DNA Breaks Research Articles

A molecular glue RBM39-degrader induces synthetic lethality in cancer cells with homologous recombination repair deficiency

E7820 and Indisulam (E7070) are sulfonamide molecular glues that modulate RNA splicing by degrading the splicing factor RBM39 via ternary complex formation with the E3 ligase adaptor DCAF15. To identify biomarkers of the antitumor efficacy of E7820, we treated patient-derived xenograft (PDX) mouse models established from 42 patients with solid tumors. The overall response rate was 38.1% (16 PDXs), and tumor regression was observed across various tumor types. Exome sequencing of the PDX genome revealed that loss-of-function mutations in genes of the homologous recombination repair (HRR) system, such as ATM, were significantly enriched in tumors that responded to E7820 (p = 4.5 × 103). Interestingly, E7820-mediated double-strand breaks in DNA were increased in tumors with BRCA2 dysfunction, and knockdown of BRCA1/2 transcripts or knockout of ATM, ATR, or BAP1 sensitized cancer cells to E7820. Transcriptomic analyses revealed that E7820 treatment resulted in the intron retention of mRNAs and decreased transcription, especially for HRR genes. This induced HRR malfunction probably leads to the synthetic lethality of tumor cells with homologous recombination deficiency (HRD). Furthermore, E7820, in combination with olaparib, exerted a synergistic effect, and E7820 was even effective in an olaparib-resistant cell line. In conclusion, HRD is a promising predictive biomarker of E7820 efficacy and has a high potential to improve the prognosis of patients with HRD-positive cancers.

Biallelic FANCA variants detected in sisters with isolated premature ovarian insufficiency.

Premature ovarian insufficiency is a common form of female infertility affecting up to 4% of women and characterised by amenorrhea with elevated gonadotropin before the age of 40. Oocytes require controlled DNA breakage and repair for homologous recombination and the maintenance of oocyte integrity. Biallelic disruption of the DNA damage repair gene, Fanconi anemia complementation group A (FANCA), is a common cause of Fanconi anaemia, a syndrome characterised by bone marrow failure, cancer predisposition, physical anomalies and POI. There is ongoing dispute about the role of heterozygous FANCA variants in POI pathogenesis, with insufficient evidence supporting causation. Here, we have identified biallelic FANCA variants in French sisters presenting with POI, including a novel missense variant of uncertain significance and a likely pathogenic deletion that initially evaded detection. Functional studies indicated no discernible effect on DNA damage sensitivity in patient lymphoblasts. These novel FANCA variants add evidence that heterozygous loss of one allele is insufficient to cause DNA damage sensitivity and POI. We propose that intragenic deletions, that are relatively common in FANCA, may be missed without careful analysis, and could explain the presumed causation of heterozygous variants. Accurate variant curation is critical to optimise patient care and outcomes.

Inotuzumab Ozogamicin: First Pediatric Approval.

Inotuzumab ozogamicin (BESPONSA™) is a CD22-targeted monoclonal antibody drug conjugate (ADC) developed by Pfizer for the treatment of CD22-postive B-cell precursor acute lymphoblastic leukaemia (ALL). Inotuzumab ozogamicin comprises a humanized IgG4 anti-CD22 monoclonal antibody covalently linked to the potent DNA-binding cytotoxic agent N-acetyl-gamma-calicheamicin dimethylhydrazide (CalichDMH) via a linker. Inotuzumab ozogamicin binds to CD22-expressing tumour cells, facilitating the delivery of conjugated CalichDMH, which after intracellular activation induces double strand DNA breaks, ultimately leading to cell cycle arrest and apoptotic cell death. Inotuzumab ozogamicin is approved in the USA, Europe and several countries worldwide for the treatment of relapsed or refractory CD22-positive B-cell precursor ALL in adults. On 6 March 2024, inotuzumab ozogamicin received its first pediatric approval in the USA for this indication in patients aged ≥1 years. Inotuzumab ozogamicin has since been approved in Japan in March 2024 for the same indication in pediatric patients. This article summarizes the milestones in the development of inotuzumab ozogamicin leading to this first approval for the treatment of relapsed or refractory CD22-positive B-cell precursor ALL in pediatric patients.

A PARP2-specific active site α-helix melts to permit DNA damage-induced enzymatic activation.

PARP1 and PARP2 recognize DNA breaks immediately upon their formation, generate a burst of local PARylation to signal their location, and are co-targeted by all current FDA-approved forms of PARP inhibitors (PARPi) used in the cancer clinic. Recent evidence indicates that the same PARPi molecules impact PARP2 differently from PARP1, raising the possibility that allosteric activation may also differ. We find that unlike for PARP1, destabilization of the autoinhibitory domain of PARP2 is insufficient for DNA damage-induced catalytic activation. Rather, PARP2 activation requires further unfolding of an active site α-helix absent in PARP1. Only one clinical PARPi, Olaparib, stabilizes the PARP2 active site α-helix, representing a structural feature with the potential to discriminate small molecule inhibitors. Collectively, our findings reveal unanticipated differences in local structure and changes in activation-coupled backbone dynamics between PARP1 and PARP2.

