Effects of mutations in lymphoid malignancy and immunodeficiency disease

Sammanfattning: Mutations are responsible for causing various human diseases, including several types of cancer and immunodeficiency syndromes. They can either be involved directly in the pathogenesis of the disease or by influencing the treatment efficacy and cause failure of the patient’s response to a specific therapy by adapting the targeted cell to resist the treatment. In paper I, we generated amino acid substitution variations of BTK at ibrutinib binding site C481, and performed functional analysis for the corresponding proteins. We have also studied various phosphorylations, which are affected by ibrutinib treatment and put our results in a structure-function context. Herein, we demonstrated that substitution of cysteine 481 by threonine (C481T) retained the kinase activity and caused ibrutinib resistance. So we identified a new escape mutant for irreversible BTK inhibitors, which we predict to be found in patients. BTK kinase is completely inactivated by amino acid (codon) replacement of C481 with arginine, phenylalanine, tryptophan or tyrosine, while the activity is severely impaired when C481 replaced by glycine. In paper II, we have compared the role of N-terminal region domains in the regulation of SYK fusion kinases in terms of phosphorylation, activation, stability and localization. Upon translocation, SYK contributes its kinase domain into two known fusion-proteins, ITK-SYK and TEL-SYK. We have also generated analogous B-cell fusion kinase, BTK-SYK for comparison. The fusion kinases showed differential activation, localization and sensitivity to various inhibitors. Here, we report the activation-mediated nuclear translocation of ITK-SYK, which is rarely seen among kinases. This unique feature of ITK-SYK is therefore of general interest, as its potential relation to ITK-SYK’s transforming capability. In paper III, we have identified a hypomorphic mutation in PGM3 gene replacing isoleucine 322 with threonine in a family with immunodeficient children, described previously. The mutation is severely destabilized and impaired the enzymatic activity of the protein, causing the described phenotype.