Kinase Domain Mutations in JAK2V617F Confer Resistance to the Novel JAK2 Inhibitor Ruxolitinib

Abstract 125

The JAK2V617F kinase mutation is frequently associated with myeloproliferative neoplasms (MPNs) and thought to be instrumental for the overproduction of myeloid lineage cells. Ruxolitinib (INCB018424) and several additional 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 cause resistance to ruxolitinib. Seven libraries of mutagenized JAK2V617F cDNA were screened to specifically identify mutations in the predicted drug-binding region that would lower sensitivity towards ruxolitinib, using a cell-based assay. We found 5 different non-synonymous point mutations that conferred drug resistance, including Y931C, G935R, R938L, I960V and E985K. Cells containing mutations had a 9 to 33-fold higher EC₅₀ for ruxolitinib. Results were analyzed by evaluating the three-dimensional structure of ruxolitinib bound to the ATP binding pocket of the monomer JAK2 kinase domain, using a docking algorithm. The inhibitor is held in the pocket by polar contacts and extensive hydrophobic interactions with several residues that line the binding pocket. A880, L855, V863 and M929 interact with the inhibitor on one side, and V911 and L983 provide hydrophobic interactions on the other side. The pyrazol ring of ruxolitinib appears to have a π-π interaction with the Y931 ring. Most mutations that were identified in our screen are either residues that interact with ruxolitinib or are in proximity to the binding pocket and hence are likely to indirectly alter inhibitor binding. The structural analysis indicates that there are additional residues in the kinase domain that may be critical for optimal binding of ruxolitinib and point mutations at these sites may also alter the affinity of the drug. Our analysis further suggests that ruxolitinib and other JAK2 inhibitors may bind in a similar way to the kinase domain. Consequently, these identified mutations conferred cross-resistance to all JAK2 kinase inhibitors tested in cell growth assays, including AZD1480 (5.6 to >7-fold higher EC₅₀), TG101348 (2.2 to 2.8-fold increase in EC₅₀), lestaurtinib (CEP-701) (2.6 to 3.3-fold increase in EC₅₀) and CYT-387 (5.1 to 7.4-fold increase in EC₅₀). Surprisingly, introduction of the ‘gatekeeper' mutation (M929I) in JAK2V617F affected only ruxolitinib sensitivity (4-fold increase in EC₅₀). In addition, the E985K and Y931C mutations reduced the sensitivity to ruxolitinib and offered a growth advantage to cells during treatment, compared to native JAK2V617F expressing cells. These results suggest that the development of point mutations in JAK2 could result in clinical resistance to a wide variety of JAK2 inhibitors currently in clinical trials in patients with myeloproliferative neoplasms.