Keya Bandyopadhyay

DNA Damage Disrupts the p14ARF-B23(Nucleophosmin) Interaction and Triggers a Transient Subnuclear Redistribution of p14ARF

The p14 alternate reading frame (ARF) tumor suppressor plays a central role in cancer by binding to mdm2 (Hdm2 in humans) and enhancing p53-mediated apoptosis following DNA damage and oncogene activation. It is unclear, however, how ARF initiates its involvement in the p53/mdm2 pathway, as p53 and mdm2 are located in the nucleoplasm, whereas ARF is largely nucleolar in tumor cells. We have used immunofluorescence and coimmunoprecipitation to examine how the subnuclear distribution and protein-protein interactions of ARF change immediately after DNA damage and over the time course of the DNA damage response in human tumor cells. We find that DNA damage disrupts the interaction of ARF with the nucleolar protein B23(nucleophosmin) and promotes a transient p53-independent translocation of ARF to the nucleoplasm, resulting in a masking of the ARF NH2 terminus that correlates with the appearance of ARF-Hdm2 complexes. The translocation also results in an unmasking of the ARF COOH terminus, suggesting that redistribution disrupts a nucleolar interaction of ARF involving this region. By 24 hours after irradiation, DNA repair has ceased and the pretreatment immunofluorescence patterns and complexes of ARF have been restored. Although the redistribution of ARF is independent of p53 and likely to be regulated by interactions other than Hdm2, ARF does not promote UV sensitization unless p53 is expressed. The results implicate the nucleolus and nucleolar interactions of the ARF, including potentially novel interactions involving its COOH terminus as sites for early DNA damage and stress-mediated cellular events.

Spermidinyl-CoA-based HAT inhibitors block DNA repair and provide cancer-specific chemo- and radiosensitization

Acetyl group turnover on specific lysine ε-amino groups of the core chromosomal histones regulates DNA accessibility function, and the acetylating and deacetylating enzymes that govern the turnover provide important targets for the development of anti-cancer drugs. Histone deacetylase (HDAC) inhibitors have been developed and evaluated extensively in clinical trials, while the development of inhibitors of histone acetyltransferase (HAT) has proceeded more slowly. Here we have examined the cellular effects of an S-substituted coenzyme A (CoA) inhibitor of histone acetylation, consisting of spermidine (Spd) linked to the S-terminus of CoA through a thioglycolic acid linkage (adduct abbreviated as Spd-CoA), as well as the effects of a truncated Spd-CoA derivative lacking the negatively charged portion of the CoA moiety. While exposure of cancer cells to Spd-CoA has little effect on cell viability, it causes a rapid inhibition of histone acetylation that correlates with a transient arrest of DNA synthesis, a transient delay in S-phase progression, and an inhibition of nucleotide excision repair and DNA double strand break repair. These effects correlate with increased cellular sensitivity to the DNA-targeted chemotherapeutic drugs, cisplatin (Platinol™) and 5-Fluorouracil, to the DNA damaging drug, camptothecin, and to UV-C irradiation. The sensitization effects of Spd-CoA are not observed in normal cells due to a barrier to uptake. The truncated Spd-CoA derivative displays similar but enhanced chemosensitization effects, suggesting that further modifications of the Spd-CoA structure could further improve potency. The results demonstrate that Spd-CoA and its truncated version are efficiently and selectively internalized into cancer cells, and suggest that the resulting inhibition of acetylation-dependent DNA repair enhances cellular sensitivity to DNA damage. These and related inhibitors of histone acetylation could therefore constitute a novel class of potent therapy sensitizers applicable to a broad range of conventional cancer treatments.

