Publications by authors named "Robert B Faryabi"

19 Publications

  • Page 1 of 1

ALC1 links chromatin accessibility to PARP inhibitor response in homologous recombination-deficient cells.

Nat Cell Biol 2021 02 18;23(2):160-171. Epub 2021 Jan 18.

Department of Cancer Biology, Penn Center for Genome Integrity, Basser Center for BRCA, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.

The response to poly(ADP-ribose) polymerase inhibitors (PARPi) is dictated by homologous recombination (HR) DNA repair and the abundance of lesions that trap PARP enzymes. It remains unclear, however, if the established role of PARP in promoting chromatin accessibility impacts viability in these settings. Using a CRISPR-based screen, we identified the PAR-binding chromatin remodeller ALC1/CHD1L as a key determinant of PARPi toxicity in HR-deficient cells. ALC1 loss reduced viability of breast cancer gene (BRCA)-mutant cells and enhanced sensitivity to PARPi by up to 250-fold, while overcoming several resistance mechanisms. ALC1 deficiency reduced chromatin accessibility concomitant with a decrease in the association of base damage repair factors. This resulted in an accumulation of replication-associated DNA damage, increased PARP trapping and a reliance on HR. These findings establish PAR-dependent chromatin remodelling as a mechanistically distinct aspect of PARPi responses and therapeutic target in HR-deficient cancers.
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http://dx.doi.org/10.1038/s41556-020-00624-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7880902PMC
February 2021

The vascular landscape of human cancer.

J Clin Invest 2021 Jan;131(2)

Department of Medicine.

Tumors depend on a blood supply to deliver oxygen and nutrients, making tumor vasculature an attractive anticancer target. However, only a fraction of patients with cancer benefit from angiogenesis inhibitors. Whether antiangiogenic therapy would be more effective if targeted to individuals with specific tumor characteristics is unknown. To better characterize the tumor vascular environment both within and between cancer types, we developed a standardized metric - the endothelial index (EI) - to estimate vascular density in over 10,000 human tumors, corresponding to 31 solid tumor types, from transcriptome data. We then used this index to compare hyper- and hypovascular tumors, enabling the classification of human tumors into 6 vascular microenvironment signatures (VMSs) based on the expression of a panel of 24 vascular "hub" genes. The EI and VMS correlated with known tumor vascular features and were independently associated with prognosis in certain cancer types. Retrospective testing of clinical trial data identified VMS2 classification as a powerful biomarker for response to bevacizumab. Thus, we believe our studies provide an unbiased picture of human tumor vasculature that may enable more precise deployment of antiangiogenesis therapy.
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http://dx.doi.org/10.1172/JCI136655DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7810485PMC
January 2021

Trib1 regulates T cell differentiation during chronic infection by restraining the effector program.

J Exp Med 2020 05;217(5)

Department of Pathology and Laboratory Medicine, Abramson Family Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA.

In chronic infections, the immune response fails to control virus, leading to persistent antigen stimulation and the progressive development of T cell exhaustion. T cell effector differentiation is poorly understood in the context of exhaustion, but targeting effector programs may provide new strategies for reinvigorating T cell function. We identified Tribbles pseudokinase 1 (Trib1) as a central regulator of antiviral T cell immunity, where loss of Trib1 led to a sustained enrichment of effector-like KLRG1+ T cells, enhanced function, and improved viral control. Single-cell profiling revealed that Trib1 restrains a population of KLRG1+ effector CD8 T cells that is transcriptionally distinct from exhausted cells. Mechanistically, we identified an interaction between Trib1 and the T cell receptor (TCR) signaling activator, MALT1, which disrupted MALT1 signaling complexes. These data identify Trib1 as a negative regulator of TCR signaling and downstream function, and reveal a link between Trib1 and effector versus exhausted T cell differentiation that can be targeted to improve antiviral immunity.
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http://dx.doi.org/10.1084/jem.20190888DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7201917PMC
May 2020

TooManyCells identifies and visualizes relationships of single-cell clades.

