JAK1-deficient Cells Research Articles

JAK1 As a Convergent Regulator of Hematopoietic Stem Cell Function and Stress Hematopoiesis

Cytokine-mediated signal transduction is critical to hematopoiesis, immune responses, and other physiological processes. Aberrant production and secretion of pro-inflammatory cytokines disturbs homeostasis and proper immune function and if persistent results in symptoms of chronic inflammation. Previous studies have illustrated the importance of JAK1 as an effector of cytokine signaling, including in immunological and neoplastic diseases such that selective JAK1 inhibition is currently being investigated in clinical trials. However, the role of Jak1 in hematopoietic stem cell (HSC) function has not been delineated. This has led us to investigate the impact of loss of Jak1 signaling on HSC function by developing a novel conditional Jak1 knockout allele (Fig. 1a). Mice with conditional deletion of Jak1 in the hematopoietic system (hereafter referred to as Jak1 KO) are characterized by leukocytosis (Jak1 KO avg. 6.34K/ul, Jak1 WT avg. 10.76K/ul, P<0.01), and reduced spleen (Jak1 KO avg. 73.76mg, Jak1 WT avg. 98.86mg, P<0.01) and thymus weights (Jak1 KO avg. 49.31mg, Jak1 WT avg. 80.82mg, P<0.01). High dimensional single cell analysis of the hematopoietic compartment of these mice using mass cytometry showed that conditional Jak1 loss in hematopoietic cells attenuates B cell and NK cell differentiation in vivo, and results in differentiation towards the myeloid lineage at the expense of lymphoid fate commitment. Further, we observed a significant reduction of lineage-Sca1+cKit+ (LSK) cells in the bone marrow of Jak1 KO mice, including a decrease in CD34-Flk2- long-term HSCs (LT-HSCs) and in CD34+Flk2- short-term HSCs (ST-HSCs) (Fig.1b). Jak1-deficient cells formed fewer colonies in colony formation unit assays, which was also seen when clonogenic assays were performed in the presence of JAK1 inhibitor GLPG0634. Most importantly, Jak1-deficient stem cells exhibited decreased competitiveness in bone marrow transplantation assays. Flow analysis at 4 weeks post transplantation showed a 3-fold reduced blood chimerism in recipients transplanted with Jak1 KO bone marrow cells and at 16 weeks, Jak1KO cells were largely outcompeted by CD45.1-positive WT cells (Fig. 1c). Jak1-deficient stem cells were also unable to rescue hematopoiesis in the setting of myelosuppressive insults leading to a worse survival of Jak1 KO mice when serially injected with 5-fluorouracil (5-FU) (Fig. 1d).Consistent with the stem cell phenotype observed in JAK1 KO mice, we found that a significant larger proportion of Jak1-deficient stem cells lacks expression of the proliferation marker Ki67 and that Jak1-deficient stem cells fail to enter the cell cycle in response to hematopoietic stress. To begin to determine the mechanism by which Jak1 regulates normal stem cell function in vivo, we assessed the impact of loss of Jak1 on transcriptional output. Gene expression profiling of LT-HSCs from Jak1 KO and WT mice identified 259 significant genes, many of which were known to be Jak1 downstream targets. Gene set enrichment analysis (GSEA) revealed that the majority of genes that were altered following deletion of Jak1 corresponded to interferon signaling and inflammatory response pathways. Consistent with these findings, our functional in vitro and in vivo assays demonstrated that Jak1-deficient cells were insensitive to type I interferons as shown by lack of Stat1 and Stat5 activation (Fig. 1e), retained Sca1 surface expression, and an unchanged cell cycle status upon IFN stimulation. Moreover, the HSC defect observed in the setting of Jak1 loss was not fully rescued by expression of a constitutively active Jak2 allele, suggesting there is non-redundant signaling in HSCs within the JAK kinase family. Together, our data suggests that Jak1 functions as a central node for interferon signaling in HSCs and reveals an essential and nonredundant role of Jak1 in HSC homeostasis and stress response. [Display omitted] DisclosuresKoppikar:Amgen: Employment. Nolan:Fluidigm: Consultancy. Levine:Novartis: Consultancy; Qiagen: Membership on an entity's Board of Directors or advisory committees.

Biallelic JAK1 mutations in immunodeficient patient with mycobacterial infection.

