Unprocessed genomic uracil as a source of DNA replication stress in cancer cells.

Alterations of bases in DNA constitute a major source of genomic instability. It is believed that base alterations trigger base excision repair (BER), generating DNA repair intermediates interfering with DNA replication. Here, we show that genomic uracil, a common type of base alteration, induces DNA replication stress (RS) without being processed by BER. In the absence of uracil DNA glycosylase (UNG), genomic uracil accumulates to high levels, DNA replication forks slow down, and PrimPol-mediated repriming is enhanced, generating single-stranded gaps in nascent DNA. ATR inhibition in UNG-deficient cells blocks the repair of uracil-induced gaps, increasing replication fork collapse and cell death. Notably, a subset of cancer cells upregulates UNG2 to suppress genomic uracil and limit RS, and these cancer cells are hypersensitive to co-treatment with ATR inhibitors and drugs increasing genomic uracil. These results reveal unprocessed genomic uracil as an unexpected source of RS and a targetable vulnerability of cancer cells.

APOBEC3A induces DNA gaps through PRIMPOL and confers gap-associated therapeutic vulnerability.

Mutation signatures associated with apolipoprotein B mRNA editing catalytic polypeptide-like 3A/B (APOBEC3A/B) cytidine deaminases are prevalent across cancers, implying their roles as mutagenic drivers during tumorigenesis and tumor evolution. APOBEC3A (A3A) expression induces DNA replication stress and increases the cellular dependency on the ataxia telangiectasia and Rad3-related (ATR) kinase for survival. Nonetheless, how A3A induces DNA replication stress remains unclear. We show that A3A induces replication stress without slowing replication forks. We find that A3A induces single-stranded DNA (ssDNA) gaps through PrimPol-mediated repriming. A3A-induced ssDNA gaps are repaired by multiple pathways involving ATR, RAD51, and translesion synthesis. Both ATR inhibition and trapping of poly(ADP-ribose) polymerase (PARP) on DNA by PARP inhibitor impair the repair of A3A-induced gaps, preferentially killing A3A-expressing cells. When used in combination, PARP and ATR inhibitors selectively kill A3A-expressing cells synergistically in a manner dependent on PrimPol-generated gaps. Thus, A3A-induced replication stress arises from PrimPol-generated ssDNA gaps, which confer a therapeutic vulnerability to gap-targeted DNA repair inhibitors.

ATR inhibition induces synthetic lethality in mismatch repair-deficient cells and augments immunotherapy.

The mismatch repair (MMR) deficiency of cancer cells drives mutagenesis and offers a useful biomarker for immunotherapy. However, many MMR-deficient (MMR-d) tumors do not respond to immunotherapy, highlighting the need for alternative approaches to target MMR-d cancer cells. Here, we show that inhibition of the ATR kinase preferentially kills MMR-d cancer cells. Mechanistically, ATR inhibitor (ATRi) imposes synthetic lethality on MMR-d cells by inducing DNA damage in a replication- and MUS81 nuclease-dependent manner. The DNA damage induced by ATRi is colocalized with both MSH2 and PCNA, suggesting that it arises from DNA structures recognized by MMR proteins during replication. In syngeneic mouse models, ATRi effectively reduces the growth of MMR-d tumors. Interestingly, the antitumor effects of ATRi are partially due to CD8+ T cells. In MMR-d cells, ATRi stimulates the accumulation of nascent DNA fragments in the cytoplasm, activating the cGAS-mediated interferon response. The combination of ATRi and anti-PD-1 antibody reduces the growth of MMR-d tumors more efficiently than ATRi or anti-PD-1 alone, showing the ability of ATRi to augment the immunotherapy of MMR-d tumors. Thus, ATRi selectively targets MMR-d tumor cells by inducing synthetic lethality and enhancing antitumor immunity, providing a promising strategy to complement and augment MMR deficiency-guided immunotherapy.

The RNA m5C modification in R-loops as an off switch of Alt-NHEJ.

