Laura A. Noël

Tolbutamide Controls Glucagon Release From Mouse Islets Differently Than Glucose: Involvement of KATP Channels From Both α-Cells and δ-Cells

We evaluated the role of ATP-sensitive K+ (KATP) channels, somatostatin, and Zn2+ in the control of glucagon secretion from mouse islets. Switching from 1 to 7 mmol/L glucose inhibited glucagon release. Diazoxide did not reverse the glucagonostatic effect of glucose. Tolbutamide decreased glucagon secretion at 1 mmol/L glucose (G1) but stimulated it at 7 mmol/L glucose (G7). The reduced glucagon secretion produced by high concentrations of tolbutamide or diazoxide, or disruption of KATP channels (Sur1−/− mice) at G1 could be inhibited further by G7. Removal of the somatostatin paracrine influence (Sst−/− mice or pretreatement with pertussis toxin) strongly increased glucagon release, did not prevent the glucagonostatic effect of G7, and unmasked a marked glucagonotropic effect of tolbutamide. Glucose inhibited glucagon release in the absence of functional KATP channels and somatostatin signaling. Knockout of the Zn2+ transporter ZnT8 (ZnT8−/− mice) did not prevent the glucagonostatic effect of glucose. In conclusion, glucose can inhibit glucagon release independently of Zn2+, KATP channels, and somatostatin. Closure of KATP channels controls glucagon secretion by two mechanisms, a direct stimulation of α-cells and an indirect inhibition via somatostatin released from δ-cells. The net effect on glucagon release results from a balance between both effects.

ETV6-PDGFRB and FIP1L1-PDGFRA stimulate human hematopoietic progenitor cell proliferation and differentiation into eosinophils: the role of nuclear factor-κB.

Background ETV6-PDGFRB (also called TEL-PDGFRB) and FIP1L1-PDGFRA are receptor-tyrosine kinase fusion genes that cause chronic myeloid malignancies associated with hypereosinophilia. The aim of this work was to gain insight into the mechanisms whereby fusion genes affect human hematopoietic cells and in particular the eosinophil lineage. Design and Methods We introduced ETV6-PDGFRB and FIP1L1-PDGFRA into human CD34+ hematopoietic progenitor and stem cells isolated from umbilical cord blood. Results Cells transduced with these oncogenes formed hematopoietic colonies even in the absence of cytokines. Both oncogenes also stimulated the proliferation of cells in liquid culture and their differentiation into eosinophils. This model thus recapitulated key features of the myeloid neoplasms induced by ETV6-PDGFRB and FIP1L1-PDGFRA. We next showed that both fusion genes activated the transcription factors STAT1, STAT3, STAT5 and nuclear factor-κB. Phosphatidylinositol-3 kinase inhibition blocked nuclear factor-κB activation in transduced progenitor cells and patients’ cells. Nuclear factor-κB was also activated in the human FIP1L1-PDGFRA-positive leukemia cell line EOL1, the proliferation of which was blocked by borte-zomib and the IκB kinase inhibitor BMS-345541. A mutant IκB that prevents nuclear translocation of nuclear factor-κB inhibited cell growth and the expression of eosinophil markers, such as the interleukin-5 receptor and eosinophil peroxidase, in progenitors transduced with ETV6-PDGFRB. In addition, several potential regulators of this process, including HES6, MYC and FOXO3 were identified using expression microarrays. Conclusions We show that human CD34+ cells expressing PDGFR fusion oncogenes proliferate autonomously and differentiate towards the eosinophil lineage in a process that requires nuclear factor-κB. These results suggest new treatment possibilities for imatinib-resistant myeloid neoplasms associated with PDGFR mutations.

Multiple oligomerization domains of KANK1-PDGFRβ are required for JAK2-independent hematopoietic cell proliferation and signaling via STAT5 and ERK

Background KANK1-PDGFRB is a fusion gene generated by the t(5;9) translocation between KANK1 and the platelet-derived growth factor receptor beta gene PDGFRB. This hybrid was identified in a myeloproliferative neoplasm featuring severe thrombocythemia, in the absence of the JAK2 V617F mutation. Design and Methods KANK1-PDGFRB was transduced into Ba/F3 cells and CD34+ human progenitor cells to gain insights into the mechanisms whereby this fusion gene transforms cells. Results Although platelet-derived growth factor receptors are capable of activating JAK2, KANK1-PDGFRβ did not induce JAK2 phosphorylation in hematopoietic cells and a JAK inhibitor did not affect KANK1-PDGFRβ-induced cell growth. Like JAK2 V617F, KANK1-PDGFRβ constitutively activated STAT5 transcription factors, but this did not require JAK kinases. In addition KANK1-PDGFRβ induced the phosphorylation of phospholipase C-γ, ERK1 and ERK2, like wild-type PDGFRβ and TEL-PDGFRβ, another hybrid protein found in myeloid malignancies. We next tested various mutant forms of KANK1-PDGFRβ in Ba/F3 cells and human CD34+ hematopoietic progenitors. The three coiled-coil domains located in the N-terminus of KANK1 were required for KANK1-PDGFRβ-induced cell growth and signaling via STAT5 and ERK. However, the coiled-coils were not essential for KANK1-PDGFRβ oligomerization, which could be mediated by another new oligomerization domain. KANK1-PDGFRβ formed homotrimeric complexes and heavier oligomers. Conclusions KANK1-PDGFRB is a unique example of a thrombocythemia-associated oncogene that does not signal via JAK2. The fusion protein is activated by multiple oligomerization domains, which are required for signaling and cell growth stimulation.

The tyrosine phosphatase SHP2 is required for cell transformation by the receptor tyrosine kinase mutants FIP1L1-PDGFRα and PDGFRα D842V

Activated forms of the platelet derived growth factor receptor alpha (PDGFRα) have been described in various tumors, including FIP1L1‐PDGFRα in patients with myeloproliferative diseases associated with hypereosinophilia and the PDGFRαD842V mutant in gastrointestinal stromal tumors and inflammatory fibroid polyps.

PDGFRB gain-of-function mutations in sporadic infantile myofibromatosis.

&NA; Infantile myofibromatosis is one of the most prevalent soft tissue tumors of infancy and childhood. Multifocal nodules with visceral lesions are associated with a poor prognosis. A few familial cases have been linked to mutations in various genes including PDGFRB. In this study, we sequenced PDGFRB, which encodes a receptor tyrosine kinase, in 16 cases of myofibromatosis or solitary myofibroma. Mutations in the coding sequence of PDGFRB were identified in 6 out of 8 patients with the sporadic