Excessive transcription-replication conflicts are a vulnerability of BRCA1-mutant cancers.

BRCA1 mutations are associated with increased breast and ovarian cancer risk. BRCA1-mutant tumors are high-grade, recurrent, and often become resistant to standard therapies. Herein, we performed a targeted CRISPR-Cas9 screen and identified MEPCE, a methylphosphate capping enzyme, as a synthetic lethal interactor of BRCA1. Mechanistically, we demonstrate that depletion of MEPCE in a BRCA1-deficient setting led to dysregulated RNA polymerase II (RNAPII) promoter-proximal pausing, R-loop accumulation, and replication stress, contributing to transcription-replication collisions. These collisions compromise genomic integrity resulting in loss of viability of BRCA1-deficient cells. We also extend these findings to another RNAPII-regulating factor, PAF1. This study identifies a new class of synthetic lethal partners of BRCA1 that exploit the RNAPII pausing regulation and highlight the untapped potential of transcription-replication collision-inducing factors as unique potential therapeutic targets for treating cancers associated with BRCA1 mutations.

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.

DNA double-strand break-capturing nuclear envelope tubules drive DNA repair.

Current models suggest that DNA double-strand breaks (DSBs) can move to the nuclear periphery for repair. It is unclear to what extent human DSBs display such repositioning. Here we show that the human nuclear envelope localizes to DSBs in a manner depending on DNA damage response (DDR) kinases and cytoplasmic microtubules acetylated by α-tubulin acetyltransferase-1 (ATAT1). These factors collaborate with the linker of nucleoskeleton and cytoskeleton complex (LINC), nuclear pore complex (NPC) protein NUP153, nuclear lamina and kinesins KIF5B and KIF13B to generate DSB-capturing nuclear envelope tubules (dsbNETs). dsbNETs are partly supported by nuclear actin filaments and the circadian factor PER1 and reversed by kinesin KIFC3. Although dsbNETs promote repair and survival, they are also co-opted during poly(ADP-ribose) polymerase (PARP) inhibition to restrain BRCA1-deficient breast cancer cells and are hyper-induced in cells expressing the aging-linked lamin A mutant progerin. In summary, our results advance understanding of nuclear structure-function relationships, uncover a nuclear-cytoplasmic DDR and identify dsbNETs as critical factors in genome organization and stability.

Emerging roles of DNA topoisomerases in the regulation of R-loops.

R-loops are comprised of a DNA:RNA hybrid and a displaced single-strand DNA (ssDNA) that reinvades the DNA duplex behind the moving RNA polymerase. Because they have several physiological functions within the cell, including gene expression, chromosomal segregation, and mitochondrial DNA replication, among others, R-loop homeostasis is tightly regulated to ensure normal functioning of cellular processes. Thus, several classes of enzymes including RNases, helicases, topoisomerases, as well as proteins involved in splicing and the biogenesis of messenger ribonucleoproteins, have been implicated in R-loop prevention, suppression, and resolution. There exist six topoisomerase enzymes encoded by the human genome that function to introduce transient DNA breaks to relax supercoiled DNA. In this mini-review, we discuss functions of DNA topoisomerases and their emerging role in transcription, replication, and regulation of R-loops, and we highlight how their role in maintaining genome stability can be exploited for cancer therapy.

BRCA1 and Metastasis: Outcome of Defective DNA Repair.

Heritable mutations in BRCA1 and BRCA2 genes are a major risk factor for breast and ovarian cancer. Inherited mutations in BRCA1 increase the risk of developing breast cancers by up to 72% and ovarian cancers by up to 69%, when compared to individuals with wild-type BRCA1. BRCA1 and BRCA2 (BRCA1/2) are both important for homologous recombination-mediated DNA repair. The link between BRCA1/2 mutations and high susceptibility to breast cancer is well established. However, the potential impact of BRCA1 mutation on the individual cell populations within a tumor microenvironment, and its relation to increased aggressiveness of cancer is not well understood. The objective of this review is to provide significant insights into the mechanisms by which BRCA1 mutations contribute to the metastatic and aggressive nature of the tumor cells.

