Proliferating Cell Nuclear Antigen (PCNA) lies at the center of the faithful duplication of eukaryotic genomes. With its distinctive doughnut-shaped molecular structure, PCNA was originally studied for its role in stimulating DNA polymerases. However, we now know that PCNA does much more than promote processive DNA synthesis. Because of the complexity of the events involved, cellular DNA replication poses major threats to genomic integrity. Whatever predicament lies ahead for the replication fork, PCNA is there to orchestrate the events necessary to handle it. Through its many protein interactions and various post-translational modifications, PCNA has far-reaching impacts on a myriad of cellular functions.

Background: PCNA mono-ubiquitination at stalled replication forks recruits translesion synthesis polymerases for fork restart. Results:The mono-ADP-ribosyltransferase PARP10 interacts with PCNA through a PIP-box. PARP10 knockdown results in DNA damage hypersensitivity and defective translesion synthesis. Conclusion: PARP10 participates in PCNA-dependent DNA damage tolerance. Significance: This is the first time that post-translational modification by mono-ADP-ribosylation is implicated in DNA repair.

Defects in DNA replication, DNA damage response, and DNA repair compromise genomic stability and promote cancer development. In particular, unrepaired DNA lesions can arrest the progression of the DNA replication machinery during S-phase, causing replication stress, mutations, and DNA breaks. HUWE1 is a HECT-type ubiquitin ligase that targets proteins involved in cell fate, survival, and differentiation. Here, we report that HUWE1 is essential for genomic stability, by promoting replication of damaged DNA. We show that HUWE1-knockout cells are unable to mitigate replication stress, resulting in replication defects and DNA breakage. Importantly, we find that this novel role of HUWE1 requires its interaction with the replication factor PCNA, a master regulator of replication fork restart, at stalled replication forks. Finally, we provide evidence that HUWE1 mono-ubiquitinates H2AX to promote signaling at stalled forks. Altogether, our work identifies HUWE1 as a novel regulator of the replication stress response.

Genomic instability, a major hallmark of cancer cells, is caused by incorrect or ineffective DNA repair. Many DNA repair mechanisms cooperate in cells to fight DNA damage, and are generally regulated by post-translational modification of key factors. Poly-ADP-ribosylation, catalyzed by PARP1, is a post-translational modification playing a prominent role in DNA repair, but much less is known about mono-ADP-ribosylation. Here we report that mono-ADP-ribosylation plays an important role in homologous recombination DNA repair, a mechanism essential for replication fork stability and double strand break repair. We show that the mono-ADP-ribosyltransferase PARP14 interacts with the DNA replication machinery component PCNA and promotes replication of DNA lesions and common fragile sites. PARP14 depletion results in reduced homologous recombination, persistent RAD51 foci, hypersensitivity to DNA damaging agents and accumulation of DNA strand breaks. Our work uncovered PARP14 as a novel factor required for mitigating replication stress and promoting genomic stability.

Two structurally related protein kinase families, the Rho kinases (ROCK) and the myotonic dystrophy kinase-related Cdc42-binding kinases (MRCK) are required for migration and invasion of cancer cells. We hypothesized that simultaneous targeting of these two kinase families might represent a novel therapeutic strategy to block the migration and invasion of metastatic cancers. To this end, we developed DJ4 as a novel small molecule inhibitor of these kinases. DJ4 potently inhibited activities of ROCK and MRCK in an ATP competitive manner. In cellular functional assays, DJ4 treatment significantly blocked stress fiber formation and inhibited migration and invasion of multiple cancer cell lines in a concentration dependent manner. Our results strongly indicate that DJ4 may be further developed as a novel anti-metastatic chemotherapeutic agent for multiple cancers.

Mitotic cells attenuate the DNA damage response (DDR) by phosphorylating 53BP1, a critical DDR mediator, to prevent its localization to damaged chromatin. Timely dephosphorylation of 53BP1 is critical for genome integrity, as premature recruitment of 53BP1 to DNA lesions impairs mitotic fidelity. Protein phosphatase 4 (PP4) dephosphorylates 53BP1 in late mitosis to allow its recruitment to DNA lesions in G1. How cells appropriately dephosphorylate 53BP1, thereby restoring DDR, is unclear. Here, we elucidate the underlying mechanism of kinetic control of 53BP1 dephosphorylation in mitosis. We demonstrate that CDK5, a kinase primarily functional in post-mitotic neurons, is active in late mitotic phases in non-neuronal cells and directly phosphorylates PP4R3β, the PP4 regulatory subunit that recognizes 53BP1. Specific inhibition of CDK5 in mitosis abrogates PP4R3β phosphorylation and abolishes its recognition and dephosphorylation of 53BP1, ultimately preventing the localization of 53BP1 to damaged chromatin. Our results establish CDK5 as a regulator of 53BP1 recruitment.

Rho-associated coiled-coil containing protein kinase (Rho kinase/ROCK) and myotonic dystrophy kinase-related Cdc42-binding kinase (MRCK) are involved in the aggressive migration of cancer cells. These are the key proteins involved in the formation of stress fibers that confer contractility to cancer cells during migration and invasion. ROCK and MRCK are primarily involved in amoeboid and mesenchymal type of migration of cancer cells respectively. Earlier studies reported that combined inhibition/silencing of ROCK and MRCK has more pronounced effect than inhibition of the individual kinases suggesting a more effective therapeutic strategy to combat metastatic cancers. With this hypothesis in mind, we developed a series of novel multikinase inhibitors and identified DJ4 as a potent ATP competitive inhibitor of ROCK1, ROCK2 and MRCKβ. DJ4 effectively inhibits the kinase activity of ROCK (ROCK1 and ROCK2) and MRCK in a cell-free system and multiple human non-small cell lung cancer cell lines. In cellular functional assays DJ4 inhibited migration of human melanoma, lung, pancreatic and breast cancer cells in a concentration-dependent manner. In live cell imaging over 20h by time-lapse microscopy, DJ4 (2.5µM ) reduced the migration rate of lung-and breast-cancer cells by 2.7 and 5.5 times compared to control cells respectively. DJ4 (5µM) also inhibited invasion of MDA-MB-231 breast cancer cells by ∼70%. Fluorescent microscopic observation also revealed that inhibition of activity of these kinases, by DJ4, causes reduction in stress fibers which leads to inhibition of cell contractility and migration. In summary, our studies demonstrate that DJ4 is a potent ATP competitive inhibitor of ROCK and MRCK, and effectively inhibits migration and invasion of multiple cancers in vitro. Together these results indicate that DJ4 has a strong potential to be an anti-metastatic drug against multiple cancer types. Citation Format: Vijaykumar P. Kale, Dhimant Desai, Taryn Dick, Katherine Choe, Ashley Colledge, Shantu Amin, Jeremy Hengst, Jong Yun. DJ4, a novel ROCK and MRCK inhibitor, potently inhibits migration and invasion of cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 736. doi:10.1158/1538-7445.AM2014-736