Publications by authors named "Dipanjan Chowdhury"

80 Publications

TIRR inhibits the 53BP1-p53 complex to alter cell-fate programs.

Mol Cell 2021 Apr 29. Epub 2021 Apr 29.

Department of Radiation Oncology, Dana-Farber Cancer Institute, Boston, MA 02115, USA; Department of Biological Chemistry & Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA; Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA. Electronic address:

53BP1 influences genome stability via two independent mechanisms: (1) regulating DNA double-strand break (DSB) repair and (2) enhancing p53 activity. We discovered a protein, Tudor-interacting repair regulator (TIRR), that associates with the 53BP1 Tudor domain and prevents its recruitment to DSBs. Here, we elucidate how TIRR affects 53BP1 function beyond its recruitment to DSBs and biochemically links the two distinct roles of 53BP1. Loss of TIRR causes an aberrant increase in the gene transactivation function of p53, affecting several p53-mediated cell-fate programs. TIRR inhibits the complex formation between the Tudor domain of 53BP1 and a dimethylated form of p53 (K382me2) that is poised for transcriptional activation of its target genes. TIRR mRNA expression levels negatively correlate with the expression of key p53 target genes in breast and prostate cancers. Further, TIRR loss is selectively not tolerated in p53-proficient tumors. Therefore, we establish that TIRR is an important inhibitor of the 53BP1-p53 complex.
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http://dx.doi.org/10.1016/j.molcel.2021.03.039DOI Listing
April 2021

CHK1 Inhibitor Blocks Phosphorylation of FAM122A and Promotes Replication Stress.

Mol Cell 2020 11 26;80(3):410-422.e6. Epub 2020 Oct 26.

Department of Radiation Oncology, Dana-Farber Cancer Institute, Boston, MA 02215, USA; Center for DNA Damage and Repair, Dana-Farber Cancer Institute, Boston, MA 02215, USA. Electronic address:

While effective anti-cancer drugs targeting the CHK1 kinase are advancing in the clinic, drug resistance is rapidly emerging. Here, we demonstrate that CRISPR-mediated knockout of the little-known gene FAM122A/PABIR1 confers cellular resistance to CHK1 inhibitors (CHK1is) and cross-resistance to ATR inhibitors. Knockout of FAM122A results in activation of PP2A-B55α, a phosphatase that dephosphorylates the WEE1 protein and rescues WEE1 from ubiquitin-mediated degradation. The resulting increase in WEE1 protein expression reduces replication stress, activates the G2/M checkpoint, and confers cellular resistance to CHK1is. Interestingly, in tumor cells with oncogene-driven replication stress, CHK1 can directly phosphorylate FAM122A, leading to activation of the PP2A-B55α phosphatase and increased WEE1 expression. A combination of a CHK1i plus a WEE1 inhibitor can overcome CHK1i resistance of these tumor cells, thereby enhancing anti-cancer activity. The FAM122A expression level in a tumor cell can serve as a useful biomarker for predicting CHK1i sensitivity or resistance.
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http://dx.doi.org/10.1016/j.molcel.2020.10.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7761918PMC
November 2020

Tumor protein expression of the DNA repair gene BRCA1 and lethal prostate cancer.

Carcinogenesis 2020 07;41(7):904-908

Department of Epidemiology, Harvard T.H. Chan School of Public Health, Boston, MA, USA.

DNA repair genes are commonly altered in metastatic prostate cancer, but BRCA1 mutations are rare. Preliminary studies suggest that higher tumor expression of the BRCA1 protein may be associated with worse prognosis. We undertook a prospective study among men with prostate cancer in the Health Professionals Follow-up Study and evaluated BRCA1 via immunohistochemical staining on tissue microarrays. BRCA1 was expressed in 60 of 589 tumors. Prevalence of BRCA1 positivity was 43% in the 14 men with metastases at diagnosis compared with 9% in non-metastatic tumors [difference, 33 percentage points; 95% confidence interval (CI), 7-59]. BRCA1-positive tumors had 2.16-fold higher Ki-67 proliferative indices (95% CI, 1.18-3.95), higher tumor aneuploidy as predicted from whole-transcriptome profiling, and higher Gleason scores. Among the 575 patients with non-metastatic disease at diagnosis, we evaluated the association between BRCA1 expression and development of lethal disease (metastasis or cancer-specific death, 69 events) during long-term follow-up (median, 18.3 years). A potential weak association of BRCA1 positivity with lethal disease (hazard ratio, 1.61; 95% CI, 0.82-3.15) was attenuated when adjusting for age, Gleason score and clinical stage (hazard ratio, 1.11; 95% CI, 0.54-2.29). In summary, BRCA1 protein expression is a feature of more proliferative and more aneuploid prostate tumors and is more common in metastatic disease. While not well suited as a prognostic biomarker in primary prostate cancer, BRCA1 protein expression may be most relevant in advanced disease.
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http://dx.doi.org/10.1093/carcin/bgaa061DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7359768PMC
July 2020

Berzosertib plus gemcitabine versus gemcitabine alone in platinum-resistant high-grade serous ovarian cancer: a multicentre, open-label, randomised, phase 2 trial.

Lancet Oncol 2020 07 15;21(7):957-968. Epub 2020 Jun 15.

Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA.

Background: High-grade serous ovarian cancers show increased replication stress, rendering cells vulnerable to ATR inhibition because of near universal loss of the G1/S checkpoint (through deleterious TP53 mutations), premature S phase entry (due to CCNE1 amplification, RB1 loss, or CDKN2A mRNA downregulation), alterations of homologous recombination repair genes, and expression of oncogenic drivers (through MYC amplification and other mechanisms). We hypothesised that the combination of the selective ATR inhibitor, berzosertib, and gemcitabine could show acceptable toxicity and superior efficacy to gemcitabine alone in high-grade serous ovarian cancer.