Nat Methods 2020 04 2;17(4):405-413. Epub 2020 Mar 2.

Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA, USA.

Identifying and visualizing transcriptionally similar cells is instrumental for accurate exploration of the cellular diversity revealed by single-cell transcriptomics. However, widely used clustering and visualization algorithms produce a fixed number of cell clusters. A fixed clustering 'resolution' hampers our ability to identify and visualize echelons of cell states. We developed TooManyCells, a suite of graph-based algorithms for efficient and unbiased identification and visualization of cell clades. TooManyCells introduces a visualization model built on a concept intentionally orthogonal to dimensionality-reduction methods. TooManyCells is also equipped with an efficient matrix-free divisive hierarchical spectral clustering different from prevalent single-resolution clustering methods. TooManyCells enables multiresolution and multifaceted exploration of single-cell clades. An advantage of this paradigm is the immediate detection of rare and common populations that outperforms popular clustering and visualization algorithms, as demonstrated using existing single-cell transcriptomic data sets and new data modeling drug-resistance acquisition in leukemic T cells.
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http://dx.doi.org/10.1038/s41592-020-0748-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7439807PMC
April 2020

Genetic Variation in Type 1 Diabetes Reconfigures the 3D Chromatin Organization of T Cells and Alters Gene Expression.

Immunity 2020 02 11;52(2):257-274.e11. Epub 2020 Feb 11.

Department of Genetics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA; Institute for Immunology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA; Epigenetics Institute, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA; Institute for Diabetes, Obesity and Metabolism, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA; Abramson Family Cancer Research Institute, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA; The Human Pancreas Analysis Program, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA, https://hpap.pmacs.upenn.edu/. Electronic address:

Genetics is a major determinant of susceptibility to autoimmune disorders. Here, we examined whether genome organization provides resilience or susceptibility to sequence variations, and how this would contribute to the molecular etiology of an autoimmune disease. We generated high-resolution maps of linear and 3D genome organization in thymocytes of NOD mice, a model of type 1 diabetes (T1D), and the diabetes-resistant C57BL/6 mice. Multi-enhancer interactions formed at genomic regions harboring genes with prominent roles in T cell development in both strains. However, diabetes risk-conferring loci coalesced enhancers and promoters in NOD, but not C57BL/6 thymocytes. 3D genome mapping of NODxC57BL/6 F1 thymocytes revealed that genomic misfolding in NOD mice is mediated in cis. Moreover, immune cells infiltrating the pancreas of humans with T1D exhibited increased expression of genes located on misfolded loci in mice. Thus, genetic variation leads to altered 3D chromatin architecture and associated changes in gene expression that may underlie autoimmune pathology.
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http://dx.doi.org/10.1016/j.immuni.2020.01.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7152927PMC
February 2020

The Pediatric Cell Atlas: Defining the Growth Phase of Human Development at Single-Cell Resolution.

Dev Cell 2019 04 28;49(1):10-29. Epub 2019 Mar 28.

Department of Biomedical Informatics, University of Cincinnati College of Medicine, and Cincinnati Children's Hospital Medical Center, Division of Biomedical Informatics, Cincinnati, OH 45229, USA.

Single-cell gene expression analyses of mammalian tissues have uncovered profound stage-specific molecular regulatory phenomena that have changed the understanding of unique cell types and signaling pathways critical for lineage determination, morphogenesis, and growth. We discuss here the case for a Pediatric Cell Atlas as part of the Human Cell Atlas consortium to provide single-cell profiles and spatial characterization of gene expression across human tissues and organs. Such data will complement adult and developmentally focused HCA projects to provide a rich cytogenomic framework for understanding not only pediatric health and disease but also environmental and genetic impacts across the human lifespan.
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http://dx.doi.org/10.1016/j.devcel.2019.03.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6616346PMC
April 2019

Oncogenic Notch Promotes Long-Range Regulatory Interactions within Hyperconnected 3D Cliques.

Mol Cell 2019 03 7;73(6):1174-1190.e12. Epub 2019 Feb 7.

Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Abramson Family Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Institute for Biomedical Informatics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Department of Cancer Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. Electronic address:

Chromatin loops enable transcription-factor-bound distal enhancers to interact with their target promoters to regulate transcriptional programs. Although developmental transcription factors such as active forms of Notch can directly stimulate transcription by activating enhancers, the effect of their oncogenic subversion on the 3D organization of cancer genomes is largely undetermined. By mapping chromatin looping genome-wide in Notch-dependent triple-negative breast cancer and B cell lymphoma, we show that beyond the well-characterized role of Notch as an activator of distal enhancers, Notch regulates its direct target genes by instructing enhancer repositioning. Moreover, a large fraction of Notch-instructed regulatory loops form highly interacting enhancer and promoter spatial clusters termed "3D cliques." Loss- and gain-of-function experiments show that Notch preferentially targets hyperconnected 3D cliques that regulate the expression of crucial proto-oncogenes. Our observations suggest that oncogenic hijacking of developmental transcription factors can dysregulate transcription through widespread effects on the spatial organization of cancer genomes.
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http://dx.doi.org/10.1016/j.molcel.2019.01.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6485942PMC
March 2019

Classes of ITD Predict Outcomes in AML Patients Treated with FLT3 Inhibitors.

Clin Cancer Res 2019 01 4;25(2):573-583. Epub 2018 Sep 4.

Department of Pathology and Laboratory Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania.

Purpose: Recurrent internal tandem duplication (ITD) mutations are observed in various cancers including acute myeloid leukemia (AML), where ITD mutations in tyrosine kinase receptor FLT3 are associated with poor prognostic outcomes. Several FLT3 inhibitors (FLT3i) are in clinical trials for high-risk -ITD-positive AML. However, the variability of survival following FLT3i treatment suggests that the mere presence of -ITD mutations might not guarantee effective clinical response. Motivated by the heterogeneity of -ITD mutations, we investigated the effects of -ITD structural features on the response of AML patients to treatment. We developed the HeatITup (HEAT diffusion for Internal Tandem dUPlication) algorithm to identify and quantitate ITD structural features including nucleotide composition. Using HeatITup, we studied the impact of ITD structural features on the clinical response to FLT3i and induction chemotherapy in -ITD-positive AML patients.

Results: HeatITup accurately identifies and classifies ITDs into newly defined categories of "typical" or "atypical" based on their nucleotide composition. A typical ITD's insert sequence completely matches the wild-type whereas an atypical ITD's insert contains nucleotides exogenous to the wild-type . Our analysis shows marked divergence between typical and atypical ITD mutation features. Furthermore, our data suggest that AML patients carrying typical -ITDs benefited significantly more from both FLT3i and induction chemotherapy treatments than patients with atypical -ITDs.

Conclusions: These results underscore the importance of structural discernment of complex somatic mutations such as ITDs in progressing toward personalized treatment of AML patients, and enable researchers and clinicians to unravel ITD complexity using the provided software..
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http://dx.doi.org/10.1158/1078-0432.CCR-18-0655DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6335170PMC
January 2019

Differential Integration of Transcriptome and Proteome Identifies Pan-Cancer Prognostic Biomarkers.

Front Genet 2018 15;9:205. Epub 2018 Jun 15.

Department of Pathology and Laboratory Medicine, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, United States.

High-throughput analysis of the transcriptome and proteome individually are used to interrogate complex oncogenic processes in cancer. However, an outstanding challenge is how to combine these complementary, yet partially disparate data sources to accurately identify tumor-specific gene products and clinical biomarkers. Here, we introduce inteGREAT for robust and scalable differential integration of high-throughput measurements. With inteGREAT, each data source is represented as a co-expression network, which is analyzed to characterize the local and global structure of each node across networks. inteGREAT scores the degree by which the topology of each gene in both transcriptome and proteome networks are conserved within a tumor type, yet different from other normal or malignant cells. We demonstrated the high performance of inteGREAT based on several analyses: deconvolving synthetic networks, rediscovering known diagnostic biomarkers, establishing relationships between tumor lineages, and elucidating putative prognostic biomarkers which we experimentally validated. Furthermore, we introduce the application of a clumpiness measure to quantitatively describe tumor lineage similarity. Together, inteGREAT not only infers functional and clinical insights from the integration of transcriptomic and proteomic data sources in cancer, but also can be readily applied to other heterogeneous high-throughput data sources. inteGREAT is open source and available to download from https://github.com/faryabib/inteGREAT.
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http://dx.doi.org/10.3389/fgene.2018.00205DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6018483PMC
June 2018

