Development And Preclinical Evaluation Of Unique First-In-Class Small Molecule Inhibitors Of The Anti-Apoptotic Protein Flip.

Abstract In cancer, evasion of cell death is a fundamental cause of resistance to therapy, prompting the development of therapeutics that reactivate cell death pathways such as Bcl-2 family inhibitors and IAP antagonists. The apoptosis modulator Cellular FLICE-like inhibitory protein (FLIP) is a non-redundant inhibitor of caspase-8 activation and is the only human pseudo-caspase. Caspase-8 is the initiator caspase for the extrinsic apoptotic pathway and is now recognized as the molecular “switch” that controls the 3 major forms of programmed cell death: apoptosis, necroptosis and pyroptosis. As such, methods to selectively activate caspase-8 in appropriate disease contexts represent an exciting new therapeutic paradigm. FLIP is frequently overexpressed in solid and haematological cancers where it is associated with poor prognosis and chemo- and radio-resistance. Moreover, by regulating caspase-8 activity, FLIP is a key determinant of cell death induced by death ligands such as TRAIL expressed by immune effector cells. Thus, targeting FLIP's interaction with caspase-8 represents a unique therapeutic opportunity for enhancing standard-of-care anti-cancer therapies and promoting anti-tumor immunity.We report the discovery and characterisation of small molecule first-in-class selective inhibitors capable of disrupting FLIP's interaction with procaspase-8 in human cancer cells. These small molecule inhibitors induce caspase-8-dependent cell death as single agents and dramatically enhance apoptosis induced by recombinant TRAIL and 2nd generation multivalent TRAIL-R2 agonists in the nM concentration range. KRAS mutant non-small cell lung cancer (NSCLC) was identified as a major sensitive disease setting for FLIP inhibitors with single digit nM activity in vitro in several models and single agent in vivo efficacy. FLIP inhibitors also demonstrated in vitro efficacy in combination with KRAS G12C inhibitors in KRAS G12C mutant NSCLC and in vitro and in vivo efficacy in combination with the 3rd generation EGFR inhibitor Osimertinib in EGFR mutant NSCLC. We have also observed single agency nM efficacy in lymphoma and leukemia models and significant efficacy in combination with standard-of-care chemotherapeutics in KRAS mutant colorectal and pancreatic cancers. In summary, we have identified novel FLIP-targeted activators of caspase-8 with a unique mechanism-of-action and the potential for use in the treatment of several human cancers and leukemias either as a single agent, or in combination with standard-of-care chemotherapeutics and other clinically-relevant targeted agents. AcknowledgementsThis work was supported by a Seeding Drug Discovery award from the Wellcome Trust. Citation Format: Catherine A. Higgins, Jennifer Fox, Jamie Roberts, Declan Doherty, Trevor Perrior, Ray Boffey, Tim Harrison, Daniel B. Longley. Development and preclinical evaluation of unique first-in-class small molecule inhibitors of the anti-apoptotic protein FLIP [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1342.