JAK1 mutation analysis in ALL
To explore possible contributions of somatic JAK1
gene mutations in ALL, genomic DNA samples from BM aspirates of adult subjects with B cell precursor ALL (B-ALL; n
= 88) and T cell ALL (T-ALL; n
= 38) obtained at diagnosis and before therapy were screened for mutations in the entire JAK1
coding region using denaturing HPLC (DHPLC). Direct sequencing of variant elution profiles allowed the identification of 37 intronic and exonic changes, including 9 nonsynonymous variants observed in 14 individuals ( and Table S1, available at http://www.jem.org/cgi/content/full/jem.20072182/DC1
). Among the missense defects, genotyping of genomic DNAs from BM obtained during remission demonstrated the somatic origin of the 1535C>T (Ser512Leu), 1901C>A (Ala634Asp), and 2171G>A (Arg724His) changes in the leukemic clones ( and ). To verify that the nonsynonymous substitutions identified in patients for whom nonleukemic DNA was not available were not gene variants occurring in the population, 335 population-matching control individuals were analyzed, and none harbored the 611A>T (Lys204Met), 2635C>A (Arg879Ser), 2635C>T (Arg879Cys), and 2636G>A (Arg879His) changes or other defects altering those codons. Although the T-ALL–restricted occurrence of three distinct substitutions affecting Arg879
(3/38 versus 0/335; Fisher's exact probability < 0.001) further supported the relevance of the substitution of this residue, we could not exclude that the 611A>T change might represent a private neutral variant. The two remaining missense changes, 184A>G (Ile62Val) and 1078C>T (Arg360Trp), were deemed nonpathogenic variants, as they were observed in nonleukemic cells of affected patients or in unaffected control subjects.
List of nonsynonymous JAK1 changes identified in subjects with ALL
Figure 1. Somatic JAK1 mutations in ALL. (A) Representative electropherograms showing the occurrence of somatically acquired JAK1 mutations in subjects with T-ALL. In all cases, mutations were observed at diagnosis (top), but were undetectable during remission (more ...)
All mutations occurred as heterozygous changes and affected conserved residues within the FERM, SH2, pseudokinase, and kinase domains (). In two cases, DHPLC profiles and electropherograms indicated that the mutant allele might be present in only a fraction of leukemic cells, suggesting that these lesions did not represent early events during leukemogenesis but were acquired during disease progression (). Remarkably, mutations were relatively common among individuals with T-ALL (18.4% of cases, 95% CI = 7.7–34.3%), whereas they occurred in only three patients with B-ALL (3.4% of cases, 95% CI = 0.7–9.6%; ). Such a difference in mutation prevalence between cohorts was statistically significant (Fisher's exact probability = 0.003). DHPLC screening performed on affected exons by using pooled DNAs excluded loss of the normal allele and a homozygous condition for a gene variant caused by mitotic recombination in all cases.
To investigate the prevalence of JAK1 mutations among pediatric ALL cases, genomic DNA from BM obtained at diagnosis was scanned for mutations in affected exons. No lesion was observed within the B-ALL cohort (n = 85), whereas a nonsynonymous 1957C>T transition (Leu653Phe) was identified in 1 of 49 subjects with T-ALL (2.0% of cases, 95% CI = 0.05–10.9%). This mutation was not observed in the BM obtained from the patient at the time of remission, indicating that it was somatically acquired in the leukemic cells.
Overall, these results indicated that JAK1 gene mutations occur in ALL and are more frequently observed among adult individuals with involvement of the T cell lineage.
Functional consequences of somatic JAK1 mutations
To examine the effects of the identified mutations on protein function, WT JAK1 or a mutant form (A634D, R724H, and R879C) was expressed transiently in JAK1-defective human fibrosarcoma U4C cells, and endogenous STAT1 phosphorylation was compared basally and after stimulation with IFN-γ (). Consistent with previous studies (10
), untransfected cells lacking functional JAK1 did not exhibit STAT1 phosphorylation in response to IFN-γ. All JAK1 mutants promoted an enhanced response to the ligand compared with WT JAK1. Of note, basal STAT1 phosphorylation was observed in cells expressing the A634D mutant, suggesting ligand-independent up-regulation of the kinase. Consistent with this, expression of the A634D mutant resulted in an essentially constitutive STAT1 transcriptional activation, whereas a statistically significant increase in STAT1 activity was observed in U4C cells expressing both the R724H and R879C mutants, basally and after stimulation ().
