Large-scale phase III clinical trials of Olaparib have revealed benefits for ovarian cancer patients with BRCA gene mutations or homologous recombination deficiency (HRD). However, fewer than 50% of ovarian cancer patients have both BRCA mutations and HRD. Therefore, improving the effect of Olaparib in HR-proficient patients is of great clinical value. Here, a combination strategy comprising Olaparib and CDK12-IN-3 effectively inhibited the growth of HR-proficient ovarian cancer in cell line, patient-derived organoid (PDO), and mouse xenograft models. Furthermore, the combination strategy induced severe DNA double-strand break (DSB) formation, increased NHEJ activity in the G2 phase, and reduced HR activity in cancer cells. Mechanistically, the combination treatment impaired Ku80 poly(ADP-ribosyl)ation (PARylation) and phosphorylation, resulting in PARP1-Ku80 complex dissociation. After dissociation, Ku80 occupancy at DSBs and the resulting Ku80-primed NHEJ activity were increased. Owing to Ku80-mediated DNA end protection, MRE11 and Rad51 foci formation was inhibited after the combination treatment, suggesting that this treatment suppressed HR activity. Intriguingly, the combination strategy expedited cGAS nuclear relocalization, further suppressing HR and, conversely, increasing genomic instability. Moreover, the inhibitory effect on cell survival persisted after drug withdrawal. These findings provide a rationale for the clinical application of CDK12-IN-3 in combination with Olaparib.

Ribosomal DNA (rDNA) encodes the ribosomal RNA genes and represents an intrinsically unstable genomic region. However, the underlying mechanisms and implications for genome integrity remain elusive. Here, we use Bloom syndrome (BS), a rare genetic disease characterized by DNA repair defects and hyper-unstable rDNA, as a model to investigate the mechanisms leading to rDNA instability. We find that in Bloom helicase (BLM) proficient cells, the homologous recombination (HR) pathway in rDNA resembles that in nuclear chromatin; it is initiated by resection, replication protein A (RPA) loading and BRCA2-dependent RAD51 filament formation. However, BLM deficiency compromises RPA-loading and BRCA1/2 recruitment to rDNA, but not RAD51 accumulation. RAD51 accumulates at rDNA despite depletion of long-range resection nucleases and rDNA damage results in micronuclei when BLM is absent. In summary, our findings indicate that rDNA is permissive to RAD51 accumulation in the absence of BLM, leading to micronucleation and potentially global genomic instability.

OBJECTIVE: To study the biological relationship between congenital lung malformations (CLMs) and malignancy.
METHODS: Biopsies of 12 CPAMs, 6 intralobar sequestrations and 2 extralobar sequestrations were analyzed through whole-genome sequencing. Blood samples from 10 patients were used to confirm or exclude somatic mosaicism. Putative somatic Single Nucleotide Variants (SNVs) were called for each malformed sample with a Panel of Normals built with control DNA samples extracted from blood. The variants were subsequently confirmed by Sanger sequencing and searched, whenever possible, in the blood samples of patients.
RESULTS: All CLMs but one presented a signature of genomic instability by means of multiple clusters of cells with gene mutations. Seven tumor transformation-related SNVs were detected in 6/20 congenital lung malformations. Four very rare in the general population SNVs were found in a region previously linked to lung cancer in 5p15.33, upstream of TERT oncogene. Furthermore, we identified missense genetic variants, whose tumorigenic role is well known, in the RET, FANCA and MET genes.
CONCLUSIONS: Genomic instability in 95% of CLMs and genetic variants linked to tumor development in 30% of them, regardless of histopathology, are predisposing factors to malignancy, that combined with exposure to carcinogens, might trigger the development of malignancy and explain the association between CLMs and lung cancer.

Collisions of the transcription and replication machineries on the same DNA strand can pose a significant threat to genomic stability. These collisions occur in part due to the formation of RNA-DNA hybrids termed R-loops, in which a newly transcribed RNA molecule hybridizes with the DNA template strand. This study investigated the role of RAD52, a known DNA repair factor, in preventing collisions by directing R-loop formation and resolution. We show that RAD52 deficiency increases R-loop accumulation, exacerbating collisions and resulting in elevated DNA damage. Furthermore, RAD52\'s ability to interact with the transcription machinery, coupled with its capacity to facilitate R-loop dissolution, highlights its role in preventing collisions. Lastly, we provide evidence of an increased mutational burden from double-strand breaks at conserved R-loop sites in human tumor samples, which is increased in tumors with low RAD52 expression. In summary, this study underscores the importance of RAD52 in orchestrating the balance between replication and transcription processes to prevent collisions and maintain genome stability.

