RNase H1 Overexpression Research Articles

ULI-ssDRIP-seq revealed R-loop dynamics during vertebrate early embryogenesis

R-loop, a chromatin structure containing one RNA:DNA hybrid and one unpaired single-stranded DNA, plays multiple biological roles. However, due to technical limitations, the landscapes and potential functions of R-loops during embryogenesis remain elusive. Here, we developed a quantitative and high-resolution ultra-low input R-loop profiling method, named ULI-ssDRIP-seq, which can map global R-loops with as few as 1000 cells. By using ULI-ssDRIP-seq, we reveal the R-loop dynamics in the zebrafish from gametes to early embryos. In oocytes, the R-loop level is relatively low in most regions of the nuclear genome, except maternal-inherited rDNA and mitochondrial genome. The correlation between R-loop and CG methylation dynamics during early development is relatively weak. Furthermore, either up- or down-regulation of global R-loops by knockdown or overexpression of RNase H1 causes a delay of embryonic development with dramatic expression changes in zygotic and maternal genes. This study provides comprehensive R-loop landscapes during early vertebrate embryogenesis and demonstrates the implication of R-loops in embryonic development.

Wiskott Aldrich syndrome protein (WASp)-deficient Th1 cells promote R-loop-driven transcriptional insufficiency and transcription-coupled nucleotide excision repair factor (TC-NER)-driven genome-instability in the pathogenesis of T cell acute lymphoblastic leukemia

BackgroundT-ALL is an aggressive hematological tumor that develops as the result of a multi-step oncogenic process which causes expansion of hematopoietic progenitors that are primed for T cell development to undergo malignant transformation and growth. Even though first-line therapy has a significant response rate, 40% of adult patients and 20% of pediatric patients will relapse. Therefore, there is an unmet need for treatment for relapsed/refractory T-ALL to develop potential targeted therapies. MethodsPediatric T-ALL patient derived T cells were grown under either nonskewingTh0 or Th1-skewing conditions to further process for ChIP-qPCR, RDIP-qPCR and other RT-PCR assays. Endogenous WASp was knocked out using CRISPR-Cas9 and was confirmed using flow cytometry and western blotting. LC-MS/MS was performed to find out proteomic dataset of WASp-interactors generated from Th1-skewed, human primary Th-cells. DNA-damage was assessed by immunofluorescence confocal-imaging and single-cell gel electrophoresis (comet assay). Overexpression of RNaseH1 was also done to restore normal Th1-transcription in WASp-deficient Th1-skewed cells. ResultsWe discovered that nuclear-WASp is required for suppressing R-loop production (RNA/DNA-hybrids) at Th1-network genes by ribonucleaseH2 (RNH2) and topoisomerase1. Nuclear-WASp is associated with the factors involved in preventing and dissolving R-loops in Th1 cells. In nuclear- WASp-reduced malignant Th1-cells, R-loops accumulate in vivo and are processed into DNA-breaks by transcription-coupled-nucleotide-excision repair (TC-NER). Several epigenetic modifications were also found to be involved at Th1 gene locus which are responsible for active/repressive marks of particular genes. By demonstrating WASp as a physiologic regulator of programmed versus unprogrammed R-loops, we suggest that the transcriptional role of WASp in vivo extends also to prevent transcription-linked DNA damage during malignancy and through modification of epigenetic dysregulations. ConclusionOur findings present a provocative possibility of resetting R-loops as a therapeutic intervention to correct both immune deficiency and malignancy in T-cell acute lymphoblastic leukemia patients and a novel role of WASp in the epigenetic regulation of T helper cell differentiation in T-ALL patients, anticipating WASp's requirement for the suppression of T-ALL progression.

Abstract 3019: Dissecting in vivo functions of R-Loops in Brca1-associated DNA replication stress and mammary tumorigenesis

Abstract Germ-line BRCA1 mutations are associated with a significantly increased incidence of breast cancer in women. Although BRCA1-associated breast tumors are basal-like and estrogen receptor α negative (ERα−), they originate from luminal progenitor cells. At the molecular level, BRCA1 is best known for its roles in resolution of DNA replication stress and promotion of homologous recombination (HR)-based double strand break (DSB) repair. In addition, BRCA1 is implicated in reduction of R-loops, a DNA-RNA hybrid structure and transcriptional byproduct associated with genome instability and transcriptional regulation. Our published findings suggest that BRCA1-dependent R-loop mitigation contributes to luminal cell-specific transcription and differentiation (Zhang et al, 2017, Nature Communications, 8:15908), which could in turn suppress Brca1-associated tumorigenesis. Using cell type-specific mouse genetics, we investigate a direct impact of R-loop removal on Brca1-associated mammary epithelial tumor formation. R-loop formation in mammary epithelial cells is efficiently reduced by over-expression of RNase H1, an R-loop-specific ribonuclease. R-loop removal does not affect mammary duct development or function. However, it drastically increases ɣH2AX foci in proliferating BRCA1-deficient epithelial cells without external DNA damage. This suggests a role of R-loops in attenuating elevated DNA replication stress caused by the loss of Brca1. While R-loop removal does not appear to affect the overall Brca1-associated mammary tumor incidence, it promotes cell differentiation from ERα− luminal progenitor cells to ERα+ mature luminal cells. Accordingly, unlike their counterparts, a significant percentage of Brca1-associated mammary tumors with RNase H1 overexpression tend to be ERα+. Thus, our findings provide in vivo evidence for a previously unappreciated role of R-loop dynamics on Brca1-associated DNA replication stress and tumor development in mammary epithelium. Citation Format: Huai-Chin Chiang, Leilei Qi, Yanfen Hu, Rong Li. Dissecting in vivo functions of R-Loops in Brca1-associated DNA replication stress and mammary tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3019.

