Activating JAK1 mutations contribute to multiple-step tumorigenesis by interacting with endogenous cytokine receptor complexes

JAK1 is a tyrosine kinase from the Janus protein kinase (JAK) family, which includes also JAK2, JAK3 and TYK2. These protein kinases are associated with cytokine receptors to mediate intracellular signaling upon cytokine binding. JAK kinases control activation of downstream signaling molecules such as STAT transcription factors to regulate proliferation, differentiation and cellular growth of target cells. We developed a two-step selection in vitro model of cellular transformation based on the IL-3-dependent pro-B cell line BaF3 that overcame defects in IL-9 receptor signal transduction by spontaneously overexpressing JAK1 during a first step of selection. We identified 18 activating de novo mutations of JAK1 as a second genetic event associated with cytokine-independent proliferation and constitutive activation of the JAK-STAT pathway. In parallel to these observations, in collaboration with Prof. Marco Tartaglia, we identified activating mutations in JAK1 in 20 % of T-cell acute lymphoblastic leukemia (T-ALL) patients and in 3% of B-ALL patients, confirming the relevance of our in vitro model-derived JAK1 mutations for human malignancies. Focusing on the mechanisms by which the JAK1 mutants might promote the development of these leukemias, we found that they need to associate with JAK1-binding cytokine receptors such as the receptors for IL-2 and IL-9, which play an important role in proliferation and differentiation of normal T lymphocytes. When the JAK1 mutants are present, these receptors are constitutively active, even without their ligand. In addition, we observed that JAK1 mutants also associate with the interferon (IFN) receptors. Therefore, tumor cells with mutated JAK1 constitutively express IFN target genes. Moreover, cells with JAK1 mutations are hypersensitive to IFN stimulation. This is particularly relevant for patients because IFN is known to have anti-proliferative activity on tumor cells. We have demonstrated the potential application of this finding by using a mouse model of acute lymphoblastic leukemia. In this model, treatment with IFN resulted in prolonged anti-tumoral protection of the mice that had tumors with JAK1 mutations, as compared to the tumors without such mutations. Based on these observations, we think that IFN- should be considered as a part of the current multi-drug protocol to treat ALL patients positive for JAK1 mutations, and maybe other cancers such as acute myeloid leukemia (AML), where JAK1 mutations occur too.JAK1 est une tyrosine kinase qui appartient, avec JAK2, JAK3 et TYK2, à la famille des Janus kinases (JAKs). Ces protéines sont associées aux récepteurs aux cytokines pour permettre leur signalisation intracellulaire. Les JAKs contrôlent l’activation de molécules de transduction du signal, comme les facteurs de transcription STATs, pour réguler la prolifération, la différenciation et la croissance des cellules cibles. Nous avons développé un modèle de tumorigenèse in vitro en deux étapes, basé sur la lignée cellulaire pro-B BaF3, dépendante de l’IL-3. Ces cellules ont, durant la première étape de sélection, surexprimé JAK1 pour pouvoir répondre à un récepteur de l’IL-9 défectueux. Nous avons pu démontrer que la deuxième étape de sélection, associée à une prolifération cellulaire autonome indépendante des cytokines et à une activation constitutive de la voie JAK-STAT, était liée à la survenue spontanée de mutations dans JAK1. 18 mutations activatrices de JAK1 ont pu être détectées dans différents clones autonomes. En parallèle à ces observations, en collaboration avec le Prof. Marco Tartaglia, nous avons identifié des mutations activatrices de JAK1 dans 20% des leucémies aigues lymphoblastiques (LLA) T de l’adulte et dans 3 % des patients atteints de LLA B. Cette observation confirme la pertinence de notre modèle cellulaire in vitro pour l’oncogenèse humaine. En se focalisant sur les mécanismes permettant aux mutants JAK1 de promouvoir la leucémogenèse, nous avons démontré qu’ils devaient s’associer à un récepteur capable de lier JAK1, comme le récepteur de l’IL-2 ou de l’IL-9. Ces récepteurs jouent un rôle important dans la prolifération et la différenciation des lymphocytes T normaux. Quand une mutation dans JAK1 est présente, ces récepteurs s’activent de manière constitutive, même sans ligand. Nous avons également observé que les mutants JAK1 se lient des récepteurs aux interférons (IFN) de type I. C’est pourquoi les cellules tumorales mutées pour JAK1 expriment constitutivement des gènes cibles des IFN. De plus, ces cellules sont hypersensibles à l’effet des IFNs. Cette observation est particulièrement relevante pour la clinique au vu de l’activité anti-proliférative exercée par les IFNs sur les cellules tumorales. Nous avons démontré l’application potentielle de ces découvertes en utilisant un modèle murin de LLA. Dans ce modèle, le traitement par IFN exerçait un effet anti-tumoral spécifique sur les cellules exprimant une forme mutée de JAK1 par rapport aux tumeurs sans mutation. Sur base de ces observations, nous pensons que l’utilisation d’IFN- devrait être ajoutée à l’arsenal thérapeutique utilisé pour traiter la LLA chez les patients mutés pour JAK1, et qu’il est envisageable d’utiliser l’IFN dans d’autres tumeurs ou des mutations de JAK1 peuvent survenir, comme dans la leucémie myéloïde aigue.(SBIM 3) -- UCL, 201

