Aberrant DNA Repair Research Articles

Ustilago maydis Trf2 ensures genome stability by antagonizing Blm-mediated telomere recombination: Fine-tuning DNA repair factor activity at telomeres through opposing regulations.

TRF2 is an essential and conserved double-strand telomere binding protein that stabilizes chromosome ends by suppressing DNA damage response and aberrant DNA repair. Herein we investigated the mechanisms and functions of the Trf2 ortholog in the basidiomycete fungus Ustilago maydis, which manifests strong resemblances to metazoans with regards to the telomere and DNA repair machinery. We showed that UmTrf2 binds to Blm in vitro and inhibits Blm-mediated unwinding of telomeric DNA substrates. Consistent with a similar inhibitory activity in vivo, over-expression of Trf2 induces telomere shortening, just like deletion of blm, which is required for efficient telomere replication. While the loss of Trf2 engenders growth arrest and multiple telomere aberrations, these defects are fully suppressed by the concurrent deletion of blm or mre11 (but not other DNA repair factors). Over-expression of Blm alone triggers aberrant telomere recombination and the accumulation of aberrant telomere structures, which are blocked by concurrent Trf2 over-expression. Together, these findings highlight the suppression of Blm as a key protective mechanism of Trf2. Notably, U. maydis harbors another double-strand telomere-binding protein (Tay1), which promotes Blm activity to ensure efficient replication. We found that deletion of tay1 partially suppresses the telomere aberration of Trf2-depleted cells. Our results thus point to opposing regulation of Blm helicase by telomere proteins as a strategy for optimizing both telomere maintenance and protection. We also show that aberrant transcription of both telomere G- and C-strand is a recurrent phenotype of telomere mutants, underscoring another potential similarity between double strand breaks and de-protected telomeres.

Epigenetic trajectory predicts development of clinical rheumatoid arthritis in ACPA+ individuals: Targeting Immune Responses for Prevention of Rheumatoid Arthritis (TIP-RA).

The presence of autoantibodies to citrullinated protein antigens (ACPAs) in the absence of clinically-apparent inflammatory arthritis (IA) identifies individuals "at-risk" for developing future clinical rheumatoid arthritis (RA). However, it is unclear why some ACPA+ individuals convert to clinical RA while others do not. We explored the possibility in the Targeting Immune Responses for Prevention of Rheumatoid Arthritis (TIP-RA) study that epigenetic remodeling is part of the trajectory from an at-risk state to clinical disease and identifies novel biomarkers associated with conversion to clinical RA. ACPA-Controls, ACPA+ At-Risk, and Early RA individuals were followed for up to 5 years, including obtaining blood samples annually and at RA diagnosis. Peripheral blood mononuclear cells (PBMCs) were separated into CD19+ B cells, memory CD4+ T cells, and naïve CD4+ T cells using antibodies and magnetic beads. Genome-wide methylation within each cell lineage was assayed using the Illumina MethylationEPIC v1.0 beadchip. ACPA+ At-Risk participants who did or did not develop RA were designated "Pre-RA" or "Non-converters", respectively.Differentially methylated loci (DML) were selected using the Limma software package. Using the Caret package, we constructed machine learning models in test and validation cohorts and identified the most predictive loci of clinical RA conversion. Cross-sectional differential methylation analysis at baseline revealed DMLs that distinguish the Pre-RA methylome from ACPA+ Non-converters, the latter which closely resembled ACPA-Controls. Genes overlapping these DMLs correspond to aberrant NOTCH signaling and DNA repair pathways in B cells. Longitudinal analysis showed that ACPA-Control and ACPA+ Non-converter methylomes are relatively constant. In contrast, the Pre-RA methylome remodeled along a dynamic "RA methylome trajectory" characterized by epigenetic changes in active regulatory elements. Clinical conversion to RA, defined based on diagnosis, marked an epigenetic inflection point for cell cycle pathways in B cells and adaptive immunity pathways in naïve T cells. Machine learning revealed individual loci associated with RA conversion. This model significantly outperformed autoantibodies plus acute phase reactants as predictors of RA conversion. DNA methylation is a dynamic process in ACPA+ individuals at-risk for developing RA that eventually transition to clinical disease. In contrast, non-converters and controls have stable methylomes. The accumulation of epigenetic marks over time prior to conversion to clinical RA conforms to pathways that are associated with immunity and can be used to identify potential pathogenic pathways for therapeutic targeting and/or use as prognostic biomarkers.

