Expression of human BRCA1Δ17–19 alternative splicing variant with a truncated BRCT domain in MCF-7 cells results in impaired assembly of DNA repair complexes and aberrant DNA damage response

Multiple myeloma (MM) is an incurable plasma cell malignancy characterized by genomic instability. MM cells present various forms of genetic instability, including chromosomal instability, microsatellite instability, and base-pair alterations, as well as changes in chromosome number. The tumor microenvironment and an abnormal DNA repair function affect genetic instability in this disease. In addition, states of the tumor microenvironment itself, such as inflammation and hypoxia, influence the DNA damage response, which includes DNA repair mechanisms, cell cycle checkpoints, and apoptotic pathways. Unrepaired DNA damage in tumor cells has been shown to exacerbate genomic instability and aberrant features that enable MM progression and drug resistance. This review provides an overview of the DNA repair pathways, with a special focus on their function in MM, and discusses the role of the tumor microenvironment in governing DNA repair mechanisms.

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Harnessing the Nucleolar DNA Damage Response in Cancer Therapy

Genes ◽

10.3390/genes12081156 ◽

2021 ◽

Vol 12 (8) ◽

pp. 1156

Author(s):

Jiachen Xuan ◽

Kezia Gitareja ◽

Natalie Brajanovski ◽

Elaine Sanij

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Cancer Therapy ◽

Dna Damage Response ◽

Rrna Genes ◽

Rrna Synthesis ◽

Pol I ◽

Clinical Investigations ◽

Damage Response ◽

Synthesis And Processing

The nucleoli are subdomains of the nucleus that form around actively transcribed ribosomal RNA (rRNA) genes. They serve as the site of rRNA synthesis and processing, and ribosome assembly. There are 400–600 copies of rRNA genes (rDNA) in human cells and their highly repetitive and transcribed nature poses a challenge for DNA repair and replication machineries. It is only in the last 7 years that the DNA damage response and processes of DNA repair at the rDNA repeats have been recognized to be unique and distinct from the classic response to DNA damage in the nucleoplasm. In the last decade, the nucleolus has also emerged as a central hub for coordinating responses to stress via sequestering tumor suppressors, DNA repair and cell cycle factors until they are required for their functional role in the nucleoplasm. In this review, we focus on features of the rDNA repeats that make them highly vulnerable to DNA damage and the mechanisms by which rDNA damage is repaired. We highlight the molecular consequences of rDNA damage including activation of the nucleolar DNA damage response, which is emerging as a unique response that can be exploited in anti-cancer therapy. In this review, we focus on CX-5461, a novel inhibitor of Pol I transcription that induces the nucleolar DNA damage response and is showing increasing promise in clinical investigations.

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Profilin-1; a novel regulator of DNA damage response and repair machinery in keratinocytes

Molecular Biology Reports ◽

10.1007/s11033-021-06210-6 ◽

2021 ◽

Author(s):

Chang-Jin Lee ◽

Min-Ji Yoon ◽

Dong Hyun Kim ◽

Tae Uk Kim ◽

Youn-Jung Kang

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Dna Damage Response ◽

Recovery Time ◽

Actin Polymerization ◽

Loss Of Function ◽

Dna Double Strand Breaks ◽

Pten Loss ◽

Damage Response ◽

Specific Regulation

AbstractProfilin-1 (PFN1) regulates actin polymerization and cytoskeletal growth. Despite the essential roles of PFN1 in cell integration, its subcellular function in keratinocyte has not been elucidated yet. Here we characterize the specific regulation of PFN1 in DNA damage response and repair machinery. PFN1 depletion accelerated DNA damage-mediated apoptosis exhibiting PTEN loss of function instigated by increased phosphorylated inactivation followed by high levels of AKT activation. PFN1 changed its predominant cytoplasmic localization to the nucleus upon DNA damage and subsequently restored the cytoplasmic compartment during the recovery time. Even though γH2AX was recruited at the sites of DNA double strand breaks in response to DNA damage, PFN1-deficient cells failed to recruit DNA repair factors, whereas control cells exhibited significant increases of these genes. Additionally, PFN1 depletion resulted in disruption of PTEN-AKT cascade upon DNA damage and CHK1-mediated cell cycle arrest was not recovered even after the recovery time exhibiting γH2AX accumulation. This might suggest PFN1 roles in regulating DNA damage response and repair machinery to protect cells from DNA damage. Future studies addressing the crosstalk and regulation of PTEN-related DNA damage sensing and repair pathway choice by PFN1 may further aid to identify new mechanistic insights for various DNA repair disorders.

