Emergence of genetic resistance against kinase inhibitors poses a great challenge for durable therapeutic response. preventing emergence of resistant variants. Most importantly, our data suggest that in order to develop resistance-free kinase inhibitors, the next-generation drug design should target the substrate-binding site. Myeloproliferative neoplasms (MPNs) are a group of hematologic malignancies that include Ph+ chronic myeloid leukemia (CML) and Ph? diseases that includes primary myelofibrosis (MF), polycythemia vera (PV), and essential thrombocythemia (ET). The discovery that constitutive ABL kinase activity is sufficient and necessary to cause CML laid KRN 633 the foundation for development of imatinib as a target-directed therapy1,2. The clinical success of BCR-ABL inhibitors for the treatment of CML KRN 633 not only revolutionized the anti-kinase therapy but also enforced the idea to identify the genetic lesions in other neoplastic diseases for therapeutic targeting2,3,4. In 2005, four groups reported kinase-activating mutations in JAK2 (JAK2-V617F) from BCR-ABL-negative MPN patients5,6,7,8. This discovery generated great interest in treating MPNs by targeting JAK2 with small-molecule kinase inhibitors. JAK2 is a cytosolic tyrosine kinase activated KRN 633 by cytokine-mediated receptor dimerization, resulting in phosphorylation of STATs required for cell proliferation, survival and myeloid development, as well as for the initial stages of the immune response9. Constitutive JAK2 signaling has been implicated in many other cancerssuch as myeloid malignancies, breast cancers and B-cell leukemias10 and lymphomas11. This provides a strong rationale for JAK2 targeting, and suggests that the resultant therapies would have broad therapeutic potential. As proof of concept, JAK2-V617F was expressed in mouse hematopoietic cells, generating a tractable mouse model of PV and MF12,13,14. In each of these disease models, treatment with small-molecule JAK2-kinase inhibitors induced apoptotic cell death and prolonged the survival of mice13,15,16,17. Collectively, these observations paved the way for clinical development of JAK2-targeted therapeutics. The JAK2 inhibitor ruxolitinib was recently approved for the treatment of MF and PV, and numerous other inhibitors are in phase-II/-III clinical trials18. Ruxolitinib and other JAK2 inhibitors have shown significant improvement in quality of life. However, KRN 633 unlike other tyrosine kinase inhibitor (TKI) therapy, they do not have clonal selectivity3,19,20,21. Given that the therapeutic response to TKI therapy is mediated by oncogene addiction, clinical and mouse studies suggest that MPNs induced by JAK2-V617F are not addicted to the driver oncogene. Three principal mechanisms i.e. genetic streamlining, oncogenic shock and synthetic lethality govern addiction to the driver oncogene22,23. There are intensive efforts to develop combination therapies KRN 633 to achieve clonal selectivity Rabbit Polyclonal to HCRTR1 for JAK2 inhibitors, perhaps by inducing synthetic lethality. In preclinical mouse models, combinations of ruxolitinib with inhibitors of PI3K, Hedgehog, HDAC, BCL2 and interferonCalpha have shown clonal selectivity for JAK2-V61724. Clinical trials are undergoing for these combinatorial treatments24. Given the prevalence of genetic resistance in response to anti-kinase therapy under selective pressure, we reasoned that genetic resistance to JAK2 inhibitors would emerge once treatment specific to the JAK2 mutant cells is established. Therefore, using JAK2-V617F-addicted cells we sought to understand patterns of resistance to JAK2 inhibitors, and to glean functional insights for further drug refinement. We performed an unbiased chemical-genetic screen using two different JAK2 inhibitors, ruxolitinib and fedratinib, to identify a comprehensive set of drug-resistant variants, in order to glean regulatory mechanisms of resistance. Our screen identified 211 resistance mutations against ruxolitinib, but a complete lack of resistance against fedratinib. The resistance mutations conferred cross-resistance to other JAK2 inhibitorsAZD1480, CYT-387 and lestaurtinib, but failed to confer resistance against fedratinib. Biochemical characterization and structural modeling revealed that fedratinib simultaneously binds to both ATP-binding and peptide-substrate-binding sites, thereby preventing emergence of resistant clones. Results Lack of genetic resistance against fedratinib We performed a ruxolitinib resistant screen using BaF3-MPL cells that showed emergence of resistant clones (data not shown). Although these clones conferred robust resistance to ruxolitinib, sequencing did not reveal mutations. characterization of these clones showed both higher IC50 and increased resistance to ruxolitinib, thus suggesting that the BaF3-MPL cells expressing JAK2-V617F are not addicted to JAK2 because MPL overexpression seemingly bypasses the JAK2 dependent survival. Therefore, we performed screening using parental BaF3 cells transduced with randomly mutagenized JAK2-V617F and two clinically relevant JAK2 inhibitors: ruxolitinib and fedratinib. Ruxolitinib-resistant clones emerged at 1, 2 and 5?M inhibitorrepresenting 10?, 25? and 50-fold increases in IC50 values for JAK2-V617F (~100?nM), respectively (Fig. 1a). In contrast, selection against fedratinib at concentrations 2-fold above IC50 (~0.9?M) did not result in any resistant clones (Fig. 1a, lower panel). From the 190 ruxolitinib-resistant.