Deciphering the correlation between Chr3q26.2 amplification through homologous recombination deficiency and improved survival in ovarian cancer under platinum-based adjuvant therapy.

e17537 Background: Discriminant copy number alterations (CNAs) have been linked to aberrant DNA repair mechanisms. This study aimed to identify CNA-based biomarkers, alongside homologous recombination deficiency (HRD) scores for ovarian cancer patients receiving platinum (Pt)-based chemotherapy, especially in BRCA1/2 wild-type or HRD-low patients. Methods: Fourteen most prevalent and significant focal CNA regions (seven amplification and seven deletion) were examined to investigate their correlations with HRD scores via SHAP values of an XGBoost model and the effectiveness of Pt-based adjuvant chemotherapy in a cohort of high-grade serous ovarian cancer (HGSC) patients (TCGA cohort, N=576), and the results were validated in a separate Chinese ovarian cancer cohort (test cohort, N=457). An in-house developed next-generation sequencing HRD pipeline (GeneseeqPrime HRD) was used to determine HRD scores. Histological subtypes, molecular features, and potential molecular mechanisms associated with the identified biomarkers were further explored. Results: Of 14 high-level CNA regions identified by GISTIC in the TCGA cohort, Chromosome 3q26.2 amplification [ Chr3(q26.2) Amp, a prevalence of 30.7%] was associated with increased HRD scores in both BRCA1/2-altered ( p=0.01) and wild-type HGSCs ( p=0.03). Consistently, in the test cohort, higher HRD scores ( p<0.01) and elevated chromosome instability scores ( p<0.01) were observed in Chr3(q26.2) amplified samples. Among 406 patients administrated with Pt-based adjuvant chemotherapy in the TCGA cohort, those with Chr3(q26.2) Amp exhibited a relatively high sensitivity rate ( p=0.09) and significantly superior disease-free survival after adjusting for HRD score, patient age, tumor stage, and residual disease [hazard ratio (HR): 0.65, 95% confidence interval (CI): 0.48–0.88] when compared to those without, representing Chr3(q26.2) Amp as an independent predictive biomarker. Similar results were obtained in 78 patients using Pt-based adjuvant chemotherapy in the test cohort (HR: 0.68, 95% CI: 0.40–1.15). Moreover, Chr3(q26.2) Amp was more prevalent in HGSCs harboring BRCA1/2 or TP53 aberrations than other histological subtypes driven by PIK3CA, PTEN or KRAS mutations ( p<0.01). In comparison to HGSCs without Chr3(q26.2) Amp, Chr3(q26.2) amplified HGSCs exhibited higher Ragnum hypoxia scores ( p<0.01) and a total of four overexpressed proteins, including p62 ( p<0.01), PIK3CA ( p<0.01), CCNB1 ( p<0.01) and TSC1 ( p<0.01), indicative of suppressed homologous recombination repair pathway and cycle cell arrest. Conclusions: Chr3(q26.2) Amp emerges as a promising biomarker for Pt-based adjuvant chemotherapy in ovarian cancer, potentially reflecting a hypoxia-induced etiology through suppressed homologous recombination repair pathway.

Mechanism of Action of KL-50, a Candidate Imidazotetrazine for the Treatment of Drug-Resistant Brain Cancers.

