Barbara Dziegielewska

Rational Design of Human DNA Ligase Inhibitors that Target Cellular DNA Replication and Repair

Based on the crystal structure of human DNA ligase I complexed with nicked DNA, computer-aided drug design was used to identify compounds in a database of 1.5 million commercially available low molecular weight chemicals that were predicted to bind to a DNA-binding pocket within the DNA-binding domain of DNA ligase I, thereby inhibiting DNA joining. Ten of 192 candidates specifically inhibited purified human DNA ligase I. Notably, a subset of these compounds was also active against the other human DNA ligases. Three compounds that differed in their specificity for the three human DNA ligases were analyzed further. L82 inhibited DNA ligase I, L67 inhibited DNA ligases I and III, and L189 inhibited DNA ligases I, III, and IV in DNA joining assays with purified proteins and in cell extract assays of DNA replication, base excision repair, and nonhomologous end-joining. L67 and L189 are simple competitive inhibitors with respect to nicked DNA, whereas L82 is an uncompetitive inhibitor that stabilized complex formation between DNA ligase I and nicked DNA. In cell culture assays, L82 was cytostatic whereas L67 and L189 were cytotoxic. Concordant with their ability to inhibit DNA repair in vitro, subtoxic concentrations of L67 and L189 significantly increased the cytotoxicity of DNA-damaging agents. Interestingly, the ligase inhibitors specifically sensitized cancer cells to DNA damage. Thus, these novel human DNA ligase inhibitors will not only provide insights into the cellular function of these enzymes but also serve as lead compounds for the development of anticancer agents.

Identification and validation of human DNA ligase inhibitors using computer-aided drug design.

Linking together of DNA strands by DNA ligases is essential for DNA replication and repair. Since many therapies used to treat cancer act by causing DNA damage, there is growing interest in the development of DNA repair inhibitors. Accordingly, virtual database screening and experimental evaluation were applied to identify inhibitors of human DNA ligase I (hLigI). When a DNA binding site within the DNA binding domain (DBD) of hLigI was targeted, more than 1 million compounds were screened from which 192 were chosen for experimental evaluation. In DNA joining assays, 10 compounds specifically inhibited hLigI, 5 of which also inhibited the proliferation of cultured human cell lines. Analysis of the 10 active compounds revealed the utility of including multiple protein conformations and chemical clustering in the virtual screening procedure. The identified ligase inhibitors are structurally diverse and have druglike physical and molecular characteristics making them ideal for further drug development studies.

T-type calcium channels blockers as new tools in cancer therapies

T-type calcium channels are involved in a multitude of cellular processes, both physiological and pathological, including cancer. T-type channels are also often aberrantly expressed in different human cancers and participate in the regulation of cell cycle progression, proliferation, migration, and survival. Here, we review the recent literature and discuss the controversies, supporting the role of T-type Ca2+ channels in cancer cells and the proposed use of channels blockers as anticancer agents. A growing number of reports show that pharmacological inhibition or RNAi-mediated downregulation of T-type channels leads to inhibition of cancer cell proliferation and increased cancer cell death. In addition to a single agent activity, experimental results demonstrate that T-type channel blockers enhance the anticancer effects of conventional radio- and chemotherapy. At present, the detailed biological mechanism(s) underlying the anticancer activity of these channel blockers is not fully understood. Recent findings and ideas summarized here identify T-type Ca2+ channels as a molecular target for anticancer therapy and offer new directions for the design of novel therapeutic strategies employing channels blockers. Physiological relevance: T-type calcium channels are often aberrantly expressed or deregulated in cancer cells, supporting their proliferation, survival, and resistance to treatment; therefore, T-type Ca2+ channels could be attractive molecular targets for anticancer therapy.

Inhibition of T-type calcium channels disrupts Akt signaling and promotes apoptosis in glioblastoma cells.