The roles of R-loops and RNA modifications in homologous recombination (HR) and other DNA double-stranded break (DSB) repair pathways remain poorly understood. Here, we find that DNA damage-induced RNA methyl-5-cytosine (m5C) modification in R-loops plays a crucial role to regulate PARP1-mediated poly ADP-ribosylation (PARylation) and the choice of DSB repair pathways at sites of R-loops. Through bisulfite sequencing, we discover that the methyltransferase TRDMT1 preferentially generates m5C after DNA damage in R-loops across the genome. In the absence of m5C, R-loops activate PARP1-mediated PARylation both in vitro and in cells. Concurrently, m5C promotes transcription-coupled HR (TC-HR) while suppressing PARP1-dependent alternative non-homologous end joining (Alt-NHEJ), favoring TC-HR over Alt-NHEJ in transcribed regions as the preferred repair pathway. Importantly, simultaneous disruption of both TC-HR and Alt-NHEJ with TRDMT1 and PARP or Polymerase θ inhibitors prevents alternative DSB repair and exhibits synergistic cytotoxic effects on cancer cells, suggesting an effective strategy to exploit genomic instability in cancer therapy.

RNF8 ubiquitylation of XRN2 facilitates R-loop resolution and restrains genomic instability in BRCA1 mutant cells.

Breast cancer linked with BRCA1/2 mutations commonly recur and resist current therapies, including PARP inhibitors. Given the lack of effective targeted therapies for BRCA1-mutant cancers, we sought to identify novel targets to selectively kill these cancers. Here, we report that loss of RNF8 significantly protects Brca1-mutant mice against mammary tumorigenesis. RNF8 deficiency in human BRCA1-mutant breast cancer cells was found to promote R-loop accumulation and replication fork instability, leading to increased DNA damage, senescence, and synthetic lethality. Mechanistically, RNF8 interacts with XRN2, which is crucial for transcription termination and R-loop resolution. We report that RNF8 ubiquitylates XRN2 to facilitate its recruitment to R-loop-prone genomic loci and that RNF8 deficiency in BRCA1-mutant breast cancer cells decreases XRN2 occupancy at R-loop-prone sites, thereby promoting R-loop accumulation, transcription-replication collisions, excessive genomic instability, and cancer cell death. Collectively, our work identifies a synthetic lethal interaction between RNF8 and BRCA1, which is mediated by a pathological accumulation of R-loops.

ATR protects ongoing and newly assembled DNA replication forks through distinct mechanisms.

The ATR kinase safeguards genomic integrity during S phase, but how ATR protects DNA replication forks remains incompletely understood. Here, we combine four distinct assays to analyze ATR functions at ongoing and newly assembled replication forks upon replication inhibition by hydroxyurea. At ongoing forks, ATR inhibitor (ATRi) increases MRE11- and EXO1-mediated nascent DNA degradation from PrimPol-generated, single-stranded DNA (ssDNA) gaps. ATRi also exposes template ssDNA through fork uncoupling and nascent DNA degradation. Electron microscopy reveals that ATRi reduces reversed forks by increasing gap-dependent nascent DNA degradation. At new forks, ATRi triggers MRE11- and CtIP-initiated template DNA degradation by EXO1, exposing nascent ssDNA. Upon PARP inhibition, ATRi preferentially exacerbates gap-dependent nascent DNA degradation at ongoing forks in BRCA1/2-deficient cells and disrupts the restored gap protection in BRCA1-deficient, PARP-inhibitor-resistant cells. Thus, ATR protects ongoing and new forks through distinct mechanisms, providing an extended view of ATR's functions in stabilizing replication forks.

Excessive transcription-replication conflicts are a vulnerability of BRCA1-mutant cancers.

BRCA1 mutations are associated with increased breast and ovarian cancer risk. BRCA1-mutant tumors are high-grade, recurrent, and often become resistant to standard therapies. Herein, we performed a targeted CRISPR-Cas9 screen and identified MEPCE, a methylphosphate capping enzyme, as a synthetic lethal interactor of BRCA1. Mechanistically, we demonstrate that depletion of MEPCE in a BRCA1-deficient setting led to dysregulated RNA polymerase II (RNAPII) promoter-proximal pausing, R-loop accumulation, and replication stress, contributing to transcription-replication collisions. These collisions compromise genomic integrity resulting in loss of viability of BRCA1-deficient cells. We also extend these findings to another RNAPII-regulating factor, PAF1. This study identifies a new class of synthetic lethal partners of BRCA1 that exploit the RNAPII pausing regulation and highlight the untapped potential of transcription-replication collision-inducing factors as unique potential therapeutic targets for treating cancers associated with BRCA1 mutations.

