Alternative DNA Research Articles

ISWI1 complex proteins facilitate developmental genome editing in Paramecium.

One of the most extensive forms of natural genome editing occurs in ciliates, a group of microbial eukaryotes. Ciliate germline and somatic genomes are contained in distinct nuclei within the same cell. During the massive reorganization process of somatic genome development, ciliates eliminate tens of thousands of DNA sequences from a germline genome copy. Recently, we showed that the chromatin remodeler ISWI1 is required for somatic genome development in the ciliate Paramecium tetraurelia Here, we describe two high similarity paralogous proteins, ICOPa and ICOPb, essential for their genome editing. ICOPa and ICOPb are highly divergent from known proteins; the only domain detected showed distant homology to the WSD (WHIM2+WHIM3) motif. We show that both ICOPa and ICOPb interact with the chromatin remodeler ISWI1. Upon ICOP knockdown, changes in alternative DNA excision boundaries and nucleosome densities are similar to those observed for ISWI1 knockdown. We thus propose that a complex comprising ISWI1 and either or both ICOPa and ICOPb are needed for Paramecium's precise genome editing.

Distinct H3K9me3 heterochromatin maintenance dynamics govern different gene programmes and repeats in pluripotent cells.

H3K9me3 heterochromatin, established by lysine methyltransferases (KMTs) and compacted by heterochromatin protein 1 (HP1) isoforms, represses alternative lineage genes and DNA repeats. Our understanding of H3K9me3 heterochromatin stability is presently limited to individual domains and DNA repeats. Here we engineered Suv39h2-knockout mouse embryonic stem cells to degrade remaining two H3K9me3 KMTs within 1 hour and found that both passive dilution and active removal contribute to H3K9me3 decay within 12-24 hours. We discovered four different H3K9me3 decay rates across the genome and chromatin features and transcription factor binding patterns that predict the stability classes. A 'binary switch' governs heterochromatin compaction, with HP1 rapidly dissociating from heterochromatin upon KMT depletion and a particular threshold level of HP1 limiting pioneer factor binding, chromatin opening and exit from pluripotency within 12 h. Unexpectedly, receding H3K9me3 domains unearth residual HP1β peaks enriched with heterochromatin-inducing proteins. Our findings reveal distinct H3K9me3 heterochromatin maintenance dynamics governing gene networks and repeats that together safeguard pluripotency.

Tendons and table salt: A recipe to preserve human DNA

For disaster victim identification, hundreds of samples of human remains have to be extracted from human bodies rapidly for later DNA analysis, often in situation with high humidity, high temperature and no access to cooling facilities. Genetic typing of these samples may be challenging due to degradation of DNA. Until today, bone and teeth are the preferred biological matrix for genetic identification of decomposed human remains. However, processing these samples is time and cost consuming. To address the challenges associated with workload, finances, and sample preservation in the genetic identification of unknown dead bodies, we tested 44 hand and foot tendons from deceased with advanced post mortem changes as alternative DNA material. In addition, we examined commercially available table salt as potential preservation method with 10 tendons over a period of 12 months. The parameters external appearance, DNA quantity and quality have been analysed. Overall, 39 of 44 tendons examined yielded valuable DNA profiles with at least ten autosomal STR loci. Storage in table salt not only increased DNA yield but also resulted in high quality STR profiles of the preserved tendons throughout the study period of 12 months compared to storage without salt. According to the results of this study, we recommend hand and foot tendons as alternative DNA material from bodies with advanced post mortem changes. We particularly recommend commercially available table salt as a storage medium for the preservation of DNA samples. Under field conditions without cooling facilities and the need to store samples prior to DNA analysis table salt is a good storage medium: it preserves DNA and is easily available.

Genome Instability Induced by Topoisomerase Misfunction.