Figure 2. Functional effects of leukemia-associated JAK1 mutations. (A) STAT1 phosphorylation assays. Basal and IFN-γ–stimulated endogenous STAT1 phosphorylation in JAK1-defective U4C cells transiently transfected with WT JAK1 or selected mutants. (more ...)
To further assess the ability of mutations to up-regulate signal flow, we transduced Ba/F3 cells with WT Jak1 or each of the selected mutants to evaluate whether their expression induced autonomous growth of cytokine-dependent cells. GFP-expressing Ba/F3 cells were selected by flow cytometry, cultured in 5 or 0.5% WEHI-3B cell conditional medium (CM) as a source of IL-3, as well as in absence of the cytokine, and counted to assess proliferation (). Three independent experiments indicated that expression of the A634D and, with less efficiency, R724H Jak1 mutants conferred IL-3–independent growth to cells, whereas cells expressing WT Jak1 or the R879C Jak1 mutant retained dependence on the cytokine for survival. Of note, cells expressing each of the three mutants exhibited enhanced growth in response to IL-3. Consistent with these findings, Ba/F3 cells expressing the A634D Jak1 mutant exhibited enhanced Stat5, Akt, and extracellular signal-regulated kinase (ERK) phosphorylation basally and after stimulation, whereas a higher phosphorylation level of these signal transducers in cells expressing the R724H Jak1 protein was observed in cultures maintained in the presence of IL-3 (). Notably, we did not observe Jak1 phosphorylation in parental and transduced Ba/F3 cells basally and cultured in 2% WEHI-3B cell CM; however, phosphorylation was appreciable in A634D and R724H Jak1-transduced cells after selection (7-d culture in absence of IL-3; unpublished data).
IL-9 protects the T cell lymphoma BW5147 cell line against dexamethasone-induced apoptosis (12
), an effect that is dependent on Jak1-mediated Stat3 and Stat5 activation (13
). To demonstrate the activating role of the ALL-associated JAK1
mutations in a different cellular system, BW5147 cells were transduced with WT Jak1
or each of the selected mutants to evaluate their effect on this stress response. GFP-expressing cells were isolated by flow cytometry, cultured in the presence of cyclosporine A and dexamethasone, with or without IL-9, and [3
H]thymidine incorporation was determined to assess proliferation (). Three independent experiments documented that expression of the A634D and R879C Jak1 mutants, but not R724H Jak1, conferred increased growth to cells basally. Of note, whereas transduced cells exhibited comparable responses to high levels of IL-9, those expressing the A634D and R879C Jak1 mutants were more responsive to low levels of the cytokine. Expression of the A634D Jak1, but not that of the R879C mutant, was associated with enhanced phosphorylation of Jak1, Stat3, and Stat5 basally (unpublished data).
Overall, these data indicated that the three selected leukemia-associated JAK1 mutants are hypermorphs, with A634D Jak1 having a seemingly stronger effect, and that different mechanisms are likely to be involved in their cell context–related gain of function.