Abstract B012: BET inhibition sensitizes preclinical models of bladder cancer to DDR inhibitors

Abstract Background: Bladder cancer (BC) remains a common and deadly disease, with 83,190 cases and 32,350 deaths projected in the U.S. in 2024. We previously showed bromodomain and extra-terminal protein inhibitors (BETi) are potent in multiple preclinical models of BC. Our bulk RNA-seq and RT-qPCR data showed BETi significantly reduces expression of homologous recombination (HR) genes, such as RAD51 and RBBP8, which could activate alternative DNA repair pathways including non-homologous end joining (NHEJ) and theta-mediated end joining (TMEJ). Specifically, DNA breaks are repaired through TMEJ by polymerase theta (Polθ) utilizing microhomology templates. Recently published data revealed Polθ inhibition is synthetically lethal when other DDR genes are concomitantly lost or inhibited. Thus, in this study, we sought to elucidate whether BETi-induced HR gene repression impacts TMEJ and POLQ expression, and to identify combinations with BETi that best leverage Polθ-mediated synthetic lethality. Methods: 5637 and J82 BC cells were pretreated with birabresib for 24 h and then transfected via electroporation with a Cas9 ribonucleoprotein, a gRNA targeting LBR2 and a 996 bp HR donor. Transfected cells were treated with birabresib for 24H, and DDR pathway function was assessed by digital droplet PCR. BC cells were synchronized at the G1/S transition with 2 mM thymidine incubated with birabresib alone. After 48 h, cells were harvested, and changes to target gene expression (i.e., POLQ) was assessed by RT-qPCR. Next, the same cells were treated with 8 ascending doses of the BETi birabresib (0.1–100 μM) alone or in combination with either the PARP inhibitor olaparib, CtIP inhibitor triapene, or RAD51 inhibitor RI-1 (10 nM–200 μM). After 72-96 h, CellTiter-Glo™ measured cell viability, and Compusyn v1 calculated combination index (CI) scores where <1.0 indicated synergism and >1.0 indicated antagonism. RT-qPCR was conducted to evaluate expression changes to POLQ after combination treatment. Results: After 48 h, birabresib significantly reduced TMEJ in both 5637 and J82 BC cells (49% reduction, P=0.008 and 41% reduction, P=0.004 both n=3), but did not significantly impact HR or NHEJ. POLQ mRNA expression was most reduced in synchronized 5637 and J82 BC cells after treatment with birabresib alone (46% reduction, P=0.006, n=3, and 38% reduction, P=0.001, n=3). Birabresib combined with olaparib was the most synergistic combination in both 5637 and J82 BC cells (CI = 0.83 and 0.29), but birabresib combined with triapene was also synergistic (CI = 0.91 and 0.90). In J82 cells, birabresib significantly reduced POLQ expression (27% reduction, P=0.008, n=3), but olaparib did not. In both 5637 and J82 cells combined birabresib and olaparib increased POLQ expression (41% increase and 5% increase, ns, n=3). Combined birabresib and triapene displayed a similar trend (46% increase and 17% increase, ns, n=3). Conclusions: These preliminary data support further exploration of POLQ expression increases caused by combined BETi+DDRi treatment in preclinical models of BC. Citation Format: Ryan M. Kemper, Bhavika C. Chirumamilla, Dennis A. Simpson, Manfred Meng, Gaorav P. Gupta, Daniel J. Crona. BET inhibition sensitizes preclinical models of bladder cancer to DDR inhibitors [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr B012.

Epigenetic and gene therapy in human and veterinary medicine.

Gene therapy is a focus of interest in both human and veterinary medicine, especially in recent years due to the potential applications of CRISPR/Cas9 technology. Another relatively new approach is that of epigenetic therapy, which involves an intervention based on epigenetic marks, including DNA methylation, histone post-translational modifications, and post-transcription modifications of distinct RNAs. The epigenome results from enzymatic reactions, which regulate gene expression without altering DNA sequences. In contrast to conventional CRISP/Cas9 techniques, the recently established methodology of epigenetic editing mediated by the CRISPR/dCas9 system is designed to target specific genes without causing DNA breaks. Both natural epigenetic processes and epigenetic editing regulate gene expression and thereby contribute to maintaining the balance between physiological functions and pathophysiological states. From this perspective, knowledge of specific epigenetic marks has immense potential in both human and veterinary medicine. For instance, the use of epigenetic drugs (chemical compounds with therapeutic potential affecting the epigenome) seems to be promising for the treatment of cancer, metabolic, and infectious diseases. Also, there is evidence that an epigenetic diet (nutrition-like factors affecting epigenome) should be considered as part of a healthy lifestyle and could contribute to the prevention of pathophysiological processes. In summary, epigenetic-based approaches in human and veterinary medicine have increasing significance in targeting aberrant gene expression associated with various diseases. In this case, CRISPR/dCas9, epigenetic targeting, and some epigenetic nutrition factors could contribute to reversing an abnormal epigenetic landscape to a healthy physiological state.