The metabolic enzyme hexokinase 2 localizes to the nucleus in AML and normal haematopoietic stem and progenitor cells to maintain stemness.

Mitochondrial metabolites regulate leukaemic and normal stem cells by affecting epigenetic marks. How mitochondrial enzymes localize to the nucleus to control stem cell function is less understood. We discovered that the mitochondrial metabolic enzyme hexokinase 2 (HK2) localizes to the nucleus in leukaemic and normal haematopoietic stem cells. Overexpression of nuclear HK2 increases leukaemic stem cell properties and decreases differentiation, whereas selective nuclear HK2 knockdown promotes differentiation and decreases stem cell function. Nuclear HK2 localization is phosphorylation-dependent, requires active import and export, and regulates differentiation independently of its enzymatic activity. HK2 interacts with nuclear proteins regulating chromatin openness, increasing chromatin accessibilities at leukaemic stem cell-positive signature and DNA-repair sites. Nuclear HK2 overexpression decreases double-strand breaks and confers chemoresistance, which may contribute to the mechanism by which leukaemic stem cells resist DNA-damaging agents. Thus, we describe a non-canonical mechanism by which mitochondrial enzymes influence stem cell function independently of their metabolic function.

Emerging roles of DNA topoisomerases in the regulation of R-loops.

R-loops are comprised of a DNA:RNA hybrid and a displaced single-strand DNA (ssDNA) that reinvades the DNA duplex behind the moving RNA polymerase. Because they have several physiological functions within the cell, including gene expression, chromosomal segregation, and mitochondrial DNA replication, among others, R-loop homeostasis is tightly regulated to ensure normal functioning of cellular processes. Thus, several classes of enzymes including RNases, helicases, topoisomerases, as well as proteins involved in splicing and the biogenesis of messenger ribonucleoproteins, have been implicated in R-loop prevention, suppression, and resolution. There exist six topoisomerase enzymes encoded by the human genome that function to introduce transient DNA breaks to relax supercoiled DNA. In this mini-review, we discuss functions of DNA topoisomerases and their emerging role in transcription, replication, and regulation of R-loops, and we highlight how their role in maintaining genome stability can be exploited for cancer therapy.

Exploiting synthetic lethality to target BRCA1/2-deficient tumors: where we stand.

The principle of synthetic lethality, which refers to the loss of viability resulting from the disruption of two genes, which, individually, do not cause lethality, has become an attractive target approach due to the development and clinical success of Poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi). In this review, we present the most recent findings on the use of PARPi in the clinic, which are currently approved for second-line therapy for advanced ovarian and breast cancer associated with mutations of BRCA1 or BRCA2 (BRCA1/2) genes. PARPi efficacy, however, appears to be limited by acquired and inherent resistance, highlighting the need for alternative and synergistic targets to eliminate these tumors. Here, we explore other identified synthetic lethal interactors of BRCA1/2, including DNA polymerase theta (POLQ), Fanconi anemia complementation group D2 (FANDC2), radiation sensitive 52 (RAD52), Flap structure-specific endonuclease 1 (FEN1), and apurinic/apyrimidinic endodeoxyribonuclease 2 (APE2), as well as other protein and nonprotein targets, for BRCA1/2-mutated cancers and their implications for future therapies. A wealth of information now exists for phenotypic and functional characterization of these novel synthetic lethal interactors of BRCA1/2, and leveraging these findings can pave the way for the development of new targeted therapies for patients suffering from these cancers.

RNF168 regulates R-loop resolution and genomic stability in BRCA1/2-deficient tumors.

Germline mutations in BRCA1 and BRCA2 (BRCA1/2) genes considerably increase breast and ovarian cancer risk. Given that tumors with these mutations have elevated genomic instability, they exhibit relative vulnerability to certain chemotherapies and targeted treatments based on poly (ADP-ribose) polymerase (PARP) inhibition. However, the molecular mechanisms that influence cancer risk and therapeutic benefit or resistance remain only partially understood. BRCA1 and BRCA2 have also been implicated in the suppression of R-loops, triple-stranded nucleic acid structures composed of a DNA:RNA hybrid and a displaced ssDNA strand. Here, we report that loss of RNF168, an E3 ubiquitin ligase and DNA double-strand break (DSB) responder, remarkably protected Brca1-mutant mice against mammary tumorigenesis. We demonstrate that RNF168 deficiency resulted in accumulation of R-loops in BRCA1/2-mutant breast and ovarian cancer cells, leading to DSBs, senescence, and subsequent cell death. Using interactome assays, we identified RNF168 interaction with DHX9, a helicase involved in the resolution and removal of R-loops. Mechanistically, RNF168 directly ubiquitylated DHX9 to facilitate its recruitment to R-loop-prone genomic loci. Consequently, loss of RNF168 impaired DHX9 recruitment to R-loops, thereby abrogating its ability to resolve R-loops. The data presented in this study highlight a dependence of BRCA1/2-defective tumors on factors that suppress R-loops and reveal a fundamental RNF168-mediated molecular mechanism that governs cancer development and vulnerability.

