The hematopoietic system provides an attractive model for studying growth factor‐controlled expansion and differentiation of cells in relation to receptor routing and its consequences for signal ...transduction. Suppressor of cytokine signaling (SOCS) proteins regulate receptor signaling partly via their ubiquitin ligase (E3)‐recruiting SOCS box domain. Whether SOCS proteins affect signaling through modulating intracellular trafficking of receptors is unknown. Here, we show that a juxtamembrane lysine residue (K632) of the granulocyte colony‐stimulating factor receptor (G‐CSFR) plays a key role in receptor routing and demonstrate that the effects of SOCS3 on G‐CSF signaling to a major extent depend on this lysine. Mutation of K632 causes accumulation of G‐CSFR in early endosomes and leads to sustained activation of signal transducer and activator of transcription 5 and ERK, but not protein kinase B. Myeloid progenitors expressing G‐CSFR mutants lacking K632 show a perturbed proliferation/differentiation balance in response to G‐CSF. This is the first demonstration of SOCS‐mediated ubiquitination and routing of a cytokine receptor and its impact on maintaining an appropriate signaling output.
Ubiquitination of the CSF3R CSF3 (colony-stimulating factor 3) receptor occurs after activated CSF3Rs are internalized and reside in early endosomes. CSF3R ubiquitination is crucial for lysosomal ...routing and degradation. The E3 ligase SOCS3 (suppressor of cytokine signalling 3) has been shown to play a major role in this process. Deubiquitinating enzymes remove ubiquitin moieties from target proteins by proteolytic cleavage. Two of these enzymes, AMSH associated molecule with the SH3 domain of STAM (signal transducing adaptor molecule) and UBPY (ubiquitin isopeptidase Y), interact with the general endosomal sorting machinery. Whether deubiquitinating enzymes control CSF3R trafficking from early towards late endosomes is unknown. In the present study, we asked whether AMSH, UBPY or a murine family of deubiquitinating enzymes could fulfil such a role. This DUB family (deubiquitin enzyme family) comprises four members (DUB1, DUB1A, DUB2 and DUB2A), which were originally described as being haematopoietic-specific and cytokine-inducible, but their function in cytokine receptor routing and signalling has remained largely unknown. We show that DUB2A expression is induced by CSF3 in myeloid 32D cells and that DUB2 decreases ubiquitination and lysosomal degradation of the CSF3R, leading to prolonged signalling. These results support a model in which CSF3R ubiquitination is dynamically controlled at the early endosome by feedback mechanisms involving CSF3-induced E3 ligase (SOCS3) and deubiquitinase (DUB2A) activities.
Background: Translocation t(12;21), resulting in the ETV6-RUNX1 fusion protein, is present in 25% of pediatric patients with B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Despite the ...favorable prognostic parameters of this B-ALL subgroup, relapse and resistance to chemotherapeutics occur and treatment-induced side effects are considerable. The molecular mechanisms underlying ETV6-RUNX1-driven leukemia are largely unknown. Increased knowledge of these mechanisms is essential to develop novel therapeutic strategies to selectively target ETV6-RUNX1-positive leukemia.
Objectives: This study aims to identify and target the molecular drivers behind ETV6-RUNX1-positive BCP-ALL.
