Danae M. Sharp

Novel Second-Generation Di-2-Pyridylketone Thiosemicarbazones Show Synergism with Standard Chemotherapeutics and Demonstrate Potent Activity against Lung Cancer Xenografts after Oral and Intravenous Administration in Vivo

We developed a series of second-generation di-2-pyridyl ketone thiosemicarbazone (DpT) and 2-benzoylpyridine thiosemicarbazone (BpT) ligands to improve the efficacy and safety profile of these potential antitumor agents. Two novel DpT analogues, Dp4e4mT and DpC, exhibited pronounced and selective activity against human lung cancer xenografts in vivo via the intravenous and oral routes. Importantly, these analogues did not induce the cardiotoxicity observed at high nonoptimal doses of the first-generation DpT analogue, Dp44mT. The Cu(II) complexes of these ligands exhibited potent antiproliferative activity having redox potentials in a range accessible to biological reductants. The activity of the copper complexes of Dp4e4mT and DpC against lung cancer cells was synergistic in combination with gemcitabine or cisplatin. It was demonstrated by EPR spectroscopy that dimeric copper compounds of the type [CuLCl](2), identified crystallographically, dissociate in solution to give monomeric 1:1 Cu:ligand complexes. These monomers represent the biologically active form of the complex.

Targeting glutamine transport to suppress melanoma cell growth

Amino acids, especially leucine and glutamine, are important for tumor cell growth, survival and metabolism. A range of different transporters deliver each specific amino acid into cells, some of which are increased in cancer. These amino acids consequently activate the mTORC1 pathway and drive cell cycle progression. The leucine transporter LAT1/4F2hc heterodimer assembles as part of a large complex with the glutamine transporter ASCT2 to transport amino acids. In this study, we show that the expression of LAT1 and ASCT2 is significantly increased in human melanoma samples and is present in both BRAFWT (C8161 and WM852) and BRAFV600E mutant (1205Lu and 451Lu) melanoma cell lines. While inhibition of LAT1 by BCH did not suppress melanoma cell growth, the ASCT2 inhibitor BenSer significantly reduced both leucine and glutamine transport in melanoma cells, leading to inhibition of mTORC1 signaling. Cell proliferation and cell cycle progression were significantly reduced in the presence of BenSer in melanoma cells in 2D and 3D cell culture. This included reduced expression of the cell cycle regulators CDK1 and UBE2C. The importance of ASCT2 expression in melanoma was confirmed by shRNA knockdown, which inhibited glutamine uptake, mTORC1 signaling and cell proliferation. Taken together, our study demonstrates that ASCT2‐mediated glutamine transport is a potential therapeutic target for both BRAFWT and BRAFV600E melanoma.

P-Glycoprotein Mediates Drug Resistance via a Novel Mechanism Involving Lysosomal Sequestration

Background: Localization of the drug transporter P-glycoprotein (Pgp) to the plasma membrane is thought to be the only contributor of Pgp-mediated multidrug resistance (MDR). Results: Lysosomal Pgp sequesters ionizable chemotherapeutics into lysosomes to prevent interaction with molecular targets, resulting in drug resistance. Conclusion: Lysosomal Pgp mediates drug resistance. Significance: Pgp-mediated sequestration of chemotherapeutics into lysosomes can be exploited pharmacologically. Localization of the drug transporter P-glycoprotein (Pgp) to the plasma membrane is thought to be the only contributor of Pgp-mediated multidrug resistance (MDR). However, very little work has focused on the contribution of Pgp expressed in intracellular organelles to drug resistance. This investigation describes an additional mechanism for understanding how lysosomal Pgp contributes to MDR. These studies were performed using Pgp-expressing MDR cells and their non-resistant counterparts. Using confocal microscopy and lysosomal fractionation, we demonstrated that intracellular Pgp was localized to LAMP2-stained lysosomes. In Pgp-expressing cells, the Pgp substrate doxorubicin (DOX) became sequestered in LAMP2-stained lysosomes, but this was not observed in non-Pgp-expressing cells. Moreover, lysosomal Pgp was demonstrated to be functional because DOX accumulation in this organelle was prevented upon incubation with the established Pgp inhibitors valspodar or elacridar or by silencing Pgp expression with siRNA. Importantly, to elicit drug resistance via lysosomes, the cytotoxic chemotherapeutics (e.g. DOX, daunorubicin, or vinblastine) were required to be Pgp substrates and also ionized at lysosomal pH (pH 5), resulting in them being sequestered and trapped in lysosomes. This property was demonstrated using lysosomotropic weak bases (NH4Cl, chloroquine, or methylamine) that increased lysosomal pH and sensitized only Pgp-expressing cells to such cytotoxic drugs. Consequently, a lysosomal Pgp-mediated mechanism of MDR was not found for non-ionizable Pgp substrates (e.g. colchicine or paclitaxel) or ionizable non-Pgp substrates (e.g. cisplatin or carboplatin). Together, these studies reveal a new mechanism where Pgp-mediated lysosomal sequestration of chemotherapeutics leads to MDR that is amenable to therapeutic exploitation.