Topoisomerases alter DNA topology by making transient DNA strand breaks (DSBs) in DNA. The DNA cleavage reaction mechanism includes the formation of a reversible protein/DNA complex that allows rapid resealing of the transient break. This mechanism allows changes in DNA topology with minimal risks of persistent DNA damage. Nonetheless, small molecules, alternate DNA structures, or mutations in topoisomerase proteins can impede the resealing of the transient breaks, leading to genome instability and potentially cell death. The consequences of high levels of enzyme/DNA adducts differ for type I and type II topoisomerases. Top1 action on DNA containing ribonucleotides leads to 2-5 nucleotide deletions in repeated sequences, while mutant Top1 enzymes can generate large deletions. By contrast, small molecules that target Top2, or mutant Top2 enzymes with elevated levels of cleavage lead to small de novo duplications. Both Top1 and Top2 have the potential to generate large rearrangements and translocations. Thus, genome instability due to topoisomerase mis-function is a potential pathogenic mechanism especially leading to oncogenic progression. Recent studies support the potential roles of topoisomerases in genetic changes in cancer cells, highlighting the need to understand how cells limit genome instability induced by topoisomerases. This review highlights recent studies that bear on these questions.

A multi-omics study of brain tissue transcription and DNA methylation revealing the genetic pathogenesis of ADHD.

Attention-deficit/hyperactivity disorder (ADHD) is a chronic psychiatric disease that often affects a patient's whole life. Research has found that genetics plays an important role in the development of ADHD. However, there is still a lack of knowledge about the tissue-specific causal effects of biological processes beyond gene expression, such as alternative splicing (AS) and DNA methylation (DNAm), on ADHD. In this paper, a multi-omics study was conducted to investigate the causal effects of the transcription and the DNAm on ADHD, by integrating ADHD genome-wide association data with quantitative trait loci data of gene expression, AS, and DNAm across 14 different brain tissues. The causal effects were estimated using four different two-sample Mendelian randomization methods. Finally, we also prioritized the expression of 866 genes showing significant causal effects, including COMMD5, ENSG00000271904, HYAL3, etc., within at least one brain tissue. We prioritized 966 unique genes that have statistically significant causal AS events, within at least one of the 14 different brain tissues. These genes include PPP1R16A, GGT7, TREM2, etc. Furthermore, through mediation analysis, 106 regulatory pathways were inferred where DNAm influences ADHD through gene expression or AS processes. Our research findings provide guidance for future experimental studies on the molecular mechanisms of ADHD development, and also put forward valuable knowledge for the prevention, diagnosis, and treatment of ADHD.

Distinct H3K9me3 heterochromatin maintenance dynamics govern different gene programs and repeats in pluripotent cells.

H3K9me3-heterochromatin, established by lysine methyltransferases (KMTs) and compacted by HP1 isoforms, represses alternative lineage genes and DNA repeats. Our understanding of H3K9me3-heterochromatin stability is presently limited to individual domains and DNA repeats. We engineered Suv39h2 KO mouse embryonic stem cells to degrade remaining two H3K9me3-KMTs within one hour and found that both passive dilution and active removal contribute to H3K9me3 decay within 12-24 hours. We discovered four different H3K9me3 decay rates across the genome and chromatin features and transcription factor binding patterns that predict the stability classes. A "binary switch" governs heterochromatin compaction, with HP1 rapidly dissociating from heterochromatin upon KMTs' depletion and a particular threshold level of HP1 limiting pioneer factor binding, chromatin opening, and exit from pluripotency within 12 hr. Unexpectedly, receding H3K9me3 domains unearth residual HP1β peaks enriched with heterochromatin-inducing proteins. Our findings reveal distinct H3K9me3-heterochromatin maintenance dynamics governing gene networks and repeats that together safeguard pluripotency.

Enriched G4 forming repeats in the human genome are associated with robust well-coordinated transcription and reduced cancer transcriptome variation

Non-B DNA G-quadruplex (G4) structures with guanine (G) runs of 2-4 repeats can trigger opposing experimental transcriptional impacts. Here, we employed bioinformatic algorithms to comprehensively assess correlations of steady-state RNA transcript levels with all putative G4 sequence (pG4) locations genome-wide in three mammalian genomes and in normal and tumor human tissues. The human pG4-containing gene set displays higher expression levels than the set without pG4, supporting and extending some prior observations. pG4 enrichment at transcription start sites (TSS) in human, but not chimpanzee and mouse genomes, suggests possible positive selection pressure for pG4 at human TSS, potentially driving genome rewiring and gene expression divergence between human and chimpanzee. Comprehensive bioinformatic analyses revealed lower pG4-containing gene set variability in humans and among different pG4 genes in tumors. As G4 stabilizers are under therapeutic consideration for cancer and pathogens, such distinctions between human normal and tumor G4s along with other species merit attention. Furthermore, in germline and cancer sequences, the most mutagenic pG4 mapped to regions promoting alternative DNA structures. Overall findings establish high pG4 at TSS as a human genome attribute statistically associated with robust well-coordinated transcription and reduced cancer transcriptome variation with implications for biology, model organisms, and medicine.