WRNIP1 prevents transcription-associated genomic instability

R-loops are non-canonical DNA structures that form during transcription and play diverse roles in various physiological processes. Disruption of R-loop homeostasis can lead to genomic instability and replication impairment, contributing to several human diseases, including cancer. Although the molecular mechanisms that protect cells against such events are not fully understood, recent research has identified fork protection factors and DNA damage response proteins as regulators of R-loop dynamics. In this study, we identify the Werner helicase-interacting protein 1 (WRNIP1) as a novel factor that counteracts transcription-associated DNA damage upon replication perturbation. Loss of WRNIP1 leads to R-loop accumulation, resulting in collisions between the replisome and transcription machinery. We observe co-localization of WRNIP1 with transcription/replication complexes and R-loops after replication perturbation, suggesting its involvement in resolving transcription-replication conflicts. Moreover, WRNIP1-deficient cells show impaired replication restart from transcription-induced fork stalling. Notably, transcription inhibition and RNase H1 overexpression rescue all the defects caused by loss of WRNIP1. Importantly, our findings highlight the critical role of WRNIP1 ubiquitin-binding zinc finger (UBZ) domain in preventing pathological persistence of R-loops and limiting DNA damage, thereby safeguarding genome integrity.

WRNIP1 prevents transcription-associated genomic instability.

R-loops are non-canonical DNA structures that form during transcription and play diverse roles in various physiological processes. Disruption of R-loop homeostasis can lead to genomic instability and replication impairment, contributing to several human diseases, including cancer. Although the molecular mechanisms that protect cells against such events are not fully understood, recent research has identified fork protection factors and DNA damage response proteins as regulators of R-loop dynamics. In this study, we identify the Werner helicase-interacting protein 1 (WRNIP1) as a novel factor that counteracts transcription-associated DNA damage upon replication perturbation. Loss of WRNIP1 leads to R-loop accumulation, resulting in collisions between the replisome and transcription machinery. We observe co-localization of WRNIP1 with transcription/replication complexes and R-loops after replication perturbation, suggesting its involvement in resolving transcription-replication conflicts. Moreover, WRNIP1-deficient cells show impaired replication restart from transcription-induced fork stalling. Notably, transcription inhibition and RNase H1 overexpression rescue all the defects caused by loss of WRNIP1. Importantly, our findings highlight the critical role of WRNIP1 ubiquitin-binding zinc finger (UBZ) domain in preventing pathological persistence of R-loops and limiting DNA damage, thereby safeguarding genome integrity.

Genome Wide siRNA Screen Identifies CGAS-Sting Pathway As a Pharmacological Target That Promotes Survival of Hematopoietic Stem Cells Deficient in Fanconi Genes

