Publications by authors named "Lee Zou"

111 Publications

The cell cycle effects of PARP inhibitors underlie their selectivity toward BRCA1/2-deficient cells.

Genes Dev 2021 Sep 12;35(17-18):1271-1289. Epub 2021 Aug 12.

Massachusetts General Hospital Cancer Center, Harvard Medical School, Charlestown, Massachusetts 02129, USA.

PARP inhibitor (PARPi) is widely used to treat BRCA1/2-deficient tumors, but why PARPi is more effective than other DNA-damaging drugs is unclear. Here, we show that PARPi generates DNA double-strand breaks (DSBs) predominantly in a cell cycle manner. During the first S phase after PARPi exposure, PARPi induces single-stranded DNA (ssDNA) gaps behind DNA replication forks. By trapping PARP on DNA, PARPi prevents the completion of gap repair until the next S phase, leading to collisions of replication forks with ssDNA gaps and a surge of DSBs. In the second S phase, BRCA1/2-deficient cells are unable to suppress origin firing through ATR, resulting in continuous DNA synthesis and more DSBs. Furthermore, BRCA1/2-deficient cells cannot recruit RAD51 to repair collapsed forks. Thus, PARPi induces DSBs progressively through cell cycle ssDNA gaps, and BRCA1/2-deficient cells fail to slow down and repair DSBs over multiple cell cycles, explaining the unique efficacy of PARPi in BRCA1/2-deficient cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/gad.348479.121DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8415318PMC
September 2021

An extending ATR-CHK1 circuitry: the replication stress response and beyond.

Curr Opin Genet Dev 2021 Jul 27;71:92-98. Epub 2021 Jul 27.

Massachusetts General Hospital Cancer Center, Harvard Medical School, Charlestown, MA 02129, USA; Department of Pathology, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA. Electronic address:

The maintenance of genomic integrity relies on the coordination of a wide range of cellular processes and efficient repair of DNA damage. Since its discovery over two decades ago, the ATR kinase has been recognized as the master regulator of the circuitry orchestrating the cellular responses to DNA damage and replication stress. Recent studies reveal that ATR additionally functions in the unperturbed cell cycle through its control of replication fork speed and stability, replication origin firing, completion of genome duplication, and chromosome segregation. Here, we discuss several recently discovered mechanisms through which ATR safeguards genomic integrity during the cell cycle, from S phase to mitosis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.gde.2021.07.003DOI Listing
July 2021

Impacts of chromatin dynamics and compartmentalization on DNA repair.

DNA Repair (Amst) 2021 Sep 19;105:103162. Epub 2021 Jun 19.

Massachusetts General Hospital Cancer Center, Harvard Medical School, Charlestown, MA, 02129, USA. Electronic address:

The proper spatial organization of DNA, RNA, and proteins is critical for a variety of cellular processes. The genome is organized into numerous functional units, such as topologically associating domains (TADs), the formation of which is regulated by both proteins and RNA. In addition, a group of chromatin-bound proteins with the ability to undergo liquid-liquid phase separation (LLPS) can affect the spatial organization and compartmentalization of chromatin, RNA, and proteins by forming condensates, conferring unique properties to specific chromosomal regions. Although the regulation of DNA repair by histone modifications and chromatin accessibility is well established, the impacts of higher-order chromatin and protein organization on the DNA damage response (DDR) have not been appreciated until recently. In this review, we will focus on the movement of chromatin during the DDR, the compartmentalization of DDR proteins via LLPS, and the roles of membraneless nuclear bodies and transcription in DNA repair. With this backdrop, we will discuss the importance of the spatial organization of chromatin and proteins for the maintenance of genome integrity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.dnarep.2021.103162DOI Listing
September 2021

RNA transcripts stimulate homologous recombination by forming DR-loops.

Nature 2021 06 12;594(7862):283-288. Epub 2021 May 12.

Massachusetts General Hospital Cancer Center, Harvard Medical School, Charlestown, MA, USA.

Homologous recombination (HR) repairs DNA double-strand breaks (DSBs) in the S and G2 phases of the cell cycle. Several HR proteins are preferentially recruited to DSBs at transcriptionally active loci, but how transcription promotes HR is poorly understood. Here we develop an assay to assess the effect of local transcription on HR. Using this assay, we find that transcription stimulates HR to a substantial extent. Tethering RNA transcripts to the vicinity of DSBs recapitulates the effects of local transcription, which suggests that transcription enhances HR through RNA transcripts. Tethered RNA transcripts stimulate HR in a sequence- and orientation-dependent manner, indicating that they function by forming DNA-RNA hybrids. In contrast to most HR proteins, RAD51-associated protein 1 (RAD51AP1) only promotes HR when local transcription is active. RAD51AP1 drives the formation of R-loops in vitro and is required for tethered RNAs to stimulate HR in cells. Notably, RAD51AP1 is necessary for the DSB-induced formation of DNA-RNA hybrids in donor DNA, linking R-loops to D-loops. In vitro, RAD51AP1-generated R-loops enhance the RAD51-mediated formation of D-loops locally and give rise to intermediates that we term 'DR-loops', which contain both DNA-DNA and DNA-RNA hybrids and favour RAD51 function. Thus, at DSBs in transcribed regions, RAD51AP1 promotes the invasion of RNA transcripts into donor DNA, and stimulates HR through the formation of DR-loops.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41586-021-03538-8DOI Listing
June 2021

Co-regulation and function of / bidirectional genes in cancer.