Structural insights into i-motif DNA structures in sequences from the insulin-linked polymorphic region

The insulin-linked polymorphic region is a variable number of tandem repeats region of DNA in the promoter of the insulin gene that regulates transcription of insulin. This region is known to form the alternative DNA structures, i-motifs and G-quadruplexes. Individuals have different sequence variants of tandem repeats and although previous work investigated the effects of some variants on G-quadruplex formation, there is not a clear picture of the relationship between the sequence diversity, the DNA structures formed, and the functional effects on insulin gene expression. Here we show that different sequence variants of the insulin linked polymorphic region form different DNA structures in vitro. Additionally, reporter genes in cellulo indicate that insulin expression may change depending on which DNA structures form. We report the crystal structure and dynamics of an intramolecular i-motif, which reveal sequences within the loop regions forming additional stabilising interactions that are critical to formation of stable i-motif structures. The outcomes of this work reveal the detail in formation of stable i-motif DNA structures, with potential for rational based drug design for compounds to target i-motif DNA.

Cell cycle arrest combined with CDK1 inhibition suppresses genome-wide mutations by activating alternative DNA repair genes during genome editing

Cells regularly repair numerous mutations. However, the effect of CRISPR/Cas9-induced dsDNA breaks on the repair processes of naturally occurring genome-wide mutations is unclear. In this study, we used TSCE5 cells with the heterozygous thymidine kinase genotype (TK+/-) to examine these effects. We strategically inserted the target sites for guide RNA (gRNA)/Cas9 and I-SceI into the functional allele and designed the experiment such that deletions of > 81bp or base substitutions within exon five disrupted the TK gene, resulting in a TK-/- genotype. TSCE5 cells in the resting state exhibited 16 genome-wide mutations that affected cellular functions. After gRNA/Cas9 editing, these cells produced 859 mutations, including 67 high-impact variants that severely affected cellular functions under standard culture conditions. Mutation profile analysis indicated a significant accumulation of C to A substitutions, underscoring the widespread induction of characteristic mutations by gRNA/Cas9. In contrast, gRNA/Cas9-edited cells under conditions of S∼G2/M arrest and cyclin-dependent kinase 1 inhibition showed only five mutations. Transcriptomic analysis revealed the downregulation of DNA replication genes and upregulation of alternative DNA repair genes, such as zinc finger protein 384 (ZNF384) and dual specificity phosphatase, under S∼G2/M conditions. Additionally, activation of nucleotide and base excision repair gene, including O-6-methylguanine-DNA methyltransferase and xeroderma pigmentosum complementation group C, was observed. This study highlights the profound impact of CRISPR/Cas9 editing on genome-wide mutation processes and underscores the emergence of novel DNA repair pathways. Finally, our findings provide significant insights into the maintenance of genome integrity during genome editing.

Flipons: The discovery of Z-DNA and soft-wired genomes

Flipons: The discovery of Z-DNA and soft-wired genomes Alan Herbert, Founder and President of InsideOutBio, discusses alternative DNA conformations and understanding of their biological functions. One of the great things about science is that every time you think you really understand things and can’t imagine how they could work differently, life surprises you. The biggest mistake you can make is believing that nature works according to your best design. Take physics, for example – first, there were the four humors, then atoms, then protons and electrons, then even more particles, and then quarks – who knows what’s next? Similarly, biology is undergoing its own progression, and we are finding that there is more to life than just Watson and Crick’s DNA.

Accelerated Screening of Alternative DNA Base‐Organic Molecule‐Base Architectures via Integrated Theory and Experiment

AbstractOrganic molecule‐mediated noncanonical DNA self‐assembly expands the standard DNA base‐pairing alphabets. However, only a very limited number of small molecules have been recognized as mediators because of the tedious and complicated experiments like crystallization and microscopy imaging. Here we present an integrative screening protocol incorporating molecular dynamics (MD) for fast theoretical simulation and native polyacrylamide gel electrophoresis for convenient experimental validation. Melamine, the molecule that was confirmed mediating noncanonical DNA base‐pairing, and 38 other candidate molecules were applied to demonstrate the feasibility of this protocol. We successfully identified seven stable noncanonical DNA duplex structures, and another eight novel structures with sub‐stability. In addition, we discovered that hairpins at both ends can significantly stabilize the noncanonical DNA structures, providing a guideline to design small organic molecule‐incorporated DNA structures. Such an efficient screening protocol will accelerate the design of alternative DNA‐molecule architectures beyond Watson–Crick pairs. Considering the wide range of potential mediators, it will also facilitate applications such as noncovalent, highly dense loading of drug molecules in DNA‐based delivery system and probe design for sensitive detection of certain molecules.