Glioblastoma multiforme (GBM) are brain tumors that are exceptionally resistant to both radio- and chemotherapy regimens and novel approaches to treatment are needed. T-type calcium channels are one type of low voltage-gated channel (LVCC) involved in embryonic cell proliferation and differentiation; however they are often over-expressed in tumors, including GBM. In this study, we found that inhibition of T-type Ca(2+) channels in GBM cells significantly reduced their survival and resistance to therapy. Moreover, either T-type selective antagonists, such as mibefradil, or siRNA-mediated knockdown of the T-type channel alpha subunits not only reduced cell viability and clonogenic potential, but also induced apoptosis. In response to channel blockade or ablation, we observed reduced phosphorylation of Akt and Rictor, suggesting inhibition of the mTORC2/Akt pathway. This was followed by reduction in phosphorylation of anti-apoptotic Bad and caspases activation. The apoptotic response was specific for T-type Ca(2+) channels, as inhibition of L-type Ca(2+) channels did not induce similar effects. Our results implicate T-type Ca(2+) channels as distinct entities for survival signaling in GBM cells and suggest that they are a novel molecular target for tumor therapy.

Phosphorylation of Human DNA Ligase I Regulates Its Interaction with Replication Factor C and Its Participation in DNA Replication and DNA Repair

ABSTRACT Human DNA ligase I (hLigI) participates in DNA replication and excision repair via an interaction with proliferating cell nuclear antigen (PCNA), a DNA sliding clamp. In addition, hLigI interacts with and is inhibited by replication factor C (RFC), the clamp loader complex that loads PCNA onto DNA. Here we show that a mutant version of hLigI, which mimics the hyperphosphorylated M-phase form of hLigI, does not interact with and is not inhibited by RFC, demonstrating that inhibition of ligation is dependent upon the interaction between hLigI and RFC. To examine the biological relevance of hLigI phosphorylation, we isolated derivatives of the hLigI-deficient cell line 46BR.1G1 that stably express mutant versions of hLigI in which four serine residues phosphorylated in vivo were replaced with either alanine or aspartic acid. The cell lines expressing the phosphorylation site mutants of hLigI exhibited a dramatic reduction in proliferation and DNA synthesis and were also hypersensitive to DNA damage. The dominant-negative effects of the hLigI phosphomutants on replication and repair are due to the activation of cellular senescence, presumably because of DNA damage arising from replication abnormalities. Thus, appropriate phosphorylation of hLigI is critical for its participation in DNA replication and repair.

T-Type Ca2+ Channel Inhibition Induces p53-Dependent Cell Growth Arrest and Apoptosis through Activation of p38-MAPK in Colon Cancer Cells

Epithelial tumor cells express T-type Ca2+ channels, which are thought to promote cell proliferation. This study investigated the cellular response to T-type Ca2+ channel inhibition either by small-molecule antagonists or by RNAi-mediated knockdown. Selective T-type Ca2+ channel antagonists caused growth inhibition and apoptosis more effectively in HCT116 cells expressing wild-type p53 (p53wt), than in HCT116 mutant p53−/− cells. These antagonists increased p53-dependent gene expression and increased genomic occupancy of p53 at specific target sequences. The knockdown of a single T-type Ca2+ channel subunit (CACNA1G) reduced cell growth and induced caspase-3/7 activation in HCT116 p53wt cells as compared with HCT116 mutant p53−/− cells. Moreover, CaCo2 cells that do not express functional p53 were made more sensitive to CACNA1G knockdown when p53wt was stably expressed. Upon T-type Ca2+ channel inhibition, p38-MAPK promoted phosphorylation at Ser392 of p53wt. Cells treated with the inhibitor SB203580 or specific RNAi targeting p38-MAPKα/β (MAPK14/MAPK11) showed resistance to T-type Ca2+ channel inhibition. Finally, the decreased sensitivity to channel inhibition was associated with decreased accumulation of p53 and decreased expression of p53 target genes, p21Cip1 (CDKN1A) and BCL2-binding component 3 (BBC3/PUMA). Implications: A novel pathway involving p53 and p38-MAPK is revealed and provides a rationale for antitumor therapies that target T-type Ca2+ channels. Mol Cancer Res; 12(3); 348–58. ©2013 AACR.

