Suppresses Replication Stress Research Articles

ATR Inhibition in Advanced Urothelial Carcinoma.

The ataxia telangiectasia and Rad3-related (ATR) checkpoint kinase 1 (CHK1) pathway is intricately involved in protecting the integrity of the human genome by suppressing replication stress and repairing DNA damage. ATR is a promising therapeutic target in cancer cells because its inhibition could lead to an accumulation of damaged DNA preventing further replication and division. ATR inhibition is being studied in multiple types of cancer, including advanced urothelial carcinoma where there remains an unmet need for novel therapies to improve outcomes. Herein, we review preclinical and clinical data evaluating 4 ATR inhibitors as monotherapy or in combination with chemotherapy. The scope of this review is focused on contemporary studies evaluating the application of this novel therapy in advanced urothelial carcinoma.

Targeting BAP1 with small compound inhibitor for colon cancer treatment

BRCA1-associated protein-1 (BAP1) is a ubiquitin C-terminal hydrolase domain-containing deubiquitinase. The gene encoding BAP1 is mutated in various human cancers, including mesothelioma, uveal melanoma and renal cell carcinoma. BAP1 plays roles in many cancer-related cellular functions, including cell proliferation, cell death, and nuclear processes crucial for genome stability, such as DNA repair and replication. While these findings suggest that BAP1 functions as a tumor suppressor, recent data also suggest that BAP1 might play tumor-promoting roles in certain cancers, such as breast cancer and hematopoietic malignancies. Here, we show that BAP1 is upregulated in colon cancer cells and tissues and that BAP1 depletion reduces colon cancer cell proliferation and tumor growth. BAP1 contributes to colon cancer cell proliferation by accelerating DNA replication and suppressing replication stress and concomitant apoptosis. A recently identified BAP1 inhibitor, TG2-179-1, which seems to covalently bind to the active site of BAP1, exhibits potent cytotoxic activity against colon cancer cells, with half-maximal inhibitory concentrations of less than 10 μM, and inhibits colon tumor growth. TG2-179-1 exerts cytotoxic activity by targeting BAP1, leading to defective replication and increased apoptosis. This work therefore shows that BAP1 acts oncogenically in colon cancer and is a potential therapeutic target for this cancer. Our work also suggests that TG2-179-1 can be developed as a potential therapeutic agent for colon cancer.

Adult T-Cell Leukemia/Lymphoma Diagnosed in North American Is Characterized By a Requirement for BCL6 and a Cell Cycle Program Associated with DNA Replication Stress

Adult T-cell leukemia/lymphoma (ATLL) is a rare and aggressive disease of malignant CD4+ T cells that develops in human T-lymphotropic virus-1 (HTLV-1) carriers. Patients diagnosed with ATLL have the worst survival among all peripheral T-cell lymphomas. At the moment, no outcome-changing targeted therapy is available for ATLL highlighting an urgent need for in-depth understanding of its pathogenesis and development of new treatment strategies. We and others have shown that ATLLs diagnosed in the Japanese (J-ATLL) and North American (NA-ATLL) patients have very different clinical behavior, with the NA-ATLL variant characterized by a higher rate of chemo-refractory disease and worse prognosis. Differences are also seen in the somatic mutation patterns and responsiveness to an immune checkpoint blockade therapy, Nivolumab. To explore additional aspects of ATLL biology where NA-ATLL and J-ATLL may exhibit distinct characteristics, we examined immunophenotypic features of 5 J-ATLL cell lines and cultures from 7 NA-ATLL patients using a panel of 17 flow cytometry markers. Our results show that, while the J-ATLL cell lines have a surface marker profile resembling classic regulatory T (Treg) cells, NA-ATLL samples are more similar to follicular T regulatory (Tfr) cells. This conclusion is also supported by GSEA analysis of of bulk RNA-seq. Consistent with a Tfr-like phenotype, NA-ATLL samples are characterized by more frequent expression of PD-1 and variable levels of BCL6. We also detected BCL6 in patient biopsy samples by IHC. Interestingly, although some J-ATLL cell lines express BCL6 as well, only NA-ATLL but not J-ATLL cell lines are sensitive to the BCL6-specific inhibitor FX1. Inactivating BCL6 by either FX1 treatment or siRNA-mediated BCL6 knock-down in NA-ATLL cells triggered rapid activation of the DNA damage response and death of S phase cells. These results suggest that in transformed NA-ATLL cells, BCL6 promotes survival by suppressing replication stress. Our experiments also demonstrate that compared to J-ATLL cell lines, NA-ATLL cell lines are more sensitive to S phase-targeting drugs including Wee1i (AZD-1775), ATRi (AZD-6738), and PARPi (Olaparib). These results support the concept that the cell cycle S phase is an Achilles’ Heel of NA-ATLL cells and an attractive therapeutic target. In summary, despite the involvement of the same HTLV-1 subtype (HTLV-1a) in ATLLs diagnosed in Japan and North America, these two ATLL variants have distinct features in clinical behavior, mutational landscape, and immunophenotype. Our experiments described here point to additional differences in the cell cycle program and roles played by BCL6. Further studies are needed to fully characterize the cellular phenotype of NA-ATLL and develop S phase-based targeting strategies to treat this devastating disease.