Strategic Training in Transdisciplinary Radiation Science for the 21st Century (STARS21): 15-Year Evaluation of an Innovative Research Training Program.

PURPOSE: To evaluate the 15-year impact of a transdisciplinary research training program for graduate students, postdoctoral fellows, and clinical trainees focused on radiation science, entitled Strategic Training in Transdisciplinary Radiation Science for the 21st Century (STARS21) with a primary objective to build capacity in radiation research. METHODS AND MATERIALS: Alumni (n = 128) and mentors (n = 41) who participated in STARS21 between 2003 and 2018 were sent an anonymized online survey designed to evaluate the program. Twelve alumni and 7 mentors also volunteered to participate in semistructured interviews. The transcribed interviews were coded and analyzed using NVivo12-Pro software. Alumni employment and publications were assessed from program records and by web-based search queries. RESULTS: Alumni are located in 11 countries, and nearly 90% are employed in a research-oriented career and continue to publish in radiation medicine- or cancer-related fields. Of those invited, 46 alumni (36%) and 12 mentors (29%) completed the online survey. Approximately 87% of alumni valued interdisciplinary collaboration, and 80% indicated that STARS21 had encouraged them to pursue such collaborations. Alumni emphasized that STARS21 assisted their career development, and the majority of alumni and mentors would recommend STARS21 to other trainees (4.48 and 4.58, respectively; 5 = strongly agree). The time invested in the program was perceived by mentors as worthwhile for the knowledge and skills gained by trainees (4.67; 5 = strongly agree), and 64% of mentors indicated that these benefits were associated with improved trainee research productivity. From the alumni and mentor perspectives, the valuable skills acquired from STARS21 included scientific communication (85% and 83%, respectively) and networking (83% and 92%, respectively). CONCLUSIONS: STARS21 is an innovative research training program that promotes interdisciplinary collaboration in radiation medicine research, which is valued by alumni and mentor respondents. Alumni can acquire important skill sets for career development, with a large proportion of alumni currently engaged in radiation research around the world.

BRCA1 and Metastasis: Outcome of Defective DNA Repair.

Heritable mutations in BRCA1 and BRCA2 genes are a major risk factor for breast and ovarian cancer. Inherited mutations in BRCA1 increase the risk of developing breast cancers by up to 72% and ovarian cancers by up to 69%, when compared to individuals with wild-type BRCA1. BRCA1 and BRCA2 (BRCA1/2) are both important for homologous recombination-mediated DNA repair. The link between BRCA1/2 mutations and high susceptibility to breast cancer is well established. However, the potential impact of BRCA1 mutation on the individual cell populations within a tumor microenvironment, and its relation to increased aggressiveness of cancer is not well understood. The objective of this review is to provide significant insights into the mechanisms by which BRCA1 mutations contribute to the metastatic and aggressive nature of the tumor cells.

Nucleolar RNA polymerase II drives ribosome biogenesis.

Proteins are manufactured by ribosomes-macromolecular complexes of protein and RNA molecules that are assembled within major nuclear compartments called nucleoli1,2. Existing models suggest that RNA polymerases I and III (Pol I and Pol III) are the only enzymes that directly mediate the expression of the ribosomal RNA (rRNA) components of ribosomes. Here we show, however, that RNA polymerase II (Pol II) inside human nucleoli operates near genes encoding rRNAs to drive their expression. Pol II, assisted by the neurodegeneration-associated enzyme senataxin, generates a shield comprising triplex nucleic acid structures known as R-loops at intergenic spacers flanking nucleolar rRNA genes. The shield prevents Pol I from producing sense intergenic noncoding RNAs (sincRNAs) that can disrupt nucleolar organization and rRNA expression. These disruptive sincRNAs can be unleashed by Pol II inhibition, senataxin loss, Ewing sarcoma or locus-associated R-loop repression through an experimental system involving the proteins RNaseH1, eGFP and dCas9 (which we refer to as 'red laser'). We reveal a nucleolar Pol-II-dependent mechanism that drives ribosome biogenesis, identify disease-associated disruption of nucleoli by noncoding RNAs, and establish locus-targeted R-loop modulation. Our findings revise theories of labour division between the major RNA polymerases, and identify nucleolar Pol II as a major factor in protein synthesis and nuclear organization, with potential implications for health and disease.

