Exploring impact of mutations in non-BRCA DNA damage response (DDR) and non-DDR genes on efficacy in phase III EMBRACA study of talazoparib (TALA) in patients (pts) with germline BRCA1/2 mutated (gBRCAm) HER2-negative (HER2-) advanced breast cancer (ABC).

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. 1018-1018
Author(s):  
Jennifer Keating Litton ◽  
Douglas Laird ◽  
Hope S. Rugo ◽  
Johannes Ettl ◽  
Sara A. Hurvitz ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Tumor Response ◽  
Brca Mutation ◽  
Phase Iii ◽  
Loss Of Function ◽  
Single Nucleotide Variants ◽  
Tp53 Mutations ◽  
Mutational Status ◽  
Damage Response

1018 Background: Loss-of-function mutations in genes encoding components of the homologous recombination DNA damage response (DDR) machinery, notably BRCA1/2, are associated with tumor sensitivity to poly(ADP-ribose) polymerase inhibitors (PARPi). In EMBRACA, the PARPi TALA showed an improvement in progression-free survival (PFS) (HR [95% CI] 0.54 [0.41-0.71], P < 0.001) vs physician's choice of chemotherapy (PCT) in g BRCAm HER2− ABC. Methods: Baseline tumor tissue from 308 pts (71%; intent-to-treat) was sequenced using the FoundationOne CDx panel. Mutations summarized below were known/likely pathogenic single-nucleotide variants, insertions, deletions, or rearrangements. Best tumor response (BOR) was using RECIST 1.1 by Investigator (confirmation of CR or PR not required). Results: 296/308 (96%) of evaluable pts exhibited ≥1 tumor BRCA mutation, with 7 of the remaining 12 exhibiting BRCA copy number alterations deemed pathogenic. Mutations in other genes implicated in DDR and/or potential sensitization to PARPi were rare, with mutations detected in BARD1, CDK12, FANCG, STAG2 (each 0.3%), ATR, BRD4, FANCC, PALB2, RAD51B (0.6%), ATM, BRIP1 (1.0%), NBN (1.3%), CHEK2, FANCA (1.6%), and ARID1A (2.3%). No association was observed between total number of DDR mutations, including BRCA1/2, and best tumor response (BOR) [odds ratio of 1 vs ≥2 DDR mutations (95% CI): TALA, 0.76 (0.31-1.87), P = 0.55; PCT, 0.98 (0.27-3.51), P = 0.97]. TP53 and PIK3CA were the most commonly mutated non- BRCA genes in BRCAm tumors (52.0 and 10.8%, respectively). TP53 mutations were more prevalent in BRCA1m vs BRCA2m tumors (85.2 vs 24.8%). PIK3CA mutations were more prevalent in BRCA2m vs BRCA1m tumors (15.9 vs 5.2%). With TALA, PFS was significantly shorter in pts with TP53 mutations than without [HR (95% CI) 1.693 (1.186-2.418), P = 0.0033]. A similar, non-significant, trend was evident with PCT [HR (95% CI) 1.439 (0.859-2.411), P = 0.1614]. PIK3CA mutational status had no impact on PFS in either arm. Conclusions: Selection based on g BRCA mutational status is appropriate to identify HER2─ ABC pts with potential for clinical benefit from TALA, with the total number of tumor mutations in BRCA1/2 and other DDR genes not impacting response (within the g BRCAm subset). TP53 mutations were associated with shorter PFS, likely reflecting the worse outcomes observed in g BRCA1m patients. Additional correlative analyses are ongoing. Clinical trial information: NCT01945775 .

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

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.

The Response to DNA Damage in CLL Cells Is Partly Determined by the Type of TP53 Mutation and Genomic Aberrations

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3119-3119
Author(s):  
Julia Mohr ◽  
Thorsten Zenz ◽  
Dirk Winkler ◽  
Andreas Bühler ◽  
Daniel Mertens ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Tp53 Mutation ◽  
Normal Karyotype ◽  
Type B ◽  
Tp53 Mutations ◽  
Damage Response ◽  
Dna Damage Response Pathway ◽  
11Q Deletion