Methods: In this multicentre, open-label, randomised, phase 2 study, 11 different centres in the US Experimental Therapeutics Clinical Trials Network enrolled women (aged ≥18 years) with recurrent, platinum-resistant high-grade serous ovarian cancer (determined histologically) and Eastern Cooperative Oncology Group performance status of 0 or 1, who had unlimited previous lines of cytotoxic therapy in the platinum-sensitive setting but no more than one line of cytotoxic therapy in the platinum-resistant setting. Eligible patients were randomly assigned (1:1) to receive intravenous gemcitabine (1000 mg/m) on day 1 and day 8, or gemcitabine plus intravenous berzosertib (210 mg/m) on day 2 and day 9 of a 21-day cycle until disease progression or intolerable toxicity. Randomisation was done centrally using the Theradex Interactive Web Response System, stratified by platinum-free interval, and with a permuted block size of six. Following central randomisation, patients and investigators were not masked to treatment assignment. The primary endpoint was investigator-assessed progression-free survival, and analyses included all patients who received at least one dose of the study drugs. The study is registered with ClinicalTrials.gov, NCT02595892, and is active but closed to enrolment.

Findings: Between Feb 14, 2017, and Sept 7, 2018, 88 patients were assessed for eligibility, of whom 70 were randomly assigned to treatment with gemcitabine alone (36 patients) or gemcitabine plus berzosertib (34 patients). At the data cutoff date (Feb 21, 2020), the median follow-up was 53·2 weeks (25·6-81·8) in the gemcitabine plus berzosertib group and 43·0 weeks (IQR 23·2-69·1) in the gemcitabine alone group. Median progression-free survival was 22·9 weeks (17·9-72·0) for gemcitabine plus berzosertib and 14·7 weeks (90% CI 9·7-36·7) for gemcitabine alone (hazard ratio 0·57, 90% CI 0·33-0·98; one-sided log-rank test p=0·044). The most common treatment-related grade 3 or 4 adverse events were decreased neutrophil count (14 [39%] of 36 patients in the gemcitabine alone group vs 16 [47%] of 34 patients in the gemcitabine plus berzosertib group) and decreased platelet count (two [6%] vs eight [24%]). Serious adverse events were observed in ten (28%) patients in the gemcitabine alone group and nine (26%) patients in the gemcitabine plus berzosertib group. There was one treatment-related death in the gemcitabine alone group due to sepsis and one treatment-related death in the gemcitabine plus berzosertib group due to pneumonitis.

Interpretation: To our knowledge, this is the first randomised study of an ATR inhibitor in any tumour type. This study shows a benefit of adding berzosertib to gemcitabine in platinum-resistant high-grade serous ovarian cancer. This combination warrants further investigation in this setting.

Funding: US National Cancer Institute.
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http://dx.doi.org/10.1016/S1470-2045(20)30180-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8023719PMC
July 2020

Endogenous oxidized DNA bases and APE1 regulate the formation of G-quadruplex structures in the genome.

Proc Natl Acad Sci U S A 2020 05 13;117(21):11409-11420. Epub 2020 May 13.

Department of Genetics, Cell Biology and Anatomy, College of Medicine, University of Nebraska Medical Center, Omaha, NE 68198;

Formation of G-quadruplex (G4) DNA structures in key regulatory regions in the genome has emerged as a secondary structure-based epigenetic mechanism for regulating multiple biological processes including transcription, replication, and telomere maintenance. G4 formation (folding), stabilization, and unfolding must be regulated to coordinate G4-mediated biological functions; however, how cells regulate the spatiotemporal formation of G4 structures in the genome is largely unknown. Here, we demonstrate that endogenous oxidized guanine bases in G4 sequences and the subsequent activation of the base excision repair (BER) pathway drive the spatiotemporal formation of G4 structures in the genome. Genome-wide mapping of occurrence of Apurinic/apyrimidinic (AP) site damage, binding of BER proteins, and G4 structures revealed that oxidized base-derived AP site damage and binding of OGG1 and APE1 are predominant in G4 sequences. Loss of APE1 abrogated G4 structure formation in cells, which suggests an essential role of APE1 in regulating the formation of G4 structures in the genome. Binding of APE1 to G4 sequences promotes G4 folding, and acetylation of APE1, which enhances its residence time, stabilizes G4 structures in cells. APE1 subsequently facilitates transcription factor loading to the promoter, providing mechanistic insight into the role of APE1 in G4-mediated gene expression. Our study unravels a role of endogenous oxidized DNA bases and APE1 in controlling the formation of higher-order DNA secondary structures to regulate transcription beyond its well-established role in safeguarding the genomic integrity.
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http://dx.doi.org/10.1073/pnas.1912355117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7260947PMC
May 2020

Immunogenomic profiling determines responses to combined PARP and PD-1 inhibition in ovarian cancer.

Nat Commun 2020 03 19;11(1):1459. Epub 2020 Mar 19.

Dana-Farber Cancer Institute, 450 Brookline Avenue, Boston, MA, 02215, USA.

Combined PARP and immune checkpoint inhibition has yielded encouraging results in ovarian cancer, but predictive biomarkers are lacking. We performed immunogenomic profiling and highly multiplexed single-cell imaging on tumor samples from patients enrolled in a Phase I/II trial of niraparib and pembrolizumab in ovarian cancer (NCT02657889). We identify two determinants of response; mutational signature 3 reflecting defective homologous recombination DNA repair, and positive immune score as a surrogate of interferon-primed exhausted CD8 + T-cells in the tumor microenvironment. Presence of one or both features associates with an improved outcome while concurrent absence yields no responses. Single-cell spatial analysis reveals prominent interactions of exhausted CD8 + T-cells and PD-L1 + macrophages and PD-L1 + tumor cells as mechanistic determinants of response. Furthermore, spatial analysis of two extreme responders shows differential clustering of exhausted CD8 + T-cells with PD-L1 + macrophages in the first, and exhausted CD8 + T-cells with cancer cells harboring genomic PD-L1 and PD-L2 amplification in the second.
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http://dx.doi.org/10.1038/s41467-020-15315-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7081234PMC
March 2020

A systemic approach to screening high-throughput RT-qPCR data for a suitable set of reference circulating miRNAs.