Accelerated Screening of Alternative DNA Base-Organic Molecule-Base Architectures via Integrated Theory and Experiment.

Organic molecule-mediated noncanonical DNA self-assembly expands the standard DNA base-pairing alphabets. However, only a very limited number of small molecules have been recognized as mediators because of the tedious and complicated experiments like crystallization and microscopy imaging. Here we present an integrative screening protocol incorporating molecular dynamics (MD) for fast theoretical simulation and native polyacrylamide gel electrophoresis for convenient experimental validation. Melamine, the molecule that was confirmed mediating noncanonical DNA base-pairing, and 38 other candidate molecules were applied to demonstrate the feasibility of this protocol. We successfully identified seven stable noncanonical DNA duplex structures, and another eight novel structures with sub-stability. In addition, we discovered that hairpins at both ends can significantly stabilize the noncanonical DNA structures, providing a guideline to design small organic molecule-incorporated DNA structures. Such an efficient screening protocol will accelerate the design of alternative DNA-molecule architectures beyond Watson-Crick pairs. Considering the wide range of potential mediators, it will also facilitate applications such as noncovalent, highly dense loading of drug molecules in DNA-based delivery system and probe design for sensitive detection of certain molecules.

Targeting S6K/NFκB/SQSTM1/Polθ signaling to suppress radiation resistance in prostate cancer

In this study we have identified POLθ-S6K-p62 as a novel druggable regulator of radiation response in prostate cancer. Despite significant advances in delivery, radiotherapy continues to negatively affect treatment outcomes and quality of life due to resistance and late toxic effects to the surrounding normal tissues such as bladder and rectum. It is essential to develop new and effective strategies to achieve better control of tumor. We found that ribosomal protein S6K (RPS6KB1) is elevated in human prostate tumors, and contributes to resistance to radiation. As a downstream effector of mTOR signaling, S6K is known to be involved in growth regulation. However, the impact of S6K signaling on radiation response has not been fully explored. Here we show that loss of S6K led to formation of smaller tumors with less metastatic ability in mice. Mechanistically we found that S6K depletion reduced NFκB and SQSTM1 (p62) reporter activity and DNA polymerase θ (POLθ) that is involved in alternate end-joining repair. We further show that the natural compound berberine interacts with S6K in a in a hitherto unreported novel mode and that pharmacological inhibition of S6K with berberine reduces Polθ and downregulates p62 transcriptional activity via NFκB. Loss of S6K or pre-treatment with berberine improved response to radiation in prostate cancer cells and prevented radiation-mediated resurgence of PSA in animals implanted with prostate cancer cells. Notably, silencing POLQ in S6K overexpressing cells enhanced response to radiation suggesting S6K sensitizes prostate cancer cells to radiation via POLQ. Additionally, inhibition of autophagy with CQ potentiated growth inhibition induced by berberine plus radiation. These observations suggest that pharmacological inhibition of S6K with berberine not only downregulates NFκB/p62 signaling to disrupt autophagic flux but also decreases Polθ. Therefore, combination treatment with radiation and berberine inhibits autophagy and alternate end-joining DNA repair, two processes associated with radioresistance leading to increased radiation sensitivity.