Elife 2021 Apr 23;10. Epub 2021 Apr 23.

Eppley Institute for Cancer Research and Fred & Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, United States.

The FOXM1 transcription factor is an oncoprotein and a top biomarker of poor prognosis in human cancer. Overexpression and activation of FOXM1 is frequent in high-grade serous carcinoma (HGSC), the most common and lethal form of human ovarian cancer, and is linked to copy number gains at chromosome 12p13.33. We show that is co-amplified and co-expressed with , a gene involved in the ATR-Chk1 signaling pathway that functions in the DNA replication stress response. We demonstrate that and are head-to-head (i.e., bidirectional) genes (BDG) regulated by a bidirectional promoter (BDP) (named F/R-BDP). FOXM1 and RHNO1 each promote oncogenic phenotypes in HGSC cells, including clonogenic growth, DNA homologous recombination repair, and poly-ADP ribosylase inhibitor resistance. FOXM1 and RHNO1 are one of the first examples of oncogenic BDG, and therapeutic targeting of FOXM1/RHNO1 BDG is a potential therapeutic approach for ovarian and other cancers.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.7554/eLife.55070DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8104967PMC
April 2021

An extended APOBEC3A mutation signature in cancer.

Nat Commun 2021 03 11;12(1):1602. Epub 2021 Mar 11.

Massachusetts General Hospital Cancer Center, Harvard Medical School, Boston, MA, USA.

APOBEC mutagenesis, a major driver of cancer evolution, is known for targeting TpC sites in DNA. Recently, we showed that APOBEC3A (A3A) targets DNA hairpin loops. Here, we show that DNA secondary structure is in fact an orthogonal influence on A3A substrate optimality and, surprisingly, can override the TpC sequence preference. VpC (non-TpC) sites in optimal hairpins can outperform TpC sites as mutational hotspots. This expanded understanding of APOBEC mutagenesis illuminates the genomic Twin Paradox, a puzzling pattern of closely spaced mutation hotspots in cancer genomes, in which one is a canonical TpC site but the other is a VpC site, and double mutants are seen only in trans, suggesting a two-hit driver event. Our results clarify this paradox, revealing that both hotspots in these twins are optimal A3A substrates. Our findings reshape the notion of a mutation signature, highlighting the additive roles played by DNA sequence and DNA structure.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-021-21891-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7952602PMC
March 2021

Targeting the DNA replication stress phenotype of KRAS mutant cancer cells.

Sci Rep 2021 Feb 11;11(1):3656. Epub 2021 Feb 11.

Department of Radiation Oncology, Massachusetts General Hospital, Harvard Medical School, 55 Fruit Street, Boston, MA, 02114, USA.

Mutant KRAS is a common tumor driver and frequently confers resistance to anti-cancer treatments such as radiation. DNA replication stress in these tumors may constitute a therapeutic liability but is poorly understood. Here, using single-molecule DNA fiber analysis, we first characterized baseline replication stress in a panel of unperturbed isogenic and non-isogenic cancer cell lines. Correlating with the observed enhanced replication stress we found increased levels of cytosolic double-stranded DNA in KRAS mutant compared to wild-type cells. Yet, despite this phenotype replication stress-inducing agents failed to selectively impact KRAS mutant cells, which were protected by CHK1. Similarly, most exogenous stressors studied did not differentially augment cytosolic DNA accumulation in KRAS mutant compared to wild-type cells. However, we found that proton radiation was able to slow fork progression and preferentially induce fork stalling in KRAS mutant cells. Proton treatment also partly reversed the radioresistance associated with mutant KRAS. The cellular effects of protons in the presence of KRAS mutation clearly contrasted that of other drugs affecting replication, highlighting the unique nature of the underlying DNA damage caused by protons. Taken together, our findings provide insight into the replication stress response associated with mutated KRAS, which may ultimately yield novel therapeutic opportunities.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-021-83142-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7878884PMC
February 2021

Alternative lengthening of telomeres is a self-perpetuating process in ALT-associated PML bodies.

Mol Cell 2021 03 15;81(5):1027-1042.e4. Epub 2021 Jan 15.

