IU Indianapolis ScholarWorks :: Browsing by Author "Turchi, John J."

Browsing by Author "Turchi, John J."

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Activation of Gcn2 by Pharmacological Agents Designed to be Inhibitors
(2023-01) Carlson, Kenneth Reed; Wek, Ronald C.; Georgiadis, Millie M.; Liu, Yunlong; Staschke, Kirk A.; Turchi, John J.
The integrated stress response (ISR) is an important mechanism by which cells confer protection against environmental stresses. Central to the ISR is a collection of related protein kinases that monitor stress conditions, such as Gcn2 (EIF2AK4) that recognizes nutrient limitations, inducing phosphorylation of eukaryotic translation initiation factor 2 (eIF2). Gcn2 phosphorylation of eIF2 lowers bulk protein synthesis, conserving energy and nutrients, coincident with preferential translation of stressadaptive gene transcripts, such as that encoding the Atf4 transcriptional regulator. While Gcn2 is central for cell protection to nutrient stress and its depletion in humans leads to pulmonary disorders, Gcn2 can also contribute to the progression of cancers and facilitate neurological disorders during chronic stress. Consequently, specific ATP-competitive inhibitors of Gcn2 protein kinase have been developed. This thesis reports that one such Gcn2 inhibitor, Gcn2iB, can activate Gcn2, probes the mechanism by which this activation occurs, and compares the mechanism of Gcn2 activation by Gcn2iB to that of uncharged tRNA. In this study, Gcn2 activation was measured in cultured human cells by immunoblot and luciferase reporter assays making use of a genetic complementation assay to assess the contribution of various Gcn2 residues to its activation. Low concentrations of Gcn2iB increase Gcn2 phosphorylation of eIF2 and enhance Atf4 expression and activity. Of importance, Gcn2iB can activate Gcn2 mutants devoid of functional regulatory domains or with certain kinase domain substitutions derived fromGcn2-deficient human patients. Other ATP-competitive inhibitors can also activate Gcn2, although there are differences in their mechanisms of activation. These results provide a cautionary note about the pharmacodynamics of eIF2 kinase inhibitors in therapeutic applications. However, compounds designed to be kinase inhibitors that instead directly activate Gcn2, even loss of function variants, may provide tools to alleviate deficiencies in Gcn2 and other regulators of the ISR.
ATR inhibition overcomes platinum tolerance associated with ERCC1- and p53-deficiency by inducing replication catastrophe
(Oxford University Press, 2023-01-11) Heyza, Joshua R.; Ekinci, Elmira; Lindquist, Jacob; Lei, Wen; Yunker, Christopher; Vinothkumar, Vilvanathan; Rowbotham, Rachelle; Polin, Lisa; Snider, Natalie G.; Van Buren, Eric; Watza, Donovan; Back, Jessica B.; Chen, Wei; Mamdani, Hirva; Schwartz, Ann G.; Turchi, John J.; Bepler, Gerold; Patrick, Steve M.; Medicine, School of Medicine
ERCC1/XPF is a heterodimeric DNA endonuclease critical for repair of certain chemotherapeutic agents. We recently identified that ERCC1- and p53-deficient lung cancer cells are tolerant to platinum-based chemotherapy. ATR inhibition synergistically re-stored platinum sensitivity to platinum tolerant ERCC1-deficient cells. Mechanistically we show this effect is reliant upon several functions of ATR including replication fork protection and altered cell cycle checkpoints. Utilizing an inhibitor of replication protein A (RPA), we further demonstrate that replication fork protection and RPA availability are critical for platinum-based drug tolerance. Dual treatment led to increased formation of DNA double strand breaks and was associated with chromosome pulverization. Combination treatment was also associated with increased micronuclei formation which were capable of being bound by the innate immunomodulatory factor, cGAS, suggesting that combination platinum and ATR inhibition may also enhance response to immunotherapy in ERCC1-deficient tumors. In vivo studies demonstrate a significant effect on tumor growth delay with combination therapy compared with single agent treatment. Results of this study have led to the identification of a feasible therapeutic strategy combining ATR inhibition with platinum and potentially immune checkpoint blockade inhibitors to overcome platinum tolerance in ERCC1-deficient, p53-mutant lung cancers.