PARP1 inhibition prevents oxidative stress in age-related hearing loss via PAR-Ca2+-AIF axis in cochlear strial marginal cells

Studies have highlighted oxidative damage in the inner ear as a critical pathological basis for sensorineural hearing loss, especially the presbycusis. Poly(ADP-ribose) polymerase-1 (PARP1) activation responds to oxidative stress-induced DNA damage with pro-repair and pro-death effects resembling two sides of the same coin. PARP1-related cell death, known as parthanatos, whose underlying mechanisms are attractive research hotspots but remain to be clarified. In this study, we observed that aged rats showed stria vascularis degeneration and oxidative damage, and PARP1-dependent cell death was prominent in age-related cochlear disorganization and dysfunction. Based on oxidative stress model of primary cultured stria marginal cells (MCs), we revealed that upregulated PARP1 and PAR (Poly(ADP-ribose)) polymers are responsible for MCs oxidative death with high mitochondrial permeability transition pore (mPTP) opening and mitochondrial membrane potential (MMP) collapse, while inhibition of PARP1 ameliorated the adverse outcomes. Importantly, the PARylation of apoptosis-inducing factor (AIF) is essential for its conformational change and translocation, which subsequently causes DNA break and cell death. Concretely, the interaction of PAR and truncated AIF (tAIF) is the mainstream in the parthanatos pathway. We also found that the effects of AIF cleavage and release were achieved through calpain activity and mPTP opening, both of which could be regulated by PARP1 via mediation of mitochondria Ca2+ concentration. In conclusion, the PAR-Ca2+-tAIF signaling pathway in parthanatos contributes to the oxidative stress damage observed in MCs. Targeting PAR-Ca2+-tAIF might be a potential therapeutic strategy for the early intervention of presbycusis and other oxidative stress-associated sensorineural deafness.

Exploring the intersection of epigenetics, DNA repair, and immunology from studies of ICF syndrome, an inborn error of immunity.

Immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome, a rare autosomal recessive disorder, manifests with hypoglobulinemia and chromosomal instability accompanied by DNA hypomethylation. Pathological variants in the DNMT3B, ZBTB24, CDCA7, or HELLS genes underlie its etiology. Activated lymphocytes from patients often display distinctive multiradial chromosomes fused via pericentromeric regions. Recent studies have provided deeper insights into how pathological variants in ICF-related proteins cause DNA hypomethylation and chromosome instability. However, the understanding of the molecular pathogenesis underlying immunodeficiency is still in its nascent stages. In the past half-decade, the roles of CDCA7, HELLS, and ZBTB24 in classical non-homologous end joining during double-strand DNA break repair and immunoglobulin class-switch recombination (CSR) have been unveiled. Nevertheless, given the decreased all classes of immunoglobulins in most patients, CSR deficiency alone cannot fully account for the immunodeficiency. The latest finding showing dysregulation of immunoglobulin signaling may provide a clue to understanding the immunodeficiency mechanism. While less common, a subgroup of patients exhibits T-cell abnormalities alongside B-cell anomalies, including reduced regulatory T-cells and increased effector memory T- and follicular helper T-cells. The dysregulation of immunoglobulin signaling in B-cells, the imbalance in T-cell subsets, and/or satellite RNA-mediated activation of innate immune response potentially explain autoimmune manifestations in a subset of patients. These findings emphasize the pivotal roles of ICF-related proteins in both B- and T-cell functions. ICF syndrome studies have illuminated many fundamental mechanisms. Further investigations will certainly continue to unveil additional mechanisms and their interplay.

Structural characterization of squash polysaccharide and its effect on STZ-induced diabetes mellitus model in MIN6 cells

A novel natural water-soluble acidic polysaccharide (PWESP-3) was isolated from squash with a molecular mass of 140.519 kDa, which was composed of arabinose (Ara, 35.30 mol%), galactose (Gal, 61.20 mol%), glucose (Glc, 1.80 mol%), and Mannuronic acid (ManA, 1.70 mol%) and contained Araf-(1→, →3)-Araf-(1→, →5)-Araf-(1→, Glcp-(1→, Galp-(1→, →3,5)-Araf-(1→, →2)-Glcp-(1→, →2)-Manp-(1→, →3)-Glcp-(1→, →4)-Galp-(1→, →3)-Galp-(1→, →6)-Galp-(1→, →3,4)-Galp-(1→, →4,6)-Galp-(1→ residues in the backbone. Moreover, the structure of PWESP-3 was identified by NMR spectra. The branch chain was connected to the main chain by the O-3 and O-4 atom of Gal. In addition, the effect of PWESP-3 on STZ-induced type I diabetes mellitus model in MIN6 cells was investigated. The results showed that PWESP-3 can increase the viability and insulin secretion of MIN6 cells and reduce the oxidative stress caused by ROS and NO. Meanwhile, PWESP-3 can also reduce the content of ATP, Ca2+, mitochondrial membrane potential and Caspase-3 activity in MIN6 cells. Furthermore, treatment with PWESP-3 can prevent single or double stranded DNA breaking to form DNA fragments and improve DNA damage in MIN6 cells, thereby avoiding apoptosis. Therefore, the above data highlight that PWESP-3 can improve the function of insulin secretion in STZ-induced MIN6 cells in vitro and can be used as an alternative food supplement to diabetes drugs.

Genotoxicity Associated with Retroviral CAR Transduction of ATM-Deficient T Cells.