Massachusetts General Hospital Cancer Center, Harvard Medical School, Charlestown, MA 02129, USA; Department of Pathology, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA. Electronic address:

Alternative lengthening of telomeres (ALT) is mediated by break-induced replication (BIR), but how BIR is regulated at telomeres is poorly understood. Here, we show that telomeric BIR is a self-perpetuating process. By tethering PML-IV to telomeres, we induced telomere clustering in ALT-associated PML bodies (APBs) and a POLD3-dependent ATR response at telomeres, showing that BIR generates replication stress. Ablation of BLM helicase activity in APBs abolishes telomere synthesis but causes multiple chromosome bridges between telomeres, revealing a function of BLM in processing inter-telomere BIR intermediates. Interestingly, the accumulation of BLM in APBs requires its own helicase activity and POLD3, suggesting that BIR triggers a feedforward loop to further recruit BLM. Enhancing BIR induces PIAS4-mediated TRF2 SUMOylation, and PIAS4 loss deprives APBs of repair proteins and compromises ALT telomere synthesis. Thus, a BLM-driven and PIAS4-mediated feedforward loop operates in APBs to perpetuate BIR, providing a critical mechanism to extend ALT telomeres.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molcel.2020.12.030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8245000PMC
March 2021

CARM1 regulates replication fork speed and stress response by stimulating PARP1.

Mol Cell 2021 02 6;81(4):784-800.e8. Epub 2021 Jan 6.

Massachusetts General Hospital Cancer Center, Harvard Medical School, Charlestown, MA 02129, USA; Department of Pathology, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02115, USA. Electronic address:

DNA replication forks use multiple mechanisms to deal with replication stress, but how the choice of mechanisms is made is still poorly understood. Here, we show that CARM1 associates with replication forks and reduces fork speed independently of its methyltransferase activity. The speeding of replication forks in CARM1-deficient cells requires RECQ1, which resolves reversed forks, and RAD18, which promotes translesion synthesis. Loss of CARM1 reduces fork reversal and increases single-stranded DNA (ssDNA) gaps but allows cells to tolerate higher replication stress. Mechanistically, CARM1 interacts with PARP1 and promotes PARylation at replication forks. In vitro, CARM1 stimulates PARP1 activity by enhancing its DNA binding and acts jointly with HPF1 to activate PARP1. Thus, by stimulating PARP1, CARM1 slows replication forks and promotes the use of fork reversal in the stress response, revealing that CARM1 and PARP1 function as a regulatory module at forks to control fork speed and the choice of stress response mechanisms.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molcel.2020.12.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7897296PMC
February 2021

cGAS suppresses genomic instability as a decelerator of replication forks.

Sci Adv 2020 Oct 14;6(42). Epub 2020 Oct 14.

Massachusetts General Hospital Cancer Center, Harvard Medical School, Boston, MA 02129, USA.

The cyclic GMP-AMP synthase (cGAS), a sensor of cytosolic DNA, is critical for the innate immune response. Here, we show that loss of cGAS in untransformed and cancer cells results in uncontrolled DNA replication, hyperproliferation, and genomic instability. While the majority of cGAS is cytoplasmic, a fraction of cGAS associates with chromatin. cGAS interacts with replication fork proteins in a DNA binding-dependent manner, suggesting that cGAS encounters replication forks in DNA. Independent of cGAMP and STING, cGAS slows replication forks by binding to DNA in the nucleus. In the absence of cGAS, replication forks are accelerated, but fork stability is compromised. Consequently, cGAS-deficient cells are exposed to replication stress and become increasingly sensitive to radiation and chemotherapy. Thus, by acting as a decelerator of DNA replication forks, cGAS controls replication dynamics and suppresses replication-associated DNA damage, suggesting that cGAS is an attractive target for exploiting the genomic instability of cancer cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/sciadv.abb8941DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7556829PMC
October 2020

Inhibition of ATR-Chk1 signaling blocks DNA double-strand-break repair and induces cytoplasmic vacuolization in metastatic osteosarcoma.

Ther Adv Med Oncol 2020 14;12:1758835920956900. Epub 2020 Sep 14.

Sarcoma Biology Laboratory, Department of Orthopaedic Surgery, David Geffen School of Medicine at UCLA, 615 Charles E. Young Dr. S., Los Angeles, CA 90095, USA.

Background: Ataxia-telangiectasia and Rad3 related protein kinase (ATR) is an essential regulator of the DNA damage response in various cancers; however, its expression and roles in osteosarcoma are unclear. We therefore chose to evaluate the significance and mechanism of ATR in metastatic osteosarcoma, as well as its potential to be a therapeutic target.

Methods: The osteosarcoma tissue microarrays constructed from 70 patient specimens underwent immunohistochemistry to quantify ATR and activated phospho-ATR (pATR) expression and their correlation with clinical outcomes. ATR sublocalization within the metastatic osteosarcoma cells was confirmed by immunofluorescence assay. Cell proliferation, apoptosis, and migration were evaluated following treatment with ATR siRNA or the selective inhibitor Berzosertib. Antitumor effects were determined with three-dimensional (3D) culture models, and the impacts on the DNA damage repair pathways were measured with Western blotting.

Results: Elevated ATR and activated pATR expression correlated with shorter patient survival and less necrosis following neoadjuvant chemotherapy. Intranuclear sublocalization of ATR and pATR suggested a mechanism related to DNA replication. ATR knockdown with siRNA or inhibition with Berzosertib suppressed cell proliferation in a time- and dose-dependent manner and induced apoptosis. In addition, ATR inhibition decreased Chk1 phosphorylation while increasing γHAX expression and PARP cleavage, consistent with the interference of DNA damage repair. The ATR inhibitor Berzosertib also produced the characteristic cytoplasmic vacuolization preceding cell death, and suppressed 3D spheroid formation and cell motility.