Protein kinase CK2 is a central regulator of topoisomerase I hyperphosphorylation and camptothecin sensitivity in cancer cell lines

Topoisomerase I (topo I) is required to unwind DNA during synthesis and provides the unique target for camptothecin-derived chemotherapeutic agents, including Irinotecan and Topotecan. While these agents are highly effective anticancer agents, some tumors do not respond due to intrinsic or acquired resistance, a process that remains poorly understood. Because of treatment toxicity, there is interest in identifying cellular factors that regulate tumor sensitivity and might serve as predictive biomarkers of therapy sensitivity. Here we identify the serine kinase, protein kinase CK2, as a central regulator of topo I hyperphosphorylation and activity and cellular sensitivity to camptothecin. In nine cancer cell lines and three normal tissue-derived cell lines we observe a consistent correlation between CK2 levels and camptothecin responsiveness. Two other topo I-targeted serine kinases, protein kinase C and cyclin-dependent kinase 1, do not show this correlation. Camptothecin-sensitive cancer cell lines display high CK2 activity, hyperphosphorylation of topo I, elevated topo I activity, and elevated phosphorylation-dependent complex formation between topo I and p14ARF, a topo I activator. Camptothecin-resistant cancer cell lines and normal cell lines display lower CK2 activity, lower topo I phosphorylation, lower topo I activity, and undetectable topo I/p14ARF complex formation. Experimental inhibition or activation of CK2 demonstrates that CK2 is necessary and sufficient for regulating these topo I properties and altering cellular responses to camptothecin. The results establish a cause and effect relationship between CK2 activity and camptothecin sensitivity and suggest that CK2, topo I phosphorylation, or topo I/p14ARF complex formation could provide biomarkers of therapy-responsive tumors.

CK2-Mediated Hyperphosphorylation of Topoisomerase I Targets Serine 506, Enhances Topoisomerase I-DNA Binding, and Increases Cellular Camptothecin Sensitivity

Topoisomerase I is the target for a potent class of chemotherapeutic drugs derived from the plant alkaloid camptothecin that includes irinotecan and topotecan. In this study we have identified a novel site of CK2-mediated topoisomerase I (topo I) phosphorylation at serine 506 (PS506) that is relevant to topo I function and to cellular responses to these topo I-targeted drugs. CK2 treatment induced hyperphosphorylation of recombinant topo I and expression of the PS506 epitope, and resulted in increased binding of topo I to supercoiled plasmid DNA. Hyperphosphorylated topo I was approximately three times more effective than the basal phosphorylated enzyme at relaxing plasmid supercoils but had similar DNA cleavage activity once bound to DNA. The PS506 epitope was expressed in cancer cell lines with elevated CK2 activity, hyperphosphorylated topo I, and increased sensitivity to camptothecin. In contrast, PS506 was not detected in normal cells or cancer cell lines with lower levels of CK2 activity. By experimentally manipulating CK2 activity in cancer cell lines, we demonstrate a cause and effect relationship between CK2 activity, PS506 expression, camptothecin-induced cellular DNA damage, and cellular camptothecin sensitivity. Our results show that the PS506 epitope is an indicator of dysregulated, hyperphosphorylated topo I in cancer cells, and may thus serve as a diagnostic or prognostic biomarker and predict tumor responsiveness to widely used topo I-targeted therapies.

The p14ARF alternate reading frame protein enhances DNA binding of topoisomerase I by interacting with the serine 506-phosphorylated core domain.

In addition to its well-characterized function as a tumor suppressor, p14ARF (ARF) is a positive regulator of topoisomerase I (topo I), a central enzyme in DNA metabolism and a target for cancer therapy. We previously showed that topo I hyperphosphorylation, a cancer-associated event mediated by elevated levels of the protein kinase CK2, increases topo I activity and the cellular sensitivity to topo I-targeted drugs. Topo I hyperphosphorylation also increases its interaction with ARF. Because the ARF−topo I interaction could be highly relevant to DNA metabolism and cancer treatment, we identified the regions of topo I involved in ARF binding and characterized the effects of ARF binding on topo I function. Using a series of topo I deletion constructs, we found that ARF interacted with the topo I core domain, which encompasses most of the catalytic and DNA-interacting residues. ARF binding increased the DNA relaxation activity of hyperphosphorylated topo I by enhancing its association with DNA, but did not affect the topo I catalytic rate. In cells, ARF promoted the chromatin association of hyperphosphorylated, but not basal phosphorylated, topo I, and increased topo I-mediated DNA nicking under conditions of oxidative stress. The aberrant nicking was found to correlate with increased formation of DNA double-strand breaks, which are precursors of many genome destabilizing events. The results suggest that the convergent actions of oxidative stress and elevated CK2 and ARF levels, which are common features of cancer cells, lead to a dysregulation of topo I that may contribute both to the cellular response to topo I-targeted drugs and to genome instability.