Evolutionary annotation of genome maintenance (GM) proteins has conventionally been established by remote relationships within protein sequence databases. However, often no significant relationship can be established. Highly sensitive approaches to attain remote homologies based on iterative profile-to-profile methods have been developed. Still, these methods have not been systematically applied in the evolutionary annotation of GM proteins. Here, by applying profile-to-profile models, we systematically survey the repertoire of GM proteins from bacteria to man. We identify multiple GM protein candidates and annotate domains in numerous established GM proteins, among other PARP, OB-fold, Macro, TUDOR, SAP, BRCT, KU, MYB (SANT), and nuclease domains. We experimentally validate OB-fold and MIS18 (Yippee) domains in SPIDR and FAM72 protein families, respectively. Our results indicate that, surprisingly, despite the immense interest and long-term research efforts, the repertoire of genome stability caretakers is still not fully appreciated.

BACKGROUND: Genomic instability is a hallmark of cancer, leading to many somatic alterations. Identifying which alterations have a system-wide impact is a challenging task. Nevertheless, this is an essential first step for prioritizing potential biomarkers. We developed CIBRA (Computational Identification of Biologically Relevant Alterations), a method that determines the system-wide impact of genomic alterations on tumor biology by integrating two distinct omics data types: one indicating genomic alterations (e.g. genomics), and another defining a system-wide expression response (e.g. transcriptomics). CIBRA was evaluated with genome-wide screens in 33 cancer types using primary and metastatic cancer data from the Cancer Genome Atlas and Hartwig Medical Foundation.
RESULTS: We demonstrate the capability of CIBRA by successfully confirming the impact of point mutations in experimentally validated oncogenes and tumor suppressor genes (0.79 AUC). Surprisingly, many genes affected by structural variants were identified to have a strong system-wide impact (30.3%), suggesting that their role in cancer development has thus far been largely under-reported. Additionally, CIBRA can identify impact with only 10 cases and controls, providing a novel way to prioritize genomic alterations with a prominent role in cancer biology. Our findings demonstrate that CIBRA can identify cancer drivers by combining genomics and transcriptomics data. Moreover, our work shows an unexpected substantial system-wide impact of structural variants in cancer. Hence, CIBRA has the potential to preselect and refine current definitions of genomic alterations to derive more nuanced biomarkers for diagnostics, disease progression, and treatment response.
METHODS: The R package CIBRA is available at https://github.com/AIT4LIFE-UU/CIBRA.

In metazoans mitochondrial DNA (mtDNA) or retrotransposon cDNA released to cytoplasm are degraded by nucleases to prevent sterile inflammation. It remains unknown whether degradation of these DNA also prevents nuclear genome instability. We used an amplicon sequencing-based method in yeast enabling analysis of millions of DSB repair products. In non-dividing stationary phase cells, Pol4-mediated non-homologous end-joining increases, resulting in frequent insertions of 1-3 nucleotides, and insertions of mtDNA (NUMTs) or retrotransposon cDNA. Yeast EndoG (Nuc1) nuclease limits insertion of cDNA and transfer of very long mtDNA ( >10 kb) to the nucleus, where it forms unstable circles, while promoting the formation of short NUMTs (~45-200 bp). Nuc1 also regulates transfer of extranuclear DNA to nucleus in aging or meiosis. We propose that Nuc1 preserves genome stability by degrading retrotransposon cDNA and long mtDNA, while short NUMTs originate from incompletely degraded mtDNA. This work suggests that nucleases eliminating extranuclear DNA preserve genome stability.

