Random Mutagenesis Reveals Residues of JAK2 Critical in Evading Inhibition by Tyrosine Kinase Inhibitors

Mutation and activation of JAK2 is a common event in Myeloproliferative Disease as JAK2 V617F and related deletion mutations are observed in Polycythemia Vera, Essential Thrombocythemia and Primary Myelofibrosis. In addition, the TEL-JAK2 chromosomal translocation is a rare event in Acute Myeloid Leukemia and Chronic Myelomonocytic Leukemia. We reasoned that activated alleles of JAK2 would develop resistance to JAK inhibitors in a clinical setting, similar to the development of Imatinib resistance in BCR-ABL-mediated Chronic Myeloid Leukemia. The objective of this study was to develop a random mutagenesis screen to isolate mutations of JAK2 resistant to tyrosine kinase inhibitors. We selected JAK Inhibitor-1 for this study, since the crystal structure of this inhibitor bound to the JAK2 JH1 kinase domain has been reported and would allow for mapping of confirmed mutations. TEL-JAK2(5–12) and JAK2 V617F were subcloned into retroviral expression vectors and random libraries of mutations were generated by transformation into XL-1 Red strain of E. coli, a strain defective in pathways of DNA repair. High titer retroviral supernatants were generated and used to transduce Ba/F3 (for TEL-JAK2) or Ba/F3-EPO-R (for JAK2 V617F). Control experiments were performed with “wild type” versions of each JAK2 allele. Inhibitor-resistant clones were identified and DNA sequencing was performed to identify JAK2 JH1 kinase domain mutations that confer resistance to inhibitor. We have restricted the analysis of JAK inhibitor-resistant mutants to those that map within the kinase domain of JAK2 for purposes of this study. We have confirmed that E864K, V881A, N909K, G935R, R975G confer resistance to JAK inhibitor-1 in growth assays. In addition, M929I (analogous to BCR-ABL T315I) mediates resistance to JAK inhibitor-1, relative to wild-type activated JAK2 alleles. All mutations result in increased phosphorylation of STAT5, Akt and Erk in the presence of inhibitor. We are currently testing the catalytic activity of each mutant to determine whether JAK2 kinase domain mutations have similar enzymatic activity or whether mutation also affects catalysis. We have mapped each mutation within the JAK2 JH1 crystal structure and models for how each mutant affects inhibitor binding will be presented. Importantly, many of these residues are highly conserved in other JAK tyrosine kinases and within BCR-ABL. We are extending these observations by testing clinically relevant inhibitors in our screen. At the conclusion of this study we will be able to identify common residues critical for resistance by JAK2 inhibitors and unique residues that are inhibitor-specific. Random mutagenesis screening offers an excellent strategy to identify JAK2 residues that may be relevant in the clinic and also serve in enhancing our knowledge regarding JAK kinase activation and regulation.