Phosphorylation of Serine 51 Regulates the Interaction of Human DNA Ligase I with Replication Factor C and Its Participation in DNA Replication and Repair

Background: Phosphorylation of human DNA ligase I (hLigI) regulates its participation in DNA replication and repair. Results: Identification of a single phosphorylation site that regulates the interaction of hLigI with replication factor C (RFC). Conclusion: Interaction between hLigI and RFC is critical for DNA replication and repair. Significance: We provide insights into the mechanism by which a single phosphorylation site regulates the cellular functions of hLigI. Human DNA ligase I (hLigI) joins Okazaki fragments during DNA replication and completes excision repair via interactions with proliferating cell nuclear antigen and replication factor C (RFC). Unlike proliferating cell nuclear antigen, the interaction with RFC is regulated by hLigI phosphorylation. To identity of the site(s) involved in this regulation, we analyzed phosphorylated hLigI purified from insect cells by mass spectrometry. These results suggested that serine 51 phosphorylation negatively regulates the interaction with RFC. Therefore, we constructed versions of hLigI in which serine 51 was replaced with either alanine (hLigI51A) to prevent phosphorylation or aspartic acid (hLigI51D) to mimic phosphorylation. hLigI51D but not hLigI51A was defective in binding to purified RFC and in associating with RFC in cell extracts. Although DNA synthesis and proliferation of hLigI-deficient cells expressing either hLig51A or hLig51 was reduced compared with cells expressing wild-type hLigI, cellular senescence was only observed in the cells expressing hLigI51D. Notably, these cells had increased levels of spontaneous DNA damage and phosphorylated CHK2. In addition, although expression of hLigI51A complemented the sensitivity of hLigI-deficient cells to a poly (ADP-ribose polymerase (PARP) inhibitor, expression of hLig151D did not, presumably because these cells are more dependent upon PARP-dependent repair pathways to repair the damage resulting from the abnormal DNA replication. Finally, neither expression of hLigI51D nor hLigI51A fully complemented the sensitivity of hLigI-deficient cells to DNA alkylation. Thus, phosphorylation of serine 51 on hLigI plays a critical role in regulating the interaction between hLigI and RFC, which is required for efficient DNA replication and repair.

T-Type Ca2+ Channel Inhibition Sensitizes Ovarian Cancer to Carboplatin.

Ovarian cancer is the deadliest gynecologic cancer, due in large part to the diagnosis of advanced stage disease, the development of platinum resistance, and inadequate treatment alternatives. Recent studies by our group and others have shown that T-type calcium (Ca2+) channels play a reinforcing role in cancer cell proliferation, cell-cycle progression, and apoptosis evasion. Therefore, we investigated whether T-type Ca2+ channels affect ovarian tumor growth and response to platinum agents. Inhibition of T-type Ca2+ channels with mibefradil or by silencing expression resulted in growth suppression in ovarian cancer cells with a simultaneous increase in apoptosis, which was accompanied by decreased expression of the antiapoptotic gene survivin (BIRC5). Analysis of intracellular signaling revealed mibefradil reduced AKT phosphorylation, increased the levels and nuclear retention of FOXO transcription factors that repress BIRC5 expression, and decreased the expression of FOXM1, which promotes BIRC5 expression. Combining carboplatin with mibefradil synergistically increased apoptosis in vitro. Importantly, mibefradil rendered platinum-resistant ovarian tumors sensitive to carboplatin in a mouse model of peritoneal metastasis. Together, the data provide rationale for future use of T-type channel antagonists together with platinum agents for the treatment of ovarian cancer. Mol Cancer Ther; 15(3); 460–70. ©2016 AACR.