NR4A1 regulates expression of immediate early genes, suppressing replication stress in cancer

Deregulation of oncogenic signals in cancer triggers replication stress. Immediate early genes (IEGs) are rapidly and transiently expressed following stressful signals, contributing to an integrated response. Here, we find that the orphan nuclear receptor NR4A1 localizes across the gene body and 3' UTR of IEGs, where it inhibits transcriptional elongation by RNA Pol II, generating R-loops and accessible chromatin domains. Acute replication stress causes immediate dissociation of NR4A1 and a burst of transcriptionally poised IEG expression. Ectopic expression of NR4A1 enhances tumorigenesis by breast cancer cells, while its deletion leads to massive chromosomal instability and proliferative failure, driven by deregulated expression of its IEG target, FOS. Approximately half of breast and other primary cancers exhibit accessible chromatin domains at IEG gene bodies, consistent with this stress-regulatory pathway. Cancers that have retained this mechanism in adapting to oncogenic replication stress may be dependent on NR4A1 for their proliferation.

MYBL2 and ATM suppress replication stress in pluripotent stem cells

Replication stress, a major cause of genome instability in cycling cells, is mainly prevented by the ATR‐dependent replication stress response pathway in somatic cells. However, the replication stress response pathway in embryonic stem cells (ESCs) may be different due to alterations in cell cycle phase length. The transcription factor MYBL2, which is implicated in cell cycle regulation, is expressed a hundred to a thousand‐fold more in ESCs compared with somatic cells. Here we show that MYBL2 activates ATM and suppresses replication stress in ESCs. Consequently, loss of MYBL2 or inhibition of ATM or Mre11 in ESCs results in replication fork slowing, increased fork stalling and elevated origin firing. Additionally, we demonstrate that inhibition of CDC7 activity rescues replication stress induced by MYBL2 loss and ATM inhibition, suggesting that uncontrolled new origin firing may underlie the replication stress phenotype resulting from loss/inhibition of MYBL2 and ATM. Overall, our study proposes that in addition to ATR, a MYBL2‐MRN‐ATM replication stress response pathway functions in ESCs to control DNA replication initiation and prevent genome instability.

WDR82/PNUTS-PP1 Prevents Transcription-Replication Conflicts by Promoting RNA Polymerase II Degradation on Chromatin

Transcription-replication (T-R) conflicts cause replication stress and loss of genome integrity. However, the transcription-related processes that restrain such conflicts are poorly understood. Here, we demonstrate that the RNA polymerase II (RNAPII) C-terminal domain (CTD) phosphatase protein phosphatase 1 (PP1) nuclear targeting subunit (PNUTS)-PP1 inhibits replication stress. Depletion of PNUTS causes lower EdU uptake, S phase accumulation, and slower replication fork rates. In addition, the PNUTS binding partner WDR82 also promotes RNAPII-CTD dephosphorylation and suppresses replication stress. RNAPII has a longer residence time on chromatin after depletion of PNUTS or WDR82. Furthermore, the RNAPII residence time is greatly enhanced by proteasome inhibition in control cells but less so in PNUTS- or WDR82-depleted cells, indicating that PNUTS and WDR82 promote degradation of RNAPII on chromatin. Notably, reduced replication is dependent on transcription and the phospho-CTD binding protein CDC73 after depletion of PNUTS/WDR82. Altogether, our results suggest that RNAPII-CTD dephosphorylation is required for the continuous turnover of RNAPII on chromatin, thereby preventing T-R conflicts.

