Pre-Clinical Application of Inhibitor of Apoptosis Protein (IAP) Antagonists in Colorectal Cancer (CRC)

Inhibitor of Apoptosis Proteins (IAPs) have diverse roles in apoptosis, acting as inhibitors of caspases and mediators of NF-κB signalling. Upregulation of IAPs has been correlated with disease progression in many cancer types whence, synthetic inhibitors of IAPs have been developed in recent years. These target IAPs by mimicking the amino-terminal (N-terminal) IAP-binding motif of Second Mitochondrial-derived Activator of Caspases (SMAC). The principal aim of this study was to provide rational understanding of the anti-tumour therapeutic effect of the IAP antagonist Birinapant in colorectal cancer (CRC), as monotherapy and in combination with 5-Fluorouracil (5FU)/Oxaliplatin or Tumour Necrosis Factor-alpha (TNFα), with emphasis on the role of the pro-inflammatory tumour microenvironment modelled by TNFα and the specific Consensus Molecular Subtypes (CMS subtypes).

An 3D ex vivo model has been established using dental sponges as culture support for CRC patient-derived explants. Active caspases levels were analysed by immunohistochemistry and compared to IAPs expression. Histological evaluation of other key markers involved in the apoptotic pathways was also carried out. Apoptosis was evaluated using a TUNEL assay and TNFα synthesis was determined by ELISA. Expression patterns of proliferation markers, Wnt signalling intermediaries and EMT mediators were also explored. CMS subtyping was performed by using gene-expression profiles and an IHC-based classifier tool and correlation between the assigned CMS and the response to Birinapant was examined.

Treatment with Birinapant and 5FU/Oxaliplatin or TNFα sensitised CRC patient-derived explants to SMAC mimetic-mediated cell death via caspase-dependant mechanisms, by triggering activation of caspase-8, -9, and -3, blocking the anti-apoptotic activity of IAPs and by stimulating the TNFα-dependant pro-apoptotic signalling. Birinapant co-treatments also reduced the expression levels of Ki-67, Zeb1, COX-2, PGE2 and β-catenin. CMS subtyping revealed that the analysed explants had a higher proportion of epithelial-like subtype CMS3 which preferably benefited from the administration of Birinapant in combination with TNFα.

The 3D system retained the in vivo tissue architecture, promising to serve as a powerful experimental platform to identify key biomarkers which could serve as predictors of treatment response. Assigning the CMS subtype to each CRC tumour also offered a patient stratification tool which provided individualised predictions of treatment outcomes.