Comparison of the enzymatic and cellular profiles of clinical JAK inhibitors for the treatment of myelofibrosis

• Ruxolitinib is the most selective and potent of the JAKinibs indicated for treating myelofibrosis. • Other JAKinibs show broader kinase inhibition, potentially contributing to off-target toxicities. Four Janus kinase inhibitors (JAKinibs) ruxolitinib, fedratinib, pacritinib, and momelotinib are indicated for myelofibrosis. All inhibit JAK2, but effects on additional kinases vary markedly, shaping their specific clinical pharmacology. Published comparative inhibitory profiles and cellular pharmacology data are incomplete and were determined here. Inhibitory potency and relative selectivity of JAKinibs were determined by full kinome profiling. JAKinib effects on signaling and cell growth were measured using JAK2-dependent and JAK2-independent cell lines. Ruxolitinib was the most potent JAK2 inhibitor (IC 50 at physiological (1 mM) ATP, 2.9 nM), followed by fedratinib (17 nM), momelotinib (29 nM), and pacritinib (39 nM), and the most selective. The other JAKinibs inhibited multiple additional kinases beyond the few typically reported for each drug in the literature. Interestingly, pacritinib and momelotinib showed only modest ACVR1 inhibition at clinical doses and physiological ATP concentrations, suggesting the role of ACVR1 inhibition in mediating their anemia-related benefits may be overestimated. The observed inhibitory levels are unlikely to translate to a clinically meaningful reduction in hepcidin levels. In JAK2-dependent cellular assays, ruxolitinib showed most potent inhibition of STAT5 phosphorylation (IC 50, 14 nM), followed by momelotinib (201 nM), pacritinib (421 nM), and fedratinib (669 nM), and was the only JAKinib that had minimal impact on growth in JAK2-independent cell lines. In conclusion, ruxolitinib was the most potent, and selective JAKinib with no relevant effects in JAK2-independent cells. In contrast, fedratinib, pacritinib, and momelotinib inhibited many other kinases and inhibited cell growth by JAK2-unrelated mechanisms at clinically relevant concentrations.