Prospective Identification of Resistance Mutants to the JAK2 Inhibitor TG101348.

Introduction: Clinical resistance to kinase inhibitors is an emerging theme in targeted, oncology therapies. For example, Gleevec resistance results due to a host of mutations in bcr-abl, the kinase associated with chronic myelogenous leukemia (CML), and have necessitated the development of 2nd generation inhibitors dasatinib and notilinib. More recently, mutations in JAK2 kinase have recently been identified as the key molecular drivers for the majority of patients with the chronic MPDs polycythemia vera, essential thrombocythemia and myelofibrosis with myeloid metaplasia. TG101348 is a selective inhibitor of JAK2 kinase and represents a novel therapy for these MPDs.

Methods: In order to better understand what mutations may occur in clinic as a result of TG101348 treatment, we used N-ethyl-N-nitrosourea (ENU)-exposed human erythroleukemia (HEL) cells to evaluate resistance mutations to TG101348. In brief, ENU pretreated HEL cells were exposed to 3 uM TG101348, a dose that is 10X the cellular IC50, for 28 days followed by genotyping to determine the incidence and location of kinase domain mutants that arose in resistant cells.

Results: Eleven different mutations were identified, with the most prevalent mutations in associated with the ATP binding pocket. In cells bearing these mutations TG101348 did not inhibit STAT5 phosphorylation, cellular proliferation or induced apoptosis at concentrations up to 3 uM, whereas in wildtype HEL cells it did. Consistent with the mutations arising specific for TG101348, JAK2 inhibitors which are derived from chemically distinct series were equally potent on wildtype or TG101348 resistant HEL cells.

Conclusions: A series of JAK2 point mutations were identified which generate resistance to the JAK2 inhibitor TG101348 in JAK2V617F-bearing cells. None of these mutations corresponded to any mutations that have been previously reported for MPD patients and therefore the clinical relevance of these mutations is not yet known. While these ENU induced mutations do not yet have clinical correlates, monitoring for their presence in any patients who do not respond to TG101348 in clinical trials may shed light on mechanisms of resistance as they occur.