Michal Zimmermann

53BP1 regulates DSB repair using Rif1 to control 5’ end resection

Fixing Broken DNA Some physiological processes, such as immunoglobulin class switching and telomere attrition, result in double-stranded DNA breaks. The DNA damage repair protein, 53BP1, prevents nucleolytic processing of these breaks, but the proteins it partners with to do this are unknown (see the Perspective by Lukas and Lukas). Di Virgilio et al. (p. 711, published online 10 January), using mass spectroscopy–based methods, and Zimmermann et al. (p. 700, published online 10 January), using a telomere-based assay, identify Rif1 as a 53BP1 phosphorylation- and DNA damage–dependent interaction partner. Mice with a B cell–specific deletion in Rif1 showed impaired immunoglobulin class switching. Rif1-deficient cells exhibited extensive 5′-3′ resection at DNA ends, with enhanced genetic instability. Thus, Rif1 partners with 53BP1 to promote the proper repair of double-stranded DNA breaks. In mammalian cells, Rap1-interacting factor 1 protects DNA ends against resection. [Also see Perspective by Lukas and Lukas] The choice between double-strand break (DSB) repair by either homology-directed repair (HDR) or nonhomologous end joining (NHEJ) is tightly regulated. Defects in this regulation can induce genome instability and cancer. 53BP1 is critical for the control of DSB repair, promoting NHEJ, and inhibiting the 5′ end resection needed for HDR. Using dysfunctional telomeres and genome-wide DSBs, we identify Rif1 as the main factor used by 53BP1 to impair 5′ end resection. Rif1 inhibits resection involving CtIP, BLM, and Exo1; limits accumulation of BRCA1/BARD1 complexes at sites of DNA damage; and defines one of the mechanisms by which 53BP1 causes chromosomal abnormalities in Brca1-deficient cells. These data establish Rif1 as an important contributor to the control of DSB repair by 53BP1.

High-Resolution CRISPR Screens Reveal Fitness Genes and Genotype-Specific Cancer Liabilities

The ability to perturb genes in human cells is crucial for elucidating gene function and holds great potential for finding therapeutic targets for diseases such as cancer. To extend the catalog of human core and context-dependent fitness genes, we have developed a high-complexity second-generation genome-scale CRISPR-Cas9 gRNA library and applied it to fitness screens in five human cell lines. Using an improved Bayesian analytical approach, we consistently discover 5-fold more fitness genes than were previously observed. We present a list of 1,580 human core fitness genes and describe their general properties. Moreover, we demonstrate that context-dependent fitness genes accurately recapitulate pathway-specific genetic vulnerabilities induced by known oncogenes and reveal cell-type-specific dependencies for specific receptor tyrosine kinases, even in oncogenic KRAS backgrounds. Thus, rigorous identification of human cell line fitness genes using a high-complexity CRISPR-Cas9 library affords a high-resolution view of the genetic vulnerabilities of a cell.

TRF1 negotiates TTAGGG repeat-associated replication problems by recruiting the BLM helicase and the TPP1/POT1 repressor of ATR signaling

The semiconservative replication of telomeres is facilitated by the shelterin component TRF1. Without TRF1, replication forks stall in the telomeric repeats, leading to ATR kinase signaling upon S-phase progression, fragile metaphase telomeres that resemble the common fragile sites (CFSs), and the association of sister telomeres. In contrast, TRF1 does not contribute significantly to the end protection functions of shelterin. We addressed the mechanism of TRF1 action using mouse conditional knockouts of BLM, TRF1, TPP1, and Rap1 in combination with expression of TRF1 and TIN2 mutants. The data establish that TRF1 binds BLM to facilitate lagging but not leading strand telomeric DNA synthesis. As the template for lagging strand telomeric DNA synthesis is the TTAGGG repeat strand, TRF1-bound BLM is likely required to remove secondary structures formed by these sequences. In addition, the data establish that TRF1 deploys TIN2 and the TPP1/POT1 heterodimers in shelterin to prevent ATR during telomere replication and repress the accompanying sister telomere associations. Thus, TRF1 uses two distinct mechanisms to promote replication of telomeric DNA and circumvent the consequences of replication stress. These data are relevant to the expression of CFSs and provide insights into TIN2, which is compromised in dyskeratosis congenita (DC) and related disorders.

Inhibition of 53BP1 favors homology-dependent DNA repair and increases CRISPR–Cas9 genome-editing efficiency

Programmable nucleases, such as Cas9, are used for precise genome editing by homology-dependent repair (HDR). However, HDR efficiency is constrained by competition from other double-strand break (DSB) repair pathways, including non-homologous end-joining (NHEJ). We report the discovery of a genetically encoded inhibitor of 53BP1 that increases the efficiency of HDR-dependent genome editing in human and mouse cells. 53BP1 is a key regulator of DSB repair pathway choice in eukaryotic cells and functions to favor NHEJ over HDR by suppressing end resection, which is the rate-limiting step in the initiation of HDR. We screened an existing combinatorial library of engineered ubiquitin variants for inhibitors of 53BP1. Expression of one variant, named i53 (inhibitor of 53BP1), in human and mouse cells, blocked accumulation of 53BP1 at sites of DNA damage and improved gene targeting and chromosomal gene conversion with either double-stranded DNA or single-stranded oligonucleotide donors by up to 5.6-fold. Inhibition of 53BP1 is a robust method to increase efficiency of HDR-based precise genome editing.