Somatic variants in DNA damage response genes such as ATM are widespread in hematologic malignancies. ATM protein is essential for double-strand DNA break repair. Germline ATM deficiencies underlie ataxia-telangiectasia (A-T), a disease manifested by radiosensitivity, immunodeficiency, and predisposition to lymphoid malignancies. Patients with A-T diagnosed with malignancies have poor tolerance to chemotherapy or radiation. In this study, we investigated chimeric antigen receptor (CAR) T cells using primary T cells from patients with A-T (ATM-/-), heterozygote donors (ATM+/-), and healthy donors. ATM-/- T cells proliferate and can be successfully transduced with CARs, though functional impairment of ATM-/- CAR T-cells was observed. Retroviral transduction of the CAR in ATM-/- T cells resulted in high rates of chromosomal lesions at CAR insertion sites, as confirmed by next-generation long-read sequencing. This work suggests that ATM is essential to preserve genome integrity of CAR T-cells during retroviral manufacturing, and its lack poses a risk of chromosomal translocations and potential leukemogenicity. Significance: CAR T-cells are clinically approved genetically modified cells, but the control of genome integrity remains largely uncharacterized. This study demonstrates that ATM deficiency marginally impairs CAR T-cell function and results in high rates of chromosomal aberrations after retroviral transduction, which may be of concern in patients with DNA repair deficiencies.

H2B oncohistones cause homologous recombination defect and genomic instability through reducing H2B monoubiquitination in Schizosaccharomyces pombe

Canonical oncohistones are histone H3 mutations in the N-terminal tail associated with tumors and affect gene expression by altering H3 post-translational modifications (PTMs) and the epigenetic landscape. Noncanonical oncohistone mutations occur in both tails and globular domains of all 4 core histones and alter gene expression by perturbing chromatin remodeling. However, the effects and mechanisms of noncanonical oncohistones remain largely unknown. Here we characterized 16 noncanonical H2B oncohistones in the fission yeast Schizosaccharomyces pombe. We found that 7 of them exhibited temperature sensitivities and 11 exhibited genotoxic sensitivities. A detailed study of 2 of these onco-mutants H2BG52D and H2BP102L revealed that they were defective in homologous recombination (HR) repair with compromised histone eviction and Rad51 recruitment. Interestingly, their genotoxic sensitivities and HR defects were rescued by inactivation of the H2BK119 deubiquitination function of Ubp8 in the Spt-Ada-Gcn5-Acetyltransferase (SAGA) complex. The levels of H2BK119 monoubiquitination (H2Bub) in the H2BG52D and H2BP102L mutants are reduced in global and local DNA break sites presumably due to enhanced recruitment of Ubp8 onto nucleosomes, and are recovered upon loss of H2B deubiquitination function of the SAGA complex. Moreover, H2BG52D and H2BP102L heterozygotes exhibit genotoxic sensitivities and reduced H2Bub in cis. We therefore conclude that H2BG52D and H2BP102L oncohistones affect HR repair and genome stability via reduction of H2Bub and propose that other noncanonical oncohistones may also affect histone PTMs to cause diseases.

In vivo genome editing via CRISPR/Cas9-mediated homology-independent targeted integration for Bietti crystalline corneoretinal dystrophy treatment

Bietti crystalline corneoretinal dystrophy (BCD) is an autosomal recessive chorioretinal degenerative disease without approved therapeutic drugs. It is caused by mutations in CYP4V2 gene, and about 80% of BCD patients carry mutations in exon 7 to 11. Here, we apply CRISPR/Cas9 mediated homology-independent targeted integration (HITI)-based gene editing therapy in HEK293T cells, BCD patient derived iPSCs, and humanized Cyp4v3 mouse model (h-Cyp4v3mut/mut) using two rAAV2/8 vectors via sub-retinal administration. We find that sgRNA-guided Cas9 generates double-strand cleavage on intron 6 of the CYP4V2 gene, and the HITI donor inserts the carried sequence, part of intron 6, exon 7-11, and a stop codon into the DNA break, achieving precise integration, effective transcription and translation both in vitro and in vivo. HITI-based editing restores the viability of iPSC-RPE cells from BCD patient, improves the morphology, number and metabolism of RPE and photoreceptors in h-Cyp4v3mut/mut mice. These results suggest that HITI-based editing could be a promising therapeutic strategy for those BCD patients carrying mutations in exon 7 to 11, and one injection will achieve lifelong effectiveness.

An Overview Of The Biological Methods Used To Assess DNA Damage In Humans

The purpose of this work is to summarize the biological methods available for assessing DNA damage in humans at present. There are several methods for determining single-strand DNA breaks, alkali-labile sites, and crosslinks, including comet assays, micronucleus assays, cytogenetics (which include sister chromatid exchange and chromosomal aberration assays), DNA repair assays, oxidative DNA damage assays (measuring oxidized bases such as 8-oxo-7, 8-dihydro-2'-deoxyguanosine), and oxidized bases. There are many factors to consider when determining the best method, including how to achieve the study's objectives, what type of DNA damage to measure, and what resources are available. Combining different techniques may also contribute to a more comprehensive understanding of DNA damage and its effects on human health. By standardizing assays and advancing technology, we will be able to determine DNA damage in humans more accurately.