Abstract A central role of the DNA damage response pathway and particularly p53 has been suggested by the prognostic role of 17p and 11q deletions in CLL. In the critical regions, two prominent genes are located that are involved in the cells’ response to DNA damage. Functional assessment of p53 may be of help to more precisely define a subgroup of patients with defects in the DNA damage response pathway. The most commonly used assays include FACS measurement of induction of p53/p21 after DNA damage, induction of p53 by nutlins or the measurement of transcriptional targets of p53 by RT-PCR or MLPA. The growing evidence of the impact of sole TP53 mutations on prognosis led us to analyse the DNA damage response in CLL with special focus on cases with TP53 mutations in the absence of 17p deletion. We investigated the response to gamma-irradiation (5Gy) in a cohort of 71 CLL cases. We enriched the cohort for the bad prognostic subgroups 17p-, 11q- and TP53 mutation in the absence of 17p deletion in order to precisely dissect the DNA damage response in these subgroups. Levels of p53 and p21 before and after treatment were determined by flow cytometry. Cells were additionally stained with a CD19 antibody to differentiate between CLL cells and CD19 negative cells (T-cells). We developed a strategy to normalize the median values of the CLL cells to the T-cells. Defects in the ATM/p53 pathway can be divided into type A and type B defects that are characterized by an impaired p21 induction and a high or a low basal p53 level, respectively (Pettitt et al. 2001). Basal p53 levels in 17p-cases (n=15) were significantly higher than for cases with TP53 mutation (P=0.012), 11q deletion (P&lt;0.01) and normal karyotype/13p deletion (P&lt;0.01), respectively). Furthermore, induction of p21 was impaired after irradiation compared to 11q del and normal/13q del (P=0.0066 and P=0.0002). In the cases with a sole TP53 mutation (n=10) we observed higher basal p53 levels compared to 11q- and normal/13q- cases (P=0.044 and P=0.007). The p21 response was heterogeneous, which may be partly explained by the presence of the mutation in subclones. Some cases with TP53 mutation (n=3) showed low p21 induction with low basal p53 levels resembling type B defects. This finding was explained by the presence of splice site and frameshift mutations that prevent formation of p53 protein. These findings suggest that not all cases with TP53 will exhibit the classical type A pattern of expression. A heterogeneous pattern of p21 induction was observed in patients with an 11q deletion (n=23) and in patients with normal karyotype or 13q deletion (n=23), the induction of 11q deleted cases being significantly lower (P=0.0102). Still, there were 11q deleted cases that responded normally to DNA damage (9/23). These cases can be hypothesized to have retained a functional ATM copy. Similarly, some of the cases with normal karyotype or 13q deletion showed impaired p21/p53 induction (n=6) and the cause for this impairment are under investigation. This study shows the importance of 17p deletions and TP53 mutations in altering the response to DNA damage in CLL. While this assay provides some insight into the functionality of the ATM/p53 pathway and the thus potentially the treatment response, the clinical and diagnostic value is currently unclear. We observed F-refractory cases showing a rather normal in vitro response to DNA damage. Along the same lines, cases with sole TP53 mutation are partly grouped with the type B defect, suggesting that TP53 mutations cannot be correctly grouped in all cases. Since the induction of p21 was not very strong, the use of other p53 targets could help to define different groups more precisely. Our findings suggest that the type of TP53 mutation will impact the DNA damage response and potentially lead to misclassification. In how far the heterogeneous pattern of 11q cases and some cases with low risk cytogenetics will help to differentiate biological and prognostic subgroups will need to be assessed in a large independent cohort with homogenous treatment. Induction of p21 and p53 in CLL with different genetic profile. Figure Figure

scholarly journals RAD51-Mediated DNA Homologous Recombination Is Independent of PTEN Mutational Status

Cancers ◽  
2020 ◽  
Vol 12 (11) ◽  
pp. 3178
Author(s):  
Asha Sinha ◽  
Ali Saleh ◽  
Raelene Endersby ◽  
Shek H. Yuan ◽  
Chirayu R. Chokshi ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Cell Lines ◽  
Dna Damage Response ◽  
Rna Expression ◽  
Tumor Cell Lines ◽  
Mutational Status ◽  
Damage Response ◽  
Repair Activity ◽  
Tissue Specimens