Guanine nucleotide binding protein like-1 (GNL1) promotes cancer cell proliferation and survival through AKT/p21 CIP1 signaling cascade.

Human guanine nucleotide binding protein like 1 (GNL1) is an evolutionary conserved putative nucleolar GTPase belonging to the HSR1_MMR1 subfamily of GTPases. GNL1 was found to be highly up-regulated in various cancers. Here, we report for the first time that GNL1 inhibits apoptosis by modulating the expression of Bcl2 family of proteins and the cleavage of caspases 7 and 8. Furthermore, GNL1 protects colon cancer cells from chemo-drug-induced apoptosis. Interestingly, GNL1 up-regulates the expression of p53 and its transcriptional target, p21 but the up-regulation of p21 was found to be p53 dependent as well as independent mechanisms. Our results further demonstrate that GNL1 promotes cell growth and survival by inducing cytoplasmic retention and stabilization of p21 through AKT-mediated phosphorylation. In addition, GNL1 failed to inhibit apoptosis under p21 knockdown conditions which suggests the critical role of p21 in GNL1-mediated cell survival. Finally, an inverse correlation of GNL1, p21, and AKT expression in primary colon and breast cancer with patient survival suggests their critical role in tumorigenesis. Collectively, our study reveals that GNL1 executes its antiapoptotic function by a novel mechanism and suggests that it may function as a regulatory component of the PI3K/AKT/p21 signaling network to promote cell proliferation and survival in cancers.

Interplay between human nucleolar GNL1 and RPS20 is critical to modulate cell proliferation.

Human Guanine nucleotide binding protein like 1 (GNL1) belongs to HSR1_MMR1 subfamily of nucleolar GTPases. Here, we report for the first time that GNL1 promotes cell cycle and proliferation by inducing hyperphosphorylation of retinoblastoma protein. Using yeast two-hybrid screening, Ribosomal protein S20 (RPS20) was identified as a functional interacting partner of GNL1. Results from GST pull-down and co-immunoprecipitation assays confirmed that interaction between GNL1 and RPS20 was specific. Further, GNL1 induced cell proliferation was altered upon knockdown of RPS20 suggesting its critical role in GNL1 function. Interestingly, cell proliferation was significantly impaired upon expression of RPS20 interaction deficient GNL1 mutant suggest that GNL1 interaction with RPS20 is critical for cell growth. Finally, the inverse correlation of GNL1 and RPS20 expression in primary colon and gastric cancers with patient survival strengthen their critical importance during tumorigenesis. Collectively, our data provided evidence that cross-talk between GNL1 and RPS20 is critical to promote cell proliferation.

GNL3L Is a Nucleo-Cytoplasmic Shuttling Protein: Role in Cell Cycle Regulation.

GNL3L is an evolutionarily conserved high molecular weight GTP binding nucleolar protein belonging to HSR1-MMR1 subfamily of GTPases. The present investigation reveals that GNL3L is a nucleo-cytoplasmic shuttling protein and its export from the nucleus is sensitive to Leptomycin B. Deletion mutagenesis reveals that the C-terminal domain (amino acids 501-582) is necessary and sufficient for the export of GNL3L from the nucleus and the exchange of hydrophobic residues (M567, L570 and 572) within the C-terminal domain impairs this process. Results from the protein-protein interaction analysis indicate that GNL3L interaction with CRM1 is critical for its export from the nucleus. Ectopic expression of GNL3L leads to lesser accumulation of cells in the 'G2/M' phase of cell cycle whereas depletion of endogenous GNL3L results in 'G2/M' arrest. Interestingly, cell cycle analysis followed by BrdU labeling assay indicates that significantly increased DNA synthesis occurs in cells expressing nuclear export defective mutant (GNL3L∆NES) compared to the wild type or nuclear import defective GNL3L. Furthermore, increased hyperphosphorylation of Rb at Serine 780 and the upregulation of E2F1, cyclins A2 and E1 upon ectopic expression of GNL3L∆NES results in faster 'S' phase progression. Collectively, the present study provides evidence that GNL3L is exported from the nucleus in CRM1 dependent manner and the nuclear localization of GNL3L is important to promote 'S' phase progression during cell proliferation.