Characterization and initial demonstration of in vivo efficacy of a novel heat-activated metalloenediyne anti-cancer agent
(Taylor & Francis, 2022) Garrett, Joy; Metzger, Erin; Dewhirst, Mark W.; Pollok, Karen E.; Turchi, John J.; Le Poole, Isabelle C.; Couch, Kira; Lew, Logan; Sinn, Anthony; Zaleski, Jeffrey M.; Dynlacht, Joseph R.; Radiation Oncology, School of Medicine
Background: Enediynes are anti-cancer agents that are highly cytotoxic due to their propensity for low thermal activation of radical generation. The diradical intermediate produced from Bergman cyclization of the enediyne moiety may induce DNA damage and cell lethality. The cytotoxicity of enediynes and difficulties in controlling their thermal cyclization has limited their clinical use. We recently showed that enediyne toxicity at 37 °C can be mitigated by metallation, but cytotoxic effects of 'metalloenediynes' on cultured tumor cells are potentiated by hyperthermia. Reduction of cytotoxicity at normothermia suggests metalloenediynes will have a large therapeutic margin, with cell death occurring primarily in the heated tumor. Based on our previous in vitro findings, FeSO4-PyED, an Fe co-factor complex of (Z)-N,N'-bis[1-pyridin-2-yl-meth-(E)-ylidene]oct-4-ene-2,6-diyne-1,8-diamine, was prioritized for further in vitro and in vivo testing in normal human melanocytes and melanoma cells. Methods: Clonogenic survival, apopotosis and DNA binding assays were used to determine mechanisms of enhancement of FeSO4-PyED cytotoxicity by hyperthermia. A murine human melanoma xenograft model was used to assess in vivo efficacy of FeSO4-PyED at 37 or 42.5 °C. Results: FeSO4-PyED is a DNA-binding compound. Enhancement of FeSO4-PyED cytotoxicity by hyperthermia in melanoma cells was due to Bergman cyclization, diradical formation, and increased apoptosis. Thermal enhancement, however, was not observed in melanocytes. FeSO4-PyED inhibited tumor growth when melanomas were heated during drug treatment, without inducing normal tissue damage. Conclusion: By leveraging the unique thermal activation properties of metalloenediynes, we propose that localized moderate hyperthermia can be used to confine the cytotoxicity of these compounds to tumors, while sparing normal tissue.
Characterization of Thermally Activated Metalloenediyne Cytotoxicity in Human Melanoma Cells
(BioOne, 2018-08) Keller, Eric J.; Porter, Meghan; Garrett, Joy E.; Varie, Meredith; Wang, Haiyan; Pollok, Karen E.; Turchi, John J.; Zaleski, Jeffrey M.; Dynlacht, Joseph R.; Radiation Oncology, School of Medicine
Enediynes are a highly cytotoxic class of compounds. However, metallation of these compounds may modulate their activation, and thus their cytotoxicity. We previously demonstrated that cytotoxicity of two different metalloenediynes, including (Z)-N,N'-bis[1-pyridyl-2-yl-meth-(E)-ylidene]octa-4-ene-2,6-diyne-1,8-diamine] (PyED), is potentiated when the compounds are administered to HeLa cells during hyperthermia treatment at concentrations that are minimally or not cytotoxic at 37°C. In this study, we further characterized the concentration, time and temperature dependence of cytotoxicity of PyED on human U-1 melanoma cells. We also investigated the potential mechanisms by which PyED cytotoxicity is enhanced during hyperthermia treatment. Cell killing with PyED was dependent on concentration, temperature during treatment and time of exposure. Potentiation of cytotoxicity was observed when cells were treated with PyED at temperatures ≥39.5°C, and enhancement of cell killing increased with temperature and with increasing time at a given temperature. All cells treated with PyED were shown to have DNA damage, but substantially more damage was observed in cells treated with PyED during heating. DNA repair was also inhibited in cells treated with the drug during hyperthermia. Thus, potentiation of PyED cytotoxicity by hyperthermia may be due to enhancement of drug-induced DNA lesions, and/or the inhibition of repair of sublethal DNA damage. While the selective thermal activation of PyED supports the potential clinical utility of metalloenediynes as cancer thermochemotherapeutic agents, therapeutic gain could be optimized