Abstract LB-262: Targeting T-type calcium channels induces the extrinsic apoptotic pathway and sensitizes glioblastoma cancer cells to radio- and chemotherapy

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL The purpose of this study is to develop a novel and effective mechanistic target for sensitizing glioblastoma tumors to anti-cancer therapy. Glioblastoma multiforme (GBM) are primary brain tumors with an exceptional resistance to radio- and chemotherapy and, therefore, have extremely poor prognosis. Conventional treatment consists of aggressive surgery, chemotherapy and/or radiotherapy; however in spite of these treatments, the median survival of GBM patients is only 9 to 12 months. T-type channels are a type of voltage-gated calcium channel important for normal cell proliferation and differentiation, but they could be also over-expressed or aberrantly activated in several human cancers, including GBM. In this study we investigate the effects of T-type channel inactivation, either by selective antagonists (mibefradil and TTL1177) or by siRNA-directed knock-down, on GBM cells survival and resistance to therapy in vitro. Our results show that blocking T-type channels not only reversibly arrests cells in G1/G0 phase of the cell cycle but also induces irreversible loss of clonogenic potential and cell death. The execution of cell death is preceded (or coincides) by induction of autophagy, as characterized by LC-3 cleavage and punctuate staining. Furthermore, we also establish that inactivation of T-type channels stimulates the extrinsic pathway of programmed cell death, as evidenced by caspase 8 activation and Bid protein cleavage. Finally, we demonstrate that inactivation of T-type calcium channels sensitizes GBM cells to radio- and chemotherapy. Taken together with the promising outcomes of the in vivo mibefradil plus temozolomide trials, our studies suggest that targeting T-type calcium channels could be a novel and very efficient therapy for glioblastoma brain tumors, either as a single modality or in combination with already established modalities. (This work was financed in part by the University of Virginia Cancer Center Pilot Grant, the Department of Radiation Oncology Pilot Grant and the Department of Chemistry Research Fund) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-262. doi:1538-7445.AM2012-LB-262

Abstract 750: Overcoming resistance to anti-EGFR therapy in non-small cell lung cancer with a T-type Ca2+ channels inhibitor

This study9s aim was to define a novel molecular target and to develop an effective agent capable of overcoming intrinsic and acquired resistance to anti-EGFR therapy. Despite introduction of new therapies, lung cancer is a major cause of cancer death. One of its characteristics is the expression and activation of epidermal growth factor receptor (EGFR). Activating mutations in EGFR are present in approximately 20% of patients with non-small cell lung cancer (NSCLC), mostly in never-smokers. While most patients initially respond to treatment with anti-EGFR therapy, essentially all patients develop acquired drug resistance. T-type calcium channels, are present in lung cancer and support cell proliferation. Importantly, their expression often coincides with abnormal expression or mutations in EGFR. In this study, we investigated the effects of T-type channel inhibition on NSCLC survival and resistance to targeted therapy in vitro. We used a panel of cell lines differing in EGFR mutation status and sensitivity to EGFR tyrosine kinase inhibitors (TKIs). Our results show that T-type channel inhibitors alone arrest cells in the G1/G0 phase of the cell cycle and induce cell death. The treatment reduces activity of both PI3K/AKT/mTOR and JAK2/STAT5 pathways, often upregulated as a mechanism of acquired resistance to TKIs. Importantly, combined treatment with TKI, gefitinib, and T-type channels inhibitor, mibefradil, resulted in synergistic growth inhibition. Our results demonstrate a connection between T-type Ca2+ channels and aberrant EGFR activity in NSCLC, and provide the rationale for future use of two targeted therapies together. Since T-type calcium channel inhibitors are already undergoing clinical trials, our discoveries could be quickly translated into clinical practice. Citation Format: Barbara Dziegielewska, Lloyd S. Gray, Jaroslaw Dziegielewski. Overcoming resistance to anti-EGFR therapy in non-small cell lung cancer with a T-type Ca2+ channels inhibitor. [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 750. doi:10.1158/1538-7445.AM2015-750