Results: Gene expression profiling of leukemic blasts of 654 ALL patients revealed that the class III PI3-kinase Vps34, an important regulator of autophagy, was exclusively up-regulated in ETV6-RUNX1-positive compared to ETV6-RUNX1-negative BCP-ALL patients (2.7-fold; p ≤ 10-30). In addition, ectopic expression of ETV6-RUNX1 in cord blood-derived hematopoietic progenitor cells (CB-HPCs) significantly induced expression of Vps34 1.3-fold already 40 hours after transduction (p ≤ 0.05). This suggests that the Vps34-autophagy pathway is activated by ETV6-RUNX1, which may mechanistically explain the leukemogenic and pro-survival properties ascribed to ETV6-RUNX1. In correspondence, Ingenuity Pathway Analysis (IPA) predicted a pro-survival and pro-proliferative phenotype in ETV6-RUNX1 transduced CB-HPCs and highlighted a network of up-regulated transcription factors, including HEY1, EGR1, GATA1 and GATA2 (2 – 25-fold up-regulation; p ≤ 0.05). Luciferase reporter assays revealed that not only the ETV6-RUNX1 fusion protein, but also the ETV6-RUNX1-induced target genes HEY1, EGR1 and GATA1 positively regulate Vps34 promoter activity (5 – 13-fold up-regulation; p ≤ 0.01).Lentiviral knockdown experiments were performed to elucidate the importance of Vps34 expression in ETV6-RUNX1-positive BCP-ALL cells. Knockdown of all Vps34 transcript variants, with two independent constructs, led to complete growth arrest of the ETV6-RUNX1-positive cell lines REH and AT2, while this only led to a decrease in proliferation of the ETV6-RUNX1-negative cell line NALM6. This growth arrest was caused by a significant induction of apoptosis (more than 4-fold 7 days after transduction; p ≤ 0.001) and a significantly reduced percentage of cycling cells (1.3-fold 7 days after transduction; p ≤ 0.05). Analysis of p62 protein expression by western blot and reverse phase protein arrays revealed that the levels of autophagy were significantly higher in ETV6-RUNX1-positive compared to ETV6-RUNX1-negative BCP-ALL patients (p ≤ 0.001). In addition, knockdown of ETV6-RUNX1 and Vps34 significantly reduced autophagy, quantified with confocal microscopy, in ETV6-RUNX1-positive cells with 50% and 84%, respectively (p ≤ 0.01). Furthermore, pharmacological inhibition of autophagy with hydroxychloroquine (HCQ) significantly reduced cell viability of BCP-ALL cell lines and primary patient-derived BCP-ALL cells (p ≤ 0.001). Treatment of the ETV6-RUNX1-positive BCP-ALL cell lines REH and AT2 with 20 µg/mL HCQ resulted in a 82% and 95% reduced cell viability, while the viability of ETV6-RUNX1-negative BCP-ALL cell lines and T-ALL cell lines were reduced to a lesser extent (NALM6: 43%; TOM-1: 50%; Loucy: 40%; Jurkat: 0%). Importantly, HCQ selectively sensitized ETV6-RUNX1-positive leukemic cells to L-asparaginase treatment in clinically relevant concentrations. Treatment of primary ETV6-RUNX1-positive patient cells with 10 µg/mL HCQ resulted in a 70% reduction in cell survival during L-asparaginase exposure (p ≤ 0.01). This sensitization was not observed in ETV6-RUNX1-negative BCP-ALL cells.
Conclusion: The ETV6-RUNX1 fusion protein activates autophagy via Vps34, which is essential for survival and proliferation of ETV6-RUNX1-positive cells. Inhibition of autophagy in primary ETV6-RUNX1-positive leukemic cells inhibited cell survival and sensitized these cells to L-asparaginase treatment. These results indicate that autophagy inhibition may provide a novel means to sensitize L-asparaginase-resistant ETV6-RUNX1-positive BCP-ALL patients.
No relevant conflicts of interest to declare.
Mutations in ELANE cause severe congenital neutropenia (SCN), but how they affect neutrophil production and contribute to leukemia predisposition is unknown. Neutropenia is alleviated by CSF3 ...(granulocyte colony-stimulating factor) therapy in most cases, but dose requirements vary between patients. Here, we show that CD34+CD45+ hematopoietic progenitor cells (HPCs) derived from induced pluripotent stem cell lines from patients with SCN that have mutations in ELANE (n = 2) or HAX1 (n = 1) display elevated levels of reactive oxygen species (ROS) relative to normal iPSC-derived HPCs. In patients with ELANE mutations causing misfolding of the neutrophil elastase (NE) protein, HPCs contained elevated numbers of promyelocyte leukemia protein nuclear bodies, a hallmark of acute oxidative stress. This was confirmed in primary bone marrow cells from 3 additional patients with ELANE-mutant SCN. Apart from responding to elevated ROS levels, PML controlled the metabolic state of these ELANE-mutant HPCs as well as the expression of ELANE, suggestive of a feed-forward mechanism of disease development. Both PML deletion and correction of the ELANE mutation restored CSF3 responses of these ELANE-mutant HPCs. These findings suggest that PML plays a crucial role in the disease course of ELANE-SCN characterized by NE misfolding, with potential implications for CSF3 therapy.