Introduction Fanconi anemia (FA) is a homozygous recessive genetic disorder due to mutation in the Fanconi family of genes. The disease is characterized by early onset bone marrow failure and a predisposition to cancer. The FA genes are involved in DNA damage sensing and repair. Mutation in these genes leads to heightened DNA damage and extreme sensitivity to cross-linking agents. Hematopoietic stem cells of FA patients are defective in differentiation and self-renewal, leading to stem cell exhaustion and bone marrow failure early in life. The current treatment for bone marrow failure is allogeneic stem cell transplant from healthy donors. Due to difficulties in finding matching donors and the inherent problems associated with allogeneic transplants, pharmacological therapies that delay or prevent bone marrow failure are of clinical importance. Other proposed therapies including TGF-b, metformin, and resveratrol have met with limited clinical success. Methodology: To identify genes that promote survival of FA cells we performed a genome wide siRNA screen using isogenic pair of cell line with FANCD2 deletions and its corrected counterpart using transfection of siRNA libraries targeting 18,119 genes from Human Drug Targets, Human Druggable Genome, and Human Genome libraries at 2000 cells/well in 384-well plates followed by cell viability measurement after 72 hrs. We measured IRF3 phosphorylation and expression of immune related genes to analyze the CGAS-STING pathway. Using peripheral blood and bone marrow derived CD34+ cells transduced with lentivirus expressing shRNA against FANCD2 we tested the effect of CGAS inhibitor in promoting CFU-GM and CFU-E. Results: A genome wide siRNA screen identified IRF7 as one of the top 5 hits whose diminished expression resulted in increased viability of F-D2 mutant PD20 cells. IRF7 along with IRF3 serve as downstream transcription factors including various Toll-like receptors, CGAS-STING pathways, and other pattern recognition innate immune receptors. Using pIRF3 as an activation readout for CGAS-STING pathway we identified that both FANCD2 and FANCA mutant cells have elevated pIRF3 levels. The elevated pIRF3 levels were further increased upon addition of mitomycin C (MMC) in FA mutant cells as compared to gene corrected cells. The pIRF3 levels decreased upon both genetic KD and small molecule inhibition of both CGAS and STING. In line with increased pIRF3 FA mutant cells showed increased gene expression of various interferon-regulated genes, which were then also reduced by CGAS inhibitor. Overexpression of RNAseH1 partially reduced pIRF3 levels in FA mutant cells indicating that the RNA:DNA hybrids whose levels are elevated in FA mutant cells is one of the ligands of CGAS. Most importantly 10µM of CGAS inhibitor RU.51 was able to rescue FA-A mutant cell line 6914 cells from low dose MMC-induced cell death showing that the sensitivity of FA mutant cells to DNA damaging agents was at least partially driven by CGAS-STING pathway. We then tested if the protective effect of CGAS inhibitor extended to human CD34+ cells deficient in FA genes. 20µM of RU.51 was able to rescue the CFU-GM and CFU-E defects of CD34+ cells carrying lentiviral integrated shRNA against FANCD2 to a level to that in cells expressing non-silencing shRNAs. Interferon-regulated gene expression was also elevated in the FANCD2-deficient CD34+ cells, which was reversed by treatment with cGAS inhibitor. Conclusions: It has previously been shown that FA patients have elevated TNF-alpha, IFN-gamma levels, and NFkbactivation, and interferon alpha administration hastens the onset of bone marrow failure in FA patients. Our results show that FA mutant cells have elevated CGAS-STING signaling resulting in heightened interferon gene expression. Elevated R-loop levels in FA cells act as trigger for the CGAS-STING pathway. CGAS inhibitor is able to rescue low dose MMC-induced cell death in FA mutant fibroblasts and also restores the CFU-GM and CFU-E forming ability of CD34+ cells. These results indicate that CGAS-STING pathway is a pharmacologically tractable target to reverse the hematopoietic stem cell exhaustion seen in FA patients.

P53-dependent R-loop formation and HPV pathogenesis

R-loops are trimeric RNA: DNA hybrids that are important physiological regulators of transcription; however, their aberrant formation or turnover leads to genomic instability and DNA breaks. High-risk human papillomaviruses (HPV) are the causative agents of genital as well as oropharyngeal cancers and exhibit enhanced amounts of DNA breaks. The levels of R-loops were found to be increased up to 50-fold in cells that maintain high-risk HPV genomes and were readily detected in squamous cell cervical carcinomas in vivo but not in normal cells. The high levels of R-loops in HPV-positive cells were present on both viral and cellular sites together with RNase H1, an enzyme that controls their resolution. Depletion of RNase H1 in HPV-positive cells further increased R-loop levels, resulting in impaired viral transcription and replication along with reduced expression of the DNA repair genes such as FANCD2 and ATR, both of which are necessary for viral functions. Overexpression of RNase H1 decreased total R-loop levels, resulting in a reduction of DNA breaks by over 50%. Furthermore, increased RNase H1 expression blocked viral transcription and replication while enhancing the expression of factors in the innate immune regulatory pathway. This suggests that maintaining elevated R-loop levels is important for the HPV life cycle. The E6 viral oncoprotein was found to be responsible for inducing high levels of R-loops by inhibiting p53's transcriptional activity. Our studies indicate that high R-loop levels are critical for HPV pathogenesis and that this depends on suppressing the p53 pathway.

Abstract 6183: PARP inhibitors preferentially sensitize splicing factor mutant myeloid neoplasms