Aberrant DNA repair is a hallmark of cancer, and many tumors display reduced DNA repair capacities that sensitize them to genotoxins. Here, we demonstrate that the differential DNA repair capacities of healthy and transformed tissue may be exploited to obtain highly selective chemotherapies. We show that the novel N3-(2-fluoroethyl)imidazotetrazine "KL-50" is a selective toxin toward tumors that lack the DNA repair protein O6-methylguanine-DNA-methyltransferase (MGMT), which reverses the formation of O6-alkylguanine lesions. We establish that KL-50 generates DNA interstrand cross-links (ICLs) by a multistep process comprising DNA alkylation to generate an O6-(2-fluoroethyl)guanine (O6FEtG) lesion, slow unimolecular displacement of fluoride to form an N1,O6-ethanoguanine (N1,O6EtG) intermediate, and ring-opening by the adjacent cytidine. The slow rate of N1,O6EtG formation allows healthy cells expressing MGMT to reverse the initial O6FEtG lesion before it evolves to N1,O6EtG, thereby suppressing the formation of toxic DNA-MGMT cross-links and reducing the amount of DNA ICLs generated in healthy cells. In contrast, O6-(2-chloroethyl)guanine lesions produced by agents such as lomustine and the N3-(2-chloroethyl)imidazotetrazine mitozolomide rapidly evolve to N1,O6EtG, resulting in the formation of DNA-MGMT cross-links and DNA ICLs in healthy tissue. These studies suggest that careful consideration of the rates of chemical DNA modification and biochemical DNA repair may lead to the identification of other tumor-specific genotoxic agents.

Aberrant DNA repair reveals a vulnerability in histone H3.3-mutant brain tumors.

Pediatric high-grade gliomas (pHGG) are devastating and incurable brain tumors with recurrent mutations in histone H3.3. These mutations promote oncogenesis by dysregulating gene expression through alterations of histone modifications. We identify aberrant DNA repair as an independent mechanism, which fosters genome instability in H3.3 mutant pHGG, and opens new therapeutic options. The two most frequent H3.3 mutations in pHGG, K27M and G34R, drive aberrant repair of replication-associated damage by non-homologous end joining (NHEJ). Aberrant NHEJ is mediated by the DNA repair enzyme polynucleotide kinase 3'-phosphatase (PNKP), which shows increased association with mutant H3.3 at damaged replication forks. PNKP sustains the proliferation of cells bearing H3.3 mutations, thus conferring a molecular vulnerability, specific to mutant cells, with potential for therapeutic targeting.

An Exploration of Small Molecules That Bind Human Single-Stranded DNA Binding Protein 1.

Human single-stranded DNA binding protein 1 (hSSB1) is critical to preserving genome stability, interacting with single-stranded DNA (ssDNA) through an oligonucleotide/oligosaccharide binding-fold. The depletion of hSSB1 in cell-line models leads to aberrant DNA repair and increased sensitivity to irradiation. hSSB1 is over-expressed in several types of cancers, suggesting that hSSB1 could be a novel therapeutic target in malignant disease. hSSB1 binding studies have focused on DNA; however, despite the availability of 3D structures, small molecules targeting hSSB1 have not been explored. Quinoline derivatives targeting hSSB1 were designed through a virtual fragment-based screening process, synthesizing them using AlphaLISA and EMSA to determine their affinity for hSSB1. In parallel, we further screened a structurally diverse compound library against hSSB1 using the same biochemical assays. Three compounds with nanomolar affinity for hSSB1 were identified, exhibiting cytotoxicity in an osteosarcoma cell line. To our knowledge, this is the first study to identify small molecules that modulate hSSB1 activity. Molecular dynamics simulations indicated that three of the compounds that were tested bound to the ssDNA-binding site of hSSB1, providing a framework for the further elucidation of inhibition mechanisms. These data suggest that small molecules can disrupt the interaction between hSSB1 and ssDNA, and may also affect the ability of cells to repair DNA damage. This test study of small molecules holds the potential to provide insights into fundamental biochemical questions regarding the OB-fold.

Overexpression of XRCC1 is Associated with Poor Survival in Patients with Head and Neck Squamous Carcinoma and Has Potential to Be Used as Targeted Therapy by Synthetic Lethality.

