BTK mutations and drug resistance structure-activity relationships

Bruton's tyrosine kinase (BTK) inhibitors are useful in a variety of human diseases from blood cancers to chronic inflammatory disorders. First generation BTK inhibitors (e.g. ibrutinib) require covalent modification of BTK to have activity. These inhibitors have become extremely useful with relatively less drug toxicity than other related agents. However, the BTK mutation C481S can evolve in the clinic, leading to BTK inhibitors being less effective. This project seeks to understand the molecular mechanisms behind this resistance, reveal the usefulness of next generation inhibitors and the process of kinome adaptive reprogramming in malignant leukaemia cells possessing the mutated BTK variant. This will be done firstly through studying intrinsic constitutive signalling within these cells, then by studying how signalling responses change to extrinsic stimuli.
The BTK mutation to be studied will be introduced into cell lines using lenti-CRISPR-Cas9 technology. The profile of active kinases in control and mutated leukaemia cells will be studied using kinomic analyses methods including mass spectrometry (MS) and nano-string arrays. Bioinformatic methods will be used to assemble signalling pathway understanding, which will be further verified through monitoring phosphosite activation as measured by appropriate Western blotting. Changes in signalling in malignant cells within patient samples that have acquired the BTK mutation associated with their therapy resistance will also be studied. This will determine the differential role of BTK inhibitors on a range of cellular effects in addition to informing the basis of the reduced drug toxicity seen in the clinic, as well as help in the design of better generations of inhibitors which will treat diseases more effectively and provide less adverse side-effects from those drugs.