JAK2R683G is an activating mutation in DS-ALL. (A) Sequence analysis shows the JAK2R683G mutation in a DS-ALL patient. (Top) Sequence obtained from bone marrow DNA at presentation and (bottom) complete remission. The A to G substitution is shown by an arrow. During remission the sequence was identical to wild-type, showing that the mutation is acquired. (B) Western blotting analysis of JAK2 and STAT5 protein in IL-3–dependent Ba/F3 cells. Extraction and Western blotting analyses were performed with the use of standard protocols. Antibodies against JAK2 and phospho-JAK2 (p-JAK2), STAT5, and phospho-STAT5 (p-STAT5) were from Cell Signaling Technology (New England Biolabs, Ipswich, MA). Lane 1 indicates untransformed Ba/F3 cells; lane 2, empty vector control (v) (with neo and puro); lane 3, empty vector + TpoR; lane 4, wild-type (wt) JAK2-transformed Ba/F3 cells; lane 5, JAK2R683G mutant–transformed Ba/F3 cells. (C) Ba/F3 proliferation after IL-3 withdrawal. The JAK2R683G mutation was generated in the IMAGE clone 6838318 containing murine JAK2 cDNA using site-directed mutagenesis (Quikchange-Xl; Stratagene; Cambridge, United Kingdom) and confirmed by full-length DNA sequencing. Ba/F3 cells were cotransduced with thrombopoietin receptor (TpoR) and wild-type or mutant (R683G) JAK2. Cells were washed 3 times in PBS and cultured at 105/mL in the absence of IL-3 for 6 days with or without the JAK2 inhibitor AG490. Cell numbers and viability were assessed in duplicate after Trypan Blue exclusion staining.