Background: Head neck squamous cell carcinoma (HNSC) is globally prevalent cancer attributed to tobacco habit. Despite the significant advances in early diagnosis and treatment of HNSC chemo-radio resistance are routinely observed in patients. Aberrant DNA repair mechanisms mainly microhomology mediated DNA end joining (MMEJ) pathway causing deleterious mutations and is implicated in treatment resistance. X-ray cross complimenting group 1 (XRCC1) has recently been shown to play an essential role in MMEJ making XRCC1 a potential therapeutic target to render tumors chemo-radiosensitive. This study analyzes the correlation between the expression level of XRCC1 gene with survival, regulation by miRNA and synthetic lethality partners in HNSCC. Materials and Methods: XRCC1 gene expression was evaluated in 520 HNSC patients and 44 of normal tissues using the UALCAN (TCGA) database and its correlation with survival outcome of HNSC patients was analyzed by Kaplan-Meier plot. Infiltration of immune cells in tumors was analyzed by “Tumor-Infiltrating Immune Estimation Resource (TIMER) and promoter methylation status of XRCC1 in samples was analysed by UALCAN. STRING was used to find gene interacting partners of XRCC1. Results: XRCC1 was significantly overexpressed in primary tumor of HNSCC and significantly increased with tumor stages and grade and associated with poor survival rate. High XRCC1 expression in HNSC was positively correlated with infiltration level of B cells naïsve, CD4+ and macrophages. Conclusion: These results indicate that XRCC1 is a prognostic marker for predicting survival in HNSC patients. Understanding how XRCC1 leads to treatment resistance and modulate immune response can lead to development of targeted therapy.

BRCA1 heterozygosity promotes DNA damage-induced senescence in a sex-specific manner following repeated mild traumatic brain injury.

Emerging evidence suggests cellular senescence, as a consequence of excess DNA damage and deficient repair, to be a driver of brain dysfunction following repeated mild traumatic brain injury (rmTBI). This study aimed to further investigate the role of deficient DNA repair, specifically BRCA1-related repair, on DNA damage-induced senescence. BRCA1, a repair protein involved in maintaining genomic integrity with multiple roles in the central nervous system, was previously reported to be significantly downregulated in post-mortem brains with a history of rmTBI. Here we examined the effects of impaired BRCA1-related repair on DNA damage-induced senescence and outcomes 1-week post-rmTBI using mice with a heterozygous knockout for BRCA1 in a sex-segregated manner. Altered BRCA1 repair with rmTBI resulted in altered anxiety-related behaviours in males and females using elevated zero maze and contextual fear conditioning. Evaluating molecular markers associated with DNA damage signalling and senescence-related pathways revealed sex-specific differences attributed to BRCA1, where females exhibited elevated DNA damage, impaired DNA damage signalling, and dampened senescence onset compared to males. Overall, the results from this study highlight sex-specific consequences of aberrant DNA repair on outcomes post-injury, and further support a need to develop sex-specific treatments following rmTBI.

Dysregulation of DNA repair genes in Fuchs endothelial corneal dystrophy

Fuchs Endothelial Corneal Dystrophy (FECD), a late-onset oxidative stress disorder, is the most common cause of corneal endothelial degeneration and is genetically associated with CTG repeat expansion in Transcription Factor 4 (TCF4). We previously reported accumulation of nuclear (nDNA) and mitochondrial (mtDNA) damage in FECD. Specifically, mtDNA damage was a prominent finding in development of disease in the ultraviolet-A (UVA) induced FECD mouse model. We hypothesize that an aberrant DNA repair may contribute to the increased DNA damage seen in FECD. We analyzed differential expression profiles of 84 DNA repair genes by real-time PCR arrays using Human DNA Repair RT-Profiler plates using cDNA extracted from Descemet's membrane-corneal endothelium (DM-CE) obtained from FECD patients with expanded (>40) or non-expanded (<40) intronic CTG repeats in TCF4 gene and from age-matched normal donors. Change in mRNA expression of <0.5- or >2.0-fold in FECD relative to normal was set as cutoff for down- or upregulation. Downregulated mitochondrial genes were further validated using the UVA-based mouse model of FECD. FECD specimens exhibited downregulation of 9 genes and upregulation of 8 genes belonging to the four major DNA repair pathways, namely, base excision repair (BER), nucleotide excision repair (NER), mismatch repair (MMR), and double strand break (DSB) repair, compared to normal donors. MMR gene MSH2 and BER gene POLB were preferentially upregulated in expanded FECD. BER genes LIG3 and NEIL2, DSB repair genes PARP3 and TOP3A, NER gene XPC, and unclassified pathway gene TREX1, were downregulated in both expanded and non-expanded FECD. MtDNA repair genes, Lig3, Neil2, and Top3a, were also downregulated in the UVA-based mouse model of FECD. Our findings identify impaired DNA repair pathways that may play an important role in DNA damage due to oxidative stress as well as genetic predisposition noted in FECD.