BMC Genomics 2020 Jan 31;21(1):111. Epub 2020 Jan 31.

Department of Biostatistics and Translational Medicine, Medical University of Lodz, Lodz, Poland.

Background: The consensus on how to choose a reference gene for serum or plasma miRNA expression qPCR studies has not been reached and none of the potential candidates have yet been convincingly validated. We proposed a new in silico approach of finding a suitable reference for human, circulating miRNAs and identified a new set of endogenous reference miRNA based on miRNA profiling experiments from Gene Expression Omnibus. We used 3 known normalization algorithms (NormFinder, BestKeeper, GeNorm) to calculate a new normalization score. We searched for a universal set of endogenous miRNAs and validated our findings on 2 new datasets using our approach.

Results: We discovered and validated a set of 13 miRNAs (miR-222, miR-92a, miR-27a, miR-17, miR-24, miR-320a, miR-25, miR-126, miR-19b, miR-199a-3p, miR-30b, miR-30c, miR-374a) that can be used to create a reliable reference combination of 3 miRNAs. We showed that on average the mean of 3 miRNAs (p = 0.0002) and 2 miRNAs (p = 0.0031) were a better reference than single miRNA. The arithmetic means of 3 miRNAs: miR-24, miR-222 and miR-27a was shown to be the most stable combination of 3 miRNAs in validation sets.

Conclusions: No single miRNA was suitable as a universal reference in serum miRNA qPCR profiling, but it was possible to designate a set of miRNAs, which consistently contributed to most stable combinations.
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http://dx.doi.org/10.1186/s12864-020-6530-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6995162PMC
January 2020

TRIP13 regulates DNA repair pathway choice through REV7 conformational change.

Nat Cell Biol 2020 01 8;22(1):87-96. Epub 2020 Jan 8.

Department of Radiation Oncology, Dana-Farber Cancer Institute, Boston, MA, USA.

DNA double-strand breaks (DSBs) are repaired through homology-directed repair (HDR) or non-homologous end joining (NHEJ). BRCA1/2-deficient cancer cells cannot perform HDR, conferring sensitivity to poly(ADP-ribose) polymerase inhibitors (PARPi). However, concomitant loss of the pro-NHEJ factors 53BP1, RIF1, REV7-Shieldin (SHLD1-3) or CST-DNA polymerase alpha (Pol-α) in BRCA1-deficient cells restores HDR and PARPi resistance. Here, we identify the TRIP13 ATPase as a negative regulator of REV7. We show that REV7 exists in active 'closed' and inactive 'open' conformations, and TRIP13 catalyses the inactivating conformational change, thereby dissociating REV7-Shieldin to promote HDR. TRIP13 similarly disassembles the REV7-REV3 translesion synthesis (TLS) complex, a component of the Fanconi anaemia pathway, inhibiting error-prone replicative lesion bypass and interstrand crosslink repair. Importantly, TRIP13 overexpression is common in BRCA1-deficient cancers, confers PARPi resistance and correlates with poor prognosis. Thus, TRIP13 emerges as an important regulator of DNA repair pathway choice-promoting HDR, while suppressing NHEJ and TLS.
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http://dx.doi.org/10.1038/s41556-019-0442-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7336368PMC
January 2020

CDK phosphorylation of TRF2 controls t-loop dynamics during the cell cycle.

Nature 2019 11 13;575(7783):523-527. Epub 2019 Nov 13.

The Francis Crick Institute, London, UK.

The protection of telomere ends by the shelterin complex prevents DNA damage signalling and promiscuous repair at chromosome ends. Evidence suggests that the 3' single-stranded telomere end can assemble into a lasso-like t-loop configuration, which has been proposed to safeguard chromosome ends from being recognized as DNA double-strand breaks. Mechanisms must also exist to transiently disassemble t-loops to allow accurate telomere replication and to permit telomerase access to the 3' end to solve the end-replication problem. However, the regulation and physiological importance of t-loops in the protection of telomere ends remains unknown. Here we identify a CDK phosphorylation site in the shelterin subunit at Ser365 of TRF2, whose dephosphorylation in S phase by the PP6R3 phosphatase provides a narrow window during which the RTEL1 helicase can transiently access and unwind t-loops to facilitate telomere replication. Re-phosphorylation of TRF2 at Ser365 outside of S phase is required to release RTEL1 from telomeres, which not only protects t-loops from promiscuous unwinding and inappropriate activation of ATM, but also counteracts replication conflicts at DNA secondary structures that arise within telomeres and across the genome. Hence, a phospho-switch in TRF2 coordinates the assembly and disassembly of t-loops during the cell cycle, which protects telomeres from replication stress and an unscheduled DNA damage response.
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http://dx.doi.org/10.1038/s41586-019-1744-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6874499PMC
November 2019

Circulating microRNAs as Biomarkers of Radiation Exposure: A Systematic Review and Meta-Analysis.

Int J Radiat Oncol Biol Phys 2020 02 23;106(2):390-402. Epub 2019 Oct 23.

Department of Biostatistics and Translational Medicine, Medical University of Lodz, Lodz, Poland; Department of Radiation Oncology, Harvard Medical School, Dana-Farber Cancer Institute, Boston, Massachusetts. Electronic address:

Purpose: MicroRNAs (miRNAs) were hypothesized to be robust and easily measured biomarkers of radiation exposure, which has led to multiple studies in various clinical and experimental scenarios. We sought to identify evolutionary conserved, radiation-induced circulating miRNAs through a multispecies, integrative systematic review and meta-analysis of miRNAs in radiation.

Methods And Materials: The systematic review was registered in the PROSPERO database (ID: 81701). We downloaded a list of studies with the query: (circulating OR plasma OR serum) AND (miRNA or microRNA) AND (radiat* OR radiotherapy OR irradiati*) from MEDLINE (103 studies), EMBASE (364 studies), and Cochrane Database of Systematic Reviews (0 studies). After deleting 116 duplicates, the remaining 351 abstracts were reviewed. Inclusion criteria were experimental study; human, mice, rat or nonhuman primate study; and serum or plasma miRNA expression measured before and after radiation exposure.