by identifying compounds that produce minimal toxicity at 37°C but which become activated and show enhancement of cytotoxicity within a tumor subjected to localized hyperthermic or thermal ablative treatment, or which might act as bifunctional agents. We thus also describe the development and initial characterization of a novel cofactor complex of PyED, platinated PyED (Pt-PyED). Pt-PyED binds to DNA-like cisplatin, and much like PyED, cytotoxicity is greatly enhanced after treatment with the drug at elevated temperatures. However, in contrast to PyED, Pt-PyED is only minimally cytotoxic at 37°C, at concentrations at which cytotoxicity is enhanced by hyperthermia. Further development of cisplatin-based enediynes may result in compounds which, when activated, will possess multiple DNA binding modalities similar to cisplatin, but produce less side effects in tissues at normothermic temperatures.
Chemical inhibitor targeting the replication protein A-DNA interaction increases the efficacy of Pt-based chemotherapy in lung and ovarian cancer
(Elsevier, 2015-01-01) Mishra, Akaash K.; Dormi, Silvana S.; Turchi, Alaina M.; Woods, Derek S.; Turchi, John J.; Department of Biochemistry and Molecular Biology, IU School of Medicine
Platinum-based chemotherapeutics exert their therapeutic efficacy via the formation of DNA adducts which interfere with DNA replication, transcription and cell division and ultimately induce cell death. Repair and tolerance of these Pt-DNA lesions by nucleotide excision repair (NER) and homologous recombination (HR) can substantially reduce the effectiveness of therapy. Inhibition of these repair pathways, therefore, holds the potential to sensitize cancer cells to Pt treatment and increase clinical efficacy. Replication Protein A (RPA) plays essential roles in both NER and HR, along with its role in DNA replication and DNA damage checkpoint activation. Each of these functions is, in part, mediated by RPA binding to single-stranded DNA (ssDNA). Here we report the synthesis and characterization of novel derivatives of RPA small molecule inhibitors and their activity in models of epithelial ovarian cancer (EOC) and non-small cell lung cancer (NSCLC). We have synthesized analogs of our previously reported RPA inhibitor TDRL-505 and determined the structure-activity relationships. These data led us to the identification of TDRL-551, which exhibited a greater than 2-fold increase in in vitro activity. TDRL-551 showed synergy with Pt in tissue culture models of EOC and in vivo efficacy, as a single agent and in combination with platinum, in a NSCLC xenograft model. These data demonstrate the utility of RPA inhibition in EOC and NSCLC and the potential in developing novel anticancer therapeutics that target RPA-DNA interactions.
Design and Structure-Guided Development of Novel Inhibitors of the Xeroderma Pigmentosum Group A (XPA) Protein–DNA Interaction
(ACS Publications, 2017-09-21) Gavande, Navnath S.; VanderVere-Carozza, Pamela; Mishra, Akaash K.; Vernon, Tyler L.; Pawelczak, Katherine S.; Turchi, John J.; Biochemistry and Molecular Biology, School of Medicine
XPA is a unique and essential protein required for the nucleotide excision DNA repair pathway and represents a therapeutic target in oncology. Herein, we are the first to develop novel inhibitors of the XPA–DNA interaction through structure-guided drug design efforts. Ester derivatives of the compounds 1 (X80), 22, and 24 displayed excellent inhibitory activity (IC50 of 0.82 ± 0.18 μM and 1.3 ± 0.22 μM, respectively) but poor solubility. We have synthesized novel amide derivatives that retain potency and have much improved solubility. Furthermore, compound 1 analogs exhibited good specificity for XPA over RPA (replication protein A), another DNA-binding protein that participates in the nucleotide excision repair (NER) pathway. Importantly, there were no significant interactions observed by the X80 class of compounds directly with DNA. Molecular docking studies revealed a mechanistic model for the interaction, and these studies could serve as the basis for continued analysis of structure–activity relationships and drug development efforts of this novel target.
