Uncovering the zinc-dependent pathways of response and resistance to THZ1 in clear cell ovarian cancer

Ovarian cancer (OC) ranks as the fifth most lethal form of cancer for women and in the US causes the most deaths of any reproductive system malignancies. The current lack of clinical tools for effective early detection results in predominantly late-stage diagnosis. Clear cell ovarian cancer (CCOC), one of the five subtypes of OC, has low rates amongst Western demographics but makes up 25-30% of all cases of OC in Eastern countries such as Japan. A key characteristic of CCOC is its high rates of resistance to standard platinum therapy, with only 20-50% of patients showing response. Thus there is an urgent need for new treatment options for CCOC that can elicit sustained responses. An exciting new area of cancer therapeutics is focused on targeting the epigenetic drivers of cancer. New epigenetic cancer therapies include drugs that target transcription-associated cyclin dependent kinases (CDKs), CDK7/12/13, which promote transcription initiation and elongation by RNA polymerase II (RNAp2). Drugs targeting these proteins, such as THZ1, induce preferential depletion of key transcripts controlling cell survival and oncogenic pathways, and therefore have potent anticancer activity. However the efficacy of targeting transcriptional CDKs in CCOC, and the pathways involved is currently unknown. CCOC lines are sensitive to THZ1 in vitro. In high-grade serous tumor cell lines, transcriptional responses to THZ1 could be largely explained by downregulated expression of master transcription factors (MTFs) SOX17 and PAX8. However, downregulation of CCOC MTFs PAX8, HNF1B and ETS2 did not explain transcriptional responses to THZ1 in the CCOC models. Instead, exposing CCOC cell line RMG1 to THZ1 showed differential enrichment of pathways related to causes and consequences of modified intracellular zinc levels. Previous findings in OC connect intracellular zinc levels with oncogenic processes including metastasis and epithelial-to-mesenchymal transition. However, the role of zinc homeostasis in response to epigenetic drugs in OC is completely novel. Treating CCOC models with zinc chelator TPEN reduced intracellular free zinc by 63-70%. Co-treating CCOC cells with TPEN and THZ1 showed a significant 23% increase in sensitivity to THZ1. THZ1 resistant CCOC models were developed by exposing CCOC lines to increasing doses of THZ1 over a period of 300 days. Surprisingly, pharmacologic reduction of intracellular zinc increased growth of THZ1-resistant cells in the presence of THZ1, suggesting that fine-tuned control of free intracellular zinc dictates cellular responses to THZ1. In addition, THZ1-resistant lines had a 25% reduction in intracellular zinc compared to THZ1-sensitive parental lines. Our ongoing work is focussed on dissecting the impact of zinc on the activity of RNAp2 in the presence of THZ1, and will define the genes and regulatory elements involved in zinc-dependent responses to THZ1.