Abstract PO5-08-06: Metabolic shift to serine pathway induced by lipids confers oncogenic properties in non-transformed breast cells

Abstract Introduction. Oncogenic factors that are local/in-breast are of great interest as they may be more specifically targetable for breast cancer prevention than systemic factors. We have identified a lipid metabolism gene signature that is enriched in breast tissue at risk for estrogen negative breast cancer (ER- BC). Utilizing the medium chain fatty acid Octanoic acid (OA) to probe lipid metabolism in non-transformed breast epithelial cells, we observed increased flux through several metabolic reactions and altered histone methylation with consequent changes in gene expression (e.g. neural genes). Neuronal signaling and regulatory circuits are observed in cancer cells of multiple origins, not just ones with ontological relationships to neurons. We hypothesize that the first and rate limiting step in the de novo serine pathway, which is catalyzed by Phosphoglycerate Dehydrogenase (PHGDH) is key to these observations. In the forward direction, PHGDH participates in the serine, one-carbon, glycine (SOG) and methionine pathways that produce the methyl donor S-adenosylmethionine (SAM) and in the reverse direction produces the oncometabolite 2-Hydroxyglycerate (2-HG). Methods. Non-transformed MCF-10A cells exposed to OA were utilized for U13C-glucose tracing. SAM and 2-HG concentrations following treatment with OA ± PHGDH inhibitor were measured by liquid chromatography. CUT&RUN for H3K4me3 was performed and genes affected by OA (PMID: 28263391) were compared with OA-responsive peaks. Single cell RNA-sequencing was carried out using breast microstructures derived from reduction mammoplasty tissue exposed to vehicle or OA. Microstructures were dissociated into single cells and sequenced using the 10x Genomics platform. The digital expression matrix file containing UMIs were analyzed with Seurat. Alkaline comet assay was performed to detect DNA breaks. Results. U13C-glucose tracing in presence of OA revealed that one-carbon-THF was redirected to the methionine cycle increasing flux to methylation. Concentrations of SAM and 2-HG increased after 15- and 30-min OA exposure, respectively; PHGDH inhibitor blocked these increases. H3K4me3 CUT&RUN revealed 661 differential peaks (FDR < 0.05) comparing OA to control. 73% of H3K4me3 OA-associated peaks were in regulatory regions of OA-induced genes (FDR < 0.01), these genes are involved in neural pathways, EMT and ER- BC. Motif analysis revealed an overrepresentation of binding sites for transcription factors ATF3/4 (p < 0.05), which are regulators of the serine pathway. Single cell RNA-sequencing revealed OA not only affected the distribution of cell subpopulations but also modulated the expression of many genes within each subcluster. The percentage of luminal progenitor subcluster 3 increased upon OA from less than 1% to about 13%. Within basal subcluster 3, OA drives the expression of ATF3, along with two of the enzymes in the de novo serine pathway: PHGDH and PSAT1. Alkaline comet assay showed DNA breaks in OA- and control 2-HG- treated cells. Conclusions. Metabolism of OA in preference to glucose and glutamine results in a metabolic shift toward the serine pathway increasing the production of SAM and 2-HG, with implications for oncogenesis: 1. SAM production results in epigenetic fostered plasticity leading to reprogramming/selecting cells that express genes consistent with a neural/neural crest-like state. These co-opted neuronal regulatory mechanisms can make critical contributions to the acquired functional capabilities that drive cancer development. 2. 2-HG exposure results in appearance of DNA breaks, which are likely consequent to the inhibition of the alpha-ketoglutarate-dependent dioxygenases KDM 4A/B by 2-HG. Their catalytic activity is required for homologous recombination repair; inhibition results in metabolic “BRCAness”. Citation Format: Mariana Bustamante Eduardo, Gannon Cottone, Shiyu Liu, Maria Paula Zappia, Elizaveta V. Benevolenskaya, Abul Bashar Mir Md. Khademul Islam, Maxim V. Frolov, Seema Khan, Susan Clare. Metabolic shift to serine pathway induced by lipids confers oncogenic properties in non-transformed breast cells [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-08-06.

Abstract PO1-08-06: Clinical-molecular correlation in breast cancer with pathogenic variants in CHEK2. Is there a relationship with HER2+ breast cancer?