Results: The screening procedure yielded 62 research studies. After verification, 30 articles contained data on miRNA expression change after irradiation. Thus, we obtained a database of 131 miRNAs from 96 pairwise post-/preirradiation comparisons reporting 2508 fold changes (FCs) of circulating miRNAs. The meta-analysis showed 28 miRNAs with significant radiation-induced change of their expression in the serum. In metaregression analysis, 7 miRNAs-miR-150 (FC = 0.40; 95% confidence interval [CI], 0.35-0.45), miR-29a (FC = 0.87; 95% CI, 0.79-0.96), miR-29b (FC = 0.85; 95% CI, 0.76-0.96), miR-30c (FC = 1.19; 95% CI, 1.09-1.30), miR-200b (FC = 1.34; 95% CI, 1.21-1.48), miR-320a (FC = 1.13; 95% CI, 1.05-1.23), and miR-30a (FC = 1.18; 95% CI, 1.07-1.30)-significantly correlated with either total or fraction dose of radiation. Additionally, miR-150, miR-320a, miR-200b, and miR-30c correlated significantly with time elapsed since irradiation.

Conclusions: Circulating miRNAs reflect the impact of ionizing radiation irrespective of the studied species, often in a dose-dependent manner. This makes circulating miRNAs promising biomarkers of radiation exposure.
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http://dx.doi.org/10.1016/j.ijrobp.2019.10.028DOI Listing
February 2020

A mitotic CDK5-PP4 phospho-signaling cascade primes 53BP1 for DNA repair in G1.

Nat Commun 2019 09 18;10(1):4252. Epub 2019 Sep 18.

Division of Radiation and Genome Stability, Department of Radiation Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, 02215, USA.

Mitotic cells attenuate the DNA damage response (DDR) by phosphorylating 53BP1, a critical DDR mediator, to prevent its localization to damaged chromatin. Timely dephosphorylation of 53BP1 is critical for genome integrity, as premature recruitment of 53BP1 to DNA lesions impairs mitotic fidelity. Protein phosphatase 4 (PP4) dephosphorylates 53BP1 in late mitosis to allow its recruitment to DNA lesions in G1. How cells appropriately dephosphorylate 53BP1, thereby restoring DDR, is unclear. Here, we elucidate the underlying mechanism of kinetic control of 53BP1 dephosphorylation in mitosis. We demonstrate that CDK5, a kinase primarily functional in post-mitotic neurons, is active in late mitotic phases in non-neuronal cells and directly phosphorylates PP4R3β, the PP4 regulatory subunit that recognizes 53BP1. Specific inhibition of CDK5 in mitosis abrogates PP4R3β phosphorylation and abolishes its recognition and dephosphorylation of 53BP1, ultimately preventing the localization of 53BP1 to damaged chromatin. Our results establish CDK5 as a regulator of 53BP1 recruitment.
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http://dx.doi.org/10.1038/s41467-019-12084-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6751209PMC
September 2019

Identification of LIMK2 as a therapeutic target in castration resistant prostate cancer.

Cancer Lett 2019 04 1;448:182-196. Epub 2019 Feb 1.

Department of Chemistry and Purdue University Center for Cancer Research, 560 Oval Drive, West Lafayette, IN, 47907, USA. Electronic address:

This study identified LIMK2 kinase as a disease-specific target in castration resistant prostate cancer (CRPC) pathogenesis, which is upregulated in response to androgen deprivation therapy, the current standard of treatment for prostate cancer. Surgical castration increases LIMK2 expression in mouse prostates due to increased hypoxia. Similarly, human clinical specimens showed highest LIMK2 levels in CRPC tissues compared to other stages, while minimal LIMK2 was observed in normal prostates. Most notably, inducible knockdown of LIMK2 fully reverses CRPC tumorigenesis in castrated mice, underscoring its potential as a clinical target for CRPC. We also identified TWIST1 as a direct substrate of LIMK2, which uncovered the molecular mechanism of LIMK2-induced malignancy. TWIST1 is strongly associated with CRPC initiation, progression and poor prognosis. LIMK2 increases TWIST1 mRNA levels upon hypoxia; and stabilizes TWIST1 by direct phosphorylation. TWIST1 also stabilizes LIMK2 by inhibiting its ubiquitylation. Phosphorylation-dead TWIST1 acts as dominant negative and fully prevents EMT and tumor formation in vivo, thereby highlighting the significance of LIMK2-TWIST1 signaling axis in CRPC. As LIMK2 null mice are viable, targeting LIMK2 should have minimal collateral toxicity, thereby improving the overall survival of CRPC patients.
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http://dx.doi.org/10.1016/j.canlet.2019.01.035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7079209PMC
April 2019

DYNLL1 binds to MRE11 to limit DNA end resection in BRCA1-deficient cells.

Nature 2018 11 31;563(7732):522-526. Epub 2018 Oct 31.

Division of Radiation and Genome Stability, Department of Radiation Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA.

Limited DNA end resection is the key to impaired homologous recombination in BRCA1-mutant cancer cells. Here, using a loss-of-function CRISPR screen, we identify DYNLL1 as an inhibitor of DNA end resection. The loss of DYNLL1 enables DNA end resection and restores homologous recombination in BRCA1-mutant cells, thereby inducing resistance to platinum drugs and inhibitors of poly(ADP-ribose) polymerase. Low BRCA1 expression correlates with increased chromosomal aberrations in primary ovarian carcinomas, and the junction sequences of somatic structural variants indicate diminished homologous recombination. Concurrent decreases in DYNLL1 expression in carcinomas with low BRCA1 expression reduced genomic alterations and increased homology at lesions. In cells, DYNLL1 limits nucleolytic degradation of DNA ends by associating with the DNA end-resection machinery (MRN complex, BLM helicase and DNA2 endonuclease). In vitro, DYNLL1 binds directly to MRE11 to limit its end-resection activity. Therefore, we infer that DYNLL1 is an important anti-resection factor that influences genomic stability and responses to DNA-damaging chemotherapy.
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http://dx.doi.org/10.1038/s41586-018-0670-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7155769PMC
November 2018

Potential of serum microRNAs as biomarkers of radiation injury and tools for individualization of radiotherapy.