Inhibition of the translesion synthesis polymerase REV1 exploits replication gaps as a cancer vulnerability.

The replication stress response, which serves as an anticancer barrier, is activated not only by DNA damage and replication obstacles but also oncogenes, thus obscuring how cancer evolves. Here, we identify that oncogene expression, similar to other replication stress-inducing agents, induces single-stranded DNA (ssDNA) gaps that reduce cell fitness. DNA fiber analysis and electron microscopy reveal that activation of translesion synthesis (TLS) polymerases restricts replication fork slowing, reversal, and fork degradation without inducing replication gaps despite the continuation of replication during stress. Consistent with gap suppression (GS) being fundamental to cancer, we demonstrate that a small-molecule inhibitor targeting the TLS factor REV1 not only disrupts DNA replication and cancer cell fitness but also synergizes with gap-inducing therapies such as inhibitors of ATR or Wee1. Our work illuminates that GS during replication is critical for cancer cell fitness and therefore a targetable vulnerability.

Abstract B36: Tracking the path of breast tumorigenesis in BRCA1 mutant breast cancer

Abstract Women with mutations in breast cancer (BrCa) gene BRCA1 are highly predisposed to BrCa. In these families, cancer develops almost exclusively in females and often at very young ages. The molecular pathogenic steps, especially the earliest ones that drive the transition of normal mammary epithelial cells (BRCA1mut/+) to tumor cells, are largely unknown. Mouse model-based studies have not successfully addressed this question, in part because, unlike women with BRCA1 (B1) mutations, B1 heterozygous mice do not form mammary tumors any faster than the wild-type (wt) mice. A mouse model that can biologically mimic what is happening in the mammary tissue of B1 mutation carriers would be an invaluable tool to study B1 mutant BrCa. Furthermore, most of the efforts to study breast tumor-associated gene changes have been based on GWAS studies or RNA-seq based analysis of the breast tumor samples. Such studies have shown that the transcriptional network is altered in the breast tumors compared to wild-type tissue. However, whether those changes are tumor initiating, i.e., “driver” changes, or are “passenger” mutations/changes is still not clear. Given the role of B1 in suppressing replication stress (RS), a tumor-promoting event, and the increased RS observed in B1 heterozygous cells, we have established an RS-driven B1 BrCa mouse model to study the early genetic changes that drive B1 mutant cancer. We find that intraductal injections of 4-nitroquinoline-1-oxide (4NQO1, a replication stress-inducing agent) leads to breast adenocarcinoma in B1flx/wt;Trp53flx/flx,K14Cre mice on average 84 days post injections unlike B1 wt mice that do not make tumors. This is the first RS-induced BrCa model that can successfully recapitulate B1 mutant BrCa. The fast timeline of tumor formation provides significant advantage for studying tumor formation, progression, and response to therapy. Furthermore, single-cell RNA sequencing analysis of the breast adenocarcinoma and tissue collected early during injections in these mice has revealed presence of a highly proliferative “transdifferentiated population” that shares properties of both luminal and basal cells (e.g., K8+K18+) and also exists in the hyperplastic tissue. No such population was found in control PBS injected mammary fat pad of B1flx/wt;Trp53flx/flx,K14Cre mice, indicating that this population might be dependent on RS. We also find that this population is enriched very early during mammary tumorigenesis and some of the top hits include cancer stem cell markers. We have also confirmed the presence of this population by immune histochemistry-based experiments. Finally, our study provides evidence for a BrCa mouse model where basal-like tumor formation is driven by RS. We also found evidence for an early precancerous transdifferentiated population that is enriched upon RS and have identified early markers that can potentially drive breast tumorigenesis in B1 mutant BrCa. Identification of such candidate genes is critical in the design of future preventive and/or therapeutic strategies. Citation Format: Joshua Rivera, Delan Khalid, Stevenson Tran, Kemmie Kibaja, Shailja Pathania. Tracking the path of breast tumorigenesis in BRCA1 mutant breast cancer [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr B36.

