Author ORCID Identifier

https://orcid.org/0000-0001-5939-0144

Year of Publication

2020

Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation

College

Pharmacy

Department

Pharmaceutical Sciences

First Advisor

Dr. Markos Leggas

Abstract

Introduction: Chromosomal translocations are common in cancer. In many cancers such as prostate cancer, leukemia and Ewing sarcoma, chromosomal translocations are the main driver of malignancy. Ewing sarcoma is a cancer diagnosed mostly in children and adolescents that has very grim outcomes for patients with metastasis and recurrent disease. Malignancy in Ewing sarcoma is due to EWS-FLI1, an aberrant transcription factor that is the result of a chromosomal translocation. EWS-FLI1 is the main driver of oncogenesis in Ewing sarcoma and has been the target of many drugs developed to treat the disease. Mithramycin (MTM) was identified as a potent inhibitor of EWS-FLI1, but despite its pre-clinical success there were serious toxicities associated with its use including hepatotoxicity. Adverse effects associated with MTM treatment resulted in treatment regimens that were not enough to decrease tumor size. Because of dose limiting toxicities there has been a collective effort to develop analogues of MTM that are less toxic but more effective against inhibiting EWS-FLI1 activity. Here I present an assessment of the unique molecular biologic activity of novel MTM analogues.

Preliminary pharmacokinetic studies have shown that in mice MTM has a shorter plasma half-life of about five hours [1]. MTM analogues MTMSA-TRP and MTMSA-TRP-TRP have extended plasma half-lives of twelve hours and over eighteen hours, respectively. Due to their longer plasma half-lives we hypothesized that MTM analogues have less affinity for organic anion transporting polypeptides (OATP) because of their physiochemical properties.

Additionally, DNA binding assays suggested that MTM analogues bind DNA and stabilize the EWS-FLI1 transcription factor reducing transcription of EWS-FLI1 target genes [2]. We hypothesize that MTM analogues bind DNA displacing and/or stabilizing EWS-ETS transcription factors. These analogues bind DNA resulting in DNA damage, we hypothesize that combining MTM analogues that cause DNA damage with PARP1 inhibitors will result in a synergistic response in cell lines expressing EWS-FLI1.

Methods: Based on preliminary cytotoxicity and pharmacokinetic studies, the ionization constant (pKa) of MTM and analogues was measured. Based on the fluorescence of MTM core aromatic rings at increasing pH, pKa values were estimated. RKO cells stably transfected with pIRES/OATP1B1 or pIRES/OATP1B3 expression vectors were used to complete competitive inhibition studies. Measuring MTM and analogue competition into OATP vs. 8- (2- [fluoresceinyl]aminoethylthio) adenosine- 3', 5'- cyclic monophosphate (8-FcA), a fluorescent substrate of OATP transporters. OATP expressing cells were also subjected to cytotoxicity studies to evaluate uptake of MTM analogues over a 72-hour assay. FVB/N Oatp1a/1b knockout (KO) and FVB/N wildtype mice were used to complete pharmacokinetic studies evaluating differences in uptake of MTM and MTMSA-TRP-TRP after pre-treatment with the OATP inhibitor rifampicin for 1 hour.

Time-resolved fluorescent energy transfer (TR-FRET) assays used CAL635 labeled DNA oligomers specific for ERG-his tagged and Sp1-his tagged peptides. Europium-labeled (EU) anti-HIS antibodies were combined with the peptide to evaluate shifts in FRET, a response to treatment with MTM or analogue. Cellular thermal shift assays (CETSA) were used to determine shifts in thermal stability of EWS-FLI1, Sp1 and RNA polymerase II (RNAPII). Protein expression studies evaluating expression of MTM analogue activity against RNAPII phosphorylated Serine 2 (p-Ser2) on its c-terminal domain (CTD). mRNA expression of DNA damage repair genes CDK12, BRCA1, FANC1 and FANCD2 in cell lines expressing EWS-FLI1 (TC-32 and A673) vs. cell lines that do not express EWS-FLI1 (PC3 and TC-32shEWS-FLI1#6) was also evaluated. Protein expression of the dsDNA damage biomarker γ-H2AX was also evaluated in TC-32 and PC3 cell lines. TC-32 and PC3 cells were also used to evaluate synergy between MTM analogues and the PARP1 inhibitor olaparib. Results from cytotoxicity studies were used to calculate the combination index (CI) values of each drug combination. Based on CI values from in vitro studies a xenograft study in female athymic nu/nu mice evaluating tumor regression after treatment with olaparib and MTMSA-TRP was conducted.

Results: Evaluation of the molecular function of MTM analogues was largely based on differences in their structure and pKa. pKa estimation studies revealed that analogues with large hydrophobic conjugations to the C3 side chain of MTM, resulted in a higher pKa than the parent compound MTM. This result also correlated with OATP mediated uptake into RKO-OATP1B1 and RKO-OATP1B3 cells in competition assays and in cell viability assays. Pharmacokinetic studies in OATP1B1 humanized mice demonstrated an increase in exposure of MTM after treatment with rifampicin.

TR-FRET and CETSA assays demonstrated pointed evidence of displacement and stabilization of EWS-FLI1 and ERG transcription factor DNA binding domains (DBD). In TR-FRET assays MTM analogues with amino acid side chains displaced ERG binding more potently than they did SP1, and CETSA assays demonstrated a physical interaction between EWS-FLI1 and MTM analogues. Sp1 CETSA experiments showed minimal shifts in thermal stability between the different analogues, suggesting that there is no physical interaction between analogue and the Sp1 transcription factor. RNAPII CETSA experiments showed that MTM and MTMSA analogues were more likely to stabilize this protein vs. MTMox analogues. Protein expression of RNAPII CTD p-Ser2 also demonstrated a concentration and EWS-FLI1 dependent decrease in expression. mRNA expression of DNA damage repair proteins CDK12, BRCA1, FANC1 and FANCD2 was assessed by qRT-PCR, indicating that MTM and analogues also induce a decrease in their expression in an EWS-FLI1 and concentration dependent manner. These interactions were the rationale to investigate the possibility of MTM analogues being DNA damaging agents and transcriptional inhibitors. MTM analogues were shown to induce DNA damage in TC-32 cells but not in PC3 cells, demonstrating a concentration and EWS-FLI1 dependent increase in γH2AX expression. Cell viability studies combining MTM, analogues and olaparib proved synergy specific to cells expressing EWS-FLI1. Xenograft synergy studies also showed that we can lower the dose of MTMSA-TRP combined with olaparib and gain a more sustained reduction in tumor size.

Conclusion: These studies in whole highlight the importance of developing and understanding the underlying mechanism of these novel analogues. Physiochemical properties of the analogues compared to MTM directly contribute to their exposure. And binding properties correlate with specificity and cytotoxicity in ETS translocation expressing cell lines. MTMSA-TRP has also shown promise in xenograft studies however, in these studies we combined it with olaparib to take advantage of the synthetic lethality imparted on cell lines that express EWS-FLI1.

Digital Object Identifier (DOI)

https://doi.org/10.13023/etd.2020.505

Available for download on Thursday, December 15, 2022

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