The Iron Chelator, Deferasirox, as a Novel Strategy for Cancer Treatment: Oral Activity Against Human Lung Tumor Xenografts and Molecular Mechanism of Action

Deferasirox is an orally effective iron (Fe) chelator currently used for the treatment of iron-overload disease and has been implemented as an alternative to the gold standard chelator, desferrioxamine (DFO). Earlier studies demonstrated that DFO exhibits anticancer activity due to its ability to deplete cancer cells of iron. In this investigation, we examined the in vitro and in vivo activity of deferasirox against cells from human solid tumors. To date, there have been no studies to investigate the effect of deferasirox on these types of tumors in vivo. Deferasirox demonstrated similar activity at inhibiting proliferation of DMS-53 lung carcinoma and SK-N-MC neuroepithelioma cell lines compared with DFO. Furthermore, deferasirox was generally similar or slightly more effective than DFO at mobilizing cellular 59Fe and inhibiting iron uptake from human transferrin depending on the cell type. However, deferasirox potently inhibited DMS-53 xenograft growth in nude mice when given by oral gavage, with no marked alterations in normal tissue histology. To understand the antitumor activity of deferasirox, we investigated its effect on the expression of molecules that play key roles in metastasis, cell cycle control, and apoptosis. We demonstrated that deferasirox increased expression of the metastasis suppressor protein N-myc downstream-regulated gene 1 and upregulated the cyclin-dependent kinase inhibitor p21CIP1/WAF1 while decreasing cyclin D1 levels. Moreover, this agent increased the expression of apoptosis markers, including cleaved caspase-3 and cleaved poly(ADP-ribose) polymerase 1. Collectively, we demonstrate that deferasirox is an orally effective antitumor agent against solid tumors.

Di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT) overcomes multidrug resistance by a novel mechanism involving the hijacking of lysosomal P-Glycoprotein (Pgp)

Background: There is a critical need for chemotherapeutics that overcome multidrug resistance (MDR). Results: Dp44mT is transported into the lysosome by Pgp, causing lysosomal targeting of Dp44mT and resulting in enhanced cytotoxicity in vitro and in vivo. Conclusion: Dp44mT overcomes MDR via utilization of lysosomal Pgp transport activity. Significance: DpT thiosemicarbazones offer a new therapeutic strategy to overcome MDR via utilization of lysosomal Pgp transport activity. Multidrug resistance (MDR) is a major obstacle in cancer treatment. More than half of human cancers express multidrug-resistant P-glycoprotein (Pgp), which correlates with a poor prognosis. Intriguingly, through an unknown mechanism, some drugs have greater activity in drug-resistant tumor cells than their drug-sensitive counterparts. Herein, we investigate how the novel anti-tumor agent di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT) overcomes MDR. Four different cell types were utilized to evaluate the effect of Pgp-potentiated lysosomal targeting of drugs to overcome MDR. To assess the mechanism of how Dp44mT overcomes drug resistance, cellular studies utilized Pgp inhibitors, Pgp silencing, lysosomotropic agents, proliferation assays, immunoblotting, a Pgp-ATPase activity assay, radiolabeled drug uptake/efflux, a rhodamine 123 retention assay, lysosomal membrane permeability assessment, and DCF (2′,7′-dichlorofluorescin) redox studies. Anti-tumor activity and selectivity of Dp44mT in Pgp-expressing, MDR cells versus drug-sensitive cells were studied using a BALB/c nu/nu xenograft mouse model. We demonstrate that Dp44mT is transported by the lysosomal Pgp drug pump, causing lysosomal targeting of Dp44mT and resulting in enhanced cytotoxicity in MDR cells. Lysosomal Pgp and pH were shown to be crucial for increasing Dp44mT-mediated lysosomal damage and subsequent cytotoxicity in drug-resistant cells, with Dp44mT being demonstrated to be a Pgp substrate. Indeed, Pgp-dependent lysosomal damage and cytotoxicity of Dp44mT were abrogated by Pgp inhibitors, Pgp silencing, or increasing lysosomal pH using lysosomotropic bases. In vivo, Dp44mT potently targeted chemotherapy-resistant human Pgp-expressing xenografted tumors relative to non-Pgp-expressing tumors in mice. This study highlights a novel Pgp hijacking strategy of the unique dipyridylthiosemicarbazone series of thiosemicarbazones that overcome MDR via utilization of lysosomal Pgp transport activity.