Molecular modeling of JAK1 and location of affected residues
To look at the structural causes resulting in JAK1 functional up-regulation, we generated a model of JAK1 structure because no crystallographic information was available for this protein. Energy-minimized models of each of the four domains were generated separately by homology to available crystallographic structures of proteins with similar sequences and overall fold. The quaternary arrangement of the four domains was then determined by superimposing the models on a predicted three-dimensional structure of JAK2 (14
). According to the superimposed structure, Ala634
are placed on the surface of the pseudokinase domain involved in the interaction with the kinase domain (). Based on the evidence supporting a negative regulatory role of the pseudokinase domain on catalytic function of JAK proteins (15
), the pathogenetic mechanism of the A634D and L653F changes is predicted to involve a looser interdomain interaction, relaxing inhibitory control on the kinase activity. Consistent with this hypothesis, substitution of two residues located in the pseudokinase domain at the interface with the kinase domain in JAK2 (Val617
) and JAK3 (Ala572
) promote increased catalytic activity basally (Fig. S1, available at http://www.jem.org/cgi/content/full/jem.20072182/DC1
). Our model also predicts that residues Lys204
would perturb the SH2–FERM interdomain interaction because they are located at the interface between these domains, approximately facing each other. This finding is noteworthy because it has been proposed that JAK1's SH2 domain does not function as a phosphotyrosyl-binding domain, but instead plays a structural role in stabilizing the conformation of the FERM domain (21
), which mediates its association to cytokine receptors and exerts an as yet uncharacterized restraint on catalytic function (22
). The molecular mechanism through which these mutations affect JAK1 function remains to be explained. Structural and functional consequences were not obvious for the activating changes affecting residues Arg724
Gene expression profile analysis in blasts with a mutated JAK1 allele
Total RNA was available from blasts of 5 JAK1
mutation-positive (S512L, A634D, and R724H) and 11 mutation-negative subjects of the T-ALL cohort. Unsupervised clustering based on 1,345 probe sets selected by nonspecific filtering clustered expression profiles of mutation-positive blasts into two clusters, suggesting contribution of JAK1
mutations to distinct major mechanisms of deregulation (Fig. S2, available at http://www.jem.org/cgi/content/full/jem.20072182/DC1
). Notably, supervised gene expression analysis revealed a distinctive expression signature shared by leukemic cells with a mutated JAK1
gene () based on the expression of 133 differentially expressed probe sets, consisting of 112 differentially expressed genes, the majority of them being overexpressed in JAK1
mutation-positive samples (Table S2). Among the up-regulated genes, those whose transcription was known to be positively modulated by JAK/STAT signaling, including IRF1
, and IRF7
, were overrepresented in all the JAK1 mutation-positive subjects, further supporting the gain-of-function role of the ALL-associated JAK1
Figure 3. Gene expression profiles and clinical relevance of somatic JAK1 mutations in adult T-ALL. (A) Supervised hierarchical clustering of gene expression profiles performed on blasts from 16 adult T-ALL patients, with (orange) or without (green) a JAK1 mutation. (more ...)
Clinical relevance of somatic JAK1 mutations
The clinical relevance of JAK1 mutations within the adult T-ALL cohort was investigated. Although no statistically significant difference was observed in white blood cell counts, gender distribution, or association with specific chromosomal rearrangements, patients with a mutated JAK1 allele tended to have a more advanced age at diagnosis (median = 40.6 vs. 24.2; P < 0.01), which was consistent with the lower prevalence of mutations identified among children and adolescents with T-ALL included in the study. Comparison of the response to therapy between JAK1 mutation-positive and -negative patients indicated a higher percentage of cases exhibiting resistance to induction therapy in the former (43 vs. 20%), although this difference did not reach statistical significance caused by the relatively small size of the study cohort. Consistent with that finding, a statistically significant reduced disease-free survival (DFS; median =8.7 vs. 20.5 mo; P = 0.01) and overall survival (OS; median = 10.6 vs. 32.5; P < 0.01) was observed among JAK1 mutation-positive patients (). Multivariate analysis confirmed the statistical significance of these associations (DFS: HR= 6.20, 95% CI = 1.32–29.09, P = 0.02; OS: HR = 2.82, 95% CI = 1.07–7.48, P = 0.04), and excluded a significant contribution of patients' age (DFS: HR = 0.99, 95% CI = 0.93–1.05, P = 0.64; OS: HR= 1.01, 95% CI = 0.97–1.05, P = 0.60).