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900 Depleting non-canonical, cell-intrinsic PD-L1 signals induces synthetic lethality to small molecule DNA damage response inhibitors in an immune independent and dependent manner

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.900 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A944-A944

Author(s):

Anand Kornepati ◽

Clare Murray ◽

Barbara Avalos ◽

Cody Rogers ◽

Kavya Ramkumar ◽

...

Keyword(s):

Ovarian Cancer ◽

Dna Damage ◽

Dna Repair ◽

Cell Lines ◽

Dna Damage Response ◽

Small Molecule ◽

Treatment Resistance ◽

Programmed Death ◽

Damage Response ◽

Response Biomarker

BackgroundTumor surface-expressed programmed death-ligand 1 (PD-L1) suppresses immunity when it engages programmed death-1 (PD-1) on anti-tumor immune cells in canonical PD-L1/PD-1.1 Non-canonical, tumour-intrinsic PD-L1 signals can mediate treatment resistance2–6 but mechanisms remain incompletely understood. Targeting non-canonical, cell-intrinsic PD-L1 signals, especially modulation of the DNA damage response (DDR), remains largely untapped.MethodsWe made PD-L1 knockout (PD-L1 KO) murine transplantable and human cell lines representing melanoma, bladder, and breast histologies. We used biochemical, genetic, and cell-biology techniques for mechanistic insights into tumor-intrinsic PD-L1 control of specific DDR and DNA repair pathways. We generated a novel inducible melanoma GEMM lacking PD-L1 only in melanocytes to corroborate DDR alterations observed in PD-L1 KO of established tumors.ResultsGenetic tumor PD-L1 depletion destabilized Chk2 and impaired ATM/Chk2, but not ATR/Chk1 DDR. PD-L1KO increased DNA damage (γH2AX) and impaired homologous recombination DNA repair (p-RPA32, BRCA1, RAD51 nuclear foci) and function (DR-GFP reporter). PD-L1 KO cells were significantly more sensitive versus controls to DDR inhibitors (DDRi) against ATR, Chk1, and PARP but not ATM in multiple human and mouse tumor models in vitro and in vivo in NSG mice. PD-1 independent, intracellular, not surface PD-L1 stabilized Chk2 protein with minimal Chek2 mRNA effect. Mechanistically, PD-L1 could directly complex with Chk2, protecting it from PIRH2-mediated polyubiquitination. PD-L1 N-terminal domains Ig-V and Ig-C but not the PD-L1 C-terminal tail co-IP’d with Chk2 and restored Chk1 inhibitor (Chk1i) treatment resistance. Tumor PD-L1 expression correlated with Chk1i sensitivity in 44 primary human small cell lung cancer cell lines, implicating tumor-intrinsic PD-L1 as a DDRi response biomarker. In WT mice, genetic PD-L1 depletion but not surface PD-L1 blockade with αPD-L1, sensitized immunotherapy-resistant, BRCA1-WT 4T1 tumors to PARP inhibitor (PARPi). PARPi effects were reduced on PD-L1 KO tumors in RAG2KO mice indicating immune-dependent DDRi efficacy. Tumor PD-L1 depletion, likely due to impaired DDR, enhanced PARPi induced tumor-intrinsic STING activation (e.g., p-TBK1, CCL5) suggesting potential to augment immunotherapies.ConclusionsWe challenge the prevailing surface PD-L1 paradigm and establish a novel mechanism for cell-intrinsic PD-L1 control of the DDR and gene product expression. We identify therapeutic vulnerabilities from tumor PD-L1 depletion utilizing small molecule DDRi currently being tested in clinical trials. Data could explain αPD-L1/DDRi treatment resistance. Intracellular PD-L1 could be a pharmacologically targetable treatment target and/or response biomarker for selective DDRi alone plus other immunotherapies.ReferencesTopalian SL, Taube JM, Anders RA, Pardoll DM. Mechanism-driven biomarkers to guide immune checkpoint blockade in cancer therapy. Nat Rev Cancer 16:275–287, doi:10.1038/nrc.2016.36 (2016).Clark CA, et al. Tumor-intrinsic PD-L1 signals regulate cell growth, pathogenesis and autophagy in ovarian cancer and melanoma. Canres 0258.2016 (2016).Gupta HB et al. Tumor cell-intrinsic PD-L1 promotes tumor-initiating cell generation and functions in melanoma and ovarian cancer. 1, 16030 (2016).Zhu H, et al. BET bromodomain inhibition promotes anti-tumor immunity by suppressing PD-L1 expression. Cell Rep 16:2829–2837, doi:10.1016/j.celrep.2016.08.032 (2016)Wu B, et al. Adipose PD-L1 modulates PD-1/PD-L1 checkpoint blockade immunotherapy efficacy in breast cancer. Oncoimmunology 7:e1500107, doi:10.1080/2162402X.2018.1500107 (2018)Liang J, et al. Verteporfin inhibits PD-L1 through autophagy and the STAT1-IRF1-TRIM28 signaling axis, exerting antitumor efficacy. Cancer Immunol Res 8:952–965, doi:10.1158/2326-6066.CIR-19-0159 (2020)