Developing small molecule inhibitors targeting Replication Protein A for platinum-based combination therapy
(2014) Mishra, Akaash K.; Turchi, John J.; Kelley, Mark Richard, 1957-; Hurley, Thomas D., 1961-; Zhang, Zhong-Yin
All platinum (Pt)-based chemotherapeutics exert their efficacy primarily via the formation of DNA adducts which interfere with DNA replication, transcription and cell division and ultimately induce cell death. Repair and tolerance of Pt-DNA lesions by nucleotide excision repair and homologous recombination (HR) can substantially reduce the effectiveness of the Pt therapy. Inhibition of these repair pathways, therefore, holds the potential to sensitize cancer cells to Pt treatment and increase clinical efficacy. Replication Protein A (RPA) plays essential roles in both NER and HR, along with its role in DNA replication and DNA damage checkpoint activation. Each of these functions requires RPA binding to single-stranded DNA (ssDNA). We synthesized structural analogs of our previously reported RPA inhibitor TDRL-505, determined the structure activity relationships and evaluated their efficacy in tissue culture models of epithelial ovarian cancer (EOC) and non-small cell lung cancer (NSCLC). These data led us to the identification of TDRL-551, which exhibited a greater than 2-fold increase in in vitro and cellular activity. TDRL-551 showed synergy with Pt in tissue culture models of EOC and in vivo efficacy, as a single agent and in combination with platinum, in a NSCLC xenograft model. These data demonstrate the utility of RPA inhibition in EOC and NSCLC and the potential in developing novel anticancer therapeutics that target RPA-DNA interactions.
Disruption-Compensation (DisCo) Network Analysis of the RNA Polymerase II Interactome
(2022-08) Burriss, Katlyn Hughes; Mosley, Amber L.; Georgiadis, Millie M.; Goebl, Mark G.; Turchi, John J.
During RNA Polymerase II (RNAPII) transcription, a dynamic network of protein-protein interactions (PPIs) coordinates the regulation of initiation, elongation, and termination. Taking a proteomics approach to study RNAPII transcription can offer a comprehensive view of the regulatory mechanisms mediated by PPIs within the transcription complex. However, traditional affinity purification mass spectrometry (APMS) methods have struggled to quantitatively capture many of the more dynamic, less abundant interactions within the elaborate RNAPII transcription interactome. To combat this challenge, we have developed and optimized a quantitative AP-MS based method termed Disruption-Compensation (DisCo) Network Analysis that we coupled with Tandem Mass Tag (TMT) labeling. In this application, TMT-DisCo was applied to investigate the PPIs that regulate RNAPII transcription. In the first study, TMT-DisCo network analysis was used to analyze how perturbation of subunits of four major transcription elongation regulators —Spt6, Spt5 (DSIF), Cdc73 (PAF-Complex), and Spt16 (FACT)— affect the RNAPII PPI network. TMT-DisCo was able to measure specific alterations of RNAPII PPIs that provide insight into the normal functions of Spt6/Spt5/Cdc73/Spt16 proteins within the RNAPII elongation complex. The observed changes in the RNAPII interactome also reveal the distinct mechanisms behind the phenotypes of each perturbation. Application of TMTDisCo provides in vivo, protein-level insights into synthetic genetic interaction data and in vitro structural data, aiding in the understanding of how dynamic PPIs regulate complex processes. The second study focused on the essential RNAPII CTD phosphatases, Ssu72 and Fcp1. TMT-DisCo captures how the ssu72-2 allele affects the ability of RNAPII to proceed through elongation, resulting in more arrested RNAPII that requires proteasomal degradation. Reduction of Ssu72 phosphatase activity shifts cells away from RNAPII reinitiation/ recycling and toward de novo expression and newly assembled RNAPII, aided by chaperones. RNAPII in fcp1-1 cells was observed to increase in interaction with the 26S proteasome, as well as TFIID and mRNA capping enzyme. These data support a model of the nuclear proteasome functioning as a chaperone during transcription initiation, as the fcp1-1 allele leads to inefficient formation of a pre-initiation complex with a hyperphosphorylated RNAPII CTD.