PTEN mutation occurs in a variety of aggressive cancers and is associated with poor patient outcomes. Recent studies have linked mutational loss of PTEN to reduced RAD51 expression and function, a key factor involved in the homologous recombination (HR) pathway. However, these studies remain controversial, as they fail to establish a definitive causal link to RAD51 expression that is PTEN-dependent, while other studies have not been able to recapitulate the relationship between the PTEN expression and the RAD51/HR function. Resolution of this apparent conundrum is essential due to the clinically-significant implication that PTEN-deficient tumors may be sensitive to poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi) commonly used in the clinical management of BRCA-mutated and other HR-deficient (HRD) tumors. Methods: Primary Pten-deficient (and corresponding wild-type) mouse embryonic fibroblasts (MEFs) and astrocytes and PTEN-null human tumor cell lines and primary cells were assessed for RAD51 expression (via the Western blot analysis) and DNA damage repair analyses (via alkali comet and γH2AX foci assays). RAD51 foci analysis was used to measure HR-dependent DNA repair. Xrcc2-deficient MEFs served as an HR-deficient control, while the stable knockdown of RAD51 (shRAD51) served to control for the relative RAD51/HR-mediated repair and the phospho-53BP1 foci analysis served to confirm and measure non-homologous end joining (NHEJ) activity in PTEN-deficient and shRAD51-expressing (HRD) lines. Cell proliferation studies were used to measure any potential added sensitivity of PTEN-null cells to the clinically-relevant PARPi, olaparib. RAD51 levels and DNA damage response signaling were assessed in PTEN-mutant brain tumor initiating cells (BTICs) derived from primary and recurrent glioblastoma multiforme (GBM) patients, while expression of RAD51 and its paralogs were examined as a function of the PTEN status in the RNA expression datasets isolated from primary GBM tumor specimens and BTICs. Results: Pten knockout primary murine cells display unaltered RAD51 expression, endogenous and DNA strand break-induced RAD51 foci and robust DNA repair activity. Defective HR was only observed in the cells lacking Xrcc2. Likewise, human glioblastoma multiforme (GBM) cell lines with known PTEN deficiency (U87, PTEN-mutated; U251 and U373, PTEN-null) show apparent expression of RAD51 and display efficient DNA repair activity. Only GBM cells stably expressing shRNAs against RAD51 (shRAD51) display dysfunctional DNA repair activity and reduced proliferative capacity, which is exacerbated by PARPi treatment. Furthermore, GBM patient-derived BTICs displayed robust RAD51 expression and intact DNA damage response signaling in spite of PTEN-inactivating mutations. RNA expression analysis of primary GBM tissue specimens and BTICs demonstrate stable levels of RAD51 and its paralogs (RAD51B, RAD51C, RAD51D, XRCC2, XRCC3, and DMC1), regardless of the PTEN mutational status. Conclusions: Our findings demonstrate definitively that PTEN loss does not alter the RAD51 expression, its paralogs, or the HR activity. Furthermore, deficiency in PTEN alone is not sufficient to impart enhanced sensitivity to PARPi associated with HRD. This study is the first to unequivocally demonstrate that PTEN deficiency is not linked to the RAD51 expression or the HR activity amongst primary neural and non-neural Pten-null cells, PTEN-deficient tumor cell lines, and primary PTEN-mutant GBM patient-derived tissue specimens and BTICs.

scholarly journals Deciphering the Genetic Aberrations in DNA Damage Response Genes and Their Possible Association with HNSCC

2020 ◽  
pp. 156-169
Author(s):  
L. Akshayaa ◽  
A. S. Smiline Girija ◽  
A. Paramasivam ◽  
J. Vijayashree Priyadharsini
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Wide Spectrum ◽  
Survival Curve ◽  
Cell Cycle Checkpoint ◽  
Single Nucleotide Variants ◽  
Differential Expression Pattern ◽  
Damage Response ◽  
Cycle Checkpoint ◽  
High Level