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Mutant huntingtin impairs PNKP and ATXN3, disrupting DNA repair and transcription

eLife ◽

10.7554/elife.42988 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 15

Author(s):

Rui Gao ◽

Anirban Chakraborty ◽

Charlene Geater ◽

Subrata Pradhan ◽

Kara L Gordon ◽

...

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Rna Polymerase Ii ◽

Dna Damage Response ◽

Functional Decline ◽

Transcriptional Elongation ◽

Damage Repair ◽

Damage Response ◽

Cyclic Amp Response Element ◽

Atm Signaling

How huntingtin (HTT) triggers neurotoxicity in Huntington’s disease (HD) remains unclear. We report that HTT forms a transcription-coupled DNA repair (TCR) complex with RNA polymerase II subunit A (POLR2A), ataxin-3, the DNA repair enzyme polynucleotide-kinase-3'-phosphatase (PNKP), and cyclic AMP-response element-binding (CREB) protein (CBP). This complex senses and facilitates DNA damage repair during transcriptional elongation, but its functional integrity is impaired by mutant HTT. Abrogated PNKP activity results in persistent DNA break accumulation, preferentially in actively transcribed genes, and aberrant activation of DNA damage-response ataxia telangiectasia-mutated (ATM) signaling in HD transgenic mouse and cell models. A concomitant decrease in Ataxin-3 activity facilitates CBP ubiquitination and degradation, adversely impacting transcription and DNA repair. Increasing PNKP activity in mutant cells improves genome integrity and cell survival. These findings suggest a potential molecular mechanism of how mutant HTT activates DNA damage-response pro-degenerative pathways and impairs transcription, triggering neurotoxicity and functional decline in HD.

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Endometrial DNA damage response is modulated in endometriosis

Human Reproduction ◽

10.1093/humrep/deaa255 ◽

2020 ◽

Author(s):

Kashmira Bane ◽

Junita Desouza ◽

Diksha Shetty ◽

Prakash Choudhary ◽

Shalaka Kadam ◽

...