Preclinical evaluation of the selective small-molecule UBA1 inhibitor, TAK-243, in acute myeloid leukemia.

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy for which new therapeutic approaches are required. One such potential therapeutic strategy is to target the ubiquitin-like modifier-activating enzyme 1 (UBA1), the initiating enzyme in the ubiquitylation cascade in which proteins are tagged with ubiquitin moieties to regulate their degradation or function. Here, we evaluated TAK-243, a first-in-class UBA1 inhibitor, in preclinical models of AML. In AML cell lines and primary AML samples, TAK-243 induced cell death and inhibited clonogenic growth. In contrast, normal hematopoietic progenitor cells were more resistant. TAK-243 preferentially bound to UBA1 over the related E1 enzymes UBA2, UBA3, and UBA6 in intact AML cells. Inhibition of UBA1 with TAK-243 decreased levels of ubiquitylated proteins, increased markers of proteotoxic stress and DNA damage stress. In vivo, TAK-243 reduced leukemic burden and targeted leukemic stem cells without evidence of toxicity. Finally, we selected populations of AML cells resistant to TAK-243 and identified missense mutations in the adenylation domain of UBA1. Thus, our data demonstrate that TAK-243 targets AML cells and stem cells and support a clinical trial of TAK-243 in this patient population. Moreover, we provide insight into potential mechanisms of acquired resistance to UBA1 inhibitors.

Ubiquitin ligase RNF8 suppresses Notch signaling to regulate mammary development and tumorigenesis.

The E3 ubiquitin ligase RNF8 plays critical roles in maintaining genomic stability by promoting the repair of DNA double-strand breaks (DSBs) through ubiquitin signaling. Abnormal activation of Notch signaling and defective repair of DSBs promote breast cancer risk. Here, we found that low expression of the full-length RNF8 correlated with poor prognosis for breast cancer patients. Our data revealed that in addition to its role in the repair of DSBs, RNF8 regulated Notch1 signaling and cell-fate determination of mammary luminal progenitors. Mechanistically, RNF8 acted as a negative regulator of Notch signaling by ubiquitylating the active NOTCH1 protein (N1ICD), leading to its degradation. Consistent with abnormal activation of Notch signaling and impaired repair of DSBs in Rnf8-mutant mammary epithelial cells, we observed increased risk of mammary tumorigenesis in mouse models for RNF8 deficiency. Notably, deficiency of RNF8 sensitized breast cancer cells to combination of pharmacological inhibitors of Notch signaling and poly(ADP-ribose) polymerase (PARP), suggesting implications for treatment of breast cancer associated with impaired RNF8 expression or function.

Risk SNP-Mediated Promoter-Enhancer Switching Drives Prostate Cancer through lncRNA PCAT19.

The prostate cancer (PCa) risk-associated SNP rs11672691 is positively associated with aggressive disease at diagnosis. We showed that rs11672691 maps to the promoter of a short isoform of long noncoding RNA PCAT19 (PCAT19-short), which is in the third intron of the long isoform (PCAT19-long). The risk variant is associated with decreased and increased levels of PCAT19-short and PCAT19-long, respectively. Mechanistically, the risk SNP region is bifunctional with both promoter and enhancer activity. The risk variants of rs11672691 and its LD SNP rs887391 decrease binding of transcription factors NKX3.1 and YY1 to the promoter of PCAT19-short, resulting in weaker promoter but stronger enhancer activity that subsequently activates PCAT19-long. PCAT19-long interacts with HNRNPAB to activate a subset of cell-cycle genes associated with PCa progression, thereby promoting PCa tumor growth and metastasis. Taken together, these findings reveal a risk SNP-mediated promoter-enhancer switching mechanism underlying both initiation and progression of aggressive PCa.