A novel activating JAK1 mutation in chronic eosinophilic leukemia

Abstract

Hypereosinophilia (HE) has been defined as persistent eosinophilia >1.5 × 109/L; it is broadly divided into primary HE (clonal or neoplastic; HEN), secondary/reactive HE (HER), or HE of undetermined significance (HEUS) when no cause is identified. The use of myeloid next-generation sequencing (NGS) panels has led to the detection of several mutations in patients previously diagnosed with HEUS, reassigning some patients to the category of HEN, specifically the World Health Organization category of chronic eosinophilic leukemia, not otherwise specified (CEL, NOS). Here, we describe a novel somatic JAK1 pseudokinase domain mutation (R629_S632delinsSA) in a patient with HE that had initially been characterized as a variant of uncertain significance. We performed functional studies that demonstrated that this mutation results in growth factor independence of Ba/F3 cells in vitro and activation of the JAK-STAT pathway. These effects were abrogated by the JAK1/JAK2 inhibitor ruxolitinib. R629_S632delinsSA is the first known somatic mutation in JAK1 linked to a clonal eosinophilic neoplasm, and highlights the importance of the JAK-STAT pathway in eosinophil survival.

Figure 1.

JAK1 pseudokinase mutations demonstrate transforming capability and result in hyperactivation of the JAK/STAT pathway. (A) Growth curves of Ba/F3 cells expressing JAK1 A634D and JAK1 R629_S632delinsSA cultured in the absence of IL-3 (graph is representative of 3 independent experiments). (B) Immunoblot analysis of Ba/F3 parental cells, Ba/F3 cells expressing JAK1 wild-type, JAK1 A634D, and JAK1 R629_S632delinsSA cultured in the presence of IL-3 and IL-3–independent Ba/F3 cells expressing JAK1 A634D, and JAK1 R629_S632delinsSA. (C) Immunoblot analysis of HEK293 cells expressing JAK1 wild type, JAK1 A634D, and JAK1 R629_S632delinsSA. p-, phosphorylated; WT, wild-type.

Figure 2.

JAK1 pseudokinase mutations demonstrate sensitivity to JAK inhibitors. (A) Representative graphs of the dose-response curves (72-hour sensitivity) for different JAK inhibitors on Ba/F3 cells expressing BCR-ABL, JAK1 A634D, and JAK1 R629_S632delinsSA (upper panels). Graphs showing mean IC₅₀ (lower left panel) and area under the dose-response curve (AUC; lower right panel) for different JAK inhibitors on Ba/F3 cells expressing BCR-ABL, JAK1 A634D, and JAK1 R629_S632delinsSA (3 independent experiments). (B) Immunoblot analysis of IL-3–independent Ba/F3 cells expressing BCR-ABL, JAK1 A634D, and JAK1 R629_S632delinsSA treated with vehicle or 50 nM or 100 nM of ruxolitinib for 4 hours (n = 2 replicates). (C) Sensitivity of peripheral blood and bone marrow specimens from the patient with JAK1 R629_S632delinsSA to JAK inhibitors, represented by IC₅₀, in comparison with control samples obtained from healthy donors (HD) and specimens from a larger cohort of patients with MPNs) (the horizontal lines denote median IC₅₀ of HD and MPN specimens). ****P < .0001, ***P < .001, 1-way analysis of variance. p-, phosphorylated.