Abstract Somatic heterozygous mutations in genes encoding for RNA splicing factors (SF) SRSF2, U2AF1, and SF3B1 are frequently mutated in patients with hematologic malignancies, representing a unique genetic vulnerability for targeted therapy. In the current study, we performed a focused drug screen with inhibitors targeting different DNA damage response and DNA metabolic pathways to identify novel therapeutic vulnerabilities generated by SF mutations. We generated a murine leukemia model by overexpressing the MLL-AF9 fusion oncogene on an Srsf2P95H/+ background, a mutational combination that is found in ~10% of MLL-rearranged leukemias. We surprisingly found that MLL-AF9 Srsf2P95H/+ mutant leukemias are sensitive to inhibitors targeting ADP-ribosyltransferases (PARP). PARP inhibitor sensitivity was also observed in isogenic murine MLL-AF9 U2af1s34/+ cells compared to MLL-AF9 U2af1+/+ cells. Second, murine Srsf2P95H leukemias showed improved prolonged survival when treated with olaparib (PARPi) compared to vehicle treatment in vivo. Third, human primary AML patient samples that harbor SF mutations are sensitive to PARPi compared to non-SF mutant samples. These data highlight that both SRSF2P95H and U2AF1S34F mutations create a common vulnerability that is dependent on PARP activity for survival. To evaluate PARP activity, we used isogenic K562 leukemia cells expressing SRSF2P95H and U2AF1S34F mutations from their endogenous loci and monitored ADP-ribosylation (ADPr) levels, a marker of PARP activity. Both SRSF2P95H and U2AF1S34F cells exhibited elevated levels of ADPr compared to wildtype cells in a PARP1- dependent manner. PARPi preferentially induced DNA damage and cell death in SF mutant cells. Surprisingly, we found that SRSF2P95H and U2AF1S34F cells are not defective in homologous recombination repair. Instead, the increased PARP1-mediated ADPr in SF-mutant cells is caused by accumulated R loops, a group of transcription intermediates containing RNA:DNA hybrids and displaced single-stranded DNA. To determine whether PARPi sensitivity is due to R-loop accumulation, we overexpressed RNase H1, an enzyme that specifically cleaves the RNA moiety within RNA:DNA hybrids in U2AF1S34F cells. Overexpression of RNase H1 significantly reduced ADPr levels and suppressed the PARPi-induced U2AF1S34F cell growth inhibition. Collectively, these results suggest that spliceosome mutants induce R-loop accumulation and elicit an R-loop-associated PARP1 response to promote cell survival. In summary, our data establish a previously unknown link between R-loop-induced PARP1 response and RNA splicing perturbation and provide a mechanistic rationale to evaluate the clinical efficacy of PARP inhibitors in spliceosome-mutant malignancies. Furthermore, our study highlights a new therapeutic potential of targeting the R-loop tolerance pathways caused by different spliceosome gene mutations. Citation Format: Dang Hai Nguyen, Sayantani Sinha, Zhiyan Silvia Liu, Maxwell Henry Bannister, Erica Arriaga-Gomez, Axia Song, Dawei Zong, Martina Sarchi, Victor Corral, Wannasiri Chiraphapphaiboon, Jennifer Yoo, Matthew McMahon, Cassandra Leibson, Derek L. Stirewalt, H Joachim Deeg, Sumit Rai, Matthew Walter, Timothy A. Graubert, Sergei Doulatov, Stanley C. Lee. PARP inhibitors preferentially sensitize splicing factor mutant myeloid neoplasms. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6183.

Spontaneous deamination of cytosine to uracil is biased to the non-transcribed DNA strand in yeast

Transcription in Saccharomyces cerevisiae is associated with elevated mutation and this partially reflects enhanced damage of the corresponding DNA. Spontaneous deamination of cytosine to uracil leads to CG>TA mutations that provide a strand-specific read-out of damage in strains that lack the ability to remove uracil from DNA. Using the CAN1 forward mutation reporter, we found that C>T and G>A mutations, which reflect deamination of the non-transcribed and transcribed DNA strands, respectively, occurred at similar rates under low-transcription conditions. By contrast, the rate of C>T mutations was 3-fold higher than G>A mutations under high-transcription conditions, demonstrating biased deamination of the non-transcribed strand (NTS). The NTS is transiently single-stranded within the ∼15 bp transcription bubble, or a more extensive region of the NTS can be exposed as part of an R-loop that can form behind RNA polymerase. Neither the deletion of genes whose products restrain R-loop formation nor the over-expression of RNase H1, which degrades R-loops, reduced the biased deamination of the NTS, and no transcription-associated R-loop formation at CAN1 was detected. These results suggest that the NTS within the transcription bubble is a target for spontaneous deamination and likely other types of DNA damage.

The RNA export and RNA decay complexes THO and TRAMP prevent transcription-replication conflicts, DNA breaks, and CAG repeat contractions.

Expansion of structure-forming CAG/CTG repetitive sequences is the cause of several neurodegenerative disorders and deletion of repeats is a potential therapeutic strategy. Transcription-associated mechanisms are known to cause CAG repeat instability. In this study, we discovered that Thp2, an RNA export factor and member of the THO (suppressors of transcriptional defects of hpr1Δ by overexpression) complex, and Trf4, a key component of the TRAMP (Trf4/5-Air1/2-Mtr4 polyadenylation) complex involved in nuclear RNA polyadenylation and degradation, are necessary to prevent CAG fragility and repeat contractions in a Saccharomyces cerevisiae model system. Depletion of both Thp2 and Trf4 proteins causes a highly synergistic increase in CAG repeat fragility, indicating a complementary role of the THO and TRAMP complexes in preventing genome instability. Loss of either Thp2 or Trf4 causes an increase in RNA polymerase stalling at the CAG repeats and other genomic loci, as well as genome-wide transcription-replication conflicts (TRCs), implicating TRCs as a cause of CAG fragility and instability in their absence. Analysis of the effect of RNase H1 overexpression on CAG fragility, RNAPII stalling, and TRCs suggests that RNAPII stalling with associated R-loops are the main cause of CAG fragility in the thp2Δ mutants. In contrast, CAG fragility and TRCs in the trf4Δ mutant can be compensated for by RPA overexpression, suggesting that excess unprocessed RNA in TRAMP4 mutants leads to reduced RPA availability and high levels of TRCs. Our results show the importance of RNA surveillance pathways in preventing RNAPII stalling, TRCs, and DNA breaks, and show that RNA export and RNA decay factors work collaboratively to maintain genome stability.

Topoisomerase 1-dependent R-loop deficiency drives accelerated replication and genomic instability.