Head and neck squamous cell carcinoma (HNSCC) includes carcinomas in the oral cavity, pharynx and larynx. It is considered as the sixth most common form of cancer in the world. Several studies have confirmed that smoking and alcohol consumption are the major risk factors for HNSCC. DNA damage response genes play an important role in the maintenance of the genome. Defects in cell cycle checkpoint and DNA repair mechanisms, such as mutation or abnormalities, may lead to the wide spectrum of human diseases. The present study employs databases and computational tools to identify the genetic abnormalities associated with DNA damage related genes which might have a direct or indirect association with HNSCC. The demographic details of HNSCC patients was obtained from The Cancer Gene Atlas (TCGA, Firehose Legacy) dataset hosted by the cBioportal database. The oncoprint data analysis revealed the highest frequency of gene alteration in the ATR gene (15%), followed by ATM, BRCA2 and CHEK2 (5%). Other genes showed less than 5% alteration. The gene expression profile of ATR gene revealed its differential expression pattern in different grades of tumor relative to normal samples. The survival curve analysis using Kaplan-Meier method revealed that a high level expression of the ATR gene leads to poor survival rate in the female HNSCC patients when compared to males. Thus the present study has identified gross and single nucleotide variants in the ATR gene which could have a putative role in the development of tumor. Further experimental research is required to confirm this association.

scholarly journals DAXX-ATRX-H3.3 Regulation of p53 Chromatin Binding and DNA Damage Response

2021 ◽  
Author(s):  
Nitish Gulve ◽  
Zhong Deng ◽  
Samantha Soldan ◽  
Olga Vladimirova ◽  
Jayamanna Wickramasinghe ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Chromatin Binding ◽  
Rna Seq ◽  
Loss Of Function ◽  
Glioblastoma Cells ◽  
Histone H3.3 ◽  
Alternative Lengthening ◽  
Damage Response ◽  
Chaperone Complex

Abstract DAXX and ATRX are tumor suppressor proteins that form a histone H3.3 chaperone complex and are frequently mutated in cancers with the alternative lengthening of telomeres (ALT), such as pediatric glioblastoma. Rapid loss of function of either DAXX or ATRX are not by themselves sufficient to induce the ALT phenotype. However, cells lacking DAXX or ATRX can be readily selected for ALT-like features. Here, we show that DAXX and ATRX null glioblastoma cells with ALT-like features have defects in p53 chromatin binding and DNA damage response regulation. RNA-seq analysis of DAXX or ATRX null U87 glioblastoma cells with ALT-like features revealed that p53 pathway is among perturbed. ALT-selected DAXX and ATRX-null cells had aberrant response to DNA damaging agent etoposide. Both DAXX and ATRX-null ALT cells showed a loss of p53 binding at a subset of response elements. Complementation of DAXX null cells with a wild-type DAXX transgene rescued p53 binding and transcription, while the tumor associated mutation L130R that disrupts ATRX binding was incapable of rescuing p53 chromatin binding. We show that histone H3.3 binding is reduced in DAXX-null cells especially at subtelomeric p53 binding sites and telomere repeats. These findings indicate that DAXX and ATRX function to enable p53 chromatin binding through modulation of histone H3.3 binding, especially at sub-telomeric sites.

scholarly journals IDH1-R132H acts as a tumor suppressor in glioma via epigenetic up-regulation of the DNA damage response

2019 ◽  
Vol 11 (479) ◽  
pp. eaaq1427 ◽  
Author(s):  
Felipe J. Núñez ◽  
Flor M. Mendez ◽  
Padma Kadiyala ◽  
Mahmoud S. Alghamri ◽  
Masha G. Savelieff ◽  
...  
Keyword(s):  
Dna Damage ◽  
Tumor Suppressor ◽  
Mouse Model ◽  
Dna Damage Response ◽  
Genetically Engineered ◽  
Low Grade ◽  
Loss Of Function ◽  
Genetically Engineered Mouse Model ◽  
Damage Response ◽  
Inactivating Mutations