Transl Res 2018 11 19;201:71-83. Epub 2018 Jun 19.

Department of Biostatistics and Translational Medicine, Medical University of Lodz, Lodz, Poland; Department of Radiation Oncology, Harvard Medical School, Dana-Farber Cancer Institute, Boston, Massachusetts, USA. Electronic address:

Due to tremendous technological advances, radiation oncologists are now capable of personalized treatment plans and deliver the dose in a highly precise manner. However, a crucial challenge is how to escalate radiation doses to cancer cells while reducing damage to surrounding healthy tissues. This determines the probability of achieving therapeutic success whilst safeguarding patients from complications. The current dose constraints rely on observational data. Therefore, incidental toxicity observed in a minority of patients limits the admissible dose thresholds for the whole population, theoretically narrowing down the curative potential of radiotherapy. Future tools for measurements of individual's radiosensitivity before and during treatment would allow proper treatment personalization. Variation in tissue tolerance is at least partially genetically-determined and recent progress in the field of molecular biology raises the possibility that novel assays will allow to predict the response to ionizing radiation. Recently, microRNAs have garnered interest as stable biomarkers of tumor radiation response and normal-tissue toxicity. Preclinical studies in mice and nonhuman primates have shown that serum circulating microRNAs can be used to accurately distinguish pre- and postirradiation states and predict the biological impact of high-dose irradiation. First reports from human studies are also encouraging, however biology-driven precision radiation oncology, which tailors treatment to individual patient's needs, still remains to be translated into clinical studies. In this review, we summarize current knowledge about the potential of serum microRNAs as biodosimeters and biomarkers for radiation injury to lung and hematopoietic cells.
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http://dx.doi.org/10.1016/j.trsl.2018.06.001DOI Listing
November 2018

Mechanism of 53BP1 activity regulation by RNA-binding TIRR and a designer protein.

Nat Struct Mol Biol 2018 07 2;25(7):591-600. Epub 2018 Jul 2.

Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN, USA.

Dynamic protein interaction networks such as DNA double-strand break (DSB) signaling are modulated by post-translational modifications. The DNA repair factor 53BP1 is a rare example of a protein whose post-translational modification-binding function can be switched on and off. 53BP1 is recruited to DSBs by recognizing histone lysine methylation within chromatin, an activity directly inhibited by the 53BP1-binding protein TIRR. X-ray crystal structures of TIRR and a designer protein bound to 53BP1 now reveal a unique regulatory mechanism in which an intricate binding area centered on an essential TIRR arginine residue blocks the methylated-chromatin-binding surface of 53BP1. A 53BP1 separation-of-function mutation that abolishes TIRR-mediated regulation in cells renders 53BP1 hyperactive in response to DSBs, highlighting the key inhibitory function of TIRR. This 53BP1 inhibition is relieved by TIRR-interacting RNA molecules, providing proof-of-principle of RNA-triggered 53BP1 recruitment to DSBs.
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http://dx.doi.org/10.1038/s41594-018-0083-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6045459PMC
July 2018

Dual HDAC and PI3K Inhibition Abrogates NFκB- and FOXM1-Mediated DNA Damage Response to Radiosensitize Pediatric High-Grade Gliomas.

Cancer Res 2018 07 14;78(14):4007-4021. Epub 2018 May 14.

Department of Radiation Oncology, Brigham and Women's Hospital, Dana-Farber Cancer Institute, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts.

Aberrant chromatin remodeling and activation of the PI3K pathway have been identified as important mediators of pediatric high-grade glioma (pHGG) and diffuse intrinsic pontine glioma (DIPG) pathogenesis. As inhibition of these pathways are promising therapeutic avenues and radiation is the only modality to prolong survival of patients with DIPG, we sought to explore radiosensitizing functions of such inhibition and to explore mechanisms of action of such agents. Here, we demonstrate that combined treatment with radiotherapy and CUDC-907, a novel first-in-class dual inhibitor of histone deacetylases (HDAC) and PI3K, evokes a potent cytotoxic response in pHGG and DIPG models. CUDC-907 modulated DNA damage response by inhibiting radiation-induced DNA repair pathways including homologous recombination and nonhomologous end joining. The radiosensitizing effects of CUDC-907 were mediated by decreased NFκB/Forkhead box M1 (FOXM1) recruitment to promoters of genes involved in the DNA damage response; exogenous expression of NFκB/FOXM1 protected from CUDC-907-induced cytotoxicity. Together, these findings reveal CUDC-907 as a novel radiosensitizer with potent antitumor activity in pHGG and DIPG and provide a preclinical rationale for the combination of CUDC-907 with radiotherapy as a novel therapeutic strategy against pHGG and DIPG. More globally, we have identified NFκB and FOXM1 and their downstream transcriptional elements as critical targets for new treatments for pHGG and DIPG. These findings describe the radiosensitizing effect of a novel agent in pediatric high-grade gliomas, addressing a critical unmet need of increasing the radiation sensitivity of these highly aggressive tumors. .
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http://dx.doi.org/10.1158/0008-5472.CAN-17-3691DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6294442PMC
July 2018

Multifaceted Impact of MicroRNA 493-5p on Genome-Stabilizing Pathways Induces Platinum and PARP Inhibitor Resistance in BRCA2-Mutated Carcinomas.