MOF Suppresses Replication Stress and Contributes to Resolution of Stalled Replication Forks.

The human MOF (hMOF) protein belongs to the MYST family of histone acetyltransferases and plays a critical role in transcription and the DNA damage response. MOF is essential for cell proliferation; however, its role during replication and replicative stress is unknown. Here we demonstrate that cells depleted of MOF and under replicative stress induced by cisplatin, hydroxyurea, or camptothecin have reduced survival, a higher frequency of S-phase-specific chromosome damage, and increased R-loop formation. MOF depletion decreased replication fork speed and, when combined with replicative stress, also increased stalled replication forks as well as new origin firing. MOF interacted with PCNA, a key coordinator of replication and repair machinery at replication forks, and affected its ubiquitination and recruitment to the DNA damage site. Depletion of MOF, therefore, compromised the DNA damage repair response as evidenced by decreased Mre11, RPA70, Rad51, and PCNA focus formation, reduced DNA end resection, and decreased CHK1 phosphorylation in cells after exposure to hydroxyurea or cisplatin. These results support the argument that MOF plays an important role in suppressing replication stress induced by genotoxic agents at several stages during the DNA damage response.

Homologous Recombination and Replication Fork Protection: BRCA2 and More!

BRCA2 is a breast and ovarian tumor suppressor that guards against genome instability, a hallmark of cancer. Significant progress has been made in improving our understanding of BRCA2 function from biochemical, cellular, and mouse studies. The knowledge gained has been actively exploited to develop therapeutic strategies, including PARP inhibition, which has shown promising clinical outcomes. Recently, tremendous excitement has been generated by the findings of the roles of BRCA2 and other proteins in suppressing replication stress through homologous recombination and in the protection of stalled replication forks. Processes such as mitotic DNA synthesis and fork reversal have taken center stage in these studies. Here, we discuss our recent findings in the context of these advances.

Mapping ubiquitination sites of S. cerevisiae Mcm10

Minichromosome maintenance protein (Mcm) 10 is a part of the eukaryotic replication machinery and highly conserved throughout evolution. As a multivalent DNA scaffold, Mcm10 coordinates the action of proteins that are indispensable for lagging strand synthesis, such as the replication clamp, proliferating cell nuclear antigen (PCNA). The binding between Mcm10 and PCNA serves an essential function during DNA elongation and is mediated by the ubiquitination of Mcm10. Here we map lysine 372 as the primary attachment site for ubiquitin on S. cerevisiae Mcm10. Moreover, we identify five additional lysines that can be ubiquitinated. Mutation of lysine 372 to arginine ablates ubiquitination of overexpressed protein and causes sensitivity to the replication inhibitor hydroxyurea in cells that are S-phase checkpoint compromised. Together, these findings reveal the high selectivity of the ubiquitination machinery that targets Mcm10 and that ubiquitination has a role in suppressing replication stress.

Genetic Interactions Implicating Postreplicative Repair in Okazaki Fragment Processing.

Ubiquitination of the replication clamp proliferating cell nuclear antigen (PCNA) at the conserved residue lysine (K)164 triggers postreplicative repair (PRR) to fill single-stranded gaps that result from stalled DNA polymerases. However, it has remained elusive as to whether cells engage PRR in response to replication defects that do not directly impair DNA synthesis. To experimentally address this question, we performed synthetic genetic array (SGA) analysis with a ubiquitination-deficient K164 to arginine (K164R) mutant of PCNA against a library of S. cerevisiae temperature-sensitive alleles. The SGA signature of the K164R allele showed a striking correlation with profiles of mutants deficient in various aspects of lagging strand replication, including rad27Δ and elg1Δ. Rad27 is the primary flap endonuclease that processes 5’ flaps generated during lagging strand replication, whereas Elg1 has been implicated in unloading PCNA from chromatin. We observed chronic ubiquitination of PCNA at K164 in both rad27Δ and elg1Δ mutants. Notably, only rad27Δ cells exhibited a decline in cell viability upon elimination of PRR pathways, whereas elg1Δ mutants were not affected. We further provide evidence that K164 ubiquitination suppresses replication stress resulting from defective flap processing during Okazaki fragment maturation. Accordingly, ablation of PCNA ubiquitination increased S phase checkpoint activation, indicated by hyperphosphorylation of the Rad53 kinase. Furthermore, we demonstrate that alternative flap processing by overexpression of catalytically active exonuclease 1 eliminates PCNA ubiquitination. This suggests a model in which unprocessed flaps may directly participate in PRR signaling. Our findings demonstrate that PCNA ubiquitination at K164 in response to replication stress is not limited to DNA synthesis defects but extends to DNA processing during lagging strand replication.