Cell Cycle Phase-Specific Drug Resistance as an Escape Mechanism of Melanoma Cells

The tumor microenvironment is characterized by cancer cell subpopulations with heterogeneous cell cycle profiles. For example, hypoxic tumor zones contain clusters of cancer cells that arrest in G1 phase. It is conceivable that neoplastic cells exhibit differential drug sensitivity based on their residence in specific cell cycle phases. In this study, we used two-dimensional and organotypic melanoma culture models in combination with fluorescent cell cycle indicators to investigate the effects of cell cycle phases on clinically used drugs. We demonstrate that G1-arrested melanoma cells, irrespective of the underlying cause mediating G1 arrest, are resistant to apoptosis induced by the proteasome inhibitor bortezomib or the alkylating agent temozolomide. In contrast, G1-arrested cells were more sensitive to mitogen-activated protein kinase pathway inhibitor-induced cell death. Of clinical relevance, pretreatment of melanoma cells with a mitogen-activated protein kinase pathway inhibitor, which induced G1 arrest, resulted in resistance to temozolomide or bortezomib. On the other hand, pretreatment with temozolomide, which induced G2 arrest, did not result in resistance to mitogen-activated protein kinase pathway inhibitors. In summary, we established a model to study the effects of the cell cycle on drug sensitivity. Cell cycle phase-specific drug resistance is an escape mechanism of melanoma cells that has implications on the choice and timing of drug combination therapies.

HDAC inhibitors restore BRAF-inhibitor sensitivity by altering PI3K and survival signalling in a subset of melanoma

Mutations in BRAF activate oncogenic MAPK signalling in almost half of cutaneous melanomas. Inhibitors of BRAF (BRAFi) and its target MEK are widely used to treat melanoma patients with BRAF mutations but unfortunately acquired resistance occurs in the majority of patients. Resistance results from mutations or non‐genomic changes that either reactivate MAPK signalling or activate other pathways that provide alternate survival and growth signalling. Here, we show the histone deacetylase inhibitor (HDACi) panobinostat overcomes BRAFi resistance in melanoma, but this is dependent on the resistant cells showing a partial response to BRAFi treatment. Using patient‐ and in vivo‐derived melanoma cell lines with acquired BRAFi resistance, we show that combined treatment with the BRAFi encorafenib and HDACi panobinostat in 2D and 3D culture systems synergistically induced caspase‐dependent apoptotic cell death. Key changes induced by HDAC inhibition included decreased PI3K pathway activity associated with a reduction in the protein level of a number of receptor tyrosine kinases, and cell line dependent upregulation of pro‐apoptotic BIM or NOXA together with reduced expression of anti‐apoptotic proteins. Independent of these changes, panobinostat reduced c‐Myc and pre‐treatment of cells with siRNA against c‐Myc reduced BRAFi/HDACi drug‐induced cell death. These results suggest that a combination of HDAC and MAPK inhibitors may play a role in treatment of melanoma where the resistance mechanisms are due to activation of MAPK‐independent pathways.

Melanoma cells in G1 phase escape proteasome inhibitor cytotoxicity

HUMMER, DOUGLAS ANDREW. Community College Presidential Change from the Department Leader’s Perspective: A Case Study. (Under the direction of Dr. Diane Chapman). Academic department leaders are the least studied level of management in higher education, yet they play a fundamental role in transforming the vision and goals of executive leadership into reality (Gonaim, 2016). Academic department leaders play such a role because as a group, they influence the largest part of the employee population at a college; the faculty (Stringer, 2002; Tierney, 1999). Since the ability to create lasting change is one of the core qualities of a highly effective community college president (The Aspen Institute & Achieving the Dream, 2013), it is important to understand how the strategies, tactics, and actions employed by the executive leadership of an institution of higher learning shape the perceptions academic department leaders have of their workplace. This study took a qualitative approach to understanding these perceptions during the first few months of new president’s administration at a large community college in the southeast US. A transcendental phenomenological (Husserl, 2004) lens and case study methodologies were used to collect and analyze structural and textural data to describe the perceptions and interpretations academic department leaders had of this presidential change phenomenon. The case study itself provided the structural description, or context of the phenomenon, and semi-structured interviews were used to give voice to the academic department leaders that participated in the study. An analysis of the data revealed a number of themes that addressed the research questions asked in this study. These themes led to the following findings in this study. Listening sessions initiated by the new president and the hiring process that brought him to the college were identified as major influencing events in creating a positive impression of this new executive and his vision for the future of the institution. This positive impression is a contributor to creating an organizational climate ready to accept change. Other themes that emerged revolved around executive leadership’s support, communication issues, the hierarchical levels of management, changes that were initiated, and the fear to speak up. The findings from this study can be used by many community colleges as they hire new presidents and prepare for the changes new executives bring. The findings can also be used by any college approaching a major change initiative. The presidential change phenomenon will affect as many as four out of every five community colleges over the next 10 years (Phillippe, 2016). Presidential changes and other major change initiatives that community colleges experience will provide many opportunities to repeat this study and share the findings with others experiencing the same phenomenon.