JAK kinases overexpression promotes in vitro cell transformation

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. Cytokine-independent 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

BACKGROUND: Activating mutations in JAK1 and JAK2 have been described in patients with various hematologic malignancies including acute lymphoblastic leukemia and myeloproliferative neoplasms, leading to clinical trials with JAK inhibitors. While there has been a tremendous effort towards the development of specific JAK inhibitors, mutations conferring resistance to such drugs have not yet been observed. DESIGN AND METHODS: Taking advantage of a model of spontaneous cellular transformation, we sequenced JAK1 in selected tumorigenic BaF3 clones and identified 25 de novo JAK1 activating mutations, including 5 mutations already described in human leukemias. We further used this library of JAK1 mutation-positive cell lines to assess their sensitivity to ATP-competitive inhibitors. RESULTS: While most JAK1 mutants were sensitive to ATP-competitive JAK inhibitors, mutations targeting Phe958 and Pro960 in the hinge region of the kinase domain rendered JAK1 constitutively active but also resistant to all tested JAK inhibitors. Furthermore, mutation of the homologous Tyr931 in JAK2 wild-type or JAK2 V617F mutant found in patients with myeloproliferative neoplasms also conferred resistance to JAK inhibitors, such as INCB018424, which is currently in clinical use. CONCLUSIONS: Our data indicate that some activating mutations not only promote autonomous cell proliferation but also confer resistance to ATP-competitive inhibitors. In vivo, such a mutation can potentially occur as primary JAK-activating mutations but also as secondary mutations combining oncogenicity with drug resistance

Acute Lymphoblastic Leukemia-associated JAK1 Mutants Activate the Janus Kinase/STAT Pathway via Interleukin-9 Receptor α Homodimers*S⃞

Activating mutations in JAK1 have been reported in acute lymphoblastic leukemias, but little is known about the mechanisms involved in their constitutive activation. Here, we studied the ability of JAK1 V658F and A634D to activate the Janus kinase (JAK)/STAT pathway upon ectopic expression in HEK293 cells alone or together with the other components of the interleukin-9 receptor complex (IL-9Rα, γc, and JAK3). Expression of JAK1 mutants alone failed to trigger STAT activation, but co-expression of the IL-9Rα chain promoted JAK1 mutant phosphorylation and STAT activation. Mutation of the FERM domain of JAK1, which is critical for cytokine receptor association, or of the single tyrosine of IL-9Rα involved in STAT recruitment abolished this activity, indicating that JAK1 mutants need to associate with a functional IL-9Rα to activate STAT factors. Several lines of evidence indicated that IL-9Rα homodimerization was involved in this process. IL-9Rα variants with mutations of the JAK-interacting BOX1 region not only failed to promote JAK1 activation but also acted as dominant negative forms reverting the effect of wild-type IL-9Rα. Coimmunoprecipitation experiments also showed the formation of IL-9Rα homodimers. Interestingly, STAT activation was partially inhibited by expression of γc, suggesting that overlapping residues are involved in IL-9Rα homodimerization and IL-9Rα/γc heterodimerization. Co-expression of wild-type JAK3 partially reverted the inhibition by γc, indicating that JAK3 cooperates with JAK1 mutants within the IL-9 receptor complex. Similar results were observed with IL-2Rβ. Taken together, our results show that IL-9Rα and IL-2Rβ homodimers efficiently mediate constitutive activation of ALL-associated JAK1 mutants

ALL-associated JAK1 mutations confer hypersensitivity to the antiproliferative effect of type I interferon

Activating mutations in JAK1 have been reported in acute lymphoblastic leukemias (ALLs). In this study, we found a type I interferon (IFN) transcriptional signature in JAK1 mutation-positive human ALL samples. This signature was recapitulated in vitro by the expression of JAK1 mutants in BW5147 and BaF3 hematopoietic cell lines. Binding of JAK1 to the IFN receptor was essential because mutations in the FERM domain abrogated this effect. Beside the constitutive activation of the type I IFN signaling cascade, JAK1 mutations also strongly potentiated the response to IFN in vitro. Typically, the proliferation of cell lines expressing JAK1(A634D) was abrogated by type I IFNs. Interestingly, we found that different JAK1 mutations differentially potentiate responses to type I IFNs or to interleukin-9, another cytokine using JAK1 to mediate its effects. This suggests that the type of mutation influences the specificity of the effect on distinct cytokine receptor signaling. Finally, we also showed in an in vivo leukemia model that cells expressing JAK1(A634D) are hypersensitive to the anti-proliferative and antitumorigenic effect of type I IFN, suggesting that type I IFNs should be considered as a potential therapy for ALL with JAK1-activating mutations. (Blood. 2010; 115(16): 3287-3295