•PML reduces ROS but stimulates metabolism and ELANE expression in iPSC-derived CD34+CD45+ cells from ELANE-SCN with NE misfolding mutations.•PML dampens granulocyte colony-stimulating factor responsiveness in a CSF3 therapy-refractory case of ELANE-SCN.
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CSF3R G-CSF (granulocyte colony-stimulating factor) receptor controls survival, proliferation and differentiation of myeloid progenitor cells via activation of multiple JAKs (Janus kinases). In ...addition to their role in phosphorylation of receptor tyrosine residues and downstream signalling substrates, JAKs have recently been implicated in controlling expression of cytokine receptors, predominantly by masking critical motifs involved in endocytosis and lysosomal targeting. In the present study, we show that increasing the levels of JAK1, JAK2 and TYK2 (tyrosine kinase 2) elevated steady-state CSF3R cell-surface expression and enhanced CSF3R protein stability in haematopoietic cells. This effect was not due to inhibition of endocytotic routing, since JAKs did not functionally interfere with the dileucine-based internalization motif or lysine-mediated lysosomal degradation of CSF3R. Rather, JAKs appeared to act on CSF3R in the biosynthetic pathway at the level of the ER (endoplasmic reticulum). Strikingly, increased JAK levels synergized with internalization- or lysosomal-routing-defective CSF3R mutants to confer growth-factor independent STAT3 (signal transducer and activator of transcription 3) activation and cell survival, providing a model for how increased JAK expression and disturbed intracellular routing of CSF3R synergize in the transformation of haematopoietic cells.
Ubiquitination of lysine (K) residues in the cytoplasmic domain of cytokine receptors plays a major role in intracellular receptor routing and control of signal duration. However, which ubiquitin ...(E3) ligases and whether specific lysines, eg. present in conserved motifs, are involved in ubiquitin-mediated routing is still largely unknown. We recently showed that SOCS3, which forms an Elongin/Cullin-based E3 ligase (ECSSOCS3), is involved in ligand-induced ubiquitination, lysosomal routing and degradation of the G-CSFR (Irandoust et al, EMBO J . 2007, 26:1782–93). Strikingly, ECSSOCS3-mediated mechanisms largely depended on ubiquitination of a single juxtamembrane lysine at position 632 (K632), even though 4 additional conserved lysines are present in the cytoplasmic domain of the G-CSFR. This juxtamembrane lysine located 5 amino acids upstream of the box1 region is conserved among several other cytokine receptors. We sought to unravel the configuration required for ubiquitination of K632 and for its function in attenuation of G-CSF signaling. First, we tested whether K632 is part of a domain that can function as an isolated motif. To this end, we fused the K632-containing domain (D) encompassing the juxtamembrane and box1 regions to the COOH-terminus of a lysine-less G-CSFR (K5R-D). Despite efficient ubiquitination of the lysine within the reallocated domain, K5R-D, similar to K5R, was severely hampered in lysosomal routing. This resulted in prolonged STAT5 activation and in G-CSF-induced hyperproliferation of myeloid 32D cells indicating that the juxtamembrane domain of the G-CSFR does not function as an isolated domain that can be shifted to a different cytoplasmic location. To delineate how imperative the positioning of K632 is for its function, we inserted 5 alanines immediately upstream or downstream of K632. However, these insertions did not affect G-CSFR signal duration suggesting that there is no stringent proximity of K632 to the cell membrane and no strict positioning relative to the SOCS3 recuitment site (Y729) to direct downregulation of G-CSFR signaling. To further study this flexibility in positioning we ‘walked' lysines through the juxtamembrane domain and the adjacent box 1-region by mutating amino acids at indicated positions to a lysine in an otherwise lysine-less G-CSFR. Mutants 628K and 630K were hardly ubiquitinated and, as predicted, were hampered in lysosomal routing. In contrast, lysines on position 631, 633, 634, 638 and 644 were ubiquitinated comparably to K632. Interestingly, while mutants 631K, 633K and 634K showed characteristics indistinguishable from 632K, mutants 638K and 644K displayed slightly decreased signal attenuation suggesting a gradual decline of functionality of lysines moved to box-1. Finally, moving the lysine downstream to position 672, which corresponds to the second lysine in the wt G-CSFR, resulted in complete loss of functionality. In conclusion, these results indicate that the positioning of a lysine within the juxtamembrane domain is crucial for ECSSOCS3-mediated signal downregulation and lysosomal routing of the G-CSFR, although a limited flexibility is tolerated towards the box1 region. The fact that inactive lysines are still ubiquitinated implies that interaction with effector proteins is involved in lysosomal routing rather than reduced or lost activity of E3 ligases. Given its high conservation among several cytokine receptors we are currently investigating a common role for the juxtamembrane lysine in cytokine receptor routing and signal attenuation.