Tumor Cell-Intrinsic Factors Underlie Heterogeneity of Immune Cell Infiltration and Response to Immunotherapy.

Immunity 2018 07 26;49(1):178-193.e7. Epub 2018 Jun 26.

Abramson Family Cancer Research Institute, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA; Department of Medicine, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA; Department of Cell and Developmental Biology, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA; Abramson Cancer Center, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA; Parker Institute for Cancer Immunotherapy, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA. Electronic address:

The biological and functional heterogeneity between tumors-both across and within cancer types-poses a challenge for immunotherapy. To understand the factors underlying tumor immune heterogeneity and immunotherapy sensitivity, we established a library of congenic tumor cell clones from an autochthonous mouse model of pancreatic adenocarcinoma. These clones generated tumors that recapitulated T cell-inflamed and non-T-cell-inflamed tumor microenvironments upon implantation in immunocompetent mice, with distinct patterns of infiltration by immune cell subsets. Co-injecting tumor cell clones revealed the non-T-cell-inflamed phenotype is dominant and that both quantitative and qualitative features of intratumoral CD8 T cells determine response to therapy. Transcriptomic and epigenetic analyses revealed tumor-cell-intrinsic production of the chemokine CXCL1 as a determinant of the non-T-cell-inflamed microenvironment, and ablation of CXCL1 promoted T cell infiltration and sensitivity to a combination immunotherapy regimen. Thus, tumor cell-intrinsic factors shape the tumor immune microenvironment and influence the outcome of immunotherapy.
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http://dx.doi.org/10.1016/j.immuni.2018.06.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6707727PMC
July 2018

MAFB enhances oncogenic Notch signaling in T cell acute lymphoblastic leukemia.

Sci Signal 2017 11 14;10(505). Epub 2017 Nov 14.

Abramson Family Cancer Research Institute, University of Pennsylvania, Philadelphia, PA 19104, USA.

Activating mutations in the gene encoding the cell-cell contact signaling protein Notch1 are common in human T cell acute lymphoblastic leukemias (T-ALLs). However, expressing mutant alleles in mice fails to efficiently induce the development of leukemia. We performed a gain-of-function screen to identify proteins that enhanced signaling by leukemia-associated Notch1 mutants. The transcription factors MAFB and ETS2 emerged as candidates that individually enhanced Notch1 signaling, and when coexpressed, they synergistically increased signaling to an extent similar to that induced by core components of the Notch transcriptional complex. In mouse models of T-ALL, MAFB enhanced leukemogenesis by the naturally occurring Notch1 mutants, decreased disease latency, and increased disease penetrance. Decreasing MAFB abundance in mouse and human T-ALL cells reduced the expression of Notch1 target genes, including and , and sustained MAFB knockdown impaired T-ALL growth in a competitive setting. MAFB bound to ETS2 and interacted with the acetyltransferases PCAF and P300, highlighting its importance in recruiting coactivators that enhance Notch1 signaling. Together, these data identify a mechanism for enhancing the oncogenic potential of weak Notch1 mutants in leukemia models, and they reveal the MAFB-ETS2 transcriptional axis as a potential therapeutic target in T-ALL.
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http://dx.doi.org/10.1126/scisignal.aam6846DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5885022PMC
November 2017

A B Cell Regulome Links Notch to Downstream Oncogenic Pathways in Small B Cell Lymphomas.