Conclusion: The faithful dependence of cells on ATR signaling for survival and progression makes it an emerging therapeutic target in metastatic osteosarcoma.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1177/1758835920956900DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7493280PMC
September 2020

Synthetic lethality by targeting the RUVBL1/2-TTT complex in mTORC1-hyperactive cancer cells.

Sci Adv 2020 Jul 31;6(31):eaay9131. Epub 2020 Jul 31.

Division of Bioengineering, Incheon National University, Incheon 22012, Republic of Korea.

Despite considerable efforts, mTOR inhibitors have produced limited success in the clinic. To define the vulnerabilities of mTORC1-addicted cancer cells and to find previously unknown therapeutic targets, we investigated the mechanism of piperlongumine, a small molecule identified in a chemical library screen to specifically target cancer cells with a hyperactive mTORC1 phenotype. Sensitivity to piperlongumine was dependent on its ability to suppress RUVBL1/2-TTT, a complex involved in chromatin remodeling and DNA repair. Cancer cells with high mTORC1 activity are subjected to higher levels of DNA damage stress via c-Myc and displayed an increased dependency on RUVBL1/2 for survival and counteracting genotoxic stress. Examination of clinical cancer tissues also demonstrated that high mTORC1 activity was accompanied by high RUVBL2 expression. Our findings reveal a previously unknown role for RUVBL1/2 in cell survival, where it acts as a functional chaperone to mitigate stress levels induced in the mTORC1-Myc-DNA damage axis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/sciadv.aay9131DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7399646PMC
July 2020

BRG1 Loss Predisposes Lung Cancers to Replicative Stress and ATR Dependency.

Cancer Res 2020 09 20;80(18):3841-3854. Epub 2020 Jul 20.

Stem Cell Program, Division of Hematology/Oncology and Division of Pulmonary Medicine, Boston Children's Hospital, Boston, Massachusetts.

Inactivation of SMARCA4/BRG1, the core ATPase subunit of mammalian SWI/SNF complexes, occurs at very high frequencies in non-small cell lung cancers (NSCLC). There are no targeted therapies for this subset of lung cancers, nor is it known how mutations in contribute to lung cancer progression. Using a combination of gain- and loss-of-function approaches, we demonstrate that deletion of BRG1 in lung cancer leads to activation of replication stress responses. Single-molecule assessment of replication fork dynamics in BRG1-deficient cells revealed increased origin firing mediated by the prelicensing protein, CDC6. Quantitative mass spectrometry and coimmunoprecipitation assays showed that BRG1-containing SWI/SNF complexes interact with RPA complexes. Finally, BRG1-deficient lung cancers were sensitive to pharmacologic inhibition of ATR. These findings provide novel mechanistic insight into BRG1-mutant lung cancers and suggest that their dependency on ATR can be leveraged therapeutically and potentially expanded to BRG1-mutant cancers in other tissues. SIGNIFICANCE: These findings indicate that inhibition of ATR is a promising therapy for the 10% of non-small cell lung cancer patients harboring mutations in SMARCA4/BRG1. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/18/3841/F1.large.jpg.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/0008-5472.CAN-20-1744DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7501156PMC
September 2020

A genome-wide and cotranscriptional suppressor of R loops.

Genes Dev 2020 07;34(13-14):863-864

Massachusetts General Hospital Cancer Center, Harvard Medical School, Charlestown, Massachusetts 02129, USA.

R loops arise from hybridization of RNA transcripts with template DNA during transcription. Unrepaired R loops lead to transcription-replication collisions, causing DNA damage and genomic instability. In this issue of , Pérez-Calero and colleagues (pp. 898-912) identify UAP56 as a cotranscriptional RNA-DNA helicase that unwinds R loops. They found that UAP56 helicase activity is required to remove R loops formed from different sources and prevent R-loop accumulation genome-wide at actively transcribed genes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/gad.339861.120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7328514PMC
July 2020

Identification of Somatically Acquired Mutations by cfDNA Analysis in Patients with Metastatic Breast Cancer.

Clin Cancer Res 2020 09 22;26(18):4852-4862. Epub 2020 Jun 22.

Massachusetts General Hospital Cancer Center, Harvard Medical School, Boston, Massachusetts.

Purpose: Plasma genotyping may identify mutations in potentially "actionable" cancer genes, such as , but their clinical significance is not well-defined. We evaluated the characteristics of somatically acquired mutations in patients with metastatic breast cancer (MBC).

Experimental Design: Patients with MBC undergoing routine cell-free DNA (cfDNA) next-generation sequencing (73-gene panel) before starting a new therapy were included. Somatic mutations were classified as known germline pathogenic mutations or novel variants, and linked to clinicopathologic characteristics. The effect of the PARP inhibitor, olaparib, was assessed , using cultured circulating tumor cells (CTCs) from a patient with a somatically acquired mutation and a second patient with an acquired mutation.

