Topoisomerase 1 Inhibition in MYC-Driven Cancer Promotes Aberrant R-Loop Accumulation to Induce Synthetic Lethality

CRISPR screening reveals topoisomerase 1 as an immediately actionable vulnerability in cancers harboring MYC as a driver oncoprotein that can be targeted with clinically approved inhibitors. MYC is a central regulator of gene transcription and is frequently dysregulated in human cancers. As targeting MYC directly is challenging, an alternative strategy is to identify specific proteins or processes required for MYC to function as a potent cancer driver that can be targeted to result in synthetic lethality. To identify potential targets in MYC-driven cancers, we performed a genome-wide CRISPR knockout screen using an isogenic pair of breast cancer cell lines in which MYC dysregulation is the switch from benign to transformed tumor growth. Proteins that regulate R-loops were identified as a potential class of synthetic lethal targets. Dysregulated MYC elevated global transcription and coincident R-loop accumulation. Topoisomerase 1 (TOP1), a regulator of R-loops by DNA topology, was validated to be a vulnerability in cells with high MYC activity. Genetic knockdown of TOP1 in MYC-transformed cells resulted in reduced colony formation compared with control cells, demonstrating synthetic lethality. Overexpression of RNaseH1, a riboendonuclease that specifically degrades R-loops, rescued the reduction in clonogenicity induced by TOP1 deficiency, demonstrating that this vulnerability is driven by aberrant R-loop accumulation. Genetic and pharmacologic TOP1 inhibition selectively reduced the fitness of MYC-transformed tumors in vivo. Finally, drug response to TOP1 inhibitors (i.e., topotecan) significantly correlated with MYC levels and activity across panels of breast cancer cell lines and patient-derived organoids. Together, these results highlight TOP1 as a promising target for MYC-driven cancers.Significance: CRISPR screening reveals topoisomerase 1 as an immediately actionable vulnerability in cancers harboring MYC as a driver oncoprotein that can be targeted with clinically approved inhibitors

Rnf8 deficiency impairs class switch recombination, spermatogenesis, and genomic integrity and predisposes for cancer

Signaling and repair of DNA double-strand breaks (DSBs) are critical for preventing immunodeficiency and cancer. These DNA breaks result from exogenous and endogenous DNA insults but are also programmed to occur during physiological processes such as meiosis and immunoglobulin heavy chain (IgH) class switch recombination (CSR). Recent studies reported that the E3 ligase RNF8 plays important roles in propagating DNA DSB signals and thereby facilitating the recruitment of various DNA damage response proteins, such as 53BP1 and BRCA1, to sites of damage. Using mouse models for Rnf8 mutation, we report that Rnf8 deficiency leads to impaired spermatogenesis and increased sensitivity to ionizing radiation both in vitro and in vivo. We also demonstrate the existence of alternative Rnf8-independent mechanisms that respond to irradiation and accounts for the partial recruitment of 53bp1 to sites of DNA damage in activated Rnf8−/− B cells. Remarkably, IgH CSR is impaired in a gene dose-dependent manner in Rnf8 mutant mice, revealing that these mice are immunodeficient. In addition, Rnf8−/− mice exhibit increased genomic instability and elevated risks for tumorigenesis indicating that Rnf8 is a novel tumor suppressor. These data unravel the in vivo pleiotropic effects of Rnf8

Inactivation of Chk2 and Mus81 Leads to Impaired Lymphocytes Development, Reduced Genomic Instability, and Suppression of Cancer

Chk2 is an effector kinase important for the activation of cell cycle checkpoints, p53, and apoptosis in response to DNA damage. Mus81 is required for the restart of stalled replication forks and for genomic integrity. Mus81Δex3-4/Δex3-4 mice have increased cancer susceptibility that is exacerbated by p53 inactivation. In this study, we demonstrate that Chk2 inactivation impairs the development of Mus81Δex3-4/Δex3-4 lymphoid cells in a cell-autonomous manner. Importantly, in contrast to its predicted tumor suppressor function, loss of Chk2 promotes mitotic catastrophe and cell death, and it results in suppressed oncogenic transformation and tumor development in Mus81Δex3-4/Δex3-4 background. Thus, our data indicate that an important role for Chk2 is maintaining lymphocyte development and that dual inactivation of Chk2 and Mus81 remarkably inhibits cancer

RNF8 ubiquitylation of XRN2 facilitates R-loop resolution and restrains genomic instability in BRCA1 mutant cells

Breast cancer linked with BRCA1/2 mutations commonly recur and resist current therapies, including PARP inhibitors. Given the lack of effective targeted therapies for BRCA1-mutant cancers, we sought to identify novel targets to selectively kill these cancers. Here, we report that loss of RNF8 significantly protects Brca1-mutant mice against mammary tumorigenesis. RNF8 deficiency in human BRCA1-mutant breast cancer cells was found to promote R-loop accumulation and replication fork instability, leading to increased DNA damage, senescence, and synthetic lethality. Mechanistically, RNF8 interacts with XRN2, which is crucial for transcription termination and R-loop resolution. We report that RNF8 ubiquitylates XRN2 to facilitate its recruitment to R-loop-prone genomic loci and that RNF8 deficiency in BRCA1-mutant breast cancer cells decreases XRN2 occupancy at R-loop-prone sites, thereby promoting R-loop accumulation, transcription-replication collisions, excessive genomic instability, and cancer cell death. Collectively, our work identifies a synthetic lethal interaction between RNF8 and BRCA1, which is mediated by a pathological accumulation of R-loops

