AbstractColorectal cancer (CRC) is a leading cause of cancer-related death and the third most common type of cancer worldwide. The five-year survival rate strongly correlates with the stage of the disease at initial diagnosis, falling from well over 90% at Stage I to less than 10% when diagnosed at Stage IV. Drug resistance is a major limitation to current treatment options for advanced CRC. Consequently, there is an unmet clinical need for novel therapeutic agents for treating patients with CRC in the advanced disease setting.
CRC is frequently associated with a pro-inflammatory tumour microenvironment in which TNFα signalling plays an important role. Inhibitor of apoptosis proteins (IAPs) are capable of converting TNFα signalling from a pro-apoptotic to a pro-survival and pro-inflammatory signal. Overexpression of IAPs is associated with chemoresistance and poor prognosis in CRC. Consequently, IAPs are an attractive target for therapeutic intervention, and IAP antagonists, such as ASTX660, have recently been developed in order to exploit their pro-survival mechanisms. When TNFα binds to its receptor TNFR1 in the presence of an IAP antagonist, it leads to the formation of a cytoplasmic death-inducing complex consisting of RIPK1, FADD and procaspase-8. Procaspase-8 undergoes homodimerization within this complex and subsequently activates effector caspases-3 and -7 to promote apoptosis. FLIP, a pseudo-caspase, can also bind to FADD and inhibit homodimerization of procaspase-8, subsequently inhibiting apoptosis.
In vitro results indicated that the CRC cell line models investigated herein harboured intrinsic resistance to IAP antagonist-mediated cell death, even in the presence of recombinant TNFα. Further investigation using siRNA-mediated silencing techniques, revealed FLIP is a major mediator of this resistance mechanism. Moreover, Entinostat, a clinically relevant Class I HDAC inhibitor, was found to downregulate FLIP expression and sensitise CRC cell line models to ASTX660. Through the use of procaspase-8 and procaspase-10 CRISPR-Cas9 knockout cell lines, the enhanced cell death observed was determined to be dependent on caspase-8, but not on its paralog, caspase-10. Furthermore, FLIP-mediated resistance, to ASTX660 and TNFα, was also overcome through the use of a novel small molecule FLIP inhibitor that targets the FLIP:FADD protein-protein interaction (PPI).
Clinical approval of IAP antagonists is not only hampered by intrinsic resistance, but also by a lack of clinical biomarkers to stratify patients who would respond well to this treatment. Herein, the potential of using the presence of a Fusobacterium nucleatum infection as a predictive biomarker for clinical positioning of these agents was investigated. F. nucleatum infection was shown to promote an upregulation of CIAP2 and TNFα in pre-clinical models and this correlated with observations made in clinical samples. Interestingly, there was evidence to suggest that FLIP expression was downregulated by F. nucleatum infection. Importantly, co-culture of F. nucleatum infected monocytic cells significantly enhanced the efficacy of IAP antagonism in a manner dependent on bacterial induction of TNFα secretion in the immune cells. Collectively, this work suggests that the presence of F. nucleatum bacteria in colorectal tumours promotes a tumour microenvironment rich in TNFα levels and may ‘prime’ tumours with elevated CIAP2 and lower FLIP expression to become sensitive to IAP antagonists.
Overall, the work presented in this thesis indicates that IAP antagonists may be most effective in pro-inflammatory, TNFα-rich CRC, but only if combined with an agent capable of overcoming FLIP-mediated resistance. Furthermore, the data presented suggest that the presence of F. nucleatum has the potential to be utilised as a predictive biomarker to identify patients who would benefit from IAP antagonistic therapy.
|Date of Award||Jul 2021|
|Supervisor||Daniel Longley (Supervisor), John E. Hallsworth (Supervisor) & Jose Bengoechea (Supervisor)|
- Colorectal cancer
- Inhibitor of Apoptosis Proteins
- Fusobacterium nucleatum