invasive Lobular Carcinoma (ILC) is the 2nd most common histotype of breast cancer, but is critically understudied. ~95% of ILC are estrogen receptor (ER) positive, and previous studies demonstrate the importance of estrogen in ILC etiology. However, retrospective studies show that anti-estrogens are substantially less effective in ILC than in ER+ Invasive Ductal Carcinoma (IDC). This strongly suggests that regulation of ER function is unique in ILC, and we hypothesize that this is due to an ILC-specific cohort of ER co-regulators. We performed Rapid Immunoprecipitation Mass Spectrometry of Endogenous Proteins (RIME) to determine ILC-specific ER-interacting proteins, and identified Mediator of DNA Damage Checkpoint 1 (MDC1) as a novel ER co-regulator in ILC cells. We confirmed ER:MDC1 interaction by co-immunoprecipitation and proximity ligation assays (PLA); interaction was specifically observed in ILC cell lines but not IDC cell lines. Consistent with co-regulator function, we found MDC1 is essential for ER-driven proliferation of ILC cells. MDC1 knockdown dysregulates transcription of ER target genes in ILC cells (e.g. IGFBP4, WNT4). Moreover, RNA-seq analysis showed that in ILC cell line MDA MB 134VI, >50% of ER target genes require MDC1 for their regulation. To understand how MDC1 controls ER transcriptional activity, we performed ChIP-qPCR and found that MDC1 controls ER binding to DNA in ILC cells. Further, MDC1 controls binding of the pioneer factor FOXA1 to DNA, and Dual PLA studies of ER:MDC1 and ER:FOXA1 interaction revealed that MDC1 knockdown decreased ER:FOXA1 interaction. MDC1 canonically functions in DNA damage response, but our preliminary data suggest MDC1 is decoupled from its canonical role in DDR in the context of ER co-regulator activity in ILC cells. Together, these data suggest MDC1, independent of its role in DDR, acts as a novel ER co-regulator in ILC and regulates ER:DNA binding and ER transcriptional function to drive ILC cell proliferation and survival.

Detection of DNA damage formation by natural organic matter using EGFP-fused MDC1-expressing cells

Studies have been conducted on the genotoxicity and carcinogenicity of disinfection by-products formed from natural organic matter (NOM) and mitigation effect for mutagens and clastogens by NOM. Whereas, reportedly, synthetic humic acid in high concentration has induced genotoxicity in human cells, and NOM samples have provoked mild oxidative and other physiological responses in aquatic organisms. Our group developed a novel detection method for DNA damage formation, namely enhanced green fluorescent protein (EGFP)-fused mediator of DNA damage checkpoint 1 (MDC1)-expressing human cells as simple and high-sensitive system. By using this method, a significant increase in the foci area was observed after 3 h, but not 24 h for 130 mgC L−1 Suwannee River fulvic acid (SRFA), 38 mgC L−1 humic acid (SRHA), and 19 mgC L−1 NOM (SRNOM). The SRNOM concentration is the original environmental one; therefore, it was suggested that the formation and repair of DNA damage associated with γ-H2AX, a biomarker for DNA double-strand breaks by mild oxidative stress, in Suwannee River (SR) were detected for the first time. The increase in the foci area was not observed for 18 mgC L−1 Lake Biwa fulvic acid (LBFA) and 50 mg L−1 catechin after both 3 h and 24 h. The difference between the SR and Lake Biwa (LB) samples may result from the differences in their electron-accepting capacity. The application of this methodology is expected to elucidate oxidative stress and toxicological effects shortly and in detail for many water samples.

Loss of the tumor suppressor BIN1 enables ATM Ser/Thr kinase activation by the nuclear protein E2F1 and renders cancer cells resistant to cisplatin

The tumor suppressor bridging integrator 1 (BIN1) is a corepressor of the transcription factor E2F1 and inhibits cell-cycle progression. BIN1 also curbs cellular poly(ADP-ribosyl)ation (PARylation) and increases sensitivity of cancer cells to DNA-damaging therapeutic agents such as cisplatin. However, how BIN1 deficiency, a hallmark of advanced cancer cells, increases cisplatin resistance remains elusive. Here, we report that BIN1 inactivates ataxia telangiectasia-mutated (ATM) serine/threonine kinase, particularly when BIN1 binds E2F1. BIN1 + 12A (a cancer-associated BIN1 splicing variant) also inhibited cellular PARylation, but only BIN1 increased cisplatin sensitivity. BIN1 prevented E2F1 from transcriptionally activating the human ATM promoter, whereas BIN1 + 12A did not physically interact with E2F1. Conversely, BIN1 loss significantly increased E2F1-dependent formation of MRE11A/RAD50/NBS1 DNA end-binding protein complex and efficiently promoted ATM autophosphorylation. Even in the absence of dsDNA breaks (DSBs), BIN1 loss promoted ATM-dependent phosphorylation of histone H2A family member X (forming γH2AX, a DSB biomarker) and mediator of DNA damage checkpoint 1 (MDC1, a γH2AX-binding adaptor protein for DSB repair). Of note, even in the presence of transcriptionally active (i.e. proapoptotic) TP53 tumor suppressor, BIN1 loss generally increased cisplatin resistance, which was conversely alleviated by ATM inactivation or E2F1 reduction. However, E2F2 or E2F3 depletion did not recapitulate the cisplatin sensitivity elicited by E2F1 elimination. Our study unveils an E2F1-specific signaling circuit that constitutively activates ATM and provokes cisplatin resistance in BIN1-deficient cancer cells and further reveals that γH2AX emergence may not always reflect DSBs if BIN1 is absent.

