JAK kinases overexpression promotes in vitro cell transformation

Abstract

Constitutive activation of the JAK-STAT pathway is frequent in cancer and contributes to oncogenesis. Here, we took advantage of the Ba/F3 cell line, a murine proB cell line dependent on IL-3 for growth, to analyse mechanisms of constitutive STAT activation in vitro. Cytokineindependent and tumorigenic Ba/F3 cell lines were derived from a two-step selection process. Cells transfected with a defective IL-9 receptor acquire IL-9 responsiveness during a first step of selection, and progress after a second selection step to autonomously growing tumorigenic cells. Microarray analysis pointed to JAK1 overexpression as a key genetic event in this transformation. Overexpression of JAK1 not only increased the sensitivity to IL-9 but also allowed a second selection step toward cytokine-independent growth with constitutive STAT activation. This progression was dependent on a functional FERM and kinase JAK1 domain. Similar results were observed after JAK2, JAK3 and TYK2 overexpression. All autonomous cell lines showed an activation of STAT5, ERK1-2 and AKT but only TYK2-overexpressing cell lines showed a constitutive activation of STAT3. Thus, JAK overexpression can be considered as one of the oncogenic events leading to the constitutive activation of the JAK-STAT pathway.

Oncogenic JAK1 and JAK2-activating mutations resistant to ATP-competitive inhibitors

“…16 In this study, we show that 80% of the autonomous BaF3 clones, selected in our in vitro model, acquired activating point mutations in the kinase or pseudokinase domain of JAK1. These JAK1 mutations provide cells with tumorigenic potential by inducing constitutive activation of the JAK-STAT pathway, which supports their autonomous proliferation.…”

“…The first step requires spontaneous upregulation of JAK1, while the mechanism involved in the second step of transformation is not known. 16 As constitutive phosphorylation of JAK1 was detectable in autonomous clones, 14 we systematically sequenced JAK1 from autonomous clones and found that the vast majority of them (139 of 164) were heterozygous for a single de novo mutation. Altogether, 25 different missense mutations were identified as listed in the Online Supplementary Table S1 and Online Supplementary Figure S1.…”

“…As expected, the total level of JAK1 was 2-3 times higher in BaF3 phe116/9 and in autonomous clones as a consequence of the first selection step, which consists of spontaneous JAK1 upregulation. 16 To confirm that the acquisition of mutations in JAK1 is responsible for autonomous BaF3 cell proliferation, we produced mutated JAK1 constructs for the first 13 of the 25 JAK1 mutations that we had found. We transduced parental BaF3 cells with either mutated or wild-type (WT) JAK1.…”

“…14, 16 We previously showed that upregulation of the endogenous JAK1 gene was associated with the first step of transformation, namely increased sensitivity of BaF3 phe116 cells to IL-9, and promotion of the second step of transformation, namely progression towards cytokine-independent BaF3 autonomous cells. 16 In this study, we show that 80% of the autonomous BaF3 clones, selected in our in vitro model, acquired activating point mutations in the kinase or pseudokinase domain of JAK1.…”

Aberrant signal transduction pathways in myeloproliferative neoplasms

“…51 In addition overexpression of several non-mutated JAK proteins, including JAK2, was shown to promote hematopoietic transformation to cytokine independence. 128 The phenotype induced by exon 12 mutants of JAK2 is more restricted to erythrocytosis, without the abnormal megakaryocyte clusters seen in the classical MPNs. 18 These in vivo data indicate that a difference must exist between signaling by JAK2 V617F and exon 12 JAK2 mutants.…”