Vijay Menon

Involvement of p53 in the repair of DNA double strand breaks: multifaceted Roles of p53 in homologous recombination repair (HRR) and non-homologous end joining (NHEJ).

p53 is a tumor suppressor protein that prevents oncogenic transformation and maintains genomic stability by blocking proliferation of cells harboring unrepaired or misrepaired DNA. A wide range of genotoxic stresses such as DNA damaging anti-cancer drugs and ionizing radiation promote nuclear accumulation of p53 and trigger its ability to activate or repress a number of downstream target genes involved in various signaling pathways. This cascade leads to the activation of multiple cell cycle checkpoints and subsequent cell cycle arrest, allowing the cells to either repair the DNA or undergo apoptosis, depending on the intensity of DNA damage. In addition, p53 has many transcription-independent functions, including modulatory roles in DNA repair and recombination. This chapter will focus on the role of p53 in regulating or influencing the repair of DNA double-strand breaks that mainly includes homologous recombination repair (HRR) and non-homologous end joining (NHEJ). Through this discussion, we will try to establish that p53 acts as an important linchpin between upstream DNA damage signaling cues and downstream cellular events that include repair, recombination, and apoptosis.

End-processing nucleases and phosphodiesterases: An elite supporting cast for the non-homologous end joining pathway of DNA double-strand break repair.

Nonhomologous end joining (NHEJ) is an error-prone DNA double-strand break repair pathway that is active throughout the cell cycle. A substantial fraction of NHEJ repair events show deletions and, less often, insertions in the repair joints, suggesting an end-processing step comprising the removal of mismatched or damaged nucleotides by nucleases and other phosphodiesterases, as well as subsequent strand extension by polymerases. A wide range of nucleases, including Artemis, Metnase, APLF, Mre11, CtIP, APE1, APE2 and WRN, are biochemically competent to carry out such double-strand break end processing, and have been implicated in NHEJ by at least circumstantial evidence. Several additional DNA end-specific phosphodiesterases, including TDP1, TDP2 and aprataxin are available to resolve various non-nucleotide moieties at DSB ends. This review summarizes the biochemical specificities of these enzymes and the evidence for their participation in the NHEJ pathway.

Nucleolar targeting by platinum: p53-independent apoptosis follows rRNA inhibition, cell-cycle arrest, and DNA compaction.

TriplatinNC is a highly positively charged, substitution-inert derivative of the phase II clinical anticancer drug, BBR3464. Such substitution-inert complexes form a distinct subset of polynuclear platinum complexes (PPCs) interacting with DNA and other biomolecules through noncovalent interactions. Rapid cellular entry is facilitated via interaction with cell surface glycosoaminoglycans and is a mechanism unique to PPCs. Nanoscale secondary ion mass spectrometry (nanoSIMS) showed rapid distribution within cytoplasmic and nucleolar compartments, but not the nucleus. In this article, the downstream effects of nucleolar localization are described. In human colon carcinoma cells, HCT116, the production rate of 47S rRNA precursor transcripts was dramatically reduced as an early event after drug treatment. Transcriptional inhibition of rRNA was followed by a robust G1 arrest, and activation of apoptotic proteins caspase-8, -9, and -3 and PARP-1 in a p53-independent manner. Using cell synchronization and flow cytometry, it was determined that cells treated while in G1 arrest immediately, but cells treated in S or G2 successfully complete mitosis. Twenty-four hours after treatment, the majority of cells finally arrest in G1, but nearly one-third contained highly compacted DNA; a distinct biological feature that cannot be associated with mitosis, senescence, or apoptosis. This unique effect mirrored the efficient condensation of tRNA and DNA in cell-free systems. The combination of DNA compaction and apoptosis by TriplatinNC treatment conferred striking activity in platinum-resistant and/or p53 mutant or null cell lines. Taken together, our results support that the biological activity of TriplatinNC reflects reduced metabolic deactivation (substitution-inert compound not reactive to sulfur nucleophiles), high cellular accumulation, and novel consequences of high-affinity noncovalent DNA binding, producing a new profile and a further shift in the structure–activity paradigms for antitumor complexes.