Cell Rep 2018 04;23(1):100-111

Department of Radiation Oncology, Division of Radiation and Genome Stability, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA. Electronic address:

BRCA1/2-mutated ovarian cancers (OCs) are defective in homologous recombination repair (HRR) of double-strand breaks (DSBs) and thereby sensitive to platinum and PARP inhibitors (PARPis). Multiple PARPis have recently received US Food and Drug Administration (FDA) approval for treatment of OCs, and resistance to PARPis is a major clinical problem. Utilizing primary and recurrent BRCA1/2-mutated carcinomas from OC patients, patient-derived lines, and an in vivo BRCA2-mutated mouse model, we identified a microRNA, miR-493-5p, that induced platinum/PARPi resistance exclusively in BRCA2-mutated carcinomas. However, in contrast to the most prevalent resistance mechanisms in BRCA mutant carcinomas, miR-493-5p did not restore HRR. Expression of miR-493-5p in BRCA2-mutated/depleted cells reduced levels of nucleases and other factors involved in maintaining genomic stability. This resulted in relatively stable replication forks, diminished single-strand annealing of DSBs, and increased R-loop formation. We conclude that impact of miR-493-5p on multiple pathways pertinent to genome stability cumulatively causes PARPi/platinum resistance in BRCA2 mutant carcinomas.
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http://dx.doi.org/10.1016/j.celrep.2018.03.038DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5908239PMC
April 2018

Serum microRNAs - potent biomarkers for radiation biodosimetry.

Oncotarget 2018 Mar 2;9(18):14038-14039. Epub 2018 Feb 2.

Department of Biostatistics and Translational Medicine, Medical University of Lodz, Lodz, Poland; Department of Radiation Oncology, Harvard Medical School, Dana-Farber Cancer Institute, Boston, Massachusetts, USA.

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http://dx.doi.org/10.18632/oncotarget.24381DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5865649PMC
March 2018

Mutational signatures reveal the role of RAD52 in p53-independent p21-driven genomic instability.

Genome Biol 2018 03 16;19(1):37. Epub 2018 Mar 16.

Molecular Carcinogenesis Group, Department of Histology and Embryology, School of Medicine, National Kapodistrian University of Athens, 75 Mikras Asias Str, GR-11527, Athens, Greece.

Background: Genomic instability promotes evolution and heterogeneity of tumors. Unraveling its mechanistic basis is essential for the design of appropriate therapeutic strategies. In a previous study, we reported an unexpected oncogenic property of p21, showing that its chronic expression in a p53-deficient environment causes genomic instability by deregulation of the replication licensing machinery.

Results: We now demonstrate that p21 can further fuel genomic instability by suppressing the repair capacity of low- and high-fidelity pathways that deal with nucleotide abnormalities. Consequently, fewer single nucleotide substitutions (SNSs) occur, while formation of highly deleterious DNA double-strand breaks (DSBs) is enhanced, crafting a characteristic mutational signature landscape. Guided by the mutational signatures formed, we find that the DSBs are repaired by Rad52-dependent break-induced replication (BIR) and single-strand annealing (SSA) repair pathways. Conversely, the error-free synthesis-dependent strand annealing (SDSA) repair route is deficient. Surprisingly, Rad52 is activated transcriptionally in an E2F1-dependent manner, rather than post-translationally as is common for DNA repair factor activation.

Conclusions: Our results signify the importance of mutational signatures as guides to disclose the repair history leading to genomic instability. We unveil how chronic p21 expression rewires the repair process and identifies Rad52 as a source of genomic instability and a candidate therapeutic target.
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http://dx.doi.org/10.1186/s13059-018-1401-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5857109PMC
March 2018

EWS/FLI Confers Tumor Cell Synthetic Lethality to CDK12 Inhibition in Ewing Sarcoma.

Cancer Cell 2018 02 18;33(2):202-216.e6. Epub 2018 Jan 18.

Department of Pediatric Oncology, Dana-Farber Cancer Institute and Boston Children's Hospital, Harvard Medical School, 450 Brookline Avenue, Boston, MA 02215, USA; The Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA. Electronic address:

Many cancer types are driven by oncogenic transcription factors that have been difficult to drug. Transcriptional inhibitors, however, may offer inroads into targeting these cancers. Through chemical genomics screening, we identified that Ewing sarcoma is a disease with preferential sensitivity to THZ1, a covalent small-molecule CDK7/12/13 inhibitor. The selective CDK12/13 inhibitor, THZ531, impairs DNA damage repair in an EWS/FLI-dependent manner, supporting a synthetic lethal relationship between response to THZ1/THZ531 and EWS/FLI expression. The combination of these molecules with PARP inhibitors showed striking synergy in cell viability and DNA damage assays in vitro and in multiple models of Ewing sarcoma, including a PDX, in vivo without hematopoietic toxicity.
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http://dx.doi.org/10.1016/j.ccell.2017.12.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5846483PMC
February 2018

A prototypical non-malignant epithelial model to study genome dynamics and concurrently monitor micro-RNAs and proteins in situ during oncogene-induced senescence.

BMC Genomics 2018 01 10;19(1):37. Epub 2018 Jan 10.

Molecular Carcinogenesis Group, Department of Histology and Embryology, School of Medicine, National & Kapodistrian University of Athens, 75 Mikras Asias St, GR-11527, Athens, Greece.

Background: Senescence is a fundamental biological process implicated in various pathologies, including cancer. Regarding carcinogenesis, senescence signifies, at least in its initial phases, an anti-tumor response that needs to be circumvented for cancer to progress. Micro-RNAs, a subclass of regulatory, non-coding RNAs, participate in senescence regulation. At the subcellular level micro-RNAs, similar to proteins, have been shown to traffic between organelles influencing cellular behavior. The differential function of micro-RNAs relative to their subcellular localization and their role in senescence biology raises concurrent in situ analysis of coding and non-coding gene products in senescent cells as a necessity. However, technical challenges have rendered in situ co-detection unfeasible until now.

Methods: In the present report we describe a methodology that bypasses these technical limitations achieving for the first time simultaneous detection of both a micro-RNA and a protein in the biological context of cellular senescence, utilizing the new commercially available SenTraGor compound. The method was applied in a prototypical human non-malignant epithelial model of oncogene-induced senescence that we generated for the purposes of the study. For the characterization of this novel system, we applied a wide range of cellular and molecular techniques, as well as high-throughput analysis of the transcriptome and micro-RNAs.