Abstract A019: Inhibition of nicotinamide adenine dinucleotide (NAD) production is a potent therapeutic strategy to inactivate homologous recombination in cancer cells

Abstract Homologous recombination (HR) is the most faithful DNA double-strand breaks (DSBs) repairing pathway. HR deficiency (HRD) can result from mutations in BRCA1, BRCA2, and PALB2, which are associated with genomic instability and cancer. HR deficient cells can survive through the use of alternative DNA repair pathways. Therefore, inhibiting these repair pathways in HRD cells trigger cancer cell death. This phenomenon is called synthetic lethality. The best-known example of synthetic lethality is that involving BRCA1/BRCA2/PALB2 deficiency with PARP-1 inhibition by Olaparib. However, ∼40% of patients will develop resistance to PARP inhibition because of several mechanisms, including a second mutation in BRCA1/2, restoring their full-length expression or RAD51 overexpression. RAD51 is one of the most important mediators of HR as it promotes the invasion of a sister chromatid allowing faithful DSB repair. There are many pieces of evidence which show that RAD51 overexpression is correlated with resistance and worse overall survival. Our hypothesis is that targeting RAD51 can trigger HR deficiency, particularly in cancer cells resistant to standard treatments. We thus sought to find inhibitors of the RAD51 recombinase. We have developed an in cellulo screening technique to test the direct effect of chemical compounds on RAD51 foci formation following irradiation. Using this technique, we identified , among 1381 chemical molecules, FK866 (Daporinad) which is a Nicotinamide Phosphoribosyltransferase Inhibitor (NAMPTi). NAMPT is involved in producing cellular Nicotinamide Adenine Dinucleotide (NAD+), which plays a crucial role in various metabolic processes within the cell, primarily in energy production and DNA repair. Remarkably, treatment of Daporinad reduced RAD51 foci formation and triggered a destabilization of the RAD51 protein by the proteasome. This destabilization seemed to be specific for RAD51, as NHEJ factors were not affected by NAMPTi. We will present data related to our objectives: i) Monitoring the effect of Daporinad on different cancer cell lines originated from cancer types which has shown resistance and worse overall survival because of RAD51 overexpression. Western blot and qPCR techniques will be used to investigate the effect of Daporinad on RAD51 protein and mRNA level and using Zeiss Celldiscoverer 7 system and Incucyte live cell imaging microscopy to observe the effect of Daporinad on cell survival; ii) To confirm the specific targeting of HR by Daporinad, we will monitor the effect of Daporinad on HR and other DNA DSB repair pathways using in cellulo reporter systems; iii) In the long term, we aim to evaluate the effect of Daporinad in vivo on tumor cell growth in pre-clinical models, such as mouse xenografts. In conclusion, small molecules affecting HR provide a source of new cancer therapies targeting RAD51 overexpression or using synthetic lethality. Citation Format: Sadaf Valeh Sheida, Thibaut Peterlini, Mélissa Thomas, Guy Poirier, Jean-Yves Masson. Inhibition of nicotinamide adenine dinucleotide (NAD) production is a potent therapeutic strategy to inactivate homologous recombination in cancer cells [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr A019.

First-in-human, phase 1/2 study of GSK4524101, an oral DNApolymerase theta inhibitor (POLQi), alone or combined with the poly(ADP-ribose) polymerase (PARP) inhibitor (PARPi) niraparib in adults with solid tumors.

TPS3174 Background: Double-stranded DNA breaks (DSBs) in human cells are typically repaired via nonhomologous end joining (NHEJ) or homologous recombination (HR). Deficiency of HR-mediated DNA repair plays a role in the initiation and progression of many tumor types. In tumors with HR deficiency, PARP inhibition leads to generation of DSBs that cannot be effectively repaired because of the HR defect, resulting in synthetic lethality. This finding has led to the clinical development and approval of several PARPi for the treatment of various tumors including certain ovarian, breast, prostate, and pancreatic cancers that are prone to display HR deficiency (HRd). DNA polymerase theta (encoded by POLQ) mediates an alternative DNA repair mechanism, microhomology-mediated end joining (MMEJ). DNA polymerase theta is generally not detectable in normal tissues but is upregulated in many tumor types. In preclinical studies, POLQi plus PARPi treatment demonstrated superior efficacy to PARPi alone in preventing the growth of HRd tumors. To evaluate the clinical potential of combining POLQi and PARPi, this first-in-human study investigates treatment with GSK4524101, an investigational POLQi, with or without the PARPi niraparib, in patients with solid tumors. Methods: This open-label, multicenter, phase 1/2 study (NCT06077877) opened in October 2023 and comprises dose-finding (part 1, including a food-effect cohort) and dose-expansion (part 2) parts. This trial aims to assess the maximum-tolerated dose, pharmacokinetics, safety, and preliminary antitumor activity of oral GSK4524101 with or without niraparib. The primary endpoints are safety (part 1) and confirmed objective response rate (part 2). Secondary endpoints include pharmacokinetics, safety, progression-free survival (part 2), and response duration (part 2). Up to 135 patients may be enrolled. To be eligible, patients must be aged ≥18 years; have an advanced or metastatic solid tumor, an Eastern Cooperative Oncology Group performance status score of 0–2, and a life expectancy of ≥3 months; and have exhausted all standard treatment options. Individuals are ineligible if they have not recovered from chemotherapy-associated adverse events or have symptomatic uncontrolled brain or leptomeningeal metastases, a history of myelodysplastic syndrome or acute myeloid leukemia, uncontrolled hypertension, or a second malignancy that has progressed or required active treatment in the previous 2 years. The study is actively recruiting in the US and Canada; as of January 1, 2024, 1 patient has been dosed. Patients enrolled in part 1 will receive GSK4524101 alone or GSK4524101 plus niraparib; patients enrolled in part 2 will be randomized to either GSK4524101 plus niraparib or niraparib alone. Part 1 of this study is expected to complete in 2025. Data presented on behalf of the original authors with their permission. Presented at the American Association for Cancer Research (AACR) Annual Meeting 2024; April 5-10, 2024; San Diego, CA. Final publication number: 9686. Reused with permission. Clinical trial information: NCT06077877 .