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Dna Damage Response ◽

Small Sample ◽

Nuclear Antigen ◽

Dna Repair Genes ◽

Eutopic Endometrium ◽

Damage Response ◽

Repair Genes

Abstract STUDY QUESTION Is the DNA damage response (DDR) dysregulated in the eutopic endometrium of women with endometriosis? SUMMARY ANSWER Endometrial expression of genes involved in DDR is modulated in women with endometriosis, compared to those without the disease. WHAT IS KNOWN ALREADY Ectopic endometriotic lesions are reported to harbour somatic mutations, thereby hinting at dysregulation of DDR and DNA repair pathways. However, it remains inconclusive whether the eutopic endometrium also manifests dysregulated DDR in endometriosis. STUDY DESIGN, SIZE, DURATION For this case–control study conducted between 2015 and 2019, eutopic endometrial (E) samples (EE- from women with endometriosis, CE- from women without endometriosis) were collected in either mid-proliferative (EE-MP, n = 23; CE-MP, n = 17) or mid-secretory (EE-MS, n = 17; CE-MS, n = 9) phases of the menstrual cycle. This study compares: (i) DNA damage marker localization, (ii) expression of DDR genes and (iii) expression of DNA repair genes in eutopic endometrial samples from women with and without endometriosis. PARTICIPANTS/MATERIALS, SETTING, METHODS The study included (i) 40 women (aged 31.9 ± 0.81 years) with endometriosis and (ii) 26 control women (aged 31.4 ± 1.02 years) without endometriosis. Eutopic endometrial samples from the two groups were divided into different parts for histological analysis, immunohistochemistry, RNA extraction, protein extraction and comet assays. Eighty-four genes of relevance in the DNA damage signalling pathway were evaluated for their expression in eutopic endometrial samples, using RT2 Profiler PCR arrays. Validations of the expression of two GADD (Growth Arrest DNA Damage Inducible) proteins - GADD45A and GADD45G were carried out by immunoblotting. DNA damage was assessed by immunohistochemical localization of γ-H2AFX (a phosphorylated variant of histone H2AX) and 8-OHdG (8-hydroxy-2′-deoxyguanosine). RNA sequencing data from mid-proliferative (EE-MP, n = 4; CE-MP, n = 3) and mid-secretory phase (EE-MS and CE-MS, n = 4 each) endometrial samples were scanned to compare the expression status of all the genes implicated in human DNA repair. PCNA (Proliferating Cell Nuclear Antigen) expression was determined to assess endometrial proliferation. Residual DNA damage in primary endometrial cells was checked by comet assays. Public datasets were also scanned for the expression of DDR and DNA repair genes as our RNASeq data were limited by small sample size. All the comparisons were made between phase-matched endometrial samples from women with and without endometriosis. MAIN RESULTS AND THE ROLE OF CHANCE Endometrial expression of DDR genes and intensity of immunolocalized γ-H2AFX were significantly (P < 0.05) higher in EE, compared to CE samples. DDR proteins, especially those belonging to the GADD family, were found to be differentially abundant in EE, as compared to CE. These patterns were evident in both mid-proliferative and mid-secretory phases. Intriguingly, higher DDR was associated with increased cell proliferation in EE-MP, compared to CE-MP. Furthermore, among the differentially expressed transcripts (DETs) encoded by DNA repair genes, the majority showed up-regulation in EE-MP, compared to CE-MP. Interestingly, CE-MP and EE-MP had a comparable percentage (P > 0.05) of cells with residual DNA damage. However, unlike the mid-proliferative phase data, many DETs encoded by DNA repair genes were down-regulated in EE-MS, compared to CE-MS. An analysis of the phase-matched control and endometriosis samples included in the GSE51981 dataset available in the Gene Expression Omnibus database also revealed significant (P < 0.05) alterations in the expression of DDR and DNA repair genes in EE, compared to CE. LARGE-SCALE DATA N/A LIMITATIONS, REASONS FOR CAUTION The study was conducted on a limited number of endometrial samples. Also, the study does not reveal the causes underlying dysregulated DDR in the eutopic endometrium of women with endometriosis. WIDER IMPLICATIONS OF THE FINDINGS Alterations in the expression of DDR and DNA repair genes indirectly suggest that eutopic endometrium, as compared to its healthy counterpart, encounters DNA damage-inducing stimuli, either of higher strength or for longer duration in endometriosis. It will be worthwhile to identify the nature of such stimuli and also explore the role of higher genomic insults and dysregulated DDR/DNA repair in the origin and/or progression of endometriosis. STUDY FUNDING/COMPETING INTEREST(S) The study was supported by the Department of Biotechnology and Indian Council of Medical Research, Government of India. No conflict of interest is declared.