Cell Rep 2017 Oct;21(3):784-797

Department of Pathology and Laboratory Medicine, Abramson Family Cancer Research Institute, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA 19104, USA. Electronic address:

Gain-of-function Notch mutations are recurrent in mature small B cell lymphomas such as mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL), but the Notch target genes that contribute to B cell oncogenesis are largely unknown. We performed integrative analysis of Notch-regulated transcripts, genomic binding of Notch transcription complexes, and genome conformation data to identify direct Notch target genes in MCL cell lines. This B cell Notch regulome is largely controlled through Notch-bound distal enhancers and includes genes involved in B cell receptor and cytokine signaling and the oncogene MYC, which sustains proliferation of Notch-dependent MCL cell lines via a Notch-regulated lineage-restricted enhancer complex. Expression of direct Notch target genes is associated with Notch activity in an MCL xenograft model and in CLL lymph node biopsies. Our findings provide key insights into the role of Notch in MCL and other B cell malignancies and have important implications for therapeutic targeting of Notch-dependent oncogenic pathways.
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http://dx.doi.org/10.1016/j.celrep.2017.09.066DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5687286PMC
October 2017

Functional proteogenomics reveals biomarkers and therapeutic targets in lymphomas.

Proc Natl Acad Sci U S A 2017 06 12;114(25):6581-6586. Epub 2017 Jun 12.

Department of Pathology and Laboratory Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104;

Identification of biomarkers and therapeutic targets is a critical goal of precision medicine. N-glycoproteins are a particularly attractive class of proteins that constitute potential cancer biomarkers and therapeutic targets for small molecules, antibodies, and cellular therapies. Using mass spectrometry (MS), we generated a compendium of 1,091 N-glycoproteins (from 40 human primary lymphomas and cell lines). Hierarchical clustering revealed distinct subtype signatures that included several subtype-specific biomarkers. Orthogonal immunological studies in 671 primary lymphoma tissue biopsies and 32 lymphoma-derived cell lines corroborated MS data. In anaplastic lymphoma kinase-positive (ALK) anaplastic large cell lymphoma (ALCL), integration of N-glycoproteomics and transcriptome sequencing revealed an ALK-regulated cytokine/receptor signaling network, including vulnerabilities corroborated by a genome-wide clustered regularly interspaced short palindromic screen. Functional targeting of IL-31 receptor β, an ALCL-enriched and ALK-regulated N-glycoprotein in this network, abrogated ALKALCL growth in vitro and in vivo. Our results highlight the utility of functional proteogenomic approaches for discovery of cancer biomarkers and therapeutic targets.
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http://dx.doi.org/10.1073/pnas.1701263114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5488937PMC
June 2017

Trib2 Suppresses Tumor Initiation in Notch-Driven T-ALL.

PLoS One 2016 18;11(5):e0155408. Epub 2016 May 18.

Department of Pathology and Laboratory Medicine, Abramson Family Cancer Research Institute, Institute of Medicine and Engineering, Institute for Immunology, Center for Personalized Diagnostics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, United States of America.

Trib2 is highly expressed in human T cell acute lymphoblastic leukemia (T-ALL) and is a direct transcriptional target of the oncogenic drivers Notch and TAL1. In human TAL1-driven T-ALL cell lines, Trib2 is proposed to function as an important survival factor, but there is limited information about the role of Trib2 in primary T-ALL. In this study, we investigated the role of Trib2 in the initiation and maintenance of Notch-dependent T-ALL. Trib2 had no effect on the growth and survival of murine T-ALL cell lines in vitro when expression was blocked by shRNAs. To test the function of Trib2 on leukemogenesis in vivo, we generated Trib2 knockout mice. Mice were born at the expected Mendelian frequencies without gross developmental anomalies. Adult mice did not develop pathology or shortened survival, and hematopoiesis, including T cell development, was unperturbed. Using a retroviral model of Notch-induced T-ALL, deletion of Trib2 unexpectedly decreased the latency and increased the penetrance of T-ALL development in vivo. Immunoblotting of primary murine T-ALL cells showed that the absence of Trib2 increased C/EBPα expression, a known regulator of cell proliferation, and did not alter AKT or ERK phosphorylation. Although Trib2 was suggested to be highly expressed in T-ALL, transcriptomic analysis of two independent T-ALL cohorts showed that low Trib2 expression correlated with the TLX1-expressing cortical mature T-ALL subtype, whereas high Trib2 expression correlated with the LYL1-expressing early immature T-ALL subtype. These data indicate that Trib2 has a complex role in the pathogenesis of Notch-driven T-ALL, which may vary between different T-ALL subtypes.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0155408PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4871414PMC
July 2017