Targeting Rab27a to suppress melanoma proliferation and invasion

HUMMER, DOUGLAS ANDREW. Community College Presidential Change from the Department Leader’s Perspective: A Case Study. (Under the direction of Dr. Diane Chapman). Academic department leaders are the least studied level of management in higher education, yet they play a fundamental role in transforming the vision and goals of executive leadership into reality (Gonaim, 2016). Academic department leaders play such a role because as a group, they influence the largest part of the employee population at a college; the faculty (Stringer, 2002; Tierney, 1999). Since the ability to create lasting change is one of the core qualities of a highly effective community college president (The Aspen Institute & Achieving the Dream, 2013), it is important to understand how the strategies, tactics, and actions employed by the executive leadership of an institution of higher learning shape the perceptions academic department leaders have of their workplace. This study took a qualitative approach to understanding these perceptions during the first few months of new president’s administration at a large community college in the southeast US. A transcendental phenomenological (Husserl, 2004) lens and case study methodologies were used to collect and analyze structural and textural data to describe the perceptions and interpretations academic department leaders had of this presidential change phenomenon. The case study itself provided the structural description, or context of the phenomenon, and semi-structured interviews were used to give voice to the academic department leaders that participated in the study. An analysis of the data revealed a number of themes that addressed the research questions asked in this study. These themes led to the following findings in this study. Listening sessions initiated by the new president and the hiring process that brought him to the college were identified as major influencing events in creating a positive impression of this new executive and his vision for the future of the institution. This positive impression is a contributor to creating an organizational climate ready to accept change. Other themes that emerged revolved around executive leadership’s support, communication issues, the hierarchical levels of management, changes that were initiated, and the fear to speak up. The findings from this study can be used by many community colleges as they hire new presidents and prepare for the changes new executives bring. The findings can also be used by any college approaching a major change initiative. The presidential change phenomenon will affect as many as four out of every five community colleges over the next 10 years (Phillippe, 2016). Presidential changes and other major change initiatives that community colleges experience will provide many opportunities to repeat this study and share the findings with others experiencing the same phenomenon.

Abstract C40: Understanding the mechanism of action of novel iron chelators on drug-resistant neoplastic cells.

Introduction: Deprivation of cellular iron (Fe) may represent a novel and effective anti-cancer strategy1. Our laboratory has developed iron chelators with marked and selective anti-tumor activity against a variety of tumor types in vitro and in vivo. One of our leading compounds, Dp44mT, demonstrated potent anti-tumor activity against drug-resistant cell lines1,2 over-expressing P-gp and MRP1. Moreover, these compounds possess high activity at releasing cellular 59Fe3. Aims: To further understand the mechanism of chelator-mediated cellular 59Fe release we investigated: The difference in iron efflux between drug-resistant MCF7-VP (MRP1 hyper-expressing) cells and their wild-type counterparts (MCF7); Whether chelator-mediated Fe release was MRP1 and/or GSH-dependent and; If chelator-mediated cellular Fe release was temperature-dependent. Results: These studies demonstrated that MRP1 over-expressing MCF7-VP cells showed reduced chelator-mediated59Fe release compared to MCF-7 cells (P Interestingly, MCF7-VP cells showed higher 59Fe uptake from 59Fe-transferrin than wild-type cells. This was due to marked transferrin receptor-1 expression in MCF7-VP compared to MCF7 cells. Additionally, the only known Fe efflux pump, ferroportin1, was down-regulated in MCF7-VP cells. This latter observation was intriguing as chelator-mediated Fe efflux was depressed in this cell type. These studies indicate that chelator-mediated 59Fe release is temperature-dependent and MRP-independent. Further studies will examine the role of MRP1 in Fe uptake and metabolism. References: 1. Whitnall, M., Howard, J., Ponka, P., and Richardson, D. R., Proc Natl Acad Sci U S A103 (40), 14901 (2006). 2. Richardson, D. R. and Milnes, K., Blood89 (8), 3025 (1997). 3. Richardson, D. R., Biochim Biophys Acta1320 (1), 45 (1997). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C40.