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. 2009 Jun 9;106(23):9414-8.
doi: 10.1073/pnas.0811761106. Epub 2009 May 22.

JAK mutations in high-risk childhood acute lymphoblastic leukemia

Charles G Mullighan  1 , Jinghui Zhang, Richard C Harvey, J Racquel Collins-Underwood, Brenda A Schulman, Letha A Phillips, Sarah K Tasian, Mignon L Loh, Xiaoping Su, Wei Liu, Meenakshi Devidas, Susan R Atlas, I-Ming Chen, Robert J Clifford, Daniela S Gerhard, William L Carroll, Gregory H Reaman, Malcolm Smith, James R Downing, Stephen P Hunger, Cheryl L Willman
Affiliations

JAK mutations in high-risk childhood acute lymphoblastic leukemia

Charles G Mullighan et al. Proc Natl Acad Sci U S A. .

Abstract

Pediatric acute lymphoblastic leukemia (ALL) is a heterogeneous disease consisting of distinct clinical and biological subtypes that are characterized by specific chromosomal abnormalities or gene mutations. Mutation of genes encoding tyrosine kinases is uncommon in ALL, with the exception of Philadelphia chromosome-positive ALL, where the t(9,22)(q34;q11) translocation encodes the constitutively active BCR-ABL1 tyrosine kinase. We recently identified a poor prognostic subgroup of pediatric BCR-ABL1-negative ALL patients characterized by deletion of IKZF1 (encoding the lymphoid transcription factor IKAROS) and a gene expression signature similar to BCR-ABL1-positive ALL, raising the possibility of activated tyrosine kinase signaling within this leukemia subtype. Here, we report activating mutations in the Janus kinases JAK1 (n = 3), JAK2 (n = 16), and JAK3 (n = 1) in 20 (10.7%) of 187 BCR-ABL1-negative, high-risk pediatric ALL cases. The JAK1 and JAK2 mutations involved highly conserved residues in the kinase and pseudokinase domains and resulted in constitutive JAK-STAT activation and growth factor independence of Ba/F3-EpoR cells. The presence of JAK mutations was significantly associated with alteration of IKZF1 (70% of all JAK-mutated cases and 87.5% of cases with JAK2 mutations; P = 0.001) and deletion of CDKN2A/B (70% of all JAK-mutated cases and 68.9% of JAK2-mutated cases). The JAK-mutated cases had a gene expression signature similar to BCR-ABL1 pediatric ALL, and they had a poor outcome. These results suggest that inhibition of JAK signaling is a logical target for therapeutic intervention in JAK mutated ALL.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Primary structure of JAK1, JAK2, and JAK3 showing the location of missense (▼) and insertion/deletion (▲) mutations. FERM, band 4.1 ezrin, radixin, and moesin domain; SH2, src-homology domain; JH2, pseudokinase domain; and JH1, kinase domain.
Fig. 2.
Fig. 2.
Functional effects of JAK mutations. (A) Ba/F3-EpoR cells were transduced with retroviruses expressing wild-type or mutant Jak2 alleles and cultured in the absence of cytokine. Each Jak2 mutant examined resulted in cytokine-independent growth. Untransduced cells and cells transduced with wild-type mJak2 remained cytokine-dependent. Mean ± SDs of triplicates are shown. (B) Transduction of Ba/F3-EpoR cells with retrovirus expressing mJak1 S646F resulted in factor independence. (C) Ba/F3-EpoR cells transduced with wild-type or mutant Jak2 were cultured without cytokine in the presence of increasing concentrations of Jak inhibitor I. Each mutation was sensitive to Jak inhibition. The BCR-ABL1-positive cell line K562 is shown as a control. (D) Growth of Ba/F3 cells transduced with S646F was inhibited by Jak inhibitor I. (E) Western blots showing activation of JAK-STAT signaling in Ba/F3-EpoR cells transduced with each mutant Jak allele. Cells were cultured without erythropoietin for 15 h and then harvested for blotting before and after 15 min of erythropoietin at 5 units/mL. Each mutation resulted in constitutive Jak2 and Stat5 phosphorylation that was augmented by pulsed erythropoietin (shown for Jak2 617F and 683G). The Jak2 kinase domain mutations showed less constitutive Jak-Stat activation than the pseudokinase domain mutations. Epo, erythropoietin; WT, wild type. (F) Western blots demonstrating abrogation of Jak-Stat activation by Jak inhibitor I. Ba/F3-EpoR cells transduced with each Jak allele were grown in the absence of cytokine, then harvested after 5 h of exposure to 5 mM Jak inhibitor I or vehicle (DMSO).
Fig. 3.
Fig. 3.
Phosphoflow cytometry analysis of Jak-Stat activation in Ba/F3-EpoR cells transduced with Jak2 retroviral constructs. Transduced cells were serum-starved and cytokine-starved and then stimulated either with erythropoietin (Epo; A and B) or pervanadate (PV; C and D), either without pharmacologic Jak inhibition (A and C) or after administration of the Jak2 inhibitor XL019 (B and D). (A) Activation of Jak-Stat phosphorylation with erythropoietin stimulation. Notably, Jak2 phosphorylation was evident for Jak2 pseudokinase mutant alleles but not the kinase domain mutants. (B) Signaling was abrogated in control and mutants treated with XL019 with subsequent erythropoietin stimulation. (C) Marked Jak2 and Stat5 phosphorylation was observed for each mutant after pervanadate stimulation. (D) Jak2 and Stat5 signaling was preferentially abrogated in mutants treated with XL019 with subsequent pervanadate stimulation.
Fig. 4.
Fig. 4.
Gene expression profile and outcome of JAK-mutated B-progenitor ALL. (A) Gene set enrichment analysis demonstrates significant enrichment of the BCR-ABL1 gene expression signature in JAK-mutated ALL. (B) Heatmap of the enriched BCR-ABL1 up-regulated gene set in the P9906 cohort, showing overexpression of BCR-ABL1 up-regulated genes in JAK-mutated ALL. Notably, several cases lacking JAK mutations also have a BCR-ABL1 signature, suggesting the presence of additional kinase mutations in these cases. (C and D) JAK mutation and IKZF1 alteration are associated with a high incidence of events (C) and relapse (D).
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