Patients with glioma whose tumors carry a mutation in isocitrate dehydrogenase 1 (IDH1R132H) are younger at diagnosis and live longer. IDH1 mutations co-occur with other molecular lesions, such as 1p/19q codeletion, inactivating mutations in the tumor suppressor protein 53 (TP53) gene, and loss-of-function mutations in alpha thalassemia/mental retardation syndrome X-linked gene (ATRX). All adult low-grade gliomas (LGGs) harboring ATRX loss also express the IDH1R132H mutation. The current molecular classification of LGGs is based, partly, on the distribution of these mutations. We developed a genetically engineered mouse model harboring IDH1R132H, TP53 and ATRX inactivating mutations, and activated NRAS G12V. Previously, we established that ATRX deficiency, in the context of wild-type IDH1, induces genomic instability, impairs nonhomologous end-joining DNA repair, and increases sensitivity to DNA-damaging therapies. In this study, using our mouse model and primary patient-derived glioma cultures with IDH1 mutations, we investigated the function of IDH1R132H in the context of TP53 and ATRX loss. We discovered that IDH1R132H expression in the genetic context of ATRX and TP53 gene inactivation (i) increases median survival in the absence of treatment, (ii) enhances DNA damage response (DDR) via epigenetic up-regulation of the ataxia-telangiectasia–mutated (ATM) signaling pathway, and (iii) elicits tumor radioresistance. Accordingly, pharmacological inhibition of ATM or checkpoint kinases 1 and 2, essential kinases in the DDR, restored the tumors’ radiosensitivity. Translation of these findings to patients with IDH1132H glioma harboring TP53 and ATRX loss could improve the therapeutic efficacy of radiotherapy and, consequently, patient survival.

scholarly journals CHD4 in the DNA-damage response and cell cycle progression: not so NuRDy now

2013 ◽  
Vol 41 (3) ◽  
pp. 777-782 ◽  
Author(s):  
Aoife O’Shaughnessy ◽  
Brian Hendrich
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Transcriptional Regulation ◽  
Dna Damage Response ◽  
Cell Cycle Progression ◽  
Cell Cycle Checkpoint ◽  
Loss Of Function ◽  
Nurd Complex ◽  
Cycle Progression ◽  
Damage Response

The CHD4 (chromodomain-helicase-DNA-binding 4) (or Mi-2β) protein is a founding component of the NuRD (nucleosome remodelling and deacetylation) complex. NuRD has long been known to function in transcriptional regulation, and is conserved throughout the animal and plant kingdoms. In recent years, evidence has steadily accumulated indicating that CHD4 can both function outside of the NuRD complex and also play important roles in cellular processes other than transcriptional regulation. A number of loss-of-function studies have identified important roles for CHD4 in the DNA-damage response and in cell cycle progression through S-phase and into G2. Furthermore, as part of NuRD, it participates in regulating acetylation levels of p53, thereby indirectly regulating the G1/S cell cycle checkpoint. Although CHD4 has a somewhat complicated relationship with the cell cycle, recent evidence indicates that CHD4 may exert some tumour-suppressor functions in human carcinogenesis. CHD4 is a defining member of the NuRD complex, but evidence is accumulating that CHD4 also plays important NuRD-independent roles in the DNA-damage response and cell cycle progression, as well as in transcriptional regulation.

scholarly journals Ubiquitin-mediated DNA damage response is synthetic lethal with G-quadruplex stabilizer CX-5461

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Tehmina Masud ◽  
Charles Soong ◽  
Hong Xu ◽  
Justina Biele ◽  
Saelin Bjornson ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Synthetic Lethality ◽  
Clinical Activity ◽  
Helicase Domain ◽  
Loss Of Function ◽  
Genome Maintenance ◽  
Synthetic Lethal ◽  
G Quadruplex ◽  
Damage Response

AbstractCX-5461 is a G-quadruplex (G4) ligand currently in trials with initial indications of clinical activity in cancers with defects in homologous recombination repair. To identify more genetic defects that could sensitize tumors to CX-5461, we tested synthetic lethality for 480 DNA repair and genome maintenance genes to CX-5461, pyridostatin (PDS), a structurally unrelated G4-specific stabilizer, and BMH-21, which binds GC-rich DNA but not G4 structures. We identified multiple members of HRD, Fanconi Anemia pathways, and POLQ, a polymerase with a helicase domain important for G4 structure resolution. Significant synthetic lethality was observed with UBE2N and RNF168, key members of the DNA damage response associated ubiquitin signaling pathway. Loss-of-function of RNF168 and UBE2N resulted in significantly lower cell survival in the presence of CX-5461 and PDS but not BMH-21. RNF168 recruitment and histone ubiquitination increased with CX-5461 treatment, and nuclear ubiquitination response frequently co-localized with G4 structures. Pharmacological inhibition of UBE2N acted synergistically with CX-5461. In conclusion, we have uncovered novel genetic vulnerabilities to CX-5461 with potential significance for patient selection in future clinical trials.

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