53BP1-RIF1-shieldin counteracts DSB resection through CST- and Polα-dependent fill-in

In DNA repair, the resection of double-strand breaks dictates the choice between homology-directed repair—which requires a 3′ overhang—and classical non-homologous end joining, which can join unresected ends1,2. BRCA1-mutant cancers show minimal resection of double-strand breaks, which renders them deficient in homology-directed repair and sensitive to inhibitors of poly(ADP-ribose) polymerase 1 (PARP1)3–8. When BRCA1 is absent, the resection of double-strand breaks is thought to be prevented by 53BP1, RIF1 and the REV7–SHLD1–SHLD2–SHLD3 (shieldin) complex, and loss of these factors diminishes sensitivity to PARP1 inhibitors4,6–9. Here we address the mechanism by which 53BP1–RIF1–shieldin regulates the generation of recombinogenic 3′ overhangs. We report that CTC1–STN1–TEN1 (CST)10, a complex similar to replication protein A that functions as an accessory factor of polymerase-α (Polα)–primase11, is a downstream effector in the 53BP1 pathway. CST interacts with shieldin and localizes with Polα to sites of DNA damage in a 53BP1- and shieldin-dependent manner. As with loss of 53BP1, RIF1 or shieldin, the depletion of CST leads to increased resection. In BRCA1-deficient cells, CST blocks RAD51 loading and promotes the efficacy of PARP1 inhibitors. In addition, Polα inhibition diminishes the effect of PARP1 inhibitors. These data suggest that CST–Polα-mediated fill-in helps to control the repair of double-strand breaks by 53BP1, RIF1 and shieldin.53BP1 and shieldin recruit the CTC1–STN1–TEN1 complex and polymerase-α to sites of DNA damage to help control the repair of double-strand breaks.

CRISPR screens identify genomic ribonucleotides as a source of PARP-trapping lesions

The observation that BRCA1- and BRCA2-deficient cells are sensitive to inhibitors of poly(ADP–ribose) polymerase (PARP) has spurred the development of cancer therapies that use these inhibitors to target deficiencies in homologous recombination1. The cytotoxicity of PARP inhibitors depends on PARP trapping, the formation of non-covalent protein–DNA adducts composed of inhibited PARP1 bound to DNA lesions of unclear origins1–4. To address the nature of such lesions and the cellular consequences of PARP trapping, we undertook three CRISPR (clustered regularly interspersed palindromic repeats) screens to identify genes and pathways that mediate cellular resistance to olaparib, a clinically approved PARP inhibitor1. Here we present a high-confidence set of 73 genes, which when mutated cause increased sensitivity to PARP inhibitors. In addition to an expected enrichment for genes related to homologous recombination, we discovered that mutations in all three genes encoding ribonuclease H2 sensitized cells to PARP inhibition. We establish that the underlying cause of the PARP-inhibitor hypersensitivity of cells deficient in ribonuclease H2 is impaired ribonucleotide excision repair5. Embedded ribonucleotides, which are abundant in the genome of cells deficient in ribonucleotide excision repair, are substrates for cleavage by topoisomerase 1, resulting in PARP-trapping lesions that impede DNA replication and endanger genome integrity. We conclude that genomic ribonucleotides are a hitherto unappreciated source of PARP-trapping DNA lesions, and that the frequent deletion of RNASEH2B in metastatic prostate cancer and chronic lymphocytic leukaemia could provide an opportunity to exploit these findings therapeutically.Mutations in all three genes encoding ribonuclease H2 sensitize cells to poly(ADP–ribose) polymerase inhibitors by compromising ribonucleotide excision repair.

Abstract PR03: High-resolution detection of fitness genes and genotype-specific cancer vulnerabilities with CRISPR-Cas9 screens

Genetic knockouts are a fundamental tool for elucidating gene function in model organisms and hold great potential for finding therapeutic targets for diseases such as cancer. The advance that pooled CRISPR-Cas9 library technology brings to human genetics sets the stage for identifying cellular fitness genes which operate either globally or specifically within a particular genetic background or environmental context. To extend the catalogue of human core and context-dependent fitness genes, we have developed the TKO (Toronto KnockOut) library, a second-generation gRNA library of 176,500 guides targeting 17,661 human protein coding genes. We used the library to screen five human cell lines, representing a cross-section of wild type and cancer tissues, to identify genes whose knockouts induced significant fitness defects. We consistently discover fivefold more fitness genes than were previously observed using systematic RNA interference, including many genes at moderate expression levels that are largely refractory to RNAi methods. We expand the known set of human core fitness genes more than fourfold to 1,580 genes, and identify dozens of essential protein complexes, both known and novel, whose heterozygous copy loss in chromosomally unstable cancers may induce a therapeutic window. We further characterize novel fitness genes of unknown function and find that they all likely exist in protein complexes with other essential genes. TKO screens accurately recapitulate pathway-specific genetic vulnerabilities induced by known oncogenes and reveal cell-type-specific dependencies for specific receptor tyrosine kinases, even in oncogenic KRAS backgrounds. We also identified a surprising and specific dependency on mitochondrial activity, which strongly supports the idea that oxidative phosphorylation dependency - a clear exception to the Warburg effect - is a targetable weakness of some tumors. Our findings demonstrate that the CRISPR-Cas9 system fundamentally alters the landscape for systematic genetics in human cells through rigorous identification of cell line essential genes, affording a high-resolution view of the genetic vulnerabilities of a cell that may represent therapeutic opportunities in cancer and that might conceivably contribute to cell plasticity and tumor progression. Citation Format: Traver Hart, Megha Chandrashekhar, Michael Aregger, Zachary Steinhart, Kevin R. Brown, Graham Macleod, Monika Mis, Michal Zimmermann, Amelie Fradet-Turcotte, Song Sun, Peter Driks, Sachdev Sidhu, Frederick P. Roth, Olivia S. Rissland, Daniel Durocher, Stephane Angers, Jason Moffat. High-resolution detection of fitness genes and genotype-specific cancer vulnerabilities with CRISPR-Cas9 screens. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr PR03.