Deacetylation induced nuclear condensation of HP1γ promotes multiple myeloma drug resistance

Acquired chemoresistance to proteasome inhibitors is a major obstacle in managing multiple myeloma but key regulators and underlying mechanisms still remain to be explored. We find that high level of HP1γ is associated with low acetylation modification in the bortezomib-resistant myeloma cells using SILAC-based acetyl-proteomics assay, and higher HP1γ level is positively correlated with poorer outcomes in the clinic. Mechanistically, elevated HDAC1 in the bortezomib-resistant myeloma cells deacetylates HP1γ at lysine 5 and consequently alleviates the ubiquitin-mediated protein degradation, as well as the aberrant DNA repair capacity. HP1γ interacts with the MDC1 to induce DNA repair, and simultaneously the deacetylation modification and the interaction with MDC1 enhance the nuclear condensation of HP1γ protein and the chromatin accessibility of its target genes governing sensitivity to proteasome inhibitors, such as CD40, FOS and JUN. Thus, targeting HP1γ stability by using HDAC1 inhibitor re-sensitizes bortezomib-resistant myeloma cells to proteasome inhibitors treatment in vitro and in vivo. Our findings elucidate a previously unrecognized role of HP1γ in inducing drug resistance to proteasome inhibitors of myeloma cells and suggest that targeting HP1γ may be efficacious for overcoming drug resistance in refractory or relapsed multiple myeloma patients.

HNRNPU facilitates antibody class-switch recombination through C-NHEJ promotion and R-loop suppression

B cells generate functionally different classes of antibodies through class-switch recombination (CSR), which requires classical non-homologous end joining (C-NHEJ) to join the DNA breaks at the donor and acceptor switch (S)regions. We show that the RNA-binding protein HNRNPU promotes C-NHEJ-mediated S-S joining through the 53BP1-shieldin DNA-repair complex. Notably, HNRNPU binds to the S region RNA/DNA G-quadruplexes, contributing to regulating R-loop and single-stranded DNA (ssDNA) accumulation. HNRNPU is an intrinsically disordered protein that interacts with both C-NHEJ and R-loop complexes in an RNA-dependent manner. Strikingly, recruitment of HNRNPU and the C-NHEJ factors is highly sensitive to liquid-liquid phase separation inhibitors, suggestive of DNA-repair condensate formation. We propose that HNRNPU facilitates CSR by forming and stabilizing the C-NHEJ ribonucleoprotein complex and preventing excessive R-loop accumulation, which otherwise would cause persistent DNA breaks and aberrant DNA repair, leading to genomic instability.

DNA damage in testicular germ cells and spermatozoa. When and how is it induced? How should we measure it? What does it mean?

This review surveys the causes and consequences of DNA damage in the male germ line from spermatogonial stem cells to fully differentiated spermatozoa. Within the stem cell population, DNA integrity is well maintained as a result of excellent DNA surveillance and repair; however, a progressive increase in background mutation rates does occur with paternal age possibly as a result of aberrant DNA repair as well as replication error. Once a germ cell has committed to spermatogenesis, it responds to genetic damage via a range of DNA repair pathways or, if this process fails, by the induction of apoptosis. When fully-differentiated spermatozoa are stressed, they also activate a truncated intrinsic apoptotic pathway which results in the activation of nucleases in the mitochondria and cytoplasm; however, the physical architecture of these cells prevents these enzymes from translocating to the nucleus to induce DNA fragmentation. Conversely, hydrogen peroxide released from the sperm midpiece during apoptosis is able to penetrate the nucleus and induce DNA damage. The base excision repair pathway responds to such damage by cleaving oxidized bases from the DNA, leaving abasic sites that are alkali-labile and readily detected with the comet assay. As levels of oxidative stress increase and these cells enter the perimortem, topoisomerase integrated into the sperm chromatin becomes activated by SUMOylation. Such activation may initially facilitate DNA repair by reannealing double strand breaks but ultimately prepares the DNA for destruction by nucleases released from the male reproductive tract. The abasic sites and oxidized base lesions found in live spermatozoa are mutagenic and may increase the mutational load carried by the offspring, particularly in the context of assisted conception. A variety of strategies are described for managing patients expressing high levels of DNA damage in their spermatozoa, to reduce the risks such lesions might pose to offspring health.