Mutations in genes encoding components of the immune system cause primary immunodeficiencies. Here, we study a patient with recurrent atypical mycobacterial infection and early-onset metastatic bladder carcinoma. Exome sequencing identified two homozygous missense germline mutations, P733L and P832S, in the JAK1 protein that mediates signalling from multiple cytokine receptors. Cells from this patient exhibit reduced JAK1 and STAT phosphorylation following cytokine stimulations, reduced induction of expression of interferon-regulated genes and dysregulated cytokine production; which are indicative of signalling defects in multiple immune response pathways including Interferon-γ production. Reconstitution experiments in the JAK1-deficient cells demonstrate that the impaired JAK1 function is mainly attributable to the effect of the P733L mutation. Further analyses of the mutant protein reveal a phosphorylation-independent role of JAK1 in signal transduction. These findings clarify JAK1 signalling mechanisms and demonstrate a critical function of JAK1 in protection against mycobacterial infection and possibly the immunological surveillance of cancer.

Interferonα Activates NF-κB in JAK1-deficient Cells through aTYK2-dependentPathway

In addition to activating members of the STAT transcription factor family, interferon alpha/beta (IFNalpha/beta) activates the NF-kappaB transcription factor. To determine the role of the Janus tyrosine kinase (JAK)-STAT pathway in NF-kappaB activation by IFN, we examined NF-kappaB activation in JAK1-deficient mutant human fibrosarcoma cells. In wild-type fibrosarcoma cells (2fTGH), IFN activates STAT1, STAT2, and STAT3, as well as NF-kappaB complexes comprised of p50 and p65. In contrast, in JAK1-deficient cells, IFN induces NF-kappaB activation and NF-kappaB dependent gene transcription but does not activate these STAT proteins and has no effect on STAT-dependent gene transcription. Expression of a catalytically inactive TYK2 tyrosine kinase in JAK1-deficient cells, as well as in the highly IFN-sensitive Daudi lymphoblastoid cell line, abrogates NF-kappaB activation by IFN. Moreover, IFN does not promote NF-kappaB activation in TYK2-deficient mutant fibrosarcoma cells. Our results demonstrate a dichotomy between the classical JAK-STAT pathway and the NF-kappaB signaling pathway. In the IFN signaling pathway leading to STAT activation, both JAK1 and TYK2 are essential, whereas NF-kappaB activation requires only TYK2.

Novel Role of Janus Kinase 1 in the Regulation of Oncostatin M Receptor Surface Expression

The oncostatin M receptor (OSMR) is part of a heterodimeric receptor complex that mediates signal transduction of the pleiotropic cytokine OSM via a signaling pathway involving Janus kinases (Jaks) and transcription factors of the signal transducers and activators of transcription (STAT) family. Upon heterologous expression of the OSMR in several cell lines, we observed that its surface expression was significantly enhanced by coexpression of the Janus kinases Jak1, Jak2, and Tyk2 but not Jak3. Chimeric receptors consisting of the extracellular region of the interleukin-5 receptor beta chain and the transmembrane and intracellular part of the OSMR were similarly up-regulated on the plasma membrane when Jak1 was coexpressed. The overall expression level of these constructs did not change significantly, but Jak1 coexpression increased the amount of endoglycosidase H-resistant, fully processed OSMR chimeras. Using mutated receptor and Jak1 constructs, we were able to demonstrate that association of Jak1 with the membrane proximal region of the receptor, but not its kinase activity, is necessary for this effect. Moreover, deletion of the OSMR box1/2 region also resulted in an improved surface expression indicating that this region may contain a signal preventing efficient receptor surface expression in the absence of associated Jaks. Finally we demonstrate that in Jak1-deficient cells, the endogenous OSMR is significantly down-regulated, an effect that can be reversed by transient expression of Jak1 in these cells.

Regulation of STAT1 nuclear export by Jak1.

Signal transducer and activator of transcription 1 (STAT1) mediates gene expression in response to cytokines and growth factors. Activation of STAT1 is achieved through its tyrosine phosphorylation, a process that involves Jak tyrosine kinases. Here we show that STAT1, although phosphorylated on Y701, is unable to localize in the nucleus in the absence of Jak1 or Jak1 kinase activity. In contrast, the nuclear accumulation of STAT1 in Tyk2-deficient cells remains intact. Nuclear presence of tyrosine-phosphorylated STAT1 could be restored in Jak1-deficient cells by leptomycin B, an inhibitor of nuclear export. Amino acids 197 to 205 of STAT1 were found to encode a leucine-rich nuclear export signal (NES). An L-->A mutation within the NES restored nuclear retention of STAT1 in Jak1-deficient cells. Impaired binding of the transcriptional coactivator CBP to tyrosine-phosphorylated STAT1 derived from Jak1-deficient cells offers a model for the intermolecular regulation of the nuclear export sequence.