ALL-associated JAK1 mutations confer hypersensitivity to the antiproliferative effect of type I interferon

Activating mutations in JAK1 have been reported in acute lymphoblastic leukemias (ALLs). In this study, we found a type I interferon (IFN) transcriptional signature in JAK1 mutation-positive human ALL samples. This signature was recapitulated in vitro by the expression of JAK1 mutants in BW5147 and BaF3 hematopoietic cell lines. Binding of JAK1 to the IFN receptor was essential because mutations in the FERM domain abrogated this effect. Beside the constitutive activation of the type I IFN signaling cascade, JAK1 mutations also strongly potentiated the response to IFN in vitro. Typically, the proliferation of cell lines expressing JAK1(A634D) was abrogated by type I IFNs. Interestingly, we found that different JAK1 mutations differentially potentiate responses to type I IFNs or to interleukin-9, another cytokine using JAK1 to mediate its effects. This suggests that the type of mutation influences the specificity of the effect on distinct cytokine receptor signaling. Finally, we also showed in an in vivo leukemia model that cells expressing JAK1(A634D) are hypersensitive to the anti-proliferative and antitumorigenic effect of type I IFN, suggesting that type I IFNs should be considered as a potential therapy for ALL with JAK1-activating mutations. (Blood. 2010; 115(16): 3287-3295

Cooperating JAK1 and JAK3 mutants increase resistance to JAK inhibitors.

The acquisition of growth signal self-sufficiency is 1 of the hallmarks of cancer. We previously reported that the murine interleukin-9-dependent TS1 cell line gives rise to growth factor-independent clones with constitutive activation of the Janus kinase (JAK)- signal transducer and activator of transcription (STAT) pathway. Here, we show that this transforming event results from activating mutations either in JAK1, JAK3, or in both kinases. Transient and stable expression of JAK1 and/or JAK3 mutants showed that each mutant induces STAT activation and that their coexpression further increases this activation. The proliferation of growth factor-independent TS1 clones can be efficiently blocked by JAK inhibitors such as ruxolitinib or CMP6 in short-term assays. However, resistant clones occur upon long-term culture in the presence of inhibitors. Surprisingly, resistance to CMP6 was not caused by the acquisition of secondary mutations in the adenosine triphosphate-binding pocket of the JAK mutant. Indeed, cells that originally showed a JAK1-activating mutation became resistant to inhibitors by acquiring another activating mutation in JAK3, whereas cells that originally showed a JAK3-activating mutation became resistant to inhibitors by acquiring another activating mutation in JAK1. These observations underline the cooperation between JAK1 and JAK3 mutants in T-cell transformation and represent a new mechanism of acquisition of resistance against JAK inhibitors

Distinct Acute Lymphoblastic Leukemia (ALL)-associated Janus Kinase 3 (JAK3) Mutants Exhibit Different Cytokine-Receptor Requirements and JAK Inhibitor Specificities

JAK1 and JAK3 are recurrently mutated in acute lymphoblastic leukemia. These tyrosine kinases associate with heterodimeric cytokine receptors such as IL-7 receptor or IL-9 receptor, in which JAK1 is appended to the specific chain, and JAK3 is appended to the common gamma chain. Here, we studied the role of these receptor complexes in mediating the oncogenic activity of JAK3 mutants. Although JAK3(V674A) and the majority of other JAK3 mutants needed to bind to a functional cytokine receptor complex to constitutively activate STAT5, JAK3(L857P) was unexpectedly found to not depend on such receptor complexes for its activity, which was induced without receptor or JAK1 co-expression. Introducing a mutation in the FERM domain that abolished JAK-receptor interaction did not affect JAK3(L857P) activity, whereas it inhibited the other receptor-dependent mutants. The same cytokine receptor independence as for JAK3(L857P) was observed for homologous Leu(857) mutations of JAK1 and JAK2 and for JAK3(L875H). This different cytokine receptor requirement correlated with different functional properties in vivo and with distinct sensitivity to JAK inhibitors. Transduction of murine hematopoietic cells with JAK3(V674A) led homogenously to lymphoblastic leukemias in BALB/c mice. In contrast, transduction with JAK3(L857P) induced various types of lymphoid and myeloid leukemias. Moreover, ruxolitinib, which preferentially blocks JAK1 and JAK2, abolished the proliferation of cells transformed by the receptor-dependent JAK3(V674A), yet proved much less potent on cells expressing JAK3(L857P). These particular cells were, in contrast, more sensitive to JAK3-specific inhibitors. Altogether, our results showed that different JAK3 mutations induce constitutive activation through distinct mechanisms, pointing to specific therapeutic perspectives.status: publishe