Osteosarcoma is the most common primary bone tumor, yet there have been no substantial advances in treatment or survival in three decades. We examined 59 tumor/normal pairs by whole-exome, whole-genome, and RNA-sequencing. Only the TP53 gene was mutated at significant frequency across all samples. The mean nonsilent somatic mutation rate was 1.2 mutations per megabase, and there was a median of 230 somatic rearrangements per tumor. Complex chains of rearrangements and localized hypermutation were detected in almost all cases. Given the intertumor heterogeneity, the extent of genomic instability, and the difficulty in acquiring a large sample size in a rare tumor, we used several methods to identify genomic events contributing to osteosarcoma survival. Pathway analysis, a heuristic analytic algorithm, a comparative oncology approach, and an shRNA screen converged on the phosphatidylinositol 3-kinase/mammalian target of rapamycin (PI3K/mTOR) pathway as a central vulnerability for therapeutic exploitation in osteosarcoma. Osteosarcoma cell lines are responsive to pharmacologic and genetic inhibition of the PI3K/mTOR pathway both in vitro and in vivo., the most common primary bone tumor, is an aggressive cancer that affects children, adolescents, and young adults. In contrast to the improvements in 5-year overall survival for childhood cancers from 58% to 82% in the past three decades, the overall survival for pediatric OS has remained static over that same time period at 60% (1, 2).Predisposition to OS is associated with germline syndromes, including hereditary retinoblastoma and Li-Fraumeni syndrome (3, 4). OS is also seen in syndromes with mutations in RECQ helicases and SQSTM1 (5, 6). However, most cases of OS develop sporadically and are characterized by complex genomics. The first genome-wide association study conducted in OS only identified two susceptibility loci implicating one gene, GRM4, a glutamate receptor (7).Linkage with hereditary retinoblastoma and Li Fraumeni led to the recognition of recurrent somatic alterations in TP53, RB1, and genes interacting with TP53 and RB1 in OS (8, 9). Candidate-gene approaches demonstrated recurrent somatic mutations, deletions, and rearrangement affecting TP53 (9). Additional mechanisms of p53 inactivation described in OS are MDM2 and COPS3 amplification (8, 9). RB1 mutations are present in 6% and deletions or structural alterations are seen in 40% of cases (10,11). CDKN2A is deleted in 10-20% of OS (9, 12). Multiple other cancer-associated genes have been reported to be altered in OS [reviewed in Kansara and Thomas (5)]. Many of these studies SignificanceWe present, to our knowledge, the first comprehensive nextgeneration sequencing of osteosarcoma in combination with a functional genomic screen in a genetically defined mouse model of osteosarcoma. Our data provide a strong rationale for targeting the phosphatidylinositol 3-kinase/mammalian target of rapamycin pathway in osteosarcoma and a foundation for rational clinical trial design. These findings present an immed...

Although DNA replication is a fundamental aspect of biology, it is not known what determines where DNA replication starts and stops in the human genome. Here we directly identify and quantitatively compare sites of replication initiation and termination in untransformed human cells. We report that replication preferentially initiates at the transcription start site of genes occupied by high levels of RNA polymerase II, and terminates at their polyadenylation sites, thus ensuring global co-directionality of transcription and replication, particularly at gene 5’ ends. During replication stress, replication initiation is stimulated downstream of genes and termination is redistributed to gene bodies; this globally re-orients replication relative to transcription around gene 3’ ends. These data suggest that replication initiation and termination are coupled to transcription in human cells, and propose a model for the impact of replication stress on genome integrity.

DNA replication stress is often defined by the slowing or stalling of replication fork progression leading to local or global DNA synthesis inhibition. Failure to resolve replication stress in a timely manner contribute toward cell cycle defects, genome instability and human disease; however, the mechanism for fork recovery remains poorly defined. Here, we show that the translesion DNA polymerase (Pol) kappa, a DinB orthologue, has a unique role in both protecting and restarting stalled replication forks under conditions of nucleotide deprivation. Importantly, Pol kappa-mediated DNA synthesis during hydroxyurea (HU)-dependent fork restart is regulated by both the Fanconi Anemia (FA) pathway and PCNA polyubiquitination. Loss of Pol kappa prevents timely rescue of stalled replication forks, leading to replication-associated genomic instability, and a p53-dependent cell cycle defect. Taken together, our results identify a previously unanticipated role for Pol kappa in promoting DNA synthesis and replication stress recovery at sites of stalled forks.