The granulocyte colony-stimulating factor receptor (G-CSF-R) induces proliferation, survival and differentiation of myeloid progenitor cells in a tightly controlled temporal fashion. These responses ...depend on multiple signaling mechanisms that are activated via distinct regions in the cytoplasmic domain of wild type (WT) G-CSF-R. About 20% of severe congenital neutropenia patients acquire mutations that truncate the C-terminus of G-CSF-R, which is often associated with disease progression to acute myeloid leukemia. Myeloid cells expressing these truncated G-CSF-R hyperproliferate and are hampered in differentiation in response to G-CSF. Multiple mechanisms have been linked to perturbed signaling of truncated G-CSF-R. Specifically, defective internalization has been associated with a prolonged activation status of the truncated G-CSF-R, which is e.g. reflected by strongly increased and sustained activation of STAT5. We found that internalized WT G-CSF-R are rapidly targeted to lysosomes, suggesting that lysosomal degradation is a major mechanism for signal attenuation. Lysine (K) residues in the cytoplasmic tail of transmembrane receptors are often determinants for ubiquitin-mediated sorting into multi-vesicular bodies and lysosomes. We observed that G-CSF-R is ubiquitinated and subsequently assessed the contribution of K residues in G-CSF-R routing and signaling. To this end, we generated mutant G-CSF-R-K5R, in which all five conserved cytoplasmic K residues were replaced by arginine (R). To study differential localization of internalized K5R and WT G-CSF-R, we introduced constitutively active Rab5 (Rab5-Q79L) to enlarge early endosomes. Under these conditions, internalized WT G-CSF-R colocalized extensively with endosomal microdomains containing the endosome-to-lysosome sorting protein Hrs. In contrast, internalized G-CSF-R-K5R was mostly found outside these domains. Consequently, lysosomal routing of G-CSF-R-K5R was severely impaired, which resulted in strongly increased receptor protein levels. Upon removal of G-CSF, downregulation of STAT5 activity in 32D/K5R cells was significantly delayed compared to 32D/WT cells, establishing that the lysine residues are not only crucial for receptor stability, but also for duration of signaling. Moreover, in the continuous presence of G-CSF, 32D/K5R cells showed strongly increased STAT5 activity and proliferation and reduced granulocytic differentiation compared to 32D/WT. Similar results were obtained in colony assays with g-csfr deficient primary bone marrow progenitors transduced with K5R or WT G-CSF-R. To substantiate a relationship between signal duration and receptor degradation, we inhibited lysosomal degradation by pharmacological inhibitors and by knockdown of Hrs and the related protein Tsg101 by RNA interference. As expected, receptor stability was significantly increased by these treatments. Surprisingly however, this did not influence STAT5 activity, suggesting that signal attenuation predominantly occurred in a pre-lysosomal compartment. We conclude from these data that ubiquitinated lysine residues of G-CSF-R are required to target internalized receptors for lysosomal degradation. However, they also appear to be involved in contracting an as yet unidentified inhibitor, perhaps a phosphatase, to a specific Hrs-positive subendosomal compartment, thereby switching off G-CSF-R already prior to its degradation in lysosomes.
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