DNA damage response (DDR) pathway engagement in cisplatin radiosensitization of non-small cell lung cancer
(Elsevier, 2016-04) Sears, Catherine R.; Cooney, Sean A.; Chin-Sinex, Helen; Mendonca, Marc S.; Turchi, John J.; Department of Medicine, School of Medicine
Non-small cell lung cancers (NSCLC) are commonly treated with a platinum-based chemotherapy such as cisplatin (CDDP) in combination with ionizing radiation (IR). Although clinical trials have demonstrated that the combination of CDDP and IR appear to be synergistic in terms of therapeutic efficacy, the mechanism of synergism remains largely uncharacterized. We investigated the role of the DNA damage response (DDR) in CDDP radiosensitization using two NSCLC cell lines. Using clonogenic survival assays, we determined that the cooperative cytotoxicity of CDDP and IR treatment is sequence dependent, requiring administration of CDDP prior to IR (CDDP-IR). We identified and interrogated the unique time and agent-dependent activation of the DDR in NSCLC cells treated with cisplatin-IR combination therapy. Compared to treatment with CDDP or IR alone, CDDP-IR combination treatment led to persistence of γH2Ax foci, a marker of DNA double-strand breaks (DSB), for up to 24h after treatment. Interestingly, pharmacologic inhibition of DDR sensor kinases revealed the persistence of γ-H2Ax foci in CDDP-IR treated cells is independent of kinase activation. Taken together, our data suggest that delayed repair of DSBs in NSCLC cells treated with CDDP-IR contributes to CDDP radiosensitization and that alterations of the DDR pathways by inhibition of specific DDR kinases can augment CDDP-IR cytotoxicity by a complementary mechanism.
DNA repair targeted therapy: The past or future of cancer treatment?
(Elsevier, 2016-04) Gavande, Navnath S.; VanderVere-Carozza, Pamela S.; Hinshaw, Hilary D.; Jalal, Shadia I.; Sears, Catherine R.; Pawelczak, Katherine S.; Turchi, John J.; Department of Medicine, School of Medicine
The repair of DNA damage is a complex process that relies on particular pathways to remedy specific types of damage to DNA. The range of insults to DNA includes small, modest changes in structure including mismatched bases and simple methylation events to oxidized bases, intra- and interstrand DNA crosslinks, DNA double strand breaks and protein-DNA adducts. Pathways required for the repair of these lesions include mismatch repair, base excision repair, nucleotide excision repair, and the homology directed repair/Fanconi anemia pathway. Each of these pathways contributes to genetic stability, and mutations in genes encoding proteins involved in these pathways have been demonstrated to promote genetic instability and cancer. In fact, it has been suggested that all cancers display defects in DNA repair. It has also been demonstrated that the ability of cancer cells to repair therapeutically induced DNA damage impacts therapeutic efficacy. This has led to targeting DNA repair pathways and proteins to develop anti-cancer agents that will increase sensitivity to traditional chemotherapeutics. While initial studies languished and were plagued by a lack of specificity and a defined mechanism of action, more recent approaches to exploit synthetic lethal interaction and develop high affinity chemical inhibitors have proven considerably more effective. In this review we will highlight recent advances and discuss previous failures in targeting DNA repair to pave the way for future DNA repair targeted agents and their use in cancer therapy.

Browsing by Author "Turchi, John J."

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