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RT2 PCR array screening reveals distinct perturbations in DNA damage response signaling in FUS-associated motor neuron disease

Molecular Brain ◽

10.1186/s13041-019-0526-4 ◽

2019 ◽

Vol 12 (1) ◽

Cited By ~ 2

Author(s):

Haibo Wang ◽

Suganya Rangaswamy ◽

Manohar Kodavati ◽

Joy Mitra ◽

Wenting Guo ◽

...

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Motor Neuron ◽

Motor Neuron Disease ◽

Dna Damage Response ◽

Spinal Cord Tissue ◽

Sporadic Als ◽

Damage Response ◽

Als Patients

AbstractAmyotrophic lateral sclerosis (ALS) is a degenerative motor neuron disease that has been linked to defective DNA repair. Many familial ALS patients harbor autosomal dominant mutations in the gene encoding the RNA/DNA binding protein ‘fused in sarcoma’ (FUS) commonly inducing its cytoplasmic mislocalization. Recent reports from our group and others demonstrate a role of FUS in maintaining genome integrity and the DNA damage response (DDR). FUS interacts with many DDR proteins and may regulate their recruitment at damage sites. Given the role of FUS in RNA transactions, here we explore whether FUS also regulates the expression of DDR factors. We performed RT2 PCR arrays for DNA repair and DDR signaling pathways in CRISPR/Cas9 FUS knockout (KO) and shRNA mediated FUS knockdown (KD) cells, which revealed significant (> 2-fold) downregulation of BRCA1, DNA ligase 4, MSH complex and RAD23B. Importantly, similar perturbations in these factors were also consistent in motor neurons differentiated from an ALS patient-derived induced pluripotent stem cell (iPSC) line with a FUS-P525L mutation, as well as in postmortem spinal cord tissue of sporadic ALS patients with FUS pathology. BRCA1 depletion has been linked to neuronal DNA double-strand breaks (DSBs) accumulation and cognitive defects. The ubiquitin receptor RAD23 functions both in nucleotide excision repair and proteasomal protein clearance pathway and is thus linked to neurodegeneration. Together, our study suggests that the FUS pathology perturbs DDR signaling via both its direct role and the effect on the expression of DDR genes. This underscors an intricate connections between FUS, genome instability, and neurodegeneration.

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Recent advances in the nucleolar responses to DNA double-strand breaks

Nucleic Acids Research ◽

10.1093/nar/gkaa713 ◽

2020 ◽

Vol 48 (17) ◽

pp. 9449-9461

Author(s):

Lea Milling Korsholm ◽

Zita Gál ◽

Blanca Nieto ◽

Oliver Quevedo ◽

Stavroula Boukoura ◽

...

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Dna Damage Response ◽

Ribosomal Dna ◽

Repetitive Sequences ◽

Dna Lesions ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Damage Response

Abstract DNA damage poses a serious threat to human health and cells therefore continuously monitor and repair DNA lesions across the genome. Ribosomal DNA is a genomic domain that represents a particular challenge due to repetitive sequences, high transcriptional activity and its localization in the nucleolus, where the accessibility of DNA repair factors is limited. Recent discoveries have significantly extended our understanding of how cells respond to DNA double-strand breaks (DSBs) in the nucleolus, and new kinases and multiple down-stream targets have been identified. Restructuring of the nucleolus can occur as a consequence of DSBs and new data point to an active regulation of this process, challenging previous views. Furthermore, new insights into coordination of cell cycle phases and ribosomal DNA repair argue against existing concepts. In addition, the importance of nucleolar-DNA damage response (n-DDR) mechanisms for maintenance of genome stability and the potential of such factors as anti-cancer targets is becoming apparent. This review will provide a detailed discussion of recent findings and their implications for our understanding of the n-DDR. The n-DDR shares features with the DNA damage response (DDR) elsewhere in the genome but is also emerging as an independent response unique to ribosomal DNA and the nucleolus.

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