The associated evolution among the extensive RNA editing, GC‐biased mutation, and PPR family expansion in the organelle genomes of Selaginellaceae

AbstractExtensive C‐to‐U editing has been reported from plastid genomes (plastomes) and mitochondrial genomes (mitogenomes) of spikemoss. While “reverse” U‐to‐C editing was recorded in other seed‐free plants such as hornworts, quillworts, and ferns, it was not observed in spikemosses. However, no comprehensive study on the association between RNA editing and other genomic features was conducted for the organelle genomes of spikemosses. Here, we report thousands of C‐to‐U editing sites from plastomes and mitogenomes of two species: 1767 and 2394 edits in Selaginella remotifolia, and 4091 and 2786 edits in Selaginella nipponica, respectively. Comparative analyses revealed two different editing frequencies among plastomes, but one similar frequency in mitogenomes. The different editing frequency in the Selaginella organelle genomes is related to the nonsynonymous substitution rate and the genome structural complexity. The high guanine and cytosine (GC) content caused by GC‐biased mutations in organelle genomes might be related to the absence of U‐to‐C editing in Selaginellaceae. Using RNA‐seq and whole‐genome data, we screened the pentatricopeptide repeat (PPR) family and discovered that the number of aspartic acid–tyrosine–tryptophan (DYW) domain‐containing PPR proteins corresponded roughly to the editing abundance in the Selaginella organelle genomes. Consequently, we hypothesize that associated evolution among RNA editing, GC‐biased mutation in organelle genomes, and the PPR protein family encoded in the nuclear genome, is probably triggered by the aberrant DNA repair system in Selaginellaceae. Our study provides new insights into the association between organelle and nuclear genomes in Selaginellaceae, which would contribute to understanding the evolution of post‐transcriptional modifications of organelle genomes in land plants.

Targets of histone H3 lysine 9 methyltransferases.

Histone H3 lysine 9 di- and trimethylation are well-established marks of constitutively silenced heterochromatin domains found at repetitive DNA elements including pericentromeres, telomeres, and transposons. Loss of heterochromatin at these sites causes genomic instability in the form of aberrant DNA repair, chromosome segregation defects, replication stress, and transposition. H3K9 di- and trimethylation also regulate cell type-specific gene expression during development and form a barrier to cellular reprogramming. However, the role of H3K9 methyltransferases extends beyond histone methylation. There is a growing list of non-histone targets of H3K9 methyltransferases including transcription factors, steroid hormone receptors, histone modifying enzymes, and other chromatin regulatory proteins. Additionally, two classes of H3K9 methyltransferases modulate their own function through automethylation. Here we summarize the structure and function of mammalian H3K9 methyltransferases, their roles in genome regulation and constitutive heterochromatin, as well as the current repertoire of non-histone methylation targets including cases of automethylation.

Early postnatal alterations in follicular stress response and survival in a mouse model of Classic Galactosemia