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.

Ultra-sensitive Tb3+/G-quadruplex fluorescence detection of oxidative DNA damage coupled with DNA glycosylase

Development of simple, sensitive and rapid sensing strategy for DNA damage induced by chemical reagents is urgent demand. In this assay, a Tb3+/G-quadruplex fluorescence system was proposed for the sensitive detection of DNA damage induced by Fenton reaction. The specific guanine-rich oligonucleotides can form G-quadruplex induced by Tb3+, leading to an enhancement of Tb3+ emission due to energy transfer from guanine to Tb3+. After a DNA damage reaction with Fenton reagents, an obvious fluorescence decrease was observed due to the structure change of Tb3+/G-quadruplex. which was also confirmed by polyacrylamide gel electrophoresis and circular dichroism. The Fe2+-mediated Fenton reaction with as low as 0.01 μM Fe2+ and 0.04 μM H2O2 induced oxidative DNA damage can be detected. Additionally, the human 8-oxoguanine DNA glycosylase (hOGG 1) was introduced to amplify DNA oxidation damage, by converting DNA oxidative nucleobases to strand break. Over 30-fold lower detection limit of 0.3 nM Fe2+ and 1.2 nM H2O2 was achieved. Alternative specific DNA damage or other DNA repair enzymes produced much less fluorescence decrease, demonstrating its good selectivity. It shows the potential to become a universal screening tool for the rapid and sensitive assessment of the genotoxicity of the overwhelming number of chemicals in environment.

Interaction of dinuclear Co(III) cylinders with higher-order DNA structures

Alternative DNA structures play critical roles in fundamental biological processes linked to human diseases. Thus, targeting and stabilizing these structures by specific ligands could affect the progression of cancer and other diseases. Here, we describe, using methods of molecular biophysics, the interactions of two oxidatively locked [Co2L3]6+ cylinders, rac-2 and meso-1, with diverse alternative DNA structures, such as junctions, G quadruplexes, and bulges. This study was motivated by earlier results demonstrating that both Co(III) cylinders exhibit potent and selective activity against cancer cells, accumulate in the nucleus of cancer cells, and prove to be efficient DNA binders. The results show that the bigger cylinder rac-2 stabilizes all DNA structures, while the smaller cylinder meso-1 stabilizes just the Y-shaped three-way junctions. Collectively, the results of this study suggest that the stabilization of alternative DNA structures by Co(III) cylinders investigated in this work might contribute to the mechanism of their biological activity.

