Jennifer L. Bryant

Overcoming Platinum Resistance in Preclinical Models of Ovarian Cancer Using the Neddylation Inhibitor MLN4924

The nearly ubiquitous development of chemoresistant disease remains a major obstacle against improving outcomes for patients with ovarian cancer. In this investigation, we evaluated the preclinical activity of MLN4924, an investigational inhibitor of the NEDD8-activating enzyme, in ovarian cancer cells. Efficacy of MLN4924 both alone and in combination with platinum was assessed. Overall, single-agent MLN4924 exhibited moderate activity in ovarian cancer cell lines. However, the combination of MLN4924 with cisplatin or carboplatin produced synergistic effects in SKOV3 and ES2 cells, as well as in primary ovarian cancer cell lines established from high-grade serous, clear cell, and serous borderline ovarian tumors. The efficacy of cisplatin plus MLN4924 was also evident in several in vitro models of platinum-resistant ovarian cancer. Mechanistically, the combination of cisplatin and MLN4924 was not associated with DNA re-replication, altered platinum-DNA adduct formation, abrogation of FANCD2 monoubiquitination, or CHK1 phosphorylation. An siRNA screen was used to investigate the contribution of each member of the cullin RING ligase (CRL) family of E3 ubiquitin ligases, the best-characterized downstream mediators of MLN4924s biologic effects. Cisplatin-induced cytotoxicity was augmented by depletion of CUL3, and antagonized by siCUL1 in both ES2 and SKOV3 ovarian cancer cells. This investigation identifies inhibition of neddylation as a novel mechanism for overcoming platinum resistance in vitro, and provides a strong rationale for clinical investigations of platinum and MLN4924 combinations in ovarian cancer. Mol Cancer Ther; 12(10); 1958–67. ©2013 AACR.

Evaluation of EVI1 and EVI1s (Δ324) as potential therapeutic targets in ovarian cancer

PURPOSE The MDS1 and EVI1 complex locus (MECOM) at 3q26 gives rise to several alternatively spliced transcripts implicated in leukemic oncogenesis. Overexpression of EVI1 in ovarian cancer has led to a proposed oncogenic role. Our objective was to evaluate the therapeutic potential of EVI1 and EVI1s (also known as Delta324) in ovarian cancer. METHODS Expression of EVI1 mRNA and protein isoforms was evaluated in ovarian cancers, normal ovaries, benign ovarian neoplasms, and fallopian tube fimbria. Effects of EVI1 isoform overexpression and knockdown on proliferation, cisplatin-induced apoptosis, and double stranded DNA breaks were investigated. RESULTS EVI1 and EVI1s mRNAs were ubiquitously expressed in ovarian cancers and benign gynecologic tissues examined, with highest expression of both isoforms noted in the cancer samples. The EVI1s to total EVI1 mRNA ratio was uniform among the examined tissues. In contrast, EVI1 protein isoform levels were undetectable in normal ovarian tissues, and highest in serous ovarian cancers. EVI1 protein expression patterns were similar between serous ovarian cancer samples, fallopian tube fimbria, and benign neoplasms. Expression of EVI1 or EVI1s did not increase proliferation in EVI1-null OVCAR8 cells. Total and isoform selective knockdown of EVI1 isoforms in EVI1 expressing ovarian cancer cells had no effect on proliferation, cisplatin-induced apoptosis, or gamma-H2AX levels in ovarian cancer cells. CONCLUSION Our data do not support a role for EVI1 or EVI1s in ovarian cancer cell proliferation or response to DNA damage. Further research is required before EVI1 can be considered an oncogene or a therapeutic target in ovarian cancer.

Sub-terahertz vibrational spectroscopy for microRNA based diagnostic of ovarian cancer

The purpose of this research is to introduce and validate novel sub-THz resonance spectroscopy combined with molecular dynamics (MD) computation as a promising approach for optical analysis and potential quantification of molecular biomarkers in ovarian (OC) cancer cells. The ability of sub-THz spectroscopy to identify and quantify biological molecules is demonstrated by interrogation of resonance features caused by atomic vibrations within biological molecules in cancer and normal samples. In vitro human cell cultures of two ovarian cancer subtypes, SK-OV-3 human epithelial and ES-2 human clear cell carcinoma, were characterized in comparison with a normal nontransformed cell line (FT131-human fallopian tube epithelial cell line). A dramatic difference between the THz absorption spectra of cancer and normal cells and cell free samples is observed with much higher absorption intensity and a strong absorption peak at frequency of ~13 cm−1 dominating the spectra from cancer samples. Comparison of experimental spectra with MD predictions of spectroscopic signatures of microRNA molecules from the miR-200 family, known to be overexpressed in ovarian cancer, suggests an origin for this pronounced spectral peak. The rest of the cancer samples’ signature is in part similar to the signatures of normal cells and represents contributions from proteins and nucleic acid polymer molecules. Even though ovarian cancer is utilized for this proof of concept, the sub-THz spectroscopy method is very general and can be as well applied to other cancer types. PAPER

Abstract A83: Profiling potent, novel protein tyrosine phosphatase 4A3 small molecule inhibitors for ovarian cancer.

