Figure 2. JAK2 L884P confers resistance to type II JAK2 inhibitors.

(A) Schematic of mutagenesis screen to identify mutations in JAK2 R683G that confer resistance to type II JAK2 inhibitors. Green cells are unable to proliferate in the absence of IL-3. Yellow cells can proliferate in the absence of IL-3 but not in the presence of JAK2 inhibitor. Red cells can proliferate both in the absence of IL-3 and in the presence of inhibitor. Cells are divided into multiple aliquots after transduction, allowing for the parallel selection of multiple clones that have independently acquired resistance. (B) Ba/F3-CRLF2 cells expressing JAK2 R683G or JAK2 R683G/L884P were treated with JAK2 inhibitor and cell proliferation was assessed after 48 hr. The same assay was performed on Ba/F3-EpoR cells expressing JAK2 V617F or JAK2 V617F/L884P. Data is presented as IC₅₀ fold-shift for cells expressing L884P compared to cells without L884P. Results are from 8 replicates across one to two independent experiments. Error bars represent SEM. (C) Immunoblotting against the indicated targets using lysates from Ba/F3-CRLF2 cells expressing JAK2 R683G or R683G/L884P. Cells were exposed to varying concentrations of JAK2 inhibitor for 2 hr. (D) X-ray structure of JAK2 JH1 domain in complex with BBT594 (Andraos et al., 2012). Ribbon representation of the JAK2 kinase domain with BBT594 illustrated as a stick model. Amino acid side chains thought to be important for stabilizing the C-helix and the P-loop are shown in green. (E) Ba/F3-CRLF2/IL7Rα cells were transduced with either wild-type JAK2 or JAK2 L884P and then cultured in media with or without TSLP. Results are from 3 replicates from a representative experiment. Error bars represent SEM. (F) Homology of JAK2 L884P to EGFR L747P insertional mutations described in non-small cell lung cancer (He et al., 2012).