Results: This experimental setting has three advantages that are presented and discussed: i) it covers a "gap" in the molecular carcinogenesis field, as almost all corresponding in vitro models are fibroblast-based, even though the majority of neoplasms have epithelial origin, ii) it recapitulates the precancerous and cancerous phases of epithelial tumorigenesis within a short time frame under the light of natural selection and iii) it uses as an oncogenic signal, the replication licensing factor CDC6, implicated in both DNA replication and transcription when over-expressed, a characteristic that can be exploited to monitor RNA dynamics.

Conclusions: Consequently, we demonstrate that our model is optimal for studying the molecular basis of epithelial carcinogenesis shedding light on the tumor-initiating events. The latter may reveal novel molecular targets with clinical benefit. Besides, since this method can be incorporated in a wide range of low, medium or high-throughput image-based approaches, we expect it to be broadly applicable.
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http://dx.doi.org/10.1186/s12864-017-4375-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5763532PMC
January 2018

Diagnostic potential for a serum miRNA neural network for detection of ovarian cancer.

Elife 2017 10 31;6. Epub 2017 Oct 31.

Harvard Medical School, Boston, United States.

Recent studies posit a role for non-coding RNAs in epithelial ovarian cancer (EOC). Combining small RNA sequencing from 179 human serum samples with a neural network analysis produced a miRNA algorithm for diagnosis of EOC (AUC 0.90; 95% CI: 0.81-0.99). The model significantly outperformed CA125 and functioned well regardless of patient age, histology, or stage. Among 454 patients with various diagnoses, the miRNA neural network had 100% specificity for ovarian cancer. After using 325 samples to adapt the neural network to qPCR measurements, the model was validated using 51 independent clinical samples, with a positive predictive value of 91.3% (95% CI: 73.3-97.6%) and negative predictive value of 78.6% (95% CI: 64.2-88.2%). Finally, biologic relevance was tested using in situ hybridization on 30 pre-metastatic lesions, showing intratumoral concentration of relevant miRNAs. These data suggest circulating miRNAs have potential to develop a non-invasive diagnostic test for ovarian cancer.
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http://dx.doi.org/10.7554/eLife.28932DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5679755PMC
October 2017

TIRR and 53BP1- partners in arms.

Cell Cycle 2017 07 21;16(13):1235-1236. Epub 2017 Jun 21.

a Department of Radiation Oncology , Dana-Farber Cancer Institute , Boston , MA , USA.

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http://dx.doi.org/10.1080/15384101.2017.1337966DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5531635PMC
July 2017

Clear cell ovarian cancers with microsatellite instability: A unique subset of ovarian cancers with increased tumor-infiltrating lymphocytes and PD-1/PD-L1 expression.

Oncoimmunology 2017;6(2):e1277308. Epub 2017 Jan 6.

Medical Gynecologic Oncology Program, Dana Farber Cancer Institute, Harvard Medical School , Boston, MA, USA.

Clear cell ovarian carcinoma (CCOC) represents a distinct histologic subtype of ovarian cancer associated with significantly worse prognosis across all stages and no effective therapeutic options. Here, we report a rare but clinically important cohort of CCOCs with microsatellite instability (MSI) (MSI-CCOCs), which are highly immunogenic and may thus be very responsive to immune checkpoint blockade. CCOCs with MSI exhibit a significantly higher number of CD8 TILs, higher CD8/CD4 ratio, and higher PD-1 TILs compared with microsatellite stable (MSS) CCOCs and compared with high grade serous ovarian cancers, which are the most common histologic subtype of ovarian cancer. Of note, PD-L1 expression in tumor cells or immune cells was noted in all cases of CCOCs with MSI. These observations open an alternative therapeutic avenue for a fraction of patients with CCOC and argue for the routine testing of CCOCs for MSI, a test that is not currently routinely performed.
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http://dx.doi.org/10.1080/2162402X.2016.1277308DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5353914PMC
January 2017

Evolutionarily conserved serum microRNAs predict radiation-induced fatality in nonhuman primates.

Sci Transl Med 2017 03;9(379)

Department of Radiation Oncology, Harvard Medical School, Dana-Farber Cancer Institute, Boston, MA 02215, USA.

Effective planning for the medical response to a radiological or nuclear accident is complex. Because of limited resources for medical countermeasures, the key would be to accurately triage and identify victims most likely to benefit from treatment. We used a mouse model system to provide evidence that serum microRNAs (miRNAs) may effectively predict the impact of radiation on the long-term viability of animals. We had previously used nonhuman primates (NHPs) to demonstrate that this concept is conserved and serum miRNA signatures have the potential to serve as prediction biomarkers for radiation-induced fatality in a human population. We identified a signature of seven miRNAs that are altered by irradiation in both mice and NHPs. Genomic analysis of these conserved miRNAs revealed that there is a combination of seven transcription factors that are predicted to regulate these miRNAs in human, mice, and NHPs. Moreover, a combination of three miRNAs (miR-133b, miR-215, and miR-375) can identify, with nearly complete accuracy, NHPs exposed to radiation versus unexposed NHPs. Consistent with historical data, female macaques appeared to be more sensitive to radiation, but the difference was not significant. Sex-based stratification allowed us to identify an interaction between gender and miR-16-2 expression, which affected the outcome of radiation exposure. Moreover, we developed a classifier based on two miRNAs (miR-30a and miR-126) that can reproducibly predict radiation-induced mortality. Together, we have obtained a five-miRNA composite signature that can identify irradiated macaques and predict their probability of survival.
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http://dx.doi.org/10.1126/scitranslmed.aal2408DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5441546PMC
March 2017

TIRR regulates 53BP1 by masking its histone methyl-lysine binding function.

Nature 2017 03 27;543(7644):211-216. Epub 2017 Feb 27.