Guanine-rich DNA sequences occur throughout the human genome and can transiently form G-quadruplex (G4) structures that may obstruct DNA replication, leading to genomic instability. Here, we apply multi-color single-molecule localization microscopy (SMLM) coupled with robust data-mining algorithms to quantitatively visualize replication fork (RF)-coupled formation and spatial-association of endogenous G4s. Using this data, we investigate the effects of G4s on replisome dynamics and organization. We show that a small fraction of active replication forks spontaneously form G4s at newly unwound DNA immediately behind the MCM helicase and before nascent DNA synthesis. These G4s locally perturb replisome dynamics and organization by reducing DNA synthesis and limiting the binding of the single-strand DNA-binding protein RPA. We find that the resolution of RF-coupled G4s is mediated by an interplay between RPA and the FANCJ helicase. FANCJ deficiency leads to G4 accumulation, DNA damage at G4-associated replication forks, and silencing of the RPA-mediated replication stress response. Our study provides first-hand evidence of the intrinsic, RF-coupled formation of G4 structures, offering unique mechanistic insights into the interference and regulation of stable G4s at replication forks and their effect on RPA-associated fork signaling and genomic instability.

DNA replication stress, defined as the slowing or stalling of replication forks, is considered an emerging hallmark of cancer and a major contributor to genomic instability associated with tumorigenesis [1]. Recent advances have been made in attempting to target DNA repair factors involved in alleviating replication stress to potentiate genotoxic treatments. Various inhibitors of ATR and Chk1, the two major kinases involved in the intra-S-phase checkpoint, are currently in Phase I and II clinical trials [2]. In addition, currently approved inhibitors of Poly-ADP Ribose Polymerase (PARP) show synthetic lethality in cells that lack double-strand break repair such as in BRCA½ deficient tumors [3]. These drugs have also been shown to exacerbate replication stress by creating a DNA-protein crosslink, termed PARP 'trapping', and this is now thought to contribute to the therapeutic efficacy. Translesion synthesis (TLS) is a mechanism whereby special repair DNA polymerases accommodate and tolerate various DNA lesions to allow for damage bypass and continuation of DNA replication [4]. This class of proteins is best characterized by the Y-family, encompassing DNA polymerases (Pols) Kappa, Eta, Iota, and Rev1. While best studied for their ability to bypass physical lesions in the DNA, there is accumulating evidence for these proteins in coping with various natural replication fork barriers and alleviating replication stress. In this mini-review, we will highlight some of these recent advances, and discuss why targeting the TLS pathway may be a mechanism of enhancing cancer associated replication stress. Exacerbation of replication stress can lead to increased genome instability, which can be toxic to cancer cells and represent a therapeutic vulnerability.

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Common fragile sites (CFSs) are breakage-prone genomic loci, and are considered to be hotspots for genomic rearrangements frequently observed in cancers. Understanding the underlying mechanisms for CFS instability will lead to better insight on cancer etiology. Here we show that Polycomb group proteins BMI1 and RNF2 are suppressors of transcriptionreplication conflicts (TRCs) and CFS instability. Cells depleted of BMI1 or RNF2 showed slower replication forks and elevated fork stalling. These phenotypes are associated with increase occupancy of RNA Pol II (RNAPII) at CFSs, suggesting that the BMI1-RNF2 complex regulate RNAPII elongation at these fragile regions. Using proximity ligase assays, we showed that depleting BMI1 or RNF2 causes increased associations between RNAPII with EdU-labeled nascent forks and replisomes, suggesting increased TRC incidences. Increased occupancy of a fork protective factor FANCD2 and R-loop resolvase RNH1 at CFSs are observed in RNF2 CRISPR-KO cells, which are consistent with increased transcription-associated replication stress in RNF2-deficient cells. Depleting FANCD2 or FANCI proteins further increased genomic instability and cell death of the RNF2-deficient cells, suggesting that in the absence of RNF2, cells depend on these fork-protective factors for survival. These data suggest that the Polycomb proteins have non-canonical roles in suppressing TRC and preserving genomic integrity.