In the adult T-ALL cohort, the mutation status for NRAS, KRAS, NOTCH1, and PTEN was also assessed (unpublished data). Among the JAK1 mutation-positive cases, no defect was observed in the NRAS, KRAS, and PTEN genes, although such mutations had a relatively low prevalence in the entire adult T-ALL cohort (RAS genes: 11% of cases, 95% CI = 2.9–24.8%; PTEN: 14% of cases, 95% CI = 4.5–28.8%). In contrast, heterozygous mutations in NOTCH1 were observed in all JAK1 mutation-positive individuals. Given the high prevalence of NOTCH1 defects observed in the cohort (70% of cases, 95% CI = 53.0–84.1%), this association was not statistically significant (P = 0.06). This observation, however, suggests that activation of JAK1 and NOTCH1 transduction pathways might cooperate in T-ALL pathogenesis and/or progression. No significant difference in response to therapy or outcome was observed between NOTCH1 mutation-positive and -negative patients. Interestingly, NOTCH1 and PTEN mutations exhibited a mutually exclusive distribution because none of the five subjects carrying PTEN lesions had a NOTCH1 defect (Fisher's exact probability = 0.001).
Aberrant JAK1 function and leukemogenesis
Functional up-regulation of two members of the JAK family, JAK2 and JAK3, has recently been discovered in myeloproliferative disorders and other malignancies of the myeloid lineage (18
). The JAK2 V617F amino acid change occurs in the majority of polycythemia vera cases and in ~50% of individuals with essential thrombocythemia or idiopathic myelofibrosis. The available data support the view that this recurrent change, which affects the pseudokinase domain of the protein, induces constitutive activation of the kinase and hypersensitivity to cytokines. Similarly, three JAK3
hypermorphic alleles promoting cytokine independence in Ba/F3 cells have been identified in acute megakaryoblastic myeloid leukemia. In this study, we showed that somatically acquired activating JAK1
mutations occur in ALL, particularly in adults, further emphasizing the importance of JAK-mediated signaling dysregulation in leukemogenesis and extending the spectrum of hematologic malignancies associated with aberrant activation of this signal transduction pathway. Even though the molecular mechanisms by which individual JAK1
mutations promote gain of function are likely to be diverse and remain to be fully characterized, modeling and biochemical data are consistent with the view that, similar to what has been observed for somatic leukemia-associated JAK2
defects, most mutations would interfere with the autoinhibitory control on the catalytic activity. For most mutations, this effect would be achieved by triggering local structural rearrangements in regions involved in interdomain interactions between the pseudokinase and kinase domains or the FERM and SH2 domains (Fig. S1).
JAK1 is expressed widely and participates in intracellular signaling elicited by class II cytokine receptors and receptors that use the gp130 or γc
receptor subunit. Although the hematopoietic defects in Jak1−/−
mice were restricted to the lymphoid cell compartment as a result of an impaired response to IL-7 (10
) and the present findings indicate a cell-context dependence of somatically acquired JAK1
mutations' contribution to leukemogenesis, we cannot exclude the involvement of this kinase in other malignancies. We speculate that a concomitant genetic event, including a mutation affecting other members of the JAK family, might synergize with the JAK1
defect to promote aberrant cell proliferation and/or survival in a cell-specific context. This hypothesis is currently under investigation.
Although 70–80% of pediatric patients with either B- or T-ALL have excellent long-term response to intensive combination chemotherapy, adult patients exhibit a less favorable outcome (4
). In B- ALL, such a poor prognosis has been associated in part with the presence of BCR
gene rearrangements. In contrast, the unfavorable outcome of adult patients with T-ALL has not conclusively been attributed to any cytogenetic lesion, albeit the prognostic relevance of aberrant ERG
gene expression and NOTCH1
mutations has recently been reported (26
). The present work provides the first evidence that JAK1
gene defects are associated with a poor response to therapy, frequent relapse of the disease, and reduced OS, identifying such mutations as a novel informative prognostic marker occurring in a sizable proportion of adult T-ALL. Although studies on larger cohorts are required to determine more precisely the clinical relevance and prognostic value of JAK1
defects in adult and pediatric ALL, our findings provide a rationale for the development of novel therapeutic approaches tailored at interfering with JAK1 signaling, encouraging studies aimed at testing the efficacy and side effects of JAK1 inhibitors in the management of adult T-ALL patients.