Ligand modulation of a dinuclear platinum compound leads to mechanistic differences in cell cycle progression and arrest

Despite similar structures and DNA binding profiles, two recently synthesized dinuclear platinum compounds are shown to elicit highly divergent effects on cell cycle progression. In colorectal HCT116 cells, BBR3610 shows a classical G2/M arrest with initial accumulation in S phase, but the derivative compound BBR3610-DACH, formed by introduction of the 1,2-diaminocyclohexane (DACH) as carrier ligand, results in severe G1/S as well as G2/M phase arrest, with nearly complete S phase depletion. The origin of this unique effect was studied. Cellular interstrand crosslinking as assayed by comet analysis was similar for both compounds, confirming previous in vitro results obtained on plasmid DNA. Immunoblotting revealed a stabilization of p53 and concomitant transient increases in p21 and p27 proteins after treatment with BBR3610-DACH. Cell viability assays and cytometric analysis of p53 and p21 null cells indicated that BBR3610-DACH-induced cell cycle arrest was p21-dependent and partially p53-dependent. However, an increase in the levels of cyclin E was observed with steady state levels of CDK2 and Cdc25A, suggesting that the G1 block occurs downstream of CDK/cyclin complex formation. The G2/M block was corroborated with decreased levels of cyclin A and cyclin B1. Surprisingly, BBR3610-DACH-induced G1 block was independent of ATM and ATR. Finally, both compounds induced apoptosis, with BBR3610-DACH showing a robust PARP-1 cleavage that was not associated with caspase-3/7 cleavage. In summary, BBR3610-DACH is a DNA binding platinum agent with unique inhibitory effects on cell cycle progression that could be further developed as a chemotherapeutic agent complementary to cisplatin and oxaliplatin.

Tracking the processing of damaged DNA double-strand break ends by ligation-mediated PCR: increased persistence of 3′-phosphoglycolate termini in SCAN1 cells

To track the processing of damaged DNA double-strand break (DSB) ends in vivo, a method was devised for quantitative measurement of 3′-phosphoglycolate (PG) termini on DSBs induced by the non-protein chromophore of neocarzinostatin (NCS-C) in the human Alu repeat. Following exposure of cells to NCS-C, DNA was isolated, and labile lesions were chemically stabilized. All 3′-phosphate and 3′-hydroxyl ends were enzymatically capped with dideoxy termini, whereas 3′-PG ends were rendered ligatable, linked to an anchor, and quantified by real-time Taqman polymerase chain reaction. Using this assay and variations thereof, 3′-PG and 3′-phosphate termini on 1-base 3′ overhangs of NCS-C-induced DSBs were readily detected in DNA from the treated lymphoblastoid cells, and both were largely eliminated from cellular DNA within 1 h. However, the 3′-PG termini were processed more slowly than 3′-phosphate termini, and were more persistent in tyrosyl-DNA phosphodiesterase 1-mutant SCAN1 than in normal cells, suggesting a significant role for tyrosyl-DNA phosphodiesterase 1 in removing 3′-PG blocking groups for DSB repair. DSBs with 3′-hydroxyl termini, which are not directly induced by NCS-C, were formed rapidly in cells, and largely eliminated by further processing within 1 h, both in Alu repeats and in heterochromatic α-satellite DNA. Moreover, absence of DNA-PK in M059J cells appeared to accelerate resolution of 3′-PG ends.

XLF/Cernunnos: An important but puzzling participant in the nonhomologous end joining DNA repair pathway

DNA double strand breaks (DSBs) are one of the most deleterious DNA lesions that promote cell death, genomic instability and carcinogenesis. The two major cellular mechanisms that repair DSBs are Nonhomologous End-Joining (NHEJ) and Homologous Recombination Repair (HRR). NHEJ is the predominant pathway, in which XLF (also called Cernunnos) is a key player. Patients with XLF mutation exhibit microcephaly, lymphopenia, and growth retardation, and are immunodeficient and radiosensitive. During NHEJ, XLF interacts with XRCC4-Ligase IV, stimulates its ligase activity, and forms DNA-binding filaments of alternating XLF and XRCC4 dimers that may serve to align broken DNA and promote ligation of noncomplementary ends. Despite its central role in NHEJ, the effects of XLF deficiency are surprisingly variable in different biological contexts, and different individual cell lines. This review summarizes the role of XLF in NHEJ, and the unexpected complexity of its interplay with other repair factors in supporting radiosurvival and V(D)J recombination.