SummaryDNA replication is a complex process tightly regulated to ensure faithful genome duplication, and its perturbation leads to DNA damage and genomic instability. Replication stress is commonly associated with slow and stalled replication forks. Recently, accelerated replication has emerged as a non-canonical form of replication stress. However, the molecular basis underlying fork acceleration is largely unknown. Here, we show that mutated HRAS activation leads to increased topoisomerase 1 (TOP1) expression, causing aberrant replication fork acceleration and DNA damage by decreasing RNA-DNA hybrids or R-loops. In these cells, restoration of TOP1 expression or mild replication inhibition rescues the perturbed replication and reduces DNA damage. Furthermore, TOP1 or RNaseH1 overexpression induces accelerated replication and DNA damage, highlighting the importance of TOP1 equilibrium in regulating R-loop homeostasis to ensure faithful DNA replication and genome integrity. Altogether, our results dissect a mechanism of oncogene-induced DNA damage by aberrant replication fork acceleration.

Mitotic DNA synthesis is caused by transcription-replication conflicts in BRCA2-deficient cells

Aberrant replication causes cells lacking BRCA2 to enter mitosis with under-replicated DNA, which activates a repair mechanism known as mitotic DNA synthesis (MiDAS). Here, we identify genome-wide the sites where MiDAS reactions occur when BRCA2 is abrogated. High-resolution profiling revealed that these sites are different from MiDAS at aphidicolin-induced common fragile sites in that they map to genomic regions replicating in the early S-phase, which are close to early-firing replication origins, are highly transcribed, and display R-loop-forming potential. Both transcription inhibition in early S-phase and RNaseH1 overexpression reduced MiDAS in BRCA2-deficient cells, indicating that transcription-replication conflicts (TRCs) and R-loops are the source of MiDAS. Importantly, the MiDAS sites identified in BRCA2-deficient cells also represent hotspots for genomic rearrangements in BRCA2-mutated breast tumors. Thus, our work provides a mechanism for how tumor-predisposing BRCA2 inactivation links transcription-induced DNA damage with mitotic DNA repair to fuel the genomic instability characteristic of cancer cells.

Topoisomerase I poison-triggered immune gene activation is markedly reduced in human small-cell lung cancers by impairment of the cGAS/STING pathway

BackgroundCurrent immunotherapy strategies have contrasting clinical results in human lung cancer patients as small-cell lung cancers (SCLC) often show features of immunological cold tumours. Topoisomerase 1 (TOP1) poisons are effective antitumor drugs with good efficacy against lung cancers.MethodsWe used molecular, genetic and bioinformatic approaches to determine the mechanism of micronuclei formation induced by two TOP1 poisons in different human cancer cells, including SCLC cell lines.ResultsTOP1 poisons stimulate similar levels of micronuclei in all tested cell lines but downstream effects can vary markedly. TOP1 poisons increase micronuclei levels with a mechanism involving R-loops as overexpression of RNaseH1 markedly reduces or abolishes both H2AX phosphorylation and micronuclei formation. TOP1 poison-induced micronuclei activate the cGAS/STING pathway leading to increased expression of immune genes in HeLa cells, but not in human SCLC cell lines, mainly due to lack of STING and/or cGAS expression. Moreover, the expression of STING and antigen-presenting machinery genes is generally downregulated in patient tumours of human lung cancer datasets.ConclusionsAltogether, our data reveal an immune signalling mechanism activated by TOP1 poisons, which is often impaired in human SCLC tumours.

DDX18 prevents R-loop-induced DNA damage and genome instability via PARP-1.

R loops occur frequently in genomes and contribute to fundamental biological processes at multiple levels. Consequently, understanding the molecular and cellular biology of R loops has become an emerging area of research. Here, it is shown that poly(ADP-ribose) polymerase-1 (PARP-1) can mediate the association of DDX18, a putative RNA helicase, with R loops thereby modulating R-loop homeostasis in endogenous R-loop-prone and DNA lesion regions. DDX18 depletion results in aberrant endogenous R-loop accumulation, which leads to DNA-replication defects. In addition, DDX18 depletion renders cells more sensitive to DNA-damaging agents and reduces RPA32 and RAD51 foci formation in response to irradiation. Notably, DDX18 depletion leads to γH2AX accumulation and genome instability, and RNase H1 overexpression rescues all the DNA-repair defects caused by DDX18 depletion. Taken together, these studies uncover a function of DDX18 in R-loop-mediated events and suggest a role for PARP-1 in mediating the binding of specific DDX-family proteins with R loops in cells.

RNase H1, the Gold Standard for R-Loop Detection.

RNase H1 has become an essential tool to uncover the physiological and pathological roles of R-loops, three-stranded structures consisting of and RNA-DNA hybrid opposite to a single DNA strand (ssDNA). RNase H1 degrades the RNA portion of the R-loops returning the two DNA strands to double-stranded form (dsDNA). Overexpression of RNase H1 in different systems has helped to address the questions of where R-loops are located, their abundance, and mechanisms of formation, stability, and degradation. In this chapter we review multiple studies that used RNase H1 as an instrument to investigate R-loops multiple functions and their relevance in health and diseases.

RNA-DNA hybrids regulate meiotic recombination.