Primary ovarian insufficiency is characterized by accelerated loss of primordial follicles, which results in ovarian failure and concomitant menopause before age 40. About 1–3% of females in the general population are diagnosed with POI; however, greater than 80% of females with the inherited disease Classic Galactosemia will develop POI. Classic Galactosemia is caused by mutations in the GALT gene encoding the enzyme galactose-1 phosphate uridylyltransferase. While dietary restriction of galactose is lifesaving in the neonatal period, the development of complications including primary ovarian insufficiency is not mitigated. Additionally, the pattern(s) of follicle loss have not been completely characterized. The chronic accumulation of aberrant metabolites such as galactose-1-phosphate and galactitol are suspected culprits in the development of the sequelae, yet the mechanisms remain elusive.Our group uses a GalT gene-trapped mouse model to study the pathophysiology of primary ovarian insufficiency in Classic Galactosemia. We recently showed that differences in the Integrated Stress Response pathway occur in mutant ovaries that likely contribute to their primary ovarian insufficiency phenotype. Using immunofluorescent staining of histological sections of ovaries at progressive ages, we saw evidence of altered Integrated Stress Response activity in granulosa cells and primordial oocytes consistent with accelerated primordial follicle growth activation, aberrant DNA damage and/or repair, and increased cellular stress/death. Overall, our findings indicate that abnormal Integrated Stress Response in the Classic Galactosemia model ovary results in accelerated primordial follicle growth activation, sometimes referred to as “burnout.” These aberrant early events help further clarify when/how the primary ovarian insufficiency phenotype arises under galactosemic conditions.

Abstract 2609: AZD9574 is a novel, brain penetrant PARP-1 selective inhibitor with activity in an orthotopic, intracranial xenograft model with aberrant DNA repair

Abstract The Poly (ADP-ribose) polymerase (PARP) family has numerous essential functions in cellular processes such as transcription, chromatin remodelling, DNA damage response and repair as well as apoptosis. PARP inhibition blocks base excision repair and results in conversion of SSBs to DNA double-strand break (DSBs). DSBs are the most deleterious form of DNA damage that can be generated by exogenous DNA damaging agents or endogenous replication stress. DSBs can be repaired by homologous recombination repair (HRR) or non-homologous end joining (NHEJ). The physiological importance of HRR is underscored by the observation of genomic instability in HRR-deficient (HRD+) cells and, importantly, the association of cancer predisposition and developmental defects with mutations in HRR genes. PARP1 and PARP2 are required for SSB repair, while PARP1 is also involved in the repair of DNA double-strand breaks (DSBs) and replication fork damage. Recent reports suggest the PARP1 inhibition is sufficient to elicit an anti-proliferative effect and that PARP2 is essential for the survival of hematopoietic stem and progenitor cells in animal models.AZD9574 is a novel brain penetrant PARP1 inhibitor that acts by selectively inhibiting and trapping PARP1 at the sites of SSBs. AZD9574 exhibited &amp;gt;8000-fold selectivity for PARP1 compared to PARP2 and other members of the PARP family (PARP2, PARP3, PARP5a and PARP6) in biochemical assays. While AZD9574 inhibited PARP1 enzymatic activity in all tested cell lines irrespective of the HRR status (IC50 range between 0.3 - 2 nM), colony formation assay in isogenic cell lines pairs confirmed higher potency and selectivity towards HRD+ models (DLD1 BRCA2-/-; SKOV-3 BRCA2-/- and SKOV-3 PALB2-/-). For example, AZD9574 IC50 in the BRCA2-/- DLD1 cells was 1.38 nM compared to IC50 &amp;gt; 40 µM BRCA2wt cells, which corresponds to a ~20,000-fold greater efficacy in the BRCA2-/-cells (ratio of AZD9574 IC50 in BRCA2wt divided by BRCA2-/-) compared to the wild type parental line. In vivo, AZD9574 demonstrated dose-dependent efficacy in a BRCA1 mutant MDA-MB-436 subcutaneous xenograft model. Anti-tumour effects of AZD9574 were manifested by pronounced growth regressions that were durable after treatment withdrawal. An intracranial xenograft model of breast cancer brain metastases was developed to assess the efficacy of AZD9574 in the context of blood-brain barrier penetrance. Treatment of animals with established intracranial lesions using a dose of 3 mg/kg AZD9574 showed sustained tumour growth suppression resulting in a significantly extended survival of tumour-bearing mice. Collectively, we believe that our data support the development of AZD9574 as a potential therapy for patients with HRD+ breast cancer whose disease has spread to the brain. Citation Format: Kunzah Jamal, Anna Staniszewska, Jacob Gordon, Shenghua Wen, Frank McGrath, Gregory Dowdell, Dominic Kabbabe, Giuditta illuzzi, Matthew Griffin, Barry R. Davies, Petra Hamerlik. AZD9574 is a novel, brain penetrant PARP-1 selective inhibitor with activity in an orthotopic, intracranial xenograft model with aberrant DNA repair [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2609.

