Molecular and Structural Basis of Pan-Resistance to BTK Degraders and Inhibitors

For this project we used CRISPR HDR to knock in the BTK A428D mutation into the endogenous locus in the TMD8 cell line. The mutation was present at a frequency of ~3%, therefore to increase its frequency prior to single cell cloning we selected the pool of cells with the BTK degrader NX-5948 0.1uM for 10 days. Following selection the frequency of the A428D allele increased from 3% to 94%. From this population we then isolated a single cell clone with BTK A428D in >98% of reads. To rule out the possibility that exposure to NX-5948 had a selected for a second BTK degrader resistance allele (i.e. cereblon mutations) we performed whole exome sequencing of the parental TMD8 cell line as well as the TMD8 BTK A428D single cell clone. The results did not reveal any known or obvious secondary resistance mutations in the single cell clone, suggesting that the BTK A428D was indeed the mutation responsible for conferring resistance to the BTK degrader.