Synergistic combinations of novel small molecule CtBP inhibitors with DR5 agonists or BH3-mimetic PROTACs targeting chemoresistant high grade serous ovarian carcinoma

Background: High Grade Serous Ovarian Carcinoma (HGSOC) is especially lethal due to frequent diagnosis at advanced stage and development of chemoresistance. The oncogenic transcription factors C-terminal binding proteins 1 and 2 (CtBP1/2; or “CtBP”) are transcriptional scaffolds/coregulators that drive tumor progression and metastasis in many solid tumors and are amplified in ~10% of HGSOC, with their frequent protein overexpression correlated with poor prognosis. Results: We have previously reported that CtBP transcriptionally represses the proapoptotic Death Receptors (DR) 4/5 in HGSOC cells, preventing activation of caspase 8-dependent apoptosis. Aside from its role as an oncogenic dependency in HGSOC, CtBP is a therapeutic target encoding an NAD-dependent dehydrogenase domain against which we have developed small molecule CtBP inhibitors (CtBPi) which exhibit significant anti-tumor activity in various solid tumor models without toxicity. Our current lead CtBPi's, NAS-2-133/134, which are naphthyl derivatives of the 1 st generation CtBP inhibitor scaffold 2-hydroxyimino-3-phenylpropanoic acid, exhibit IC 50 values of 4-17 µM in multiple HGSOC cell lines, while A2780 cells with homozygous deletion of CtBP1/2 (A2780/DKO) were resistant to NAS-2-134 relative to parental A2780 cells (IC 50 s = >>25 µM [beyond titration] vs. 20 µM, respectively), demonstrating that NAS-2-134 exhibits potent on target cytotoxic selectivity. In addition, NAS-2-134 induced caspase-8 dependent apoptosis in HGSOC cells via upregulation of DR5 and exhibited synergistic cytotoxicity when combined with the DR5 agonists MD5.1 or Biomyifi using Bliss methodology (Bliss synergy scores: MD5.1=15.8, BIOYMIFI=15.3). Further mechanistic studies revealed NAS-2-134 decreased Bcl-2 levels, which prompted evaluation of potential synergy of NAS-2-134 with BH3-mimetics venetoclax (ABT-199) and navitoclax (ABT-263), which target Bcl-2 and Bcl-2/X L , respectively. Though NAS-2-134 and ABT-199 did not exhibit synergistic cell killing in HGSOC cells, NAS-2-134 and ABT-263 exhibited strong synergy with a Bliss score of 47. Importantly, both NAS-2-134 and the closely related CtBPi NAS-2-133 demonstrated potent in vivo antitumor activity, abrogating growth of OVCAR3 xenografts in immune deficient mice. Conclusion: We demonstrate that NAS-2-134 exhibits target selective cytotoxicity in HGSOC cells via induction of caspase-8/DR5 dependent apoptosis in vitro and both NAS-2-134 and NAS-2-133 exhibit potent anti-HGSOC tumor activity in vivo; and 2) concerted targeting of CtBP and DR5 or Bcl-2/X L is synergistically cytotoxic in HGSOC cells and this strategy may lead to a future low toxicity precision therapy for chemoresistant HGSOC.