PTP4A3 (also known as PRL-3) is a unique protein tyrosine phosphatase that is highly expressed in human ovarian cancer patient samples. Genetic knockdown studies implicate PTP4A3 in ovarian cancer tumorigenesis and the maintenance of the malignant phenotype. Interestingly, cBioportal data mining reveals that PTP4A3 is one of the most highly amplified genes in ovarian cancer, implicating PTP4A3 as a novel, and to date, largely underexplored potential molecular therapeutic target. Unfortunately, the lack of potent and selective PTP4A3 inhibitors impedes the validation of PTP4A3 as a drug target for ovarian cancer. To drive target validation studies and complement our ongoing cell-based genetic PTP4A3 knockdown strategies, we have developed novel PTP4A3 small molecule inhibitors to delineate its mechanistic role in ovarian cancer and therapeutic potential as a drug target. We resynthesized the most potent published PTP4A3 inhibitor (thienopyridone or JMS-631-050). Concurrently, we discovered a new, more active thienopyridone analog, JMS-631-053. Additionally, we identified a structurally related inactive analog, JMS-557-038, which could be used as a pharmacologically powerful control. JMS-631-050 and JMS-631-053 inhibited recombinant PTP4A3 in vitro phosphatase activity with IC50 values of 138 and 7 nM, respectively. We then profiled the impact of JMS-631-050, JMS-631-053 and JMS-557-038 on cell adhesion-based survival using a panel of eight ovarian cancer cell lines, which included a representative high serous grade ovarian cancer cell line (i.e., OVCAR4) and several drug sensitive/resistant cell line pairs. In all ovarian cancer cells lines tested, both JMS-631-050 and JMS-631-053 inhibited cell adhesion-mediated survival. However, JMS-631-053 displayed more potent cell-based effects than JMS-631-050 in all cell lines evaluated. A2780 cells were the most sensitive to JMS-631-053 effects (EC50=0.60±0.2 μM). Significantly, OVCAR4 cells were also highly sensitive to JMS-631-053 with an EC50=1.5±0.3 μM. Both JMS-631-050 and JMS-631-053 maintained cell-based activity when evaluated in three dimensional cell culture models. As expected the PTP4A3 inactive analog, JMS-557-038 had no effect on cell-adhesion-mediated survival (EC50s>50 μM) in any of the ovarian cancer cell lines tested. Thus, JMS-631-050 and JMS-631-053 are attractive chemotypes for further evaluation as single agents for ovarian cancer. Moreover, as the drug resistant cell lines A2780CP20 and HeyA8MDR retained sensitivity to JMS-631-050 and JMS-631-053, these chemotypes are under evaluation in combination studies using existing ovarian cancer therapies. Hence, JMS-631-050 and JMS-631-053 will be valuable reagents for further validation of PTP4A3 as an ovarian cancer target as well as potential leads for future drug dis. Citation Format: Elizabeth R. Sharlow, Joseph M. Salamoun, Kelley E. McQueeney, Sophie Lewandowski, Jennifer Bryant, Alex Cheung, Paula Pekic, Charles N. Landen, Jr., Peter Wipf, John S. Lazo. Profiling potent, novel protein tyrosine phosphatase 4A3 small molecule inhibitors for ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A83.

Abstract 3380: Overcoming platinum resistance in ovarian cancer using the novel compound MLN4924.