Department of Radiation Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts 02115, USA.

P53-binding protein 1 (53BP1) is a multi-functional double-strand break repair protein that is essential for class switch recombination in B lymphocytes and for sensitizing BRCA1-deficient tumours to poly-ADP-ribose polymerase-1 (PARP) inhibitors. Central to all 53BP1 activities is its recruitment to double-strand breaks via the interaction of the tandem Tudor domain with dimethylated lysine 20 of histone H4 (H4K20me2). Here we identify an uncharacterized protein, Tudor interacting repair regulator (TIRR), that directly binds the tandem Tudor domain and masks its H4K20me2 binding motif. Upon DNA damage, the protein kinase ataxia-telangiectasia mutated (ATM) phosphorylates 53BP1 and recruits RAP1-interacting factor 1 (RIF1) to dissociate the 53BP1-TIRR complex. However, overexpression of TIRR impedes 53BP1 function by blocking its localization to double-strand breaks. Depletion of TIRR destabilizes 53BP1 in the nuclear-soluble fraction and alters the double-strand break-induced protein complex centring 53BP1. These findings identify TIRR as a new factor that influences double-strand break repair using a unique mechanism of masking the histone methyl-lysine binding function of 53BP1.
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http://dx.doi.org/10.1038/nature21358DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5441565PMC
March 2017

Circulating miR-29a and miR-150 correlate with delivered dose during thoracic radiation therapy for non-small cell lung cancer.

Radiat Oncol 2016 Apr 27;11:61. Epub 2016 Apr 27.

Harvard Medical School, 25 Shattuck St, Boston, MA, 02115, USA.

Background: Risk of normal tissue toxicity limits the amount of thoracic radiation therapy (RT) that can be routinely prescribed to treat non-small cell lung cancer (NSCLC). An early biomarker of response to thoracic RT may provide a way to predict eventual toxicities-such as radiation pneumonitis-during treatment, thereby enabling dose adjustment before the symptomatic onset of late effects. MicroRNAs (miRNAs) were studied as potential serological biomarkers for thoracic RT. As a first step, we sought to identify miRNAs that correlate with delivered dose and standard dosimetric factors.

Methods: We performed miRNA profiling of plasma samples obtained from five patients with Stage IIIA NSCLC at five dose-points each during radical thoracic RT. Candidate miRNAs were then assessed in samples from a separate cohort of 21 NSCLC patients receiving radical thoracic RT. To identify a cellular source of circulating miRNAs, we quantified in vitro miRNA expression intracellularly and within secreted exosomes in five NSCLC and stromal cell lines.

Results: miRNA profiling of the discovery cohort identified ten circulating miRNAs that correlated with delivered RT dose as well as other dosimetric parameters such as lung V20. In the validation cohort, miR-29a-3p and miR-150-5p were reproducibly shown to decrease with increasing radiation dose. Expression of miR-29a-3p and miR-150-5p in secreted exosomes decreased with radiation. This was concomitant with an increase in intracellular levels, suggesting that exosomal export of these miRNAs may be downregulated in both NSCLC and stromal cells in response to radiation.

Conclusions: miR-29a-3p and miR-150-5p were identified as circulating biomarkers that correlated with delivered RT dose. miR-150 has been reported to decrease in the circulation of mammals exposed to radiation while miR-29a has been associated with fibrosis in the human heart, lungs, and kidneys. One may therefore hypothesize that outlier levels of circulating miR-29a-3p and miR-150-5p may eventually help predict unexpected responses to radiation therapy, such as toxicity.
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http://dx.doi.org/10.1186/s13014-016-0636-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4847218PMC
April 2016

Association and prognostic significance of BRCA1/2-mutation status with neoantigen load, number of tumor-infiltrating lymphocytes and expression of PD-1/PD-L1 in high grade serous ovarian cancer.

Oncotarget 2016 Mar;7(12):13587-98

Medical Gynecologic Oncology Program, Dana Farber Cancer Institute, Harvard Medical School, Boston, MA, USA.

Immune checkpoint inhibitors (e.g., anti-PD-1 and anti-PD-L1 antibodies) have demonstrated remarkable efficacy against hypermutated cancers such as melanomas and lung carcinomas. One explanation for this effect is that hypermutated lesions harbor more tumor-specific neoantigens that stimulate recruitment of an increased number of tumor-infiltrating lymphocytes (TILs), which is counterbalanced by overexpression of immune checkpoints such as PD-1 or PD-L1. Given that BRCA1/2-mutated high grade serous ovarian cancers (HGSOCs) exhibit a higher mutational load and a unique mutational signature with an elevated number of larger indels up to 50 bp, we hypothesized that they may also harbor more tumor-specific neoantigens, and, therefore, exhibit increased TILs and PD-1/PD-L1 expression. Here, we report significantly higher predicted neoantigens in BRCA1/2-mutated tumors compared to tumors without alterations in homologous recombination (HR) genes (HR-proficient tumors). Tumors with higher neoantigen load were associated with improved overall survival and higher expression of immune genes associated with tumor cytotoxicity such as genes of the TCR, the IFN-gamma and the TNFR pathways. Furthermore, immunohistochemistry studies demonstrated that BRCA1/2-mutated tumors exhibited significantly increased CD3+ and CD8+ TILs, as well as elevated expression of PD-1 and PD-L1 in tumor-associated immune cells compared to HR-proficient tumors. Survival analysis showed that both BRCA1/2-mutation status and number of TILs were independently associated with outcome. Of note, two distinct groups of HGSOCs, one with very poor prognosis (HR proficient with low number of TILs) and one with very good prognosis (BRCA1/2-mutated tumors with high number of TILs) were defined. These findings support a link between BRCA1/2-mutation status, immunogenicity and survival, and suggesting that BRCA1/2-mutated HGSOCs may be more sensitive to PD-1/PD-L1 inhibitors compared to HR-proficient HGSOCs.
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http://dx.doi.org/10.18632/oncotarget.7277DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4924663PMC
March 2016