title: Kinase domain mutations confer resistance to novel inhibitors targeting JAK2V617F in myeloproliferative neoplasms.
abstract: The transforming JAK2V617F kinase is frequently associated with myeloproliferative neoplasms and thought to be instrumental for the overproduction of myeloid lineage cells. Several small molecule drugs targeting JAK2 are currently in clinical development for treatment in these diseases. We performed a high-throughput in vitro screen to identify point mutations in JAK2V617F that would be predicted to have potential clinical relevance and associated with drug resistance to the JAK2 inhibitor ruxolitinib (INCB018424). Seven libraries of mutagenized JAK2V617F cDNA were screened to specifically identify mutations in the predicted drug-binding region that would confer resistance to ruxolitinib, using a BaF3 cell-based assay. We identified five different non-synonymous point mutations that conferred drug resistance. Cells containing mutations had a 9- to 33-fold higher EC(50) for ruxolitinib compared with native JAK2V617F. Our results further indicated that these mutations also conferred cross-resistance to all JAK2 kinase inhibitors tested, including AZD1480, TG101348, lestaurtinib (CEP-701) and CYT-387. Surprisingly, introduction of the 'gatekeeper' mutation (M929I) in JAK2V617F affected only ruxolitinib sensitivity (fourfold increase in EC(50)). These results suggest that JAK2 inhibitors currently in clinical trials may be prone to resistance as a result of point mutations and caution should be exercised when administering these drugs.
authors.affiliation: Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA.
authors.forename: A
authors.initials: A
authors.lastname: Deshpande
authors.forename: M M
authors.initials: MM
authors.lastname: Reddy
authors.forename: G O M
authors.initials: GO
authors.lastname: Schade
authors.forename: A
authors.initials: A
authors.lastname: Ray
authors.forename: T K
authors.initials: TK
authors.lastname: Chowdary
authors.forename: J D
authors.initials: JD
authors.lastname: Griffin
authors.forename: M
authors.initials: M
authors.lastname: Sattler
mesh_terms: D000818:Animals; D019008:Drug Resistance, Neoplasm; D057166:High-Throughput Screening Assays; D006801:Humans; D053614:Janus Kinase 2 / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics*; D051379:Mice; D009196:Myeloproliferative Disorders / Q000188:drug therapy* / Q000235:genetics*; D009570:Nitriles; D017354:Point Mutation; D047428:Protein Kinase Inhibitors / Q000627:therapeutic use*; D017434:Protein Structure, Tertiary; D011720:Pyrazoles / Q000627:therapeutic use*; D011743:Pyrimidines
chemical_list: D009570:Nitriles; D047428:Protein Kinase Inhibitors; D011720:Pyrazoles; D011743:Pyrimidines; C540383:ruxolitinib; C507924:JAK2 protein, human; D053614:Janus Kinase 2