Abstract B032: PhIP-seq assessment of the serum antibody repertoire before and after immune-related adverse events in four melanoma patients treated with checkpoint blockade immunotherapy

Abstract Phage immunoprecipitation sequencing (PhIP-seq) is a technique to profile the epitope specificities of an antibody repertoire by phage display of a peptide library followed by immunoprecipitation and next-generation sequencing (1). We have developed phage libraries corresponding to all 36-mer peptides in the human proteome (approximately 413,000 peptides), as well as libraries of peptides found in viruses, bacteria, and toxins. Here, in a pilot study to understand if PhIP-seq might be used to identify predictive biomarkers for immune-related adverse events (irAE) in the context of checkpoint blockade immunotherapy, we applied the human proteome library to probe the self-directed IgG response in sera from four melanoma patients receiving checkpoint blockade who experienced irAE. In total, 16 serum samples acquired at the pre-treatment, post-treatment / pre-irAE, or post-irAE timepoints were assayed from these patients. We additionally analyzed samples from six melanoma patients who received vaccines targeting the MART-1 and NY-ESO-1 antigens. Using a stringent confidence threshold, we identified a median 31 self-directed antibody specificities (hits) at the pre-immunotherapy timepoint in these cancer patients and 41.5 hits post-therapy, compared to a median 6.5 hits in healthy donors. Patients receiving combination checkpoint blockade showed more hits than those treated with monotherapy. For a patient who received combination nivolumab and ipilimumab and developed a hepatic irAE, a cluster of 25 hits (of 161 total) was detected uniquely in a sample taken within two months subsequent to the adverse event. This cluster included a peptide from the C-Reactive Protein (CRP) gene and other genes expressed in the liver (EHBP1, VSTM2L), or across tissues (SAFB2), but also included genes with low expression in the liver (SPTBN4, DMBT1, IQGAP3, SPTBN4). In a patient who received ipilimumab and nivolumab and developed myalgia, hits against targets associated with autoimmune disease were found including NUMA1 (connective tissue autoimmunity), TRPM1 (melanoma-associated retinopathy), as well as three epitopes in the Mediator Of DNA Damage Checkpoint 1 (MDC1) gene. Finally, for three patients receiving a peptide vaccine targeting MART-1 and NY-ESO-1 with adjuvant poly-ICLC and montanide, we detected an IgG response against NY-ESO-1, but not MART-1, in each patient. No response to either protein was observed in three patients receiving a dendritic cell vaccine against the same antigens.Our small study suggests that PhIP-seq readily detects changes in the epitope specificities of the serum antibody repertoire in the course of immunotherapy. A key limitation of PhIP-seq is that the method can only detect antibodies with specificities for linear (as opposed to conformational) epitopes. Nevertheless, the assay’s low cost (approximately $30 per sample), small sample size requirement (1 µL serum), and ability to work with dried blood spot samples may make it an attractive technology to discover serological predictors of irAE in larger cohorts.

Ribosomal protein L6 (RPL6) is recruited to DNA damage sites in a poly(ADP-ribose) polymerase–dependent manner and regulates the DNA damage response