DYRK1A regulates the recruitment of 53BP1 to the sites of DNA damage in part through interaction with RNF169

Human DYRK1A gene encoding Dual-specificity tyrosine (Y)- Regulated Kinase 1A (DYRK1A) is a dosage-dependent gene whereby either trisomy or haploinsufficiency result in developmental abnormalities. However, the function and regulation of this important protein kinase are not fully understood. Here we report proteomic analysis of DYRK1A in human cells that revealed a novel role of DYRK1A in the DNA double-strand break (DSB) repair signaling. This novel function of DYRK1A is mediated in part by its interaction with ubiquitin-binding protein RNF169 that regulates the choice between homologous recombination (HR) and non-homologous end joining (NHEJ) DSB repair. Accumulation of RNF169 at the DSB sites promotes homologous recombination (HR) by limiting the recruitment of the scaffold protein 53BP1 that promotes NHEJ by protecting the DNA ends from resection. Inducible overexpression of active, but not the kinase inactive, DYRK1A in U-2 OS cells inhibited accumulation of 53BP1 at the DSB sites in RNF169-dependent manner. Mutation of DYRK1A phosphorylation sites in RNF169 or pharmacological inhibition of DYRK1A using harmine decreased the ability of RNF169 to displace 53BP1 from radiation-induced DSB sites. In order to further investigate the role of DYRK1A in regulation of DNA repair, we used CRISPR-Cas9 mediated knockout of DYRK1A in human and mouse cells. Interestingly, knockout of DYRK1A also caused a defect in 53BP1 DSB recruitment that was independent of RNF169, suggesting that dosage of DYRK1A can influence the DNA repair processes through several mechanisms. U-2 OS cells devoid of DYRK1A displayed an increased DNA repair and HR efficiency, and showed a decreased sensitivity to the PARP inhibitor olaparib when compared to control cells. Given evidence of its altered expression in human cancers, DYRK1A levels could represent a significant determinant of the DNA damaging therapy response.

2. Polynuclear Platinum Complexes. Structural Diversity and DNA Binding

Polynuclear platinum complexes (PPCs) represent a discrete structural class of DNA-binding agents with excellent antitumor properties. The use of at least two platinum coordinating units automatically means that multifunctional DNA binding modes are possible. The structural variability inherent in a polynuclear platinum structure can be harnessed to produce discrete modes of DNA binding, with conformational changes distinct from and indeed inaccessible to, the mononuclear agents such as cisplatin. Since our original contributions in this field a wide variety of dinuclear complexes especially have been prepared, their DNA binding studied, and potential relevance to cytotoxicity examined. This chapter focuses on how DNA structure and reactivity is modulated through interactions with PPCs with emphasis on novel aspects of such structure and reactivity. How these major changes are further reflected in damaged DNA-protein binding and cellular effects are reviewed. We further review, for the first time, the great structural diversity achieved in PPC complex design and summarize their major DNA binding effects.

Abstract B10: The role of Down syndrome's DYRK1A kinase in repair of the DNA double strand breaks