Targeting aberrant replication and DNA repair events for treating breast cancers

The major limitations of DNA-targeting chemotherapy drugs include life-threatening toxicity, acquired resistance and occurrence of secondary cancers. Here, we report a small molecule, Carbazole Blue (CB), that binds to DNA and inhibits cancer growth and metastasis by targeting DNA-related processes that tumor cells use but not the normal cells. We show that CB inhibits the expression of pro-tumorigenic genes that promote unchecked replication and aberrant DNA repair that cancer cells get addicted to survive. In contrast to chemotherapy drugs, systemic delivery of CB suppressed breast cancer growth and metastasis with no toxicity in pre-clinical mouse models. Using PDX and ex vivo explants from estrogen receptor (ER) positive, ER mutant and TNBC patients, we further demonstrated that CB effectively blocks therapy-sensitive and therapy-resistant breast cancer growth without affecting normal breast tissue. Our data provide a strong rationale to develop CB as a viable therapeutic for treating breast cancers.

The Role of DNA Repair in Genomic Instability of Multiple Myeloma.

Multiple Myeloma (MM) is a B cell malignancy marked by genomic instability that arises both through pathogenesis and during disease progression. Despite recent advances in therapy, MM remains incurable. Recently, it has been reported that DNA repair can influence genomic changes and drug resistance in MM. The dysregulation of DNA repair function may provide an alternative explanation for genomic instability observed in MM cells and in cells derived from MM patients. This review provides an overview of DNA repair pathways with a special focus on their involvement in MM and discusses the role they play in MM progression and drug resistance. This review highlights how unrepaired DNA damage due to aberrant DNA repair response in MM exacerbates genomic instability and chromosomal abnormalities, enabling MM progression and drug resistance.

The antidepressant imipramine inhibits breast cancer growth by targeting estrogen receptor signaling and DNA repair events

Aberrant activities of various cell cycle and DNA repair proteins promote cancer growth and progression and render them resistant to therapies. Here, we demonstrate that the anti-depressant imipramine blocks growth of triple-negative (TNBC) and estrogen receptor-positive (ER+) breast cancers by inducing cell cycle arrest and by blocking heightened homologous recombination (HR) and non-homologous end joining-mediated (NHEJ) DNA repair activities. Our results reveal that imipramine inhibits the expression of several cell cycle- and DNA repair-associated proteins including E2F1, CDK1, Cyclin D1, and RAD51. In addition, we show that imipramine inhibits the growth of ER + breast cancers by inhibiting the estrogen receptor- α (ER-α) signaling. Our studies in preclinical mouse models and ex vivo explants from breast cancer patients show that imipramine sensitizes TNBC to the PARP inhibitor olaparib and endocrine resistant ER + breast cancer to anti-estrogens. Our studies suggest that repurposing imipramine could enhance routine care for breast cancer patients. Based on these results, we designed an ongoing clinical trial, where we are testing the efficacy of imipramine for treating patients with triple-negative and estrogen receptor-positive breast cancer. Since aberrant DNA repair activity is used by many cancers to survive and become resistant to therapy, imipramine could be used alone and/or with currently used drugs for treating many aggressive cancers.

The hematopoietic compartment is sufficient for lupus development resulting from the POLB-Y265C mutation.

Systemic lupus erythematosus is a chronic disease characterized by autoantibodies, renal and cutaneous disease, and immune complex formation. Emerging evidence suggests that aberrant DNA repair is an underlying mechanism of lupus development. We previously showed that the POLBY265C/C mutation, which results in development of an aberrant immune repertoire, leads to lupus-like disease in mice. To address whether the hematopoietic compartment is sufficient for lupus development, we transplanted bone marrow cells from POLBY265C/C and POLB+/+ into wild-type congenic mice. Only mice transplanted with the POLBY265C/C bone marrow develop high levels of antinuclear antibodies and renal disease. In conclusion, we show that the hematopoietic compartment harvested from the POLBY265C/C mice is sufficient for development of autoimmune disease.