RNA-DNA hybrids are often associated with genome instability and also function as a cellular regulator in many biological processes. In this study, we show that accumulated RNA-DNA hybrids cause multiple defects in budding yeast meiosis, including decreased sporulation efficiency and spore viability. Further analysis shows that these RNA-DNA hybrid foci colocalize with RPA/Rad51 foci on chromosomes. The efficient formation of RNA-DNA hybrid foci depends on Rad52 and ssDNA ends of meiotic DNA double-strand breaks (DSBs), and their number is correlated with DSB frequency. Interestingly, RNA-DNA hybrid foci and recombination foci show similar dynamics. The excessive accumulation of RNA-DNA hybrids around DSBs competes with Rad51/Dmc1, impairs homolog bias, and decreases crossover and noncrossover recombination. Furthermore, precocious removal of RNA-DNA hybrids by RNase H1 overexpression also impairs meiotic recombination similarly. Taken together, our results demonstrate that RNA-DNA hybrids form at ssDNA ends of DSBs to actively regulate meiotic recombination.

R-Loop Associated Mitotic Stress Confers Vulnerabilities in Splicing Factor Mutant Leukemia

The RNA splicing factor SF3B1 is one of the most frequently mutated genes in chronic lymphocytic leukemia (CLL). Co-expression of mutant Sf3b1 and Atm deletionin murine B cellsresults in CLL development, in which CLL cells exhibit RNA splicing dysregulation, increased DNA damage and recurrent chromosomal amplification. These findings suggest a strong link between splicing factormutation, chromosomal instability (CIN), and oncogenesis, although the mechanism has yet to be elucidated.CIN in cells with defective RNA processing is triggered, at least in part, by excessive R-loop (RNA:DNA hybrid with displaced single strand DNA). Using dotblot assay, we observed R-loop augmentation in SF3B1 mutant (MT) compared to wild-type (WT) cells in both human cell lines and murine CLL cells. Along with R-loop accumulation, SF3B1 MT increased DNA double strand breaks (DSB) (measured by comet assay) and mis-segregated chromosomes during mitosis (quantified by immunofluorescence using anti-phospho-histone H3 and anti-αtubulin antibodies). Overexpression of R-loop resolving enzyme RNaseH1 in SF3B1 MT cell lines reduced DSB (t-test p-value <0.0001) and mitotic stress (t-test p-value <0.0001), confirming the causative role of R-loop in CIN.To elucidate the mechanisms underlying SF3B1 MT-associated CIN, we used DNA:RNA hybrid immunoprecipitation (DRIP) coupled with sequencing to map R-loop in SF3B1 MT and WT Nalm6 cells. Although SF3B1 MT caused minimal changes in overall R-loop distribution, it led to increased R-loop formation at centromeric regions. Proper centromeric R-loop (cen-R-loop) formation is essential for accurate chromosome segregation. Those R-loop are coated by phosphorylated replication protein A (p-RPA). p-RPA and centromere immunofluorescence (IF) co-staining revealed increased cen-R-loop in SF3B1 MT cells. Intriguingly, overexpression of RNaseH1 reduced both levels of centromeric p-RPA and percentage of cells with defective mitosis, suggesting SF3B1 MT associated cen-R-loop impacts CIN.To further discern the molecular underpinnings of R-loop mediated CIN, we investigated the effect of their accumulation on spindle geometry in metaphase cells. By analyzing the total body of chromosomes two-dimensional area, we observed one fold greater magnitude of chromosome oscillations in SF3B1 Nalm6 MT cells. Accordingly, compared to WT cells, SF3B1 MT cells displayed longer and wider spindles to accommodate an aneuploid chromosomal content. Removal of R-loops rescued mitotic spindle architecture and CIN in SF3B1 MT cells, highlighting a direct contribution of R-loop to kinetochore-microtubule organization and stability.As there is no evidence of strong binding of SF3B1 to centromeres, we tested the possibility that SF3B1 MT contributes to cen-R-loop formation through alternative splice variants. Through overlapping conserved SF3B1 MT-associated splice variants across isogenic cell lines and murine model with available R-loop interactome data, we identified SERBP1, which encodes an RNA-binding protein, as a target candidate . SF3B1 MT induces loss-of-function in SERBP1 through alternative splicing. Knockdown of SERBP1 triggered R-loop accumulation, increased centromeric p-RPA coating, and aberrant mitosis, which recapitulated the SF3B1 MT phenotype. Importantly, overexpression of SERBP1 WT alleviated both DSB and R-loop accumulation in SF3B1 MT cells. These observations strongly suggest that SF3B1 MT augments R-loop formation through dysregulated splicing of SERBP1 and possibly other genes involved in R-loop metabolism.We also investigated the possibility that the impact of RNA splicing defect on R-loop biology is a generalizable phenomenon. Examing a panel of leukemia-associated splicing factors (U2AF1, SRSF2, ZRSR2, RNU1), we found that these mutations all display R-loop dependent mitotic stress. Thus, mitotic stress caused by aberrant R-loop formation could be a convergent mechanism in splicing factor mutated leukemia cells.Altogether, we demonstrated that SF3B1 mutation promotes CIN by destabilizing mitotic spindles through aberrant R-loop accumulation at the centromere, via dysregulated splicing in genes such as SERBP1. Our study highlights an unrecognized role of cen-R-loop as a critical link between RNA splicing dysregulation and CIN, providing an opportunity for therapeutic targeting of R-loop in splicing factor mutant leukemias. DisclosuresWu: Pharmacyclics: Research Funding; BioNTech: Current equity holder in publicly-traded company.

