Inhibition of the Pax8 Transcription Factor as a Novel Therapeutic Approach for Ovarian and Endometrial Cancer

The PAX8 transcription factor is essential for ovarian cancer proliferation, and its silencing in ovarian and endometrial cancer cells leads to apoptosis 1,2. PAX8 expression in other tissues is restricted to non-essential organs, suggesting that it is a good target for therapy with minimal side effects 3,4. We propose a novel anti-ovarian and -endometrial cancer therapy based on small molecule binders of PAX8 that inhibit its activity. This, population shift-based approach, involving the stabilization of a PAX8 conformation that cannot bind DNA, is readily applicable also to other transcription factors linked to other types of cancers.
Ovarian cancer is the deadliest gynecologic malignancy in the western world, with 30-40% 5-year overall survival. The mainstay of treatment is surgery and chemotherapy, and most patients initially respond, but in 80% of the cases the disease eventually relapses and the patients succumb to their illness 5. Targeted therapies are scarce and mostly effective in a small percentage of patients. Therefore, novel ovarian cancer therapies are a significant unmet need. Endometrial cancer is the most common gynecologic malignancy, and is usually highly curable with surgery alone 6. However, about 10% of patients have an aggressive subtype, called uterine serous papillary cancer, that can be fatal and would usually require systemic therapy. PAX8 is of special importance in this aggressive subtype 1. 

A wide variety of DNA-binding transcription factors are linked to cancer, and are considered top targets for anti-cancer therapy. We suggest that the difficulty to inhibit transcription factors is because the effort to inhibit transcription factor activity has been focused only on the DNA-bound conformation, which is difficult to inhibit, because it does not have a druggable binding pocket. Here, we examine for the first time an alternative approach, using the transcription factor PAX8:
 • We conducted a virtual screen of small molecules that would stabilize PAX8 conformations that cannot bind DNA. 
 • Stabilizer molecules cause a shift in the PAX8 protein population towards a non-DNA binding conformation, thereby reducing PAX8       transcriptional activity.
Our preliminary in-silico screen resulted in a family of compounds that were ordered off-the-shelf, tested in-vitro, and optimized through a series of in-silico and in-vitro optimization iterations. This family includes four compounds that inhibit PAX8 transcriptional activity and ovarian cancer proliferation with an IC50 of about 3 µM. Furthermore, we utilized and analyzed results of a high throughput screen, and discovered two additional compounds that inhibit PAX8 activity with an IC50 of about 3 µM. Reassuringly, several of these compounds showed an ability to dissociate the binding of PAX8 to DNA. Recently, we developed a stable high-throughput screen of PAX8 inhibitors in collaboration with the Blavatnik Center for Drug Discovery. This screen provided independent validation of our current hits, and is currently used for a much larger screen. In addition, we have developed a novel bacteria-based screen for new PAX8 inhibitors. In this screen, PAX8 expression kills bacteria and the inhibitors rescues it. This, positive selection approach, provides further validation of our current hits. It will also be used in a high throughput manner. 

 • Up to date our solution is the first that allows a direct treatment of ovarian and endometrial cancer via targeting of PAX8.
 • Due to predicted specificity, we vision a very low rate of side effects.
 • We have a detailed reproducible multi-method platform to test novel PAX8 inhibitors. 

Our inhibitors might affect other members of the PAX family which share high sequence similarity with PAX8. We have not yet tested our compounds for specificity against other PAX proteins. If needed, we will optimize specificity using similar in-silico and in-vitro platforms that will be tailored for other PAX proteins, or link our inhibitors to a targeting antibody. 

 • Use our high throughput assays to find more hits
 • Apply standard medicinal chemistry and structural biology in a hit-to-lead phase and come up with novel chemical entity inhibitors with IC50 in the sub micro-molar range
 • Test these inhibitors in mice model 

1. Fares, B. et al. PAX8 plays an essential antiapoptotic role in uterine serous papillary cancer. Oncogene 40, 5275–5285 (2021).
2. Ghannam-Shahbari, D. et al. PAX8 activates a p53-p21-dependent pro-proliferative effect in high grade serous ovarian carcinoma. Oncogene 37, 2213 (2018).
3. Bowen, N. J. et al. Emerging roles for PAX8 in ovarian cancer and endosalpingeal development. Gynecol. Oncol. 104, 331–337 (2007).
4. Laury, A. R. et al. A comprehensive analysis of PAX8 expression in human epithelial tumors. Am. J. Surg. Pathol. 35, 816–826 (2011).
5. Perets, R. & Drapkin, R. It’s Totally Tubular….Riding The New Wave of Ovarian Cancer Research. Cancer Res. 76, 10–17 (2016).
6. Siegel, R. L., Miller, K. D., Fuchs, H. E. & Jemal, A. Cancer statistics, 2022. CA. Cancer J. Clin. 72, 7–33 (2022).

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