Purpose: The objective of this investigation was to evaluate the preclinical activity of MLN4924, an investigational inhibitor of the NEDD8-activating enzyme, against chemoresistant and chemosensitive ovarian cancer cells. ExperimentalDesign: Efficacy of MLN4924 both alone and in combination with cisplatin was assessed using short-term tetrazolium based and longer term focus formation assays. Mechanistic studies included cell cycle analysis, evaluation of DNA-platinum adduct formation (using an ELISA), and evaluation of DNA damage repair pathways. In addition, we employed an siRNA screen to assess the contribution of each member of the Cullin RING-Ligase (CRL) family of E3 ubiquitin ligases, the best characterized downstream mediators of MLN4924’s biological effects. Results: Single agent MLN4924 exhibited moderate activity in ovarian cancer cell lines. The combination of MLN4924 with cisplatin or carboplatin resulted in significant platinum sensitization in SKOV3 and ES2 cells, as well as, in several primary ovarian cancer cell lines established from high grade serous, clear cell, and serous borderline ovarian tumors. Furthermore, the platinum sensitizing effect of MLN4924 was also observed in several in vitro models of platinum resistant ovarian cancer (using both established and primary chemoresistant ovarian cancer cells). Mechanistically, we show that depletion of CUL3 and CUL5 augment, while knockdown of CUL1 and CUL4A antagonize cisplatin-induced cytotoxicity in ovarian cancer cells. The combination of cisplatin and MLN4924 was not associated with DNA re-replication, altered platinum-DNA adduct formation, or abrogation of FANCD2 monoubiquitination. Conclusions: Our investigation reveals a novel mechanism for overcoming platinum resistance in vitro, and provides a strong rationale for clinical investigations of platinum and MLN4924 combinations in ovarian cancer. This work also highlights that different CRL ubiquitination pathways can have distinct and opposing effects on platinum sensitivity and resistance in ovarian cancer cells. Citation Format: Amir A. Jazaeri, Etsuko Shibata, Jonghoon Park, Jennifer Bryant, Mark R. Conaway, Susan C. Modesitt, Peter G. Smith, Michael Milhollen, Allison J. Berger, Anindya Dutta. Overcoming platinum resistance in ovarian cancer using the novel compound MLN4924. [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 3380. doi:10.1158/1538-7445.AM2013-3380

Abstract 801: Comparison of cisplatin induced changes in serous ovarian cancer and normal fallopian tube cells

Platinum drugs are the most active agents for treatment of ovarian cancer. While most patients initially respond to this treatment, the majority eventually become resistant and ultimately succumb to the disease. Despite much investigation, in vitro elucidated mechanisms of platinum resistance have not correlated with those involved in clinical chemoresistance. Traditionally, in vitro models have consisted of established ovarian cancer cell lines that have been propagated in culture for decades. Chemoresistance is then modeled by growing cells in the presence of increasing concentrations of platinum or other cytotoxic drugs. As an alternative approach, we developed primary cell lines from normal human fallopian tube epithelial cells (n=5) and serous ovarian cancers (n=15). The latter group included tumors isolated from chemo-naive (n=9) and chemo-exposed (n=6) patients. Each primary cell line was treated with vehicle and cisplatin (50uM) for 24hrs and expression profiling was performed using Affymetrix arrays. Differentially expressed genes were identified using BRB ArrayTools (P 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 801. doi:1538-7445.AM2012-801

Abstract 608: Oncogenic role of cullin 4 E3 ubiquitin ligase (CRL4-CDT2) in epithelial ovarian cancer

Objective: Ubiquitination is a key posttranslational modification involved in the tight regulation of biological processes including cell-cycle regulation and DNA replication and repair. Our objective was to investigate the expression and function of CDT2, a substrate recognition factor for the CUL4 E3 ubiquitin ligase family (CRL4-CDT2) in ovarian cancer. Methods: CDT2 expression was evaluated using realtime RT-PCR in 26 serous ovarian cancers 11 normal ovaries, and 14 fallopian tube fimbria specimens and confirmed by Western blotting on a subset of the above. Immunohistochemistry was performed using tissue microarrays consisting of over 130 independent ovarian cancer samples of various histologies, as well as, normal fallopian tube samples using antibodies to CDT2 and p21. Each samples was represented at least in triplicate and scored semi-quantitatively. CDT2 knockdown was accomplished using siRNA and overexpression via retroviral transduction. Cell growth was evaluated using MTT assays. Statistical analysis were performed using either parametric (t test) as well as non-parametric (Mann-Whitney and Kruskal Wallis) methods as indicated. Comparison of p21 and CDT2 expression was performed using Spearman9s correlation. Results: CDT2 mRNA expression was approximately 9 fold higher in serous ovarian cancers compared to normal ovary and fallopian tube fimbria samples (P Conclusions: The results of this ongoing investigation are the first to suggest an oncogenic role for the CRL4-CDT2 ubiquitin ligase in ovarian cancer. The mechanism of action remains unknown but does not appear to involve p21 degradation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 608. doi:10.1158/1538-7445.AM2011-608