BACKGROUND: Nijmegen breakage syndrome (NBS) is an autosomal recessive DNA repair disorder that manifests through increased genomic instability, malignancy, and cellular and humoral immunodeficiencies. The prognosis for NBS patients is poor due to their increased susceptibility to fatal infections and lymphoproliferative malignancies. Currently, there is no specific treatment for NBS, though allogeneic hematopoietic stem cell transplantation (HSCT) has been performed and documented as case series to demonstrate the utility of transplantation.
METHODS: A 14-year-old girl with NBS and haploidentical HSCT from her older brother due to recurrent lung infection was referred for liver transplantation (LT) due to liver cirrhosis, hepatopulmonary syndrome (HPS), and suspicion of liver malignancy. It was decided to perform LT using the living donor who had previously donated for HSCT.
RESULTS: Living donor left lobe LT was successfully performed from her brother. The patient experienced no complications in the early postoperative period and was discharged on the seventh postoperative day. Pathological examination of extracted liver has shown \"intermediate cell carcinoma\" in two foci. After 1 year LT, the patient has had an uneventful course in terms of LT complications and infection, with minimal immunosuppression.
CONCLUSIONS: NBS patients have an increased prevalence of malignancies, including primary hepatic malignancy, but most are managed medically or with limited resections. Transplantation in these patients can be curative for hepatic malignancy with a favorable safety profile.

Pituitary neuroendocrine tumors (PitNET) represent the vast majority of sellar masses. Some behave aggressively, growing rapidly and invading surrounding tissues, with high rates of recurrence and resistance to therapy. Our aim was to establish patterns of genomic, transcriptomic and methylomic evolution throughout time in primary and recurrent tumors from the same patient. Therefore, we performed transcriptome- and exome-sequencing and methylome microarrays of aggressive, primary, and recurrent PitNET from the same patient. Primary and recurrent tumors showed a similar exome profile, potentially indicating a stable genome over time. In contrast, the transcriptome of primary and recurrent PitNET was dissimilar. Gonadotroph, silent corticotroph, as well as metastatic corticotroph and a somatotroph PitNET expressed genes related to fatty acid biosynthesis and metabolism, phosphatidylinositol signaling, glycerophospholipid and phospholipase D signaling, respectively. Diacylglycerol kinase gamma (DGKG), a key enzyme in glycerophospholipid metabolism and phosphatidylinositol signaling pathways, was differentially expressed between primary and recurrent PitNET. These alterations did not seem to be regulated by DNA methylation, but rather by several transcription factors. Molecular docking showed that dasatinib, a small molecule tyrosine kinase inhibitor used in the treatment of chronic lymphocytic and acute lymphoblastic leukemia, could target DGKG. Dasatinib induced apoptosis and decreased proliferation in GH3 cells. Our data indicate that pituitary tumorigenesis could be driven by transcriptomically heterogeneous clones, and we describe alternative pharmacological therapies for aggressive and recurrent PitNET.

The τ-subunit of the clamp loader complex (CLC) physically interacts with both the DnaB helicase and the polymerase III (Pol III) core α-subunit through Domains IV and V, respectively. This interaction is proposed to help maintain rapid and efficient DNA synthesis rates with high genomic fidelity and plasticity, facilitating enzymatic coupling within the replisome. To test this hypothesis, CRISPR-Cas9 editing was used to create site-directed genomic mutations within the dnaX gene at the C-terminus of τ predicted to interact with the α-subunit of Pol III. Perturbation of the α-τ binding interaction in vivo resulted in cellular and genomic stress markers that included reduced growth rates, fitness, and viabilities. Specifically, dnaX:mut strains showed increased cell filamentation, mutagenesis frequencies, and activated SOS. In situ fluorescence flow cytometry and microscopy quantified large increases in the amount of single-stranded DNA (ssDNA) gaps present. Removal of the C-terminus of τ (I618X) still maintained its interactions with DnaB and stimulated unwinding but lost its interaction with Pol III, resulting in significantly reduced rolling circle DNA synthesis. Intriguingly, dnaX:L635P/D636G had the largest induction of SOS, high mutagenesis, and the most prominent ssDNA gaps, which can be explained by an impaired ability to regulate the unwinding speed of DnaB resulting in a faster rate of in vitro rolling circle DNA replication, inducing replisome decoupling. Therefore, τ stimulated DnaB unwinding and physical coupling with Pol III acts to enforce replisome plasticity to maintain an efficient rate of synthesis and prevent genomic instability.