Ribosomal proteins are the building blocks of ribosome biogenesis. Beyond their known participation in ribosome assembly, the ribosome-independent functions of ribosomal proteins are largely unknown. Here, using immunoprecipitation, subcellular fractionation, His-ubiquitin pulldown, and immunofluorescence microscopy assays, along with siRNA-based knockdown approaches, we demonstrate that ribosomal protein L6 (RPL6) directly interacts with histone H2A and is involved in the DNA damage response (DDR). We found that in response to DNA damage, RPL6 is recruited to DNA damage sites in a poly(ADP-ribose) polymerase (PARP)-dependent manner, promoting its interaction with H2A. We also observed that RPL6 depletion attenuates the interaction between mediator of DNA damage checkpoint 1 (MDC1) and H2A histone family member X, phosphorylated (γH2AX), impairs the accumulation of MDC1 at DNA damage sites, and reduces both the recruitment of ring finger protein 168 (RNF168) and H2A Lys-15 ubiquitination (H2AK15ub). These RPL6 depletion-induced events subsequently inhibited the recruitment of the following downstream repair proteins: tumor protein P53-binding protein 1 (TP53BP1) and BRCA1, DNA repair-associated (BRCA1). Moreover, the RPL6 knockdown resulted in defects in the DNA damage-induced G2-M checkpoint, DNA damage repair, and cell survival. In conclusion, our study identifies RPL6 as a critical regulatory factor involved in the DDR. These findings expand our knowledge of the extraribosomal functions of ribosomal proteins in cell physiology and deepen our understanding of the molecular mechanisms underlying DDR regulation.

Knockdown of RNF2 enhances the radiosensitivity of squamous cell carcinoma in lung.

A previous study has reported that knockdown of RING finger protein 2 (RNF2) increases the radiosensitivity of esophageal cancer cells both in vitro and in vivo. However, the effect of RNF2 knockdown on radiosensitivity in squamous cell carcinoma (SqCC) remains unknown. For this, NCI-H226 and SK-MES-1 cells were exposed to X-ray irradiation and then RNF2 levels were determined. RNF2 was knocked-down and stable transfectants were selected. Radiosensitivity, cell proliferation, apoptosis, cell cycle, and γ-H2AX foci formation were evaluated. Interaction among ataxia telangiectasia mutated protein (ATM), mediator of DNA damage checkpoint 1 (MDC1), and H2AX were examined. Xenograft models were used to explore the effect of RNF2 knockdown on radiosensitivity in vivo. The results showed that RNF2 expression was significantly increased by X-ray irradiation. RNF2 knockdown combined with X-ray irradiation markedly inhibited cell proliferation, caused cell cycle arrest at the G1 phase, and induced cell apoptosis. In addition, RNF2 knockdown enhanced the radiosensitivity of SqCC cells, inhibited irradiation-induced γ-H2AX foci formation, and impaired the interactions among ATM, MDC1, and H2AX. Furthermore, combination of RNF2 knockdown and X-ray irradiation suppressed tumor growth and promoted tumor cell apoptosis in vivo. RNF2 may be a new therapeutic target to enhance the radiosensitivity of SqCC cells in lung.

Tyrosine kinase c-Abl couples RNA polymerase II transcription to DNA double-strand breaks.

DNA is constantly exposed to endogenous and exogenous damage. Various types of DNA repair counteract highly toxic DNA double-strand breaks (DSBs) to maintain genome stability. Recent findings suggest that the human DNA damage response (DDR) utilizes small RNA species, which are produced as long non-coding (nc)RNA precursors and promote recognition of DSBs. However, regulatory principles that control production of such transcripts remain largely elusive. Here we show that the Abelson tyrosine kinase c-Abl/ABL1 causes formation of RNA polymerase II (RNAPII) foci, predominantly phosphorylated at carboxy-terminal domain (CTD) residue Tyr1, at DSBs. CTD Tyr1-phosphorylated RNAPII (CTD Y1P) synthetizes strand-specific, damage-responsive transcripts (DARTs), which trigger formation of double-stranded (ds)RNA intermediates via DNA–RNA hybrid intermediates to promote recruitment of p53-binding protein 1 (53BP1) and Mediator of DNA damage checkpoint 1 (MDC1) to endogenous DSBs. Interference with transcription, c-Abl activity, DNA–RNA hybrid formation or dsRNA processing impairs CTD Y1P foci formation, attenuates DART synthesis and delays recruitment of DDR factors and DSB signalling. Collectively, our data provide novel insight in RNA-dependent DDR by coupling DSB-induced c-Abl activity on RNAPII to generate DARTs for consequent DSB recognition.

53BP1 regulates cell cycle arrest in esophageal cancer model.