Results: Among 215 patients with MBC, 29 (13.5%) had somatic cfDNA mutations [nine (4%) known germline pathogenic and rest (9%) novel variants]. Known germline pathogenic mutations were common in younger patients ( = 0.008), those with triple-negative disease ( = 0.022), and they were more likely to be protein-truncating alterations and be associated with mutations. Functional analysis of a CTC culture harboring a somatic mutation demonstrated high sensitivity to PARP inhibition, while another CTC culture harboring a somatic mutation showed no differential sensitivity. Across the entire cohort, APOBEC mutational signatures (COSMIC Signatures 2 and 13) and the "BRCA" mutational signature (COSMIC Signature 3) were present in mutant and wild-type cases, demonstrating the high mutational burden associated with advanced MBC.

Conclusions: Somatic mutations are readily detectable in MBC by cfDNA analysis, and may be present as both known germline pathogenic and novel variants.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/1078-0432.CCR-20-0638DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7501190PMC
September 2020

Quantification of ongoing APOBEC3A activity in tumor cells by monitoring RNA editing at hotspots.

Nat Commun 2020 06 12;11(1):2971. Epub 2020 Jun 12.

Department of Biological Chemistry, Center for Epigenetics and Metabolism, Chao Family Comprehensive Cancer Center, University of California Irvine, Irvine, CA, USA.

APOBEC3A is a cytidine deaminase driving mutagenesis, DNA replication stress and DNA damage in cancer cells. While the APOBEC3A-induced vulnerability of cancers offers an opportunity for therapy, APOBEC3A protein and mRNA are difficult to quantify in tumors due to their low abundance. Here, we describe a quantitative and sensitive assay to measure the ongoing activity of APOBEC3A in tumors. Using hotspot RNA mutations identified from APOBEC3A-positive tumors and droplet digital PCR, we develop an assay to quantify the RNA-editing activity of APOBEC3A. This assay is superior to APOBEC3A protein- and mRNA-based assays in predicting the activity of APOBEC3A on DNA. Importantly, we demonstrate that the RNA mutation-based APOBEC3A assay is applicable to clinical samples from cancer patients. Our study presents a strategy to follow the dysregulation of APOBEC3A in tumors, providing opportunities to investigate the role of APOBEC3A in tumor evolution and to target the APOBEC3A-induced vulnerability in therapy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-020-16802-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7293259PMC
June 2020

mC modification of mRNA serves a DNA damage code to promote homologous recombination.

Nat Commun 2020 06 5;11(1):2834. Epub 2020 Jun 5.

Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, UPMC Hillman Cancer Center, 5117 Centre Ave., Pittsburgh, PA, 15213, USA.

Recruitment of DNA repair proteins to DNA damage sites is a critical step for DNA repair. Post-translational modifications of proteins at DNA damage sites serve as DNA damage codes to recruit specific DNA repair factors. Here, we show that mRNA is locally modified by mC at sites of DNA damage. The RNA methyltransferase TRDMT1 is recruited to DNA damage sites to promote mC induction. Loss of TRDMT1 compromises homologous recombination (HR) and increases cellular sensitivity to DNA double-strand breaks (DSBs). In the absence of TRDMT1, RAD51 and RAD52 fail to localize to sites of reactive oxygen species (ROS)-induced DNA damage. In vitro, RAD52 displays an increased affinity for DNA:RNA hybrids containing mC-modified RNA. Loss of TRDMT1 in cancer cells confers sensitivity to PARP inhibitors in vitro and in vivo. These results reveal an unexpected TRDMT1-mC axis that promotes HR, suggesting that post-transcriptional modifications of RNA can also serve as DNA damage codes to regulate DNA repair.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-020-16722-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7275041PMC
June 2020

Alternative lengthening of telomeres: from molecular mechanisms to therapeutic outlooks.

Cell Biosci 2020 10;10:30. Epub 2020 Mar 10.

Massachusetts General Hospital Cancer Center, Harvard Medical School, Charlestown, MA 02129 USA.

To escape replicative senescence, cancer cells have to overcome telomere attrition during DNA replication. Most of cancers rely on telomerase to extend and maintain telomeres, but 4-11% of cancers use a homologous recombination-based pathway called alternative lengthening of telomeres (ALT). ALT is prevalent in cancers from the mesenchymal origin and usually associates with poor clinical outcome. Given its critical role in protecting telomeres and genomic integrity in tumor cells, ALT is an Achilles heel of tumors and an attractive target for cancer therapy. Here, we review the recent progress in the mechanistic studies of ALT, and discuss the emerging therapeutic strategies to target ALT-positive cancers.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s13578-020-00391-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7063710PMC
March 2020

An R-loop-initiated CSB-RAD52-POLD3 pathway suppresses ROS-induced telomeric DNA breaks.

Nucleic Acids Res 2020 02;48(3):1285-1300

Massachusetts General Hospital Cancer Center, Harvard Medical School, Charlestown, MA 02129, USA.

