Mechanisms of Drug Resistance Against JAK2 Inhibitors

Abstract 1426

The discovery of activating somatic mutations of JAK2 kinase as a contributing factor in the pathogenesis of myeloproliferative neoplasms (MPNs) —polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (MF)—prompted the development of JAK2-targeted therapies for these diseases^1–3. Therapy with JAK2 inhibitors (INCB018424 and TG101348) induces rapid and marked reductions in spleen size and can lead to remarkable improvements in constitutional symptoms and overall quality of life suggesting that therapeutic targeting of JAK2 holds great promise for the treatment of JAK2 mediated disorders. It is likely that these two clinical agents will soon be approved by the FDA for the treatment of these diseases. Given our experience in development of resistance in tyrosine kinase inhibitor therapy (TKI) especially in the context of BCR/ABL targeting by Gleevec, we anticipate that resistance to JAK2 inhibitors will develop as well. To understand the mechanism of resistance against JAK2 targeted therapies, we performed mutagenesis studies and in vitro screening for resistance as previously reported for selection of resistance mutation in BCR/ABL (Azam et. al. (2003), Cell and Azam et. al. (2006), PNAS.).

We sequenced the resistant clones isolated from INCB018424 resistant screening and identified 125 different amino substitutions spanning across FERM, SH2, pseudokinase and kinase domains. Frequency of resistant mutations demonstrated several major hot-spots clustered in the FERM, pseudokinase and kinase domain. We observed sixty different mutations in the kinase domain that clustered into eight major hot-spots with several minor-spots in regulatory regions including the hinge region, catalytic-loop, activation-loop and in the helical region of c-lobe. Structural modeling of these mutations suggests that the JAK2 kinase, like ABL kinase, is subject to auto-inhibited regulatory mechanisms and selects for drug resistant mutations from allosteric domains (FERM, SH2 and Pseudokinase domain). We anticipate that these results will be valuable in diagnosis of clinical resistance and in the identification and design of next generation small molecule JAK2 inhibitors