Gli1 Protein Regulates the S-phase Checkpoint in Tumor Cells via Bid Protein, and Its Inhibition Sensitizes to DNA Topoisomerase 1 Inhibitors

Aberrant expression of hedgehog molecules, particularly Gli1, is common in cancers of many tissues and is responsible for their aggressive behavior and chemoresistance. Here we demonstrate a novel and tumor-specific role for aberrant Gli1 in the regulation of the S-phase checkpoint that suppresses replication stress and resistance to chemotherapy. Inhibition of Gli1 in tumor cells induced replication stress-mediated DNA damage response, attenuated their clonogenic potential, abrogated camptothecin (CPT)-induced Chk1 phosphorylation, and potentiated its cytotoxicity. However, in normal fibroblasts, Gli1 siRNAs showed no significant changes in CPT-induced Chk1 phosphorylation. Further analysis of ataxia telangiectasia and Rad3-related protein (ATR)/Chk1 signaling cascade genes in tumor cells revealed an unexpected mechanism whereby Gli1 regulates ATR-mediated Chk1 phosphorylation by transcriptional regulation of the BH3-only protein Bid. Consistent with its role in DNA damage response, Bid down-regulation in tumor cells abolished CPT-induced Chk1 phosphorylation and sensitized them to CPT. Correspondingly, Gli1 inhibition affected the expression of Bid and the association of replication protein A (RPA) with the ATR- interacting protein (ATRIP)-ATR complex, and this compromised the S-phase checkpoint. Conversely, complementation of Bid in Gli1-deficient cells restored CPT-induced Chk1 phosphorylation. An in silico analysis of the Bid promoter identified a putative Gli1 binding site, and further studies using luciferase reporter assays confirmed Gli1-dependent promoter activity. Collectively, our studies established a novel connection between aberrant Gli1 and Bid in the survival of tumor cells and their response to chemotherapy, at least in part, by regulating the S-phase checkpoint. Importantly, our data suggest a novel drug combination of Gli1 and Top1 inhibitors as an effective therapeutic strategy in treating tumors that expresses Gli1.

Novel Role for PTEN in Regulating Non Homologous End‐Joining Factor 53BP1 to Suppress Replication Fork Instability (LB120)

PTEN is one of the most frequently inactivated tumor suppressors with unresolved connections to DNA repair processes that prevent genomic instability. Using single molecule DNA spreading, we here show that PTEN suppresses replication fork instability by promoting efficient replication restart of stalled replication forks, a phenotype mirroring ATM and BLMs helicase functions. Moreover, we find that similar to BLMs, PTEN suppresses replication stress dependent DNA bridges. Previous studies show non‐homologous end‐joining (NHEJ) protein and ATM phosphorylation target 53BP1 in complex with BLMs at stalled fork, an interaction requiring phosphorylation of 53BP1. Yet, the role of 53BP1 at stalled forks remains ill‐defined. Strikingly, 53BP1 is highly phosphorylated in PTEN proficient cells upon replication stalling, but not in cells lacking PTEN. We further find 53BP1 to be readily recruited to nuclear foci upon replication stalling. Yet foci formation is compromised in PTEN defective cells, implying PTEN controls a function for 53BP1 when replication forks are stalling. Lastly, we reveal a direct requirement for 53BP1 in restarting stalled replication forks. These data reveal a surprising function for NHEJ factors controlled by PTEN to suppress replication fork stability and genomic instability.Grant Funding Source: Supported by 1K22CA175262‐01 (K.S.) and UO1CA164188 (H.W.)