DNA-damage-induced differentiation of leukaemic cells as an anti-cancer barrier.

Nature 2014 Oct 27;514(7520):107-11. Epub 2014 Jul 27.

Laboratory of Genome Integrity, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.

Self-renewal is the hallmark feature both of normal stem cells and cancer stem cells. Since the regenerative capacity of normal haematopoietic stem cells is limited by the accumulation of reactive oxygen species and DNA double-strand breaks, we speculated that DNA damage might also constrain leukaemic self-renewal and malignant haematopoiesis. Here we show that the histone methyl-transferase MLL4, a suppressor of B-cell lymphoma, is required for stem-cell activity and an aggressive form of acute myeloid leukaemia harbouring the MLL-AF9 oncogene. Deletion of MLL4 enhances myelopoiesis and myeloid differentiation of leukaemic blasts, which protects mice from death related to acute myeloid leukaemia. MLL4 exerts its function by regulating transcriptional programs associated with the antioxidant response. Addition of reactive oxygen species scavengers or ectopic expression of FOXO3 protects MLL4(-/-) MLL-AF9 cells from DNA damage and inhibits myeloid maturation. Similar to MLL4 deficiency, loss of ATM or BRCA1 sensitizes transformed cells to differentiation, suggesting that myeloid differentiation is promoted by loss of genome integrity. Indeed, we show that restriction-enzyme-induced double-strand breaks are sufficient to induce differentiation of MLL-AF9 blasts, which requires cyclin-dependent kinase inhibitor p21(Cip1) (Cdkn1a) activity. In summary, we have uncovered an unexpected tumour-promoting role of genome guardians in enforcing the oncogene-induced differentiation blockade in acute myeloid leukaemia.
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http://dx.doi.org/10.1038/nature13483DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4410707PMC
October 2014

53BP1 mediates productive and mutagenic DNA repair through distinct phosphoprotein interactions.

Cell 2013 Jun 30;153(6):1266-80. Epub 2013 May 30.

Laboratory of Genome Integrity, National Cancer Institute, NIH, Bethesda, MD 20892, USA.

The DNA damage response (DDR) protein 53BP1 protects DNA ends from excessive resection in G1, and thereby favors repair by nonhomologous end-joining (NHEJ) as opposed to homologous recombination (HR). During S phase, BRCA1 antagonizes 53BP1 to promote HR. The pro-NHEJ and antirecombinase functions of 53BP1 are mediated in part by RIF1, the only known factor that requires 53BP1 phosphorylation for its recruitment to double-strand breaks (DSBs). Here, we show that a 53BP1 phosphomutant, 53BP18A, comprising alanine substitutions of the eight most N-terminal S/TQ phosphorylation sites, mimics 53BP1 deficiency by restoring genome stability in BRCA1-deficient cells yet behaves like wild-type 53BP1 with respect to immunoglobulin class switch recombination (CSR). 53BP18A recruits RIF1 but fails to recruit the DDR protein PTIP to DSBs, and disruption of PTIP phenocopies 53BP18A. We conclude that 53BP1 promotes productive CSR and suppresses mutagenic DNA repair through distinct phosphodependent interactions with RIF1 and PTIP.
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http://dx.doi.org/10.1016/j.cell.2013.05.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3713552PMC
June 2013

Identification of early replicating fragile sites that contribute to genome instability.

Cell 2013 Jan 24;152(3):620-32. Epub 2013 Jan 24.