This study aims to investigate effects of checkpoint kinase, mediator of DNA damage checkpoint 1 (MDC1) and p53-binding protein 1 (53BP1) silencing on p53, checkpoint kinase 1 and 2 (CHK1 and CHK2), and CHK2-T68 expression. Eca109 cells were divided into untransfected Eca109, Blank-vector, MDC1-RNAi transfection, and 53BP1-RNAi transfection group. Streptavidin-peroxidase (SP) immunohistochemical assay was used to examine CHK2-T68 expression. About 4 groups were used to establish esophageal carcinoma nude-mouse models, and assigned as Eca-109 control (or Eca-109 plus 15 Gy γ-rays irradiation, Eca-109+IR), Blank-vector (or Blank-vecor+IR), 53BP1-RNAi (or 53BP1-RNAi+IR), and MDC1-RNAi group (or MDC1-RNAi+IR group) by injecting. The expression of p53, CHK1, CHK2 were evaluated using SP immunohistochemical assay. 53BP1 and MDC1 down-regulation significantly inhibited expression of CHK2-T68 in Eca-109 cells compared to untreated group (p<0.05). There were significant differences for CHK2-T68 expressions in different time and groups (p<0.05). 53BP1 down-regulation significantly reduced p53 and enhanced CHK1 and CHK2 expression compared to that of Eca-109 control group (p<0.05) in Eca-109 cells. 53BP1 down-regulation significantly regulated CHK1, CHK2, and p53 in xenograft nude mice models exposed to γ-ray irradiation compared to that of untreated group (p<0.05). p53 was negatively correlated with CHK1 and CHK2 in xenograft nude mice models. 53BP1 regulated the cell cycle arrest by modulating p53, CHK1, and CHK2 expression in both Eca-109 cells and xenograft nude mice models.

Somatic MDC1 Mutation in Putative Pre-Leukaemic Stem Cell of a Biclonal Case of Chronic Lymphocytic Leukaemia

Chronic lymphocytic leukaemia (CLL) is an inherently heterogeneous disease in which founding lesions and cell of origin remain a subject of debate. This case of truly biclonal CLL provides evidence of the presence of a pre-leukaemic stem cell, supporting other lines of evidence that pathogenic genetic and epigenetic lesions in CLL arise early in haematopoiesis.An individual with trisomy 12 containing CLL, present at a frequency of 30%, was identified. Two distinct CD5/19-positive CLL clones were separated by flow cytometry, based on bimodal expression of CD49d. Each leukaemic clone was subjected to targeted immunoglobulin heavy chain variable region gene (IGHV) sequencing, whole exome sequencing (WES) and single-nucleotide polymorphism (SNP) microarray. CD5-positive T cells and a non-malignant superficial skin biopsy sample from the same individual were analysed as comparators.The CD49d-positive and CD49d-negative fractions harboured completely different IGHV rearrangements and mutational statuses (V4-34*02, hypermutated, and V3-21*02, unmutated, respectively) indicative of two unique leukaemic clones. Furthermore, trisomy 12, a relatively common aneuploidy in CLL, was present at high frequency (89.9%) in the CD49d-positive fraction but was absent in the CD49-negative and T-cell fractions. A mutation in the known CLL driver SF3B1 (c.1866G>T) was present in the CD49-negative, unmutated clone alone, along with a 17p deleted subclone in around 20% of this fraction. The presence of a 17p deletion is highly clinically relevant in this disease, and it is noteworthy that this subclone was not detected by routine fluorescence in situ hybridisation (FISH) at diagnosis. Two putative driver mutations in KMT2D and BCL11B were detected in the CD49d-positive, trisomy 12 clone alone (c.15256C>T and c.1387G>A respectively). A TET2 mutation (c.3609C>G) was found in all three fractions. The mutation in this epigenetic regulator has previously been described in clonal haematopoiesis of indeterminate potential (CHIP) and was hypothesised to have been acquired in a haematopoietic stem cell predisposing the individual to the development of haematological malignancy. However, the presence of this mutation was confirmed by Sanger sequencing of genomic DNA extracted from the skin sample, and is therefore of germline origin. The role of germline TET2 variants has not been described in lymphoid malignancy.Importantly, eight coding mutations were found common to both leukaemic clones but were absent from T-cells, implicating a common cell of origin prior to IGHV rearrangement but following B-lineage commitment (that is, a putative pre-leukaemic stem cell). One of these mutations was a disruptive 123 base pair deletion (c.4824_4946del) in MDC1 (mediator of DNA damage checkpoint 1) and was present at a variant allele frequency (VAF) of approximately 50% in both CLL clones, suggesting it was a founder mutation prior to subclone divergence. MDC1 is a mediator in the Ataxia Telangiectasia Mutated (ATM)-dependent DNA damage repair pathway and has not been previously implicated in CLL pathogenesis unlike ATM, which is frequently lost in del11q CLL. The proposed evolution of CLL in this case is presented in Figure 1.In summary, this highly unusual case of CLL provides insights into the evolution of CLL and adds another line of evidence to support the proposed early origins of CLL in haematopoiesis. The role that MDC1 may contribute to CLL pathogenesis, in particular its association with ATM dysregulation, remains to be elucidated. Further work to delineate critical pathways based on the discovered mutations is underway, and includes global transciptomic analysis of the purified leukaemic clones by RNAseq. DisclosuresNo relevant conflicts of interest to declare.