Abstract 5593: A cancer-associated hyperphosphorylation site on topoisomerase I serine 506 as a biomarker for camptothecin sensitivity.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Topoisomerase I (topo I) plays a central role in cancer as the target of a potent class of chemotherapeutic drugs derived from the plant alkaloid camptothecin that includes irinotecan and topotecan. We have identified a novel cancer-specific phosphorylation site at serine 506 in topo I (PS506), targeted by protein kinase CK2, that identifies cancer cells with elevated CK2, hyperphosphorylated topo I, increased topo I activity, and increased sensitivity to camptothecin. CK2 treatment of recombinant topo I in vitro also induces topo I hyperphosphorylation and expression of the PS506 epitope, and results in increased binding of topo I to supercoiled plasmid DNA. The hyperphosphorylated, PS506-form of topo I is more effective than the basal phosphorylated enzyme at relaxing plasmid supercoils but has a similar catalytic rate as the basal phosphorylated enzyme once bound to DNA. The PS506 epitope is not detected in normal cells or cancer cell lines with lower levels of CK2 activity. By experimentally manipulating CK2 activity in cancer cell lines, we have demonstrated a cause and effect relationship between CK2 activity, PS506 expression, camptothecin-induced cellular DNA damage, and cellular camptothecin sensitivity. The PS506 epitope is therefore an indicator of dysregulated, hyperphosphorylated topo I in cancer cells, and holds promise as a novel diagnostic biomarker that predicts tumor responsiveness to widely used topo I-targeted therapies. Citation Format: Keya Bandyopadhyay, Pingchuan Li, Ruth A. Gjerset. A cancer-associated hyperphosphorylation site on topoisomerase I serine 506 as a biomarker for camptothecin sensitivity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5593. doi:10.1158/1538-7445.AM2013-5593

Abstract 5444: Polyamine-based HAT inhibitors as therapy sensitizers

Histone acetylation plays an important role in regulating chromatin structure and is tightly controlled through the actions of histone acetyltransferases (HAT) and histone deacetylases (HDAC). Deregulation of either HAT or HDAC activity can affect chromatin remodeling needed to carry out its functions and contribute to cancer. While HATs are potential targets for anticancer therapeutics, the development of effective HAT inhibitors has been slow. We have examined the cellular effects of an S-substituted coenzyme A (CoA) inhibitor of histone acetylation, consisting of spermidine (Spd) linked to the S-terminus of CoA through a thioglycolic acid linkage (adduct abbreviated as Spd-CoA), as well as the effects of a truncated Spd-CoA derivative lacking the negatively charged portion of the CoA moiety. While exposure of cancer cells to Spd-CoA has little effect on cell viability, it causes a rapid inhibition of specific acetylated lysines, H3-K9 and H3-K56. That inhibition correlates with a transient arrest of DNA synthesis, a transient delay in S-phase progression, and an inhibition of nucleotide excision repair and DNA double strand break repair. Furthermore, Spd-CoA synergizes with a variety of commonly used DNA damaging chemotherapeutic agents, cisplatin (Platinol TM ) and 5-fluorouracil, camptothecin, as well as UV-C radiation to induce cancer cell killing. This suggests that a common mechanism, relevant to DNA damage, underlies the ability of histone acetylation inhibition to synergize with drugs and radiation. Normal human fibroblasts and epithelial cells are not sensitized to DNA damage by Spd-CoA due to a barrier to uptake, indicating that this differential uptake can be exploited to achieve cancer cell-specific sensitization. Furthermore, therapy sensitization occurs in both p53-null cancer cells and in cancer cells expressing wild-type p53, indicating that p53-mediated apoptosis is not required. The truncated Spd-CoA derivative displays similar but enhanced chemosensitization effects, suggesting that this class of inhibitors may be amenable to further refinement and have considerable clinical potential as a novel class of potent therapy sensitizers applicable to a broad range of conventional cancer treatments, particularly to reduce therapy toxicity and reverse therapy resistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5444.