The function of DYRK1A protein kinase is regulated by its gene dosage whereby both gains and losses of one copy of DYRK1A gene on chromosome 21 result in developmental abnormalities. In order to better understand the function and regulation of DYRK1A, we applied a highly sensitive MudPIT proteomic approach to identify DYRK1A-interacting proteins in human cells. Four biological replicate MudPIT experiments were performed and the proteins reproducibly detected in the DYRK1A immunoprecipitates but not in the controls, were identified. Six proteins detected in all four biological replicate experiments were also most highly enriched in the DYRK1A immunoprecipitates, suggesting that these proteins form stable and abundant complexes with DYRK1A. One of these proteins, RNF169, has been recently characterized as a component of ubiquitin-mediated cascade involved in the repair of DNA double-strand breaks (DSBs). DSBs are deleterious DNA lesions that are repaired by hierarchical and orchestrated recruitment of multiple different proteins to a modified chromatin in the vicinity of the DNA damage sites. Presence of specific chromatin marks including ubiquitination regulates the choice between two major DSB repair pathways: homologous recombination repair (HRR) and non-homologous end joining (NHEJ), mediated by recruitment of chromatin-binding DNA damage response proteins including 53BP1 and RNF169. Binding of 53BP1 could prevent the resection of the DNA strands near the damage site necessary for the HRR while RNF169 is thought to limit the 53BP1 accumulation and therefore, to promote the HRR. To determine whether DYRK1A plays a role in these processes, we knocked out its expression in human and mouse cell lines using CRISPR-Cas9 approach. We found that initial accumulation of 53BP1 at the gamma-irradiation induced foci (IRIFs) was similar in the DYRK1A-null and in the control cells. However, both the number of the 53BP1 IRIFs and their persistence over time were significantly reduced in the cell lines that lacked DYRK1A. This effect was dependent on the presence of RNF169, suggesting that DYRK1A regulates the ability of RNF169 to limit 53BP19s accumulation at the DSBs. Next, we sought to determine the mechanism of this regulation and found that RNF169 is phosphorylated by DYRK1A at two sites located in a highly conserved domain with no known function. Interestingly, the phospho mimetic mutant of RNF169 displayed a decreased ability to inhibit 53BP1 IRIF formation when compared to the wild type or the non-phosphorylatable RNF169 alleles. We also determined the effect of DYRK1A overexpression on the recruitment of RNF169 and 53BP1 to the sites of IR-induced DNA damage. Surprisingly, accumulation of both RNF169 and 53BP1 in the IRIFs was diminished upon overexpression of active, but not the kinase-inactive DYRK1A. Domain-mapping of DYRK1A-RNF169 binding demonstrated that the ability of DYRK1A to abolish the RNF169 IRIF formation is independent of their interaction, suggesting that overexpression of functional DYRK1A could affect other factors in the cell that play a role in the DNA damage response. Since loss of DYRK1A could be relevant to cancer due to its widespread gene copy number losses, we determined the effect of DYRK1A loss on the ability of the cells to repair their DNA. Using the DR-GFP reporter of HRR and the neutral comet assays, we found that CRISPR-Cas9-mediated depletion of DYRK1A results in an increased efficiency of the DNA DSB repair. Our findings implicate DYRK1A in the critical processes of DNA damage response and characterize a novel function of this important protein kinase. Citation Format: Vijay R. Menon, Varsha Ananthapadmanabhan, Larisa Litovchick. The role of Down syndrome9s DYRK1A kinase in repair of the DNA double strand breaks [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr B10.

Abstract 1022: DYRK1A kinase regulates mTOR signaling via modulating the TSC complex

The Dual specificity Tyrosine-phosphorylation-Regulated Kinase 1A (DYRK1A) kinase is encoded on chromosome 21 and is involved in the pathogenesis of Down syndrome. Earlier studies have shown that DYRK1A is essential for cells to undergo growth arrest following serum starvation, at least in part by promoting the assembly of the DREAM repressor complex. However, the function of this important kinase is not fully understood. Using MudPIT MS/MS proteomic analysis of DYRK1A-interacting proteins followed by immunoprecipitation-Western blotting, we characterized the binding between DYRK1A and the subunits of the TSC tumor suppressor protein complex, a major regulator of the mTORC1 pathway. This interaction was mapped to the kinase domain of DYRK1A, suggesting that the TSC could serve as a substrate for DYRK1A phosphorylation. Indeed, overexpression of DYRK1A increased phosphorylation of both TSC1 and TSC2; this effect was fully reversed by the DYRK1A inhibitor, harmine. Functional studies revealed that loss of DYRK1A in two different cancer cell lines resulted in increased phosphorylation of mTORC1 substrates S6K1 and 4E-BP1. This effect of DYRK1A knockdown was inhibited by rapamycin, confirming the involvement of mTORC1 signaling. Thus, our work revealed novel function of DYRK1A in the regulation of mTOR signaling that controls cell growth, proliferation, and survival. Citation Format: Vijay R. Menon, Larisa Litovchick. DYRK1A kinase regulates mTOR signaling via modulating the TSC complex. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1022. doi:10.1158/1538-7445.AM2015-1022