Abstract Background: The CHEK2 gene codes for checkpoint protein Kinase 2 (CHK2), an effector in the ATM-CHK2-p53 DNA damage repair pathway in double-stranded DNA breaks. Initially, germline pathogenic variants were associated with moderate risk for breast carcinoma, but later studies linked alterations in the gene to other types of cancer. The aim of our study was to establish a clinical-molecular correlation of profiles of patients carrying pathogenic variants and variants of uncertain significance (VUS) in CHEK2 with a diagnosis of breast carcinoma, in patients treated in our hospital. And to describe whether the pathogenic variant c.1100del present different clinical or immunohistochemical characteristics than the rest, also considering the particularity of being an island population. Methodology: We have collected data from genetic studies performed in patients from our hospital, covering a population of 580,000 inhabitants, in Santa Cruz de Tenerife ( Canary Islands, Spain), between 2017 and 2023.Out of 952 patients with suspected hereditary cancer, 81 had CHEK2 mutations. In patients with CHEK2 mutations and breast cancer we established three groups for clinical molecular analysis: 1) pathogenic variant c.1100del, 2) other pathogenic variants and 3) VUS, to be correlated with age, histology, ER, PR, Ki, grade (G), HER2, second tumour or recurrence, subtype, HER2 low, aggressive clinic, neuropathy, haematological and digestive toxicity. The germline genetic study performed covered 77 genes related to hereditary cancer using the SureSelect HS library preparation kit (Agilent Technologies). The classification of the variants identified was performed according to the American College of Medical Genetics (ACMG) criteria. Results: In the 952 genetic studies performed, 8.5% of cases had CHEK2 mutations, 2.62% were related to breast carcinoma (N=26) of which 54% had the pathogenic variant “c.1100del”, 16% “other” pathogenic variants and 30% VUS. The mean age at diagnosis was 42 years, being lower than 38 years in pathogenic variants other than c.1100del. Histologically, infiltrating ductal carcinoma (85%) and mucinous carcinomas (12%), ER+ (93%), PR+ (77%), HER 2+ (31%), HER2 low (44%), G2-3 (84%) and Ki >15% (70%) were predominant. Second diagnoses were tumour or recurrence in 40%, being higher in the group of pathogenic variants other than c.1100del (50%), and showing aggressive clinical course in 26%. The predominant subtype was luminal B (50%), HER2+ (31%), luminal A (12 %) and triple negative (7%), although in the cluster analysis patients with pathogenic variant c.1100del HER2+ were higher (38%) and luminal B (38%), while in pathogenic variants other than c.1100del and VUS the luminal subtype was predominant (75%). Chemotherapy toxicity: neuropathy (35%), haematological and digestive toxicity (50%). Conclusion: Our results are consistent with other reported series, being related with all breast carcinoma subtypes, mainly luminal B and HER2+ and with triple negative at a lesser extend. Considering HER2 low patients were about 75% of patients with CHEK2 mutations, a molecular mechanism that correlates a truncating mutation in CHK2 and HER2 overexpression can be hypothesized. In the cluster analysis, HER2+ (38%), and luminal B (38%) subtypes predominate in patients with pathogenic variant c.1100del, while in pathogenic variants other than c.1100del and VUS the luminal B subtype predominates (75%), as well as recurrence or second tumours in 50% of cases. On the other hand, considering that this is an island population, the possible effect of geographical isolation and the resulting inbreeding in the past, these facts do not seem to elicit differences comparing to the rest of the continental population, although the increase in pathogenic variants in CHEK2 were observed in patients from the small island of La Gomera. The relationship between CHEK2 and HER2 in breast cancer is an area of active research. Relative frequency in groups of CHEK2 variants Relative frequency in CHEK2 “ pathogenic variants c.1100 del (53%)”, “other”pathogenic variants no c.1100 del (15 %)" and VUS (30%). Relative frequency of breast cancer subtypes according to CHEK2 variants. HER2+: 30,8%. Luminal A: 11,5% . Luminal B: 50%. Triple negative: 7,7% . Histology, ER, PR, Ki, HER2 and HER2 low Histology , infiltrating ductal carcinoma (84%), ER+ (92%), PR (77%), Ki >15% (70%), HER2+ (31%), HER2 low (45%) Citation Format: Natalia Pérez-Rodríguez, Carol Prieto-Morin, Maria del Carmen Maeso, Lina Pérez-Mendez. Clinical-molecular correlation in breast cancer with pathogenic variants in CHEK2. Is there a relationship with HER2+ breast cancer? [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-08-06.

SNP-Associated Substitutions of Amino Acid Residues in the dNTP Selection Subdomain Decrease Polβ Polymerase Activity.

In the cell, DNA polymerase β (Polβ) is involved in many processes aimed at maintaining genome stability and is considered the main repair DNA polymerase participating in base excision repair (BER). Polβ can fill DNA gaps formed by other DNA repair enzymes. Single-nucleotide polymorphisms (SNPs) in the POLB gene can affect the enzymatic properties of the resulting protein, owing to possible amino acid substitutions. For many SNP-associated Polβ variants, an association with cancer, owing to changes in polymerase activity and fidelity, has been shown. In this work, kinetic analyses and molecular dynamics simulations were used to examine the activity of naturally occurring polymorphic variants G274R, G290C, and R333W. Previously, the amino acid substitutions at these positions have been found in various types of tumors, implying a specific role of Gly-274, Gly-290, and Arg-333 in Polβ functioning. All three polymorphic variants had reduced polymerase activity. Two substitutions-G274R and R333W-led to the almost complete disappearance of gap-filling and primer elongation activities, a decrease in the deoxynucleotide triphosphate-binding ability, and a lower polymerization constant, due to alterations of local contacts near the replaced amino acid residues. Thus, variants G274R, G290C, and R333W may be implicated in an elevated level of unrepaired DNA damage.