Reactive oxygen species (ROS) inflict multiple types of lesions in DNA, threatening genomic integrity. How cells respond to ROS-induced DNA damage at telomeres is still largely unknown. Here, we show that ROS-induced DNA damage at telomeres triggers R-loop accumulation in a TERRA- and TRF2-dependent manner. Both ROS-induced single- and double-strand DNA breaks (SSBs and DSBs) contribute to R-loop induction, promoting the localization of CSB and RAD52 to damaged telomeres. RAD52 is recruited to telomeric R-loops through its interactions with both CSB and DNA:RNA hybrids. Both CSB and RAD52 are required for the efficient repair of ROS-induced telomeric DSBs. The function of RAD52 in telomere repair is dependent on its ability to bind and recruit POLD3, a protein critical for break-induced DNA replication (BIR). Thus, ROS-induced telomeric R-loops promote repair of telomeric DSBs through CSB-RAD52-POLD3-mediated BIR, a previously unknown pathway protecting telomeres from ROS. ROS-induced telomeric SSBs may not only give rise to DSBs indirectly, but also promote DSB repair by inducing R-loops, revealing an unexpected interplay between distinct ROS-induced DNA lesions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gkz1114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7026659PMC
February 2020

Induction of BRCAness in Triple-Negative Breast Cancer by a CDK12/13 Inhibitor Improves Chemotherapy.

Cancer Cell 2019 11;36(5):461-463

Massachusetts General Hospital Cancer Center, Harvard Medical School, Charlestown, MA 02129, USA; Department of Pathology, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA. Electronic address:

In this issue of Cancer Cell, Quereda and colleagues report that a newly developed specific inhibitor of CDK12/13, SR-4835, sensitizes triple-negative breast cancer cells to PARP inhibitors and DNA-damaging chemotherapeutics by reducing expression of the genes in the DNA damage response pathway.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ccell.2019.10.012DOI Listing
November 2019

ATR Protects the Genome against R Loops through a MUS81-Triggered Feedback Loop.

Mol Cell 2020 02 7;77(3):514-527.e4. Epub 2019 Nov 7.

Massachusetts General Hospital Cancer Center, Harvard Medical School, Charlestown, MA 02129, USA; Department of Pathology, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA. Electronic address:

R loops arising during transcription induce genomic instability, but how cells respond to the R loop-associated genomic stress is still poorly understood. Here, we show that cells harboring high levels of R loops rely on the ATR kinase for survival. In response to aberrant R loop accumulation, the ataxia telangiectasia and Rad3-related (ATR)-Chk1 pathway is activated by R loop-induced reversed replication forks. In contrast to the activation of ATR by replication inhibitors, R loop-induced ATR activation requires the MUS81 endonuclease. ATR protects the genome from R loops by suppressing transcription-replication collisions, promoting replication fork recovery, and enforcing a G2/M cell-cycle arrest. Furthermore, ATR prevents excessive cleavage of reversed forks by MUS81, revealing a MUS81-triggered and ATR-mediated feedback loop that fine-tunes MUS81 activity at replication forks. These results suggest that ATR is a key sensor and suppressor of R loop-induced genomic instability, uncovering a signaling circuitry that safeguards the genome against R loops.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molcel.2019.10.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7007873PMC
February 2020

The CHK1 Inhibitor Prexasertib Exhibits Monotherapy Activity in High-Grade Serous Ovarian Cancer Models and Sensitizes to PARP Inhibition.

Clin Cancer Res 2019 10 13;25(20):6127-6140. Epub 2019 Aug 13.

Center for DNA Damage and Repair, Dana-Farber Cancer Institute, Boston, Massachusetts.

Purpose: PARP inhibitors are approved for the treatment of high-grade serous ovarian cancers (HGSOC). Therapeutic resistance, resulting from restoration of homologous recombination (HR) repair or replication fork stabilization, is a pressing clinical problem. We assessed the activity of prexasertib, a checkpoint kinase 1 (CHK1) inhibitor known to cause replication catastrophe, as monotherapy and in combination with the PARP inhibitor olaparib in preclinical models of HGSOC, including those with acquired PARP inhibitor resistance.

Experimental Design: Prexasertib was tested as a single agent or in combination with olaparib in 14 clinically annotated and molecularly characterized luciferized HGSOC patient-derived xenograft (PDX) models and in a panel of ovarian cancer cell lines. The ability of prexasertib to impair HR repair and replication fork stability was also assessed.

Results: Prexasertib monotherapy demonstrated antitumor activity across the 14 PDX models. Thirteen models were resistant to olaparib monotherapy, including 4 carrying mutation. The combination of olaparib with prexasertib was synergistic and produced significant tumor growth inhibition in an olaparib-resistant model and further augmented the degree and durability of response in the olaparib-sensitive model. HGSOC cell lines, including those with acquired PARP inhibitor resistance, were also sensitive to prexasertib, associated with induction of DNA damage and replication stress. Prexasertib also sensitized these cell lines to PARP inhibition and compromised both HR repair and replication fork stability.

Conclusions: Prexasertib exhibits monotherapy activity in PARP inhibitor-resistant HGSOC PDX and cell line models, reverses restored HR and replication fork stability, and synergizes with PARP inhibition.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/1078-0432.CCR-19-0448DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6801076PMC
October 2019

Loss of Slug Compromises DNA Damage Repair and Accelerates Stem Cell Aging in Mammary Epithelium.

