Understanding the role of IKKα in the tumour microenvironment and establishing it as a novel therapeutic target in colorectal cancer

Professor Joanne Edwards, Dr Lesley Stark, Dr Nigel Jamieson

Colorectal cancer (CRC) remains the 2nd most common cancer and 3rd most common cause of cancer-related death globally. Despite advancements in treatment of disease through recent adoption of immunotherapies, novel therapies are required to improve patient outcomes. IKKα represents a promising therapeutic target and is involved in non-canonical NF-κB signalling. This pathway is activated by either RANK/RANKL, CD40/CD40L, LTbR/ LTb or BAFF/BAFFR ligand receptor interaction, leading to subsequent activation of NIK and IKKα. This results in phosphorylation and ubiquitination of the p100/RelB complex, which translocates to the nucleus and acts as a master regulator of genes associated with the hallmarks of cancer. We have demonstrated that high expression of IKKα is associated with reduced prognosis in prostate, breast, and right-sided colon cancer (1-3). Evidence from other groups utilising CRC cell lines suggests that IKKα may also be involved in the canonical NF-κB pathway. This project aims to further our understanding of IKKα role in NF-κB signal transduction within the tumour microenvironment (TME), investigate downstream pathways of IKKα activation and explore the therapeutic potential of combining IKKα inhibitors with chemotherapy for colon cancer and radiotherapy for rectal cancer through in vitro studies.

To study IKKα in the TME we will use retrospective discovery, validation and clinical trial CRC cohorts, which will be stained via multiplex immunohistochemistry (mIF) for total IKKα expression, phosphorylated IKKα at serine 176 and tyrosine 23 (markers of IKKα activation), aSMA, pCK, CD3, CD68 and CD66b. Data will be analysed digitally using Visiopharm®. Outputs will be assessed for association with clinical characteristics and outcomes to identify which markers (eg total IKKα or phosphorylated IKKα at serine 176 and tyrosine) and in which cells (specific immune cells e.g. CD3 positive or CD68 positive) have the highest prognostic power. Nanostring GeoMx spatial transcriptomics will be performed on the immune, fibroblast and tumour cell populations that express total or phosphorylated IKKα to identify up or down stream pathways of interest. Pathways of interest will be explored/validated in both cohorts from mIF experiments to assess expression in patient tissue. Cell pellets and patient tissue will be stained via chromogenic IHC to validate these data at the protein level. Drug screening with first-in-class IKKα inhibitors with fluorouracil (5FU) or 0-6Gys radiation will be performed on 2D monocultures of colon (HT29, HCT116) and rectal cells (SW837, CaCo2) respectively. Responses will be measured using WST-1 cell viability assays, clonogenics and functional assays identified as important through RNASeq experiments. Drug screening will also be performed with cell lines grown as cocultures with fibroblasts, macrophages or lymphocytes depending on mIF results, and in a small cohort (n=10) of patient-derived organoids.

This project will further our understanding of the role of IKKα in the TME and which subpopulation of CRC patients may benefit from therapeutic inhibition of IKKα in CRC. Finally, the use of IKKα inhibitors in combination with standard of care chemo/chemoradiotherapy will be investigated in recapitulative preclinical models as a step towards moving this novel targeted therapy towards clinical trials for patients with CRC.

Lab websites

Edwards websites:

https://www.gla.ac.uk/research/az/translationalcancerpathology/

https://www.gla.ac.uk/researchinstitutes/cancersciences/staff/joanneedwards/

Stark website: https://www.ed.ac.uk/cancer-centre/research/stark-group

Jamieson website: https://www.gla.ac.uk/researchinstitutes/cancersciences/staff/nigeljamieson/