Abstract C5: FLIP protein-protein interaction inhibitors enhance sensitivity of colorectal cancer cells to chemotherapy and TRAIL
Authors: Fox Jennifer P. , Majkut Joanna , Higgins Catherine , Nemeth Zsuzsanna , Malik Adnan , Scott Christopher J. , Blurton Peter , Boffey Ray J. , Perrior Trevor R. , Harrison Timothy , Longley Daniel B. 
Published: May 2016

Background

Colorectal Cancer (CRC) is the second most common cause of cancer death, with 40% of patients with this disease obtaining no benefit from current chemotherapy. Novel therapeutic strategies are needed to improve CRC patient response rates and survival. FLIP is an inhibitor of the extrinsic apoptotic pathway that binds to FADD at death-inducing signalling complexes (DISCs), such as those formed by the TNF-α-related apoptosis inducing ligand (TRAIL) receptors TRAIL-R1 and TRAIL-R2, thereby blocking homodimerization and activation of procaspase-8 and inhibiting apoptosis induction. We previously reported that FLIP blocks apoptosis induced by TRAIL and standard-of-care chemotherapeutics (5-Fluorouracil, oxaliplatin and SN38) in CRC models. Moreover, FLIP is frequently overexpressed in CRC and its overexpression correlates with poor prognosis. Subsequently, we have developed novel small molecule inhibitors that target FLIP's critical protein-protein interactions, preventing its interaction with FADD and therefore promoting activation of caspase-8 and apoptosis induction.

Methods

A DISC recruitment assay was used to assess levels of FLIP at the TRAIL-R2 DISC. Caspase activity, cell viability and apoptosis induction were assessed in CRC models treated with FLIP inhibitors alone and in combination with TRAIL or standard-of-care chemotherapeutics. Mechanism-of-action was assessed using caspase-8-targeted siRNA and FLIP overexpressing models.

Results

Using CRC cell line models, it was demonstrated that FLIP recruitment to the DISC is inhibited by FLIP inhibitors from the lead series. As a result, TRAIL-induced caspase-8 and caspase-3/7 activity were enhanced and increased levels of apoptosis cells were observed. Decreased cell viability was observed that was proportionate to the levels of apoptosis induced. Cell death triggered by FLIP inhibitors was shown to be caspase-8-dependent consistent with the expected mechanism-of-action. Importantly, inhibitors of FLIP also enhanced apoptosis induction in response to 5-Fluorouracil, oxaliplatin and SN38.

Conclusion

We have developed inhibitors of FLIP that decrease its recruitment to the TRAIL-R2 DISC and increase TRAIL-induced caspase activation and apoptosis. Moreover, these inhibitors synergise with 5-Fluorouracil, oxaliplatin and SN38, suggesting that this novel class of agents has therapeutic potential in CRC when used in conjunction with standard-of-care chemotherapeutic agents.

Acknowledgements

This work was supported by a Seeding Drug Discovery award from the Wellcome Trust (reference: 099470).

Citation Format: Jennifer P. Fox, Joanna Majkut, Catherine Higgins, Zsuzsanna Nemeth, Adnan Malik, Christopher J. Scott, Peter Blurton, Ray J. Boffey, Trevor R. Perrior, Timothy Harrison, Daniel B. Longley. FLIP protein-protein interaction inhibitors enhance sensitivity of colorectal cancer cells to chemotherapy and TRAIL. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C5.