Cell Rep 2019 07;28(2):394-407.e6

Department of Developmental, Molecular, & Chemical Biology, Sackler School of Graduate Biomedical Sciences, Tufts University, Boston, MA 02111, USA; Raymond and Beverly Sackler Convergence Laboratory, Tufts University School of Medicine, Boston, MA 02111, USA. Electronic address:

DNA damage activates checkpoints that limit the replicative potential of stem cells, including differentiation. These checkpoints protect against cancer development but also promote tissue aging. Because mice lacking Slug/Snai2 exhibit limited stem cell activity, including luminobasal differentiation, and are protected from mammary cancer, we reasoned that Slug might regulate DNA damage checkpoints in mammary epithelial cells. Here, we show that Slug facilitates efficient execution of RPA32-mediated DNA damage response (DDR) signaling. Slug deficiency leads to delayed phosphorylation of ataxia telangiectasia mutated and Rad3-related protein (ATR) and its effectors RPA32 and CHK1. This leads to impaired RAD51 recruitment to DNA damage sites and persistence of unresolved DNA damage. In vivo, Slug/Snai2 loss leads to increased DNA damage and premature aging of mammary epithelium. Collectively, our work demonstrates that the mammary stem cell regulator Slug controls DDR checkpoints by dually inhibiting differentiation and facilitating DDR repair, and its loss causes unresolved DNA damage and accelerated aging.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2019.06.043DOI Listing
July 2019

Myc targeted CDK18 promotes ATR and homologous recombination to mediate PARP inhibitor resistance in glioblastoma.

Nat Commun 2019 07 2;10(1):2910. Epub 2019 Jul 2.

Molecular Neurosurgery Laboratory and the Brain Tumor Research Center and Department of Neurosurgery, Massachusetts General Hospital and Harvard Medical School, Boston, 02114, MA, USA.

PARP inhibitors (PARPis) have clinical efficacy in BRCA-deficient cancers, but not BRCA-intact tumors, including glioblastoma (GBM). We show that MYC or MYCN amplification in patient-derived glioblastoma stem-like cells (GSCs) generates sensitivity to PARPi via Myc-mediated transcriptional repression of CDK18, while most tumors without amplification are not sensitive. In response to PARPi, CDK18 facilitates ATR activation by interacting with ATR and regulating ATR-Rad9/ATR-ETAA1 interactions; thereby promoting homologous recombination (HR) and PARPi resistance. CDK18 knockdown or ATR inhibition in GSCs suppressed HR and conferred PARPi sensitivity, with ATR inhibitors synergizing with PARPis or sensitizing GSCs. ATR inhibitor VE822 combined with PARPi extended survival of mice bearing GSC-derived orthotopic tumors, irrespective of PARPi-sensitivity. These studies identify a role of CDK18 in ATR-regulated HR. We propose that combined blockade of ATR and PARP is an effective strategy for GBM, even for low-Myc GSCs that do not respond to PARPi alone, and potentially other PARPi-refractory tumors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-019-10993-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6606647PMC
July 2019

Passenger hotspot mutations in cancer driven by APOBEC3A and mesoscale genomic features.

Science 2019 06;364(6447)

Massachusetts General Hospital Cancer Center, Harvard Medical School, Boston, MA, USA.

Cancer drivers require statistical modeling to distinguish them from passenger events, which accumulate during tumorigenesis but provide no fitness advantage to cancer cells. The discovery of driver genes and mutations relies on the assumption that exact positional recurrence is unlikely by chance; thus, the precise sharing of mutations across patients identifies drivers. Examining the mutation landscape in cancer genomes, we found that many recurrent cancer mutations previously designated as drivers are likely passengers. Our integrated bioinformatic and biochemical analyses revealed that these passenger hotspot mutations arise from the preference of APOBEC3A, a cytidine deaminase, for DNA stem-loops. Conversely, recurrent APOBEC-signature mutations not in stem-loops are enriched in well-characterized driver genes and may predict new drivers. This demonstrates that mesoscale genomic features need to be integrated into computational models aimed at identifying mutations linked to diseases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/science.aaw2872DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6731024PMC
June 2019

Calcium Influx Guards Replication Forks against Exonuclease 1.

Mol Cell 2019 06;74(6):1103-1105

Massachusetts General Hospital Cancer Center, Harvard Medical School, Charlestown, MA 02129, USA; Department of Pathology, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02115, USA. Electronic address:

In this issue, Li et al. (2019) report a previously unknown Ca-CaMKK2-AMPK signaling cascade that protects stalled forks from degradation by phosphorylating and inhibiting the EXO1 nuclease, revealing a surprising role for Ca influx in the maintenance of genomic stability.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molcel.2019.06.005DOI Listing
June 2019

Targeting R-loop-associated ATR response in myelodysplastic syndrome.

Oncotarget 2019 Apr 5;10(27):2581-2582. Epub 2019 Apr 5.