Smoking‐induced genetic and epigenetic alterations in infertile men

Male fertility rates have shown a progressive decrease in both developing and industrialised countries in the past 50 years. Clinical and epidemiological studies have demonstrated controversial results about the harmful effects of cigarette smoking on seminal parameters. Some studies could not establish a negative effect by tobacco smoking on sperm quality and function, whereas others have found a significant reduction in sperm quality and function. This study reviews the components in cigarette smoke and discusses the effects of smoking on male fertility by focusing extensively on smoking‐induced genetic and epigenetic alterations in infertile men. Chromosomal aneuploidies, sperm DNA fragmentation and gene mutations are discussed in the first section, while changes in DNA methylation, chromatin remodelling and noncoding RNAs are discussed in the second section as part of epigenetic alterations

Caspase-8 is essential for maintaining chromosomal stability and suppressing B-cell lymphomagenesis

International audienc

Synergistic Interaction of Rnf8 and p53 in the Protection against Genomic Instability and Tumorigenesis

10.1371/journal.pgen.1003259PLoS Genetics91e100325

RNF168 ubiquitylates 53BP1 and controls its response to DNA double-strand breaks

Defective signaling or repair of DNA double-strand breaks has been associated with developmental defects and human diseases. The E3 ligase RING finger 168 (RNF168), mutated in the human radiosensitivity, immunodeficiency, dysmorphic features, and learning difficulties syndrome, was shown to ubiquitylate H2A-type histones, and this ubiquitylation was proposed to facilitate the recruitment of p53-binding protein 1 (53BP1) to the sites of DNA double-strand breaks. In contrast to more upstream proteins signaling DNA double-strand breaks (e.g., RNF8), deficiency of RNF168 fully prevents both the initial recruitment to and retention of 53BP1 at sites of DNA damage; however, the mechanism for this difference has remained unclear. Here, we identify mechanisms that regulate 53BP1 recruitment to the sites of DNA double-strand breaks and provide evidence that RNF168 plays a central role in the regulation of 53BP1 functions. RNF168 mediates K63-linked ubiquitylation of 53BP1 which is required for the initial recruitment of 53BP1 to sites of DNA double-strand breaks and for its function in DNA damage repair, checkpoint activation, and genomic integrity. Our findings highlight the multistep roles of RNF168 in signaling DNA damage

Radioresistance and increased proliferation of <i>Rnf8^−/−</i> cells in the absence of p53.

<p>(A) Freshly isolated thymocytes from <i>Rnf8^−/−p53^−/−</i> mice and control littermates were irradiated with 2 Gy and cell death was determined 12 hours later. Data are presented as the means SD of at least 5 independent experiments. * represents significant difference (P<0.05; student t-test) compared to <i>WT</i> and <i>Rnf8^−/−</i> controls. No difference was observed between <i>p53^−/−</i> and <i>Rnf8^−/−p53^−/−</i> thymoctes. (B) Freshly isolated splenocytes were irradiated (2 Gy) and cell death was determined 24 hours later. Data are presented as the means SD of at least 5 independent experiments. * represents statistical significance (P<0.05; student t-test) compared to <i>WT</i> and <i>Rnf8^−/−</i> controls. No significant difference was observed between <i>p53^−/−</i> and <i>Rnf8^−/−p53^−/−</i> splenocytes. (C) Cumulative growth curve of <i>Rnf8^−/−p53^−/−</i> MEFs. Passage 2 <i>Rnf8^−/−p53^−/−</i> MEFs and controls were plated in 60 mm plates at a density of 3×10⁵ cells/plate. Cells were replated at the same density every 3 days and cumulative cell growth was calculated. Data are presented as the log10 of means ± SD of at least 5 independent experiments.</p

p53 deficiency rescues the senescence of <i>Rnf8^−/−</i> MEFs.

<p>(A) Western blot analysis of <i>Rnf8^−/−p53^−/−</i> and control MEFs for p21 and p19^ARF under untreated conditions and following 8 Gy ionizing radiation. β-actin is used as a loading control. * indicates a non specific band. (B) Western blot analysis of phosphorylated Ser-p53 and total p53 in untreated or γ-irradiated <i>Rnf8^−/−p53^−/−</i> and control MEFs. β-actin was used as a loading control. * indicates a non specific band. (C) Senescence-associated β-galactosidase staining of <i>Rnf8^−/−</i> MEFs and controls. MEFs were fixed and stained with X-gal solutions. The cells were then visualized using a brightfield microscope. These images are representative of three different experiments. (D) Quantification of the percentages of β-galactosidase-positive cells for 3 different experiments. * indicates P<0.05. Bar: 500 µm.</p