Spliceosome Mutant Myeloid Malignancies Are Preferentially Sensitive to PARP Inhibition

▪Somatic heterozygous mutations in spliceosome genes SRSF2, U2AF1, and SF3B1 commonly occur in patients with myeloid malignancies such as myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). Moreover, SRSF2 and U2AF1 mutations associate with poor survival and high risk of progression to AML, representing a unique genetic vulnerability for targeted therapy. We and others previously found that R-loops, a group of transcription intermediates containing RNA:DNA hybrids and displaced single-stranded DNA, are a source of genomic instability induced by different spliceosome mutants. We further showed that inhibition of ATR kinase activity preferentially kills spliceosome mutant cells in an R-loop-dependent manner. Inspired by ATR inhibition results, we performed a focused drug screen with inhibitors targeting additional DNA damage response pathways to identify novel therapeutic vulnerabilities generated by spliceosome mutations. We generated a murine leukemia model by overexpressing the MLL-AF9 fusion oncogene on an Srsf2 P95H/+background, a mutational combination that is found in ~10% of MLL-rearranged leukemias. Surprisingly, we found that Srsf2 P95H/+cells are more sensitive to five inhibitors targeting ADP-ribosyltransferases or PARP (olaparib, talazoparib, rucaparib, niraparib, veliparib) (Figs 1A-B). Olaparib (PARPi)-treated Srsf2 P95H/+cells exhibited increased apoptosis compared to Srsf2 +/+ cells as determined by AnnexinV (Fig 1C). PARPi sensitivity was also observed in isogenic murine MLL-AF9 U2af1 S34F/+cells compared to MLL-AF9 U2af1 +/+ cells (Fig 1D). These data highlight that both SRSF2 P95H and U2AF1 S34F mutations create a common vulnerability that is dependent on PARP activity for survival.To evaluate PARP activity in cells, we used isogenic K562 leukemia cells expressing SRSF2 P95H and U2AF1 S34F mutations from their endogenous loci and monitored PAR (poly(ADP-ribose)) chain levels, a marker of PARP activity. Both SRSF2 P95H and U2AF1 S34F cells exhibited elevated PAR levels compared to wildtype cells (Figs 1E-F). PARPi treatment significantly suppressed PAR signals in SRSF2 P95H and U2AF1 S34F cells. PARP inhibitors target both PARP1 and PARP2 enzymes, of which PARP1 plays a key role in DNA damage response. We used CRISPR-Cas9 to knockout PARP1 gene to determine the major PARP responsible for elevated PAR level in these leukemia cells. PARP1 deletion abrogated elevated PAR levels in U2AF1 S34F (Fig 1G) and SRSF2 P95H cells (data not shown). Altogether, we demonstrated that SRSF2 P95H and U2AF1 S34F cells trigger a PARP1 response critical for cell survival.To test whether increased PAR level arises from U2AF1 S34F-induced R-loops, we generated U2AF1 S34F cells that inducibly express RNaseH1, an enzyme that specifically cleaves the RNA moiety within RNA:DNA hybrids. Induction of RNaseH1 in U2AF1 S34F cells significantly reduced PAR levels, showing that U2AF1 S34F-induced PAR chains is R-loop-dependent (Fig 1H). Moreover, RNaseH1 overexpression suppressed the growth inhibition of PARPi-treated U2AF1 S34F cells (Fig 1I). Collectively, these results suggest that U2AF1 S34F mutants induce R-loop accumulation and elicit an R-loop-associated PARP1 signaling to promote cell survival.We next tested whether combining ATR inhibitor (ATRi) can further exacerbate PARPi sensitivity in spliceosome mutant cells. To examine ATR activity, we monitored phosphorylated RPA (Replication Protein A, or pRPA), a known ATR substrate. pRPA level was enhanced in PARPi-treated SRSF2 P95H cells compared to PARPi-treated SRSF2 WT cells but was suppressed when treated with ATRi (Fig 1J), suggesting that splicing factor mutant cells are more reliant on ATR function in the context of PARPi. Importantly, the combination of PARPi with ATRi, but not with ATMi, significantly promoted cell growth inhibition in SRSF2 P95H cells compared to SRSF2 WT cells or to SRSF2 P95H cells treated with individual compounds alone (Fig 1K).Collectively, these data provide a pre-clinical rationale that splicing factor mutant leukemias are preferentially sensitive to PARP1 modulation compared to their wildtype counterpart. Moreover, combining PARPi and ATRi may further sensitize spliceosome mutant cells and could represent a new therapeutic strategy in myeloid leukemia patients harboring these mutations (Fig 1L). [Display omitted] DisclosuresGraubert: Calico: Research Funding; Janssen: Research Funding; astrazeneca: Research Funding.

Nonsense-Mediated RNA Decay Is a Unique Vulnerability of Cancer Cells Harboring SF3B1 or U2AF1 Mutations.