Lee Zou: Massachusetts General Hospital Cancer Center, Harvard Medical School, Charlestown, MA, USA; Department of Pathology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.18632/oncotarget.26851DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6498995PMC
April 2019

Analysis of DNA Damage Response Gene Alterations and Tumor Mutational Burden Across 17,486 Tubular Gastrointestinal Carcinomas: Implications for Therapy.

Oncologist 2019 10 30;24(10):1340-1347. Epub 2019 Apr 30.

Foundation Medicine, Inc., Cambridge, Massachusetts, USA.

Background: Alterations in the DNA damage response (DDR) pathway confer sensitivity to certain chemotherapies, radiation, and other DNA damage repair targeted therapies. are the most well-studied DDR genes, but recurrent alterations are described in other DDR pathway members across cancers. Deleterious DDR alterations may sensitize tumor cells to poly (ADP-ribose) polymerase inhibition, but there are also increasing data suggesting that there may also be synergy with immune checkpoint inhibitors. The relevance of DDR defects in gastrointestinal (GI) cancers is understudied. We sought to characterize DDR-defective GI malignancies and to explore genomic context and tumor mutational burden (TMB) to provide a platform for future rational investigations.

Materials And Methods: Tumor samples from 17,486 unique patients with advanced colorectal, gastroesophageal, or small bowel carcinomas were assayed using hybrid-capture-based comprehensive genomic profiling including sequencing of 10 predefined DDR genes: , , , , , , , and . TMB (mutations per megabase [mut/Mb]) was calculated from up to 1.14 Mb of sequenced DNA. Clinicopathologic features were extracted and descriptive statistics were used to explore genomic relationships among identified subgroups.

Results: DDR alterations were found in 17% of cases: gastric adenocarcinoma 475/1,750 (27%), small bowel adenocarcinoma 148/666 (22%), esophageal adenocarcinoma 467/2,501 (19%), and colorectal cancer 1,824/12,569 (15%). (9.2%) and (4.7%) were the most commonly altered DDR genes in this series, followed by (2.3%), (1.1%), (1.0%), (0.8%), (0.7%), (0.6%), (0.1%) and (0.1%). More than one DDR gene alteration was found in 24% of cases. High microsatellite instability (MSI-H) and high TMB (TMB-H, ≥20 mut/Mb) were found in 19% and 21% of DDR-altered cases, respectively. Of DDR-altered/TMB-H cases, 87% were also MSI-H. However, even in the microsatellite stable (MSS)/DDR-wild-type (WT) versus MSS/DDR-altered, TMB-high was seen more frequently (0.4% vs. 3.3%, < .00001.) Median TMB was 5.4 mut/Mb in the MSS/DDR-altered subset versus 3.8 mut/Mb in the MSS/DDR-WT subset ( ≤ .00001), and alterations were enriched in the MSS/TMB-high cases.

Conclusion: This is the largest study to examine selected DDR defects in tubular GI cancers and confirms that DDR defects are relatively common and that there is an association between the selected DDR defects and a high TMB in more than 20% of cases. Microsatellite stable DDR-defective tumors with elevated TMB warrant further exploration.

Implications For Practice: Deleterious DNA damage response (DDR) alterations may sensitize tumor cells to poly (ADP-ribose) polymerase inhibition, but also potentially to immune checkpoint inhibitors, owing to accumulation of mutations in DDR-defective tumors. The relevance of DDR defects in gastrointestinal (GI) cancers is understudied. This article characterizes DDR-defective GI malignancies and explores genomic context and tumor mutational burden to provide a platform for future rational investigations.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1634/theoncologist.2019-0034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6795150PMC
October 2019

Critical questions in ovarian cancer research and treatment: Report of an American Association for Cancer Research Special Conference.

Cancer 2019 06 5;125(12):1963-1972. Epub 2019 Mar 5.

University of Texas MD Anderson Cancer Center, Houston, Texas.

Substantial progress has been made in understanding ovarian cancer at the molecular and cellular level. Significant improvement in 5-year survival has been achieved through cytoreductive surgery, combination platinum-based chemotherapy, and more effective treatment of recurrent cancer, and there are now more than 280,000 ovarian cancer survivors in the United States. Despite these advances, long-term survival in late-stage disease has improved little over the last 4 decades. Poor outcomes relate, in part, to late stage at initial diagnosis, intrinsic drug resistance, and the persistence of dormant drug-resistant cancer cells after primary surgery and chemotherapy. Our ability to accelerate progress in the clinic will depend on the ability to answer several critical questions regarding this disease. To assess current answers, an American Association for Cancer Research Special Conference on "Critical Questions in Ovarian Cancer Research and Treatment" was held in Pittsburgh, Pennsylvania, on October 1-3, 2017. Although clinical, translational, and basic investigators conducted much of the discussion, advocates participated in the meeting, and many presentations were directly relevant to patient care, including treatment with poly adenosine diphosphate ribose polymerase (PARP) inhibitors, attempts to improve immunotherapy by overcoming the immune suppressive effects of the microenvironment, and a better understanding of the heterogeneity of the disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/cncr.32004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6557260PMC
June 2019
-->