Exploring the Roles of Different DNA Repair Proteins in Short Inverted Repeat Mediated Genomic Instability: A Pilot Study

Repetitive DNA sequences are abundant in the human genome and can adopt alternative (i.e., non-B) DNA structures. These sequences contribute to diverse biological functions, including genomic instability. Previously, we found that Z-DNA-, H-DNA- and cruciform DNA-forming sequences are mutagenic, implicating them in cancer etiology. These sequences can stimulate the formation of DNA double-strand breaks (DSBs), causing deletions via cleavage by the endonuclease ERCC1-XPF. Interestingly, the activity of ERCC1-XPF in H-DNA-induced mutagenesis is nucleotide excision repair (NER)-dependent, but its role in Z-DNA-induced mutagenesis is NER-independent. Instead, Z-DNA is processed by ERCC1-XPF in a mechanism dependent on the mismatch repair (MMR) complex, MSH2-MSH3. These observations indicate distinct mechanisms of non-B-induced genomic instability. However, the roles of NER and MMR proteins, as well as additional nucleases (CtIP and MRE11), in the processing of cruciform DNA remain unknown. Here, we present data on the processing of cruciform-forming short inverted repeats (IRs) by DNA repair proteins using mammalian cell-based systems. From this pilot study, we show that, in contrast to H-DNA and Z-DNA, short IRs are processed in a NER- and MMR-independent manner, and the nucleases CtIP and MRE11 suppress short IR-induced genomic instability in mammalian cells.

Abstract CT169: First-in-human, phase 1/2 study of GSK4524101, an oral DNA polymerase theta inhibitor (POLQi), alone or combined with the poly(ADP-ribose) polymerase (PARP) inhibitor (PARPi) niraparib in adults with solid tumors

Abstract Background: Double-stranded DNA breaks (DSBs) in human cells are typically repaired via nonhomologous end joining (NHEJ) or homologous recombination (HR). Deficiency of HR-mediated DNA repair plays a role in the initiation and progression of many tumor types. In tumors with HR deficiency, PARP inhibition leads to generation of DSBs that cannot be effectively repaired because of the HR defect, resulting in synthetic lethality. This finding has led to the clinical development and approval of several PARPi for the treatment of various tumors including certain ovarian, breast, prostate, and pancreatic cancers that are prone to display HR deficiency (HRd). DNA polymerase theta (encoded by POLQ) mediates an alternative DNA repair mechanism, microhomology-mediated end joining (MMEJ). DNA polymerase theta is generally not detectable in normal tissues but is upregulated in many tumor types. In preclinical studies, POLQi plus PARPi treatment demonstrated superior efficacy to PARPi alone in preventing the growth of HRd tumors. To evaluate the clinical potential of combining POLQi and PARPi, this first-in-human study investigates treatment with GSK4524101, an investigational POLQi, with or without the PARPi niraparib, in patients with solid tumors. Methods: This open-label, multicenter, phase 1/2 study (NCT06077877) opened in October 2023 and comprises dose-finding (part 1, including a food-effect cohort) and dose-expansion (part 2) parts. This trial aims to assess the maximum-tolerated dose, pharmacokinetics, safety, and preliminary antitumor activity of oral GSK4524101 with or without niraparib. The primary endpoints are safety (part 1) and confirmed objective response rate (part 2). Secondary endpoints include pharmacokinetics, safety, progression-free survival (part 2), and response duration (part 2). Up to 135 patients may be enrolled. To be eligible, patients must be aged ≥18 years; have an advanced or metastatic solid tumor, an Eastern Cooperative Oncology Group performance status score of 0-2, and a life expectancy of ≥3 months; and have exhausted all standard treatment options. Individuals are ineligible if they have not recovered from chemotherapy-associated adverse events or have symptomatic uncontrolled brain or leptomeningeal metastases, a history of myelodysplastic syndrome or acute myeloid leukemia, uncontrolled hypertension, or a second malignancy that has progressed or required active treatment in the previous 2 years. The study is actively recruiting in the US and Canada; as of January 1, 2024, 1 patient has been dosed. Patients enrolled in part 1 will receive GSK4524101 alone or GSK4524101 plus niraparib; patients enrolled in part 2 will be randomized to either GSK4524101 plus niraparib or niraparib alone. Part 1 of this study is expected to complete in 2025. Citation Format: Vivek Samnotra, Veronica Moroz, Luda Shtessel, Mita Kuchimanchi, Patrick Hanafin, Malar Pannirselvam, Aishwarya Bhaskar, Andrew Liu, Haluk Yuzugullu, Minal Barve, David Sommerhalder, Timothy Yap, Michele Sanicola-Nadel. First-in-human, phase 1/2 study of GSK4524101, an oral DNA polymerase theta inhibitor (POLQi), alone or combined with the poly(ADP-ribose) polymerase (PARP) inhibitor (PARPi) niraparib in adults with solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT169.