Nonsense-mediated RNA decay (NMD) is recognized as an RNA surveillance pathway that targets aberrant mRNAs with premature translation termination codons (PTC) for degradation, however, its molecular mechanisms and roles in health and disease remain incompletely understood. In this study, we developed a novel reporter system to accurately measure NMD activity in individual cells. A genome-wide CRISPR-Cas9 knockout screen using this reporter system identified novel NMD-promoting factors, including multiple components of the SF3B complex and other U2 spliceosome factors. Interestingly, cells with mutations in the spliceosome genes SF3B1 and U2AF1, which are commonly found in myelodysplastic syndrome (MDS) and cancers, have overall attenuated NMD activity. Compared with wild-type (WT) cells, SF3B1- and U2AF1-mutant cells were more sensitive to NMD inhibition, a phenotype that is accompanied by elevated DNA replication obstruction, DNA damage, and chromosomal instability. Remarkably, the sensitivity of spliceosome mutant cells to NMD inhibition was rescued by overexpression of RNase H1, which removes R-loops in the genome. Together, these findings shed new light on the functional interplay between NMD and RNA splicing and suggest a novel synthetic lethal strategy for the treatment of MDS and cancers with spliceosome mutations. SIGNIFICANCE: This study has developed a novel NMD reporter system and identified a potential therapeutic approach of targeting the NMD pathway to treat cancer with spliceosome gene mutations.

The S100A9/Fto Axis Induces Formation of RNA:DNA Hybrids That Serve As Damps Initiating Myeloid Skewing & Genetic Injury in MDS

We previously reported that S100A9 activates the NLRP3 inflammasome to promote pyroptosis, ineffective hematopoiesis and the development of MDS in a feed forward fashion. The critical role of S100A8/9 was recently confirmed by others, demonstrating its importance as a key pathogenic driver of disease. Nonetheless, the precise mechanism by which S100A9 may foster DNA damage in MDS is unclear. We recently showed that S100A9 directs overexpression of the fat-mass and obesity-associated gene (FTO) encoding an m6A RNA demethylase, which leads to nuclear exclusion of SRSF2. Removal of SRSF2 from its functional domain in the nucleosome leads to stalling of RNA polymerase II and formation of the nucleic acid R-loops, comprising DNA:RNA hybrids and the associated non-template single-stranded DNA. S100A9/FTO axis activation leads to SRSF2 deregulation through suppression of its main nuclear transport protein RanBP2, thereby stalling transcription with resulting accumulation of nuclear R-loops and cytosolic/extracellular RNA:DNA hybrids. Persistent R-loops induce DNA damage while also compromising DNA repair. Here we identify an S100A9/FTO-regulated pathway responsible for induction of genomic instability through the accumulation of cytoplasmic RNA:DNA hybrids. While accumulation of these damage associated molecular pattern (DAMP) molecules coincides with the formation of double stranded breaks (DSB), their precise role in the age-induced inflammatory process initiated by S100A9 in MDS pathogenesis remains unknown. We first investigated which components of the S100A9/FTO axis are critical to hematopoiesis and those that are important for both the development of RNA:DNA hybrids and γH2AX activation. We analyzed the contribution of RanBP2 and the effects of elimination of R-loop formation via overexpression of RNAse H1, an enzyme that removes stalled R-loops by degrading DNA-hybridized RNA, thereby reducing accumulation of cytoplasmic RNA:DNA hybrids. CRISPR knock-down of RanBP2 showed that the protein is critical for accumulation of DSB as evidenced by upregulation of γH2AX foci and RNA:DNA hybrid accumulation. Importantly, overexpression of RNAse H1 degraded R-loops and restored colony-forming capacity, indicating that RNA:DNA hybrids induced by S100A9/FTO have profound effects on hematopoietic potential. We hypothesized that the RNA:DNA hybrid DAMPs have an important role in the pathogenesis of MDS through engagement and activation of its two principal receptors, TLR9 and NLRP3. Both are upregulated in MDS, while the NLRP3 inflammasome plays a critical role in MDS pathobiology through the induction of pyroptosis, self-renewal and cell swelling (Basiorka S, et. al. Blood 2016). CRISPR knock-downof each receptor in MDS bone marrow (BM) mononuclear cells (MNC) had distinct effects: (1) proportional reduction in γH2AX with TLR9 knock-down, and (2) reduction in RNA:DNA hybrids with NLRP3 suppression. Using synthetic RNA:DNA hybrids, transfection (to simulate internal DAMPs) of healthy BM MNC or passive uptake (to simulate their extracellular effect) induced myeloid skewing and reduced colony formation indicating that S100A9/FTO-induced RNA:DNA hybrids are critical intracellular DAMPs that contribute to disease pathobiology. These findings were validated by flow cytometric analysis showing that exposure to synthetic RNA:DNA hybrids directly induced the accumulation of γH2AX in normal hematopoietic stem cells. We conclude that S100A9/FTO-induced RNA:DNA-hybrids lead to genomic instability, representing a previously uncharacterized mechanism contributing to MDS pathogenesis. Our studies provide evidence that targeting this cascade offers significant potential for development of novel, biologically rational therapeutics for MDS. DisclosuresSallman:Celgene: Research Funding. Padron:Incyte: Honoraria, Research Funding.