Laboratory of Genome Integrity, National Cancer Institute, NIH, Bethesda, Maryland 20892, USA.

DNA double-strand breaks (DSBs) in B lymphocytes arise stochastically during replication or as a result of targeted DNA damage by activation-induced cytidine deaminase (AID). Here we identify recurrent, early replicating, and AID-independent DNA lesions, termed early replication fragile sites (ERFSs), by genome-wide localization of DNA repair proteins in B cells subjected to replication stress. ERFSs colocalize with highly expressed gene clusters and are enriched for repetitive elements and CpG dinucleotides. Although distinct from late-replicating common fragile sites (CFS), the stability of ERFSs and CFSs is similarly dependent on the replication-stress response kinase ATR. ERFSs break spontaneously during replication, but their fragility is increased by hydroxyurea, ATR inhibition, or deregulated c-Myc expression. Moreover, greater than 50% of recurrent amplifications/deletions in human diffuse large B cell lymphoma map to ERFSs. In summary, we have identified a source of spontaneous DNA lesions that drives instability at preferred genomic sites.
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http://dx.doi.org/10.1016/j.cell.2013.01.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3629730PMC
January 2013

The DNA damage- and transcription-associated protein paxip1 controls thymocyte development and emigration.

Immunity 2012 Dec 15;37(6):971-85. Epub 2012 Nov 15.

Laboratory of Genome Integrity, National Cancer Institute, Center for Cancer Research, National Institutes of Health, Bethesda MD 20892 USA.

Histone 3 lysine 4 trimethylation (H3K4me3) is associated with promoters of active genes and found at hot spots for DNA recombination. Here we have shown that PAXIP1 (also known as PTIP), a protein associated with MLL3 and MLL4 methyltransferase and the DNA damage response, regulates RAG-mediated cleavage and repair during V(D)J recombination in CD4(+) CD8(+) DP thymocytes. Loss of PAXIP1 in developing thymocytes diminished Jα H3K4me3 and germline transcription, suppressed double strand break formation at 3' Jα segments, but resulted in accumulation of unresolved T cell receptor α-chain gene (Tcra) breaks. Moreover, PAXIP1 was essential for release of mature single positive (SP) αβ T cells from the thymus through transcriptional activation of sphingosine-1-phosphate receptor S1pr1 as well as for natural killer T cell development. Thus, in addition to maintaining genome integrity during Tcra rearrangements, PAXIP1 controls distinct transcriptional programs during DP differentiation necessary for Tcra locus accessibility, licensing mature thymocytes for trafficking and natural killer T cell development.
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http://dx.doi.org/10.1016/j.immuni.2012.10.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3525809PMC
December 2012

BRCA1 functions independently of homologous recombination in DNA interstrand crosslink repair.

Mol Cell 2012 Apr 22;46(2):125-35. Epub 2012 Mar 22.

Laboratory of Genome Integrity, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.

Brca1 is required for DNA repair by homologous recombination (HR) and normal embryonic development. Here we report that deletion of the DNA damage response factor 53BP1 overcomes embryonic lethality in Brca1-nullizygous mice and rescues HR deficiency, as measured by hypersensitivity to polyADP-ribose polymerase (PARP) inhibition. However, Brca1,53BP1 double-deficient cells are hypersensitive to DNA interstrand crosslinks (ICLs), indicating that BRCA1 has an additional role in DNA crosslink repair that is distinct from HR. Disruption of the nonhomologous end-joining (NHEJ) factor, Ku, promotes DNA repair in Brca1-deficient cells; however deletion of either Ku or 53BP1 exacerbates genomic instability in cells lacking FANCD2, a mediator of the Fanconi anemia pathway for ICL repair. BRCA1 therefore has two separate roles in ICL repair that can be modulated by manipulating NHEJ, whereas FANCD2 provides a key activity that cannot be bypassed by ablation of 53BP1 or Ku.
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http://dx.doi.org/10.1016/j.molcel.2012.02.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3340543PMC
April 2012