Effect of miR-181b Treatment on Senescent Human Aortic Smooth Muscle Cells

Large Artery Stiffness (LAS) is independently associated with increased cardiovascular morbidity and mortality and its development is multifactorial and has been associated with the onset of cellular senescence (CS). CS is defined as a stable cell cycle arrest, characterized by an altered cell phenotype with impaired function and detrimental secretory profile. Vascular smooth muscle cells (VSMCs) are present in two main phenotypes: the contractile phenotype seen in young populations, and the synthetic phenotype related to aging and senescent VSMCs. Micro-RNAs (miR) are non-coding RNAs that can alter protein expression. miR-181b has been demonstrated to undergo downregulation with aging with a close to 80% decrease, this correlates with an upregulation of markers of senescence. To test the hypothesis that miR-181b could modulate the senescent phenotype in VSMCs and promote a more contractile phenotype, a cellular model comprising young and old human aortic smooth muscle cells (HASMCs) was employed. These cells were transfected with either a miR-181b mimic or a scrambled miR control (SCR), resulting in an 11-fold increase in the young and a 3.3-fold increase in the old. Cell proliferation was measured using electric cell-substrate impedance sensing (ECIS) technology, where it was found to be lower in older HASMCs (1.05±0.001) compared to the young (1.1±0.001, p<0.05). When miR-181b was introduced, there was a significant increase in proliferation for both age groups by 0.03-fold compared to SCR (p<0.05). Old HASMCs demonstrated a decreased average ionomycin-induced contractile response (3.65%±0.46) when compared to the young (7.17%±0.60, p<0.05). Lipofectamine-mediated transfection of HASMCs with miR-181b resulted in an increased contractility response in the old HASMCs (5.46%±0.91) compared to scrambled control (1.41%±0.39, p<0.05), while there was no significant difference for the young cells. The percentage of Senescence-Associated-β-galactosidase (Saβ-gal) positive cells, a marker of senescence, was higher in old (39.74%±3.04) vs young (4.35%±0.72, p<0.05). The percentage of Saβ-gal positive cells tended to decrease in old VSMCs after miR-181b transfection (SCR 79.65%±4.38 vs Old181b 70.39±4.03, p=0.2.02), with no effect on young cells. Immunofluorescence in situ hybridization (IF-FISH) staining for the double-stranded DNA break marker (53BP1) was lower in the young (4.65%) compared to old HASMCs (32.69%). Likewise, the percentage of cells with one or more 53BP1 foci colocalization in telomeres, referred to as Telomere dysfunction-induced foci (TIF), was higher in old (11.54%) compared to young (2.33%) VSMCs (p=0.002). However, miR-181b transfection did not impact these markers regardless of age (p=0.94). Transfection of HASMCs with miR-181b did not impact senescent cell abundance, but improved contractility and cellular proliferation. Combined with our prior data demonstrating a senomorphic effect of miR-181b transfection to improve cellular stiffness and favorably modulate of senescence-associated secretory factors, our results demonstrate an enhancement in the phenotypic characteristics of the old HASMCs group, suggesting that miR-181b participates in the modulation of the senescent VSMCs phenotype. This translates to a potential improvement in HASMC health that could reduce the development of LAS. National Institutes of Health Awards: K08 AG070281 (ET), T32 HL139451 (DM), R01 AG048366 (LAL), R01 AG060395 (AJD), and Western Institute for Veterans Research (ET). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Twin Prime Editing Mediated Exon Skipping/Reinsertion for Restored Collagen VII Expression in Recessive Dystrophic Epidermolysis Bullosa

Gene editing nucleases, base editors, and prime editors are potential locus specific genetic treatment strategies for recessive dystrophic epidermolysis bullosa (RDEB); however, many RDEB COL7A1 mutations are unique, making the development of personalized editing reagents challenging. 270 of the ∼320 COL7A1 EB mutations reside in exons that can be skipped, and antisense oligonucleotides (ASO) and gene editing nucleases have been used to create in-frame deletions. ASOs are transient and nucleases generate deleterious double stranded DNA breaks (DSB) and uncontrolled mixtures of allele products. We developed a twin prime editing (twinPE) strategy using the PEmax and recently evolved PE6 prime editors and dual prime editing guide RNAs flanking COL7A1 exon five. Prime editing-mediated deletion of exon 5 with a homozygous premature stop codon was achieved in RDEB fibroblasts, keratinocytes, and iPSC with minimal DSBs, and collagen type VII (C7) protein was restored. TwinPE can replace the target exon with recombinase attachment sequences, and we exploited this to re-insert a normal copy of exon 5 using the Bxb1 recombinase. These findings demonstrate that twinPE can facilitate locus-specific, predictable, in-frame deletions and sequence replacement with few DSBs as a strategy that may enable a single therapeutic agent to treat multiple RDEB patient cohorts.

AORTIC FUNCTION AND NTRATIVE STRESS IN A RAT MODEL OF POLYCYSTIC OVARY SYNDROME

Objective: Polycystic ovary syndrome (PCOS) represents the most prevalent endocrine disorder in women of fertile age; besides infertility, an elevated cardiovascular risk was described. Vitamin D hypovitaminosis affects about 80-90% of PCOS patients. Our team assessed aortic function and nitrative stress in a hyperandrogenic rat model and the effectiveness of Vitamin D substitution. Design and method: Female Wistar rats at the age of 3-4 weeks were randomly assigned into four groups. Eight rats received no treatment (controls, C), eight rats received weekly Vitamin D per os (140 IU/100 g body weight, group D), eight received cutaneous testosterone daily (3.3 mg/100 g, T) and eight received Vitamin D and testosterone (T+D). At the eighth week of the treatment, aortic rings were isolated for analysis: they were cut into 3-4 mm long segments for myography, other segments were formaldehyde fixed and paraffin embedded to assess, by immunohistochemical methods, nitrative stress markers (nitrotyrosine, NT) and DNA breaks signaled by nuclear poly(ADP-ribose) polymer (PAR) detection. Results: The weight gains of the two testosterone-treated groups were significantly higher between the 6-8 weeks compared to the other two groups, Vitamin D treatment did not influence body weight. Rats in the C and D groups had 3 or 4 estruses in the last 14 days. T rats had 1 or 2 estruses during the same period. T+D rats had 1-2 (N=3), or 3-4 (N=5) estruses. Aortic contraction, induced by the alpha1-adrenergic agonist phenylephrine, was significantly elevated in T and T+D animals in comparison to controls and Vitamin D treated rats. The endothelium-mediated aortic relaxation, as a response to acetyl-choline and insulin, was unaltered; however, estrogen-induced relaxation was significantly impaired in T and T+D groups. According to resorcin-fuchsin staining, no fibrotic alteration was detected. Nitrative stress and poly(ADP-ribosyl)ation were similar in all four groups. Conclusions: Eight weeks of testosterone administration caused increased weight gain and sex cycle lengthening. The latter was reversible by Vitamin D supplementation in 62.5% of the rats. Testosterone or Vitamin D did not alter vascular histology. Hyperandrogenism increased alpha1-adrenerg mediated vasoconstriction and compromised estrogen-mediated relaxation without altering nitrative stress.

MitoQ Treatment Mitigates Senescence Burden in DOXO-Induced Senescent Endothelial Cells Through Reductions in mtROS and DNA Damage

Cardiovascular toxicity is one of the adverse consequences of chemotherapy that limits treatment. Specifically, chemotherapeutics such as doxorubicin (DOXO) can lead to endothelial dysfunction through the induction of reactive oxygen species (ROS) and mitochondrial ROS (mtROS) production. MtROS are chemically reactive molecules generated as byproducts of oxidative phosphorylation during mitochondrial respiration. DOXO exacerbates its cardiotoxic effects by amplifying mtROS production, leading to an increase in DNA damage and cell senescence, particularly in endothelial cells (ECs). MitoQ is a mitochondria-targeted antioxidant designed to accumulate within the mitochondria and mitigate oxidative damage by neutralizing mtROS. However, whether MitoQ can rescue mtROS-induced DNA damage and senescence remains elusive. Here, we tested the hypothesis that MitoQ will attenuate DOXO-induced mtROS thus blunting DNA damage and subsequent EC senescence. Human umbilical vein endothelial cells (HUVECs) were simultaneously subjected to 24-hour treatments with DOXO (250nM) and MitoQ (250nM). To evaluate the antioxidative effect of the MitoQ, we examined mitochondrial superoxide using MitoSOX. The results demonstrated that DOXO increases mitochondrial superoxide by 40% (Fig1, p=0.019), whereas addition of MitoQ (MitoQ+DOXO) attenuated mitochondrial superoxide significantly compared to the DOXO group (Fig1, p=0.033). Gene expression of senescence markers, p16 and p21, and Senescence Associated Secretory Phenotypes (SASPs) factors were also examined to assess the effect of MitoQ on senescent endothelial cells. p16 was significantly reduced in the MitoQ+DOXO treatment group compared to the DOXO alone (Fig2, p=0.0090). MitoQ+DOXO lowered p21 expression by 35%, although this difference did not reach significance (Fig2, p=0.340). Moreover, MitoQ+DOXO reduced the SASP factors, IL-8, IL-6 and MCP-1, by more than 54% in comparison to the DOXO group (Fig2, all p≤0.027). Senescent endothelial cells were examined for 53BP1, a double-stranded DNA break marker, using an immunofluorescence (IF) assay. MitoQ+DOXO reduced 53BP1 by 32.6% compared to the DOXO-treated group (Fig3, p=0.011). We also assessed cell proliferation using Bromodeoxyuridine (BrdU) assay. In line with other results, the BrdU assay indicates that the proliferative capability of MitoQ+DOXO-treated cells was improved by more than 20% compared to DOXO-induced senescent cells (Fig4, p=0.008). Our findings suggest that MitoQ can mitigate DOXO-induced mtROS, attenuate DNA damage, and ameliorate senescence in endothelial cells. This provides mechanistic insight suggesting that mtROS may be a driver of cellular senescence in ECs via an increase in DNA damage and MitoQ may be an effcacious therapeutic to treat chemotherapy-induced cardiovascular toxicity. Funded in part by awards from National Institutes of Health Awards R01 AG060395, R01 AG048366, T32 HL139451, F31 AG076312, R01 AG077751, R01 AG076748, Nora Eccles Treadwell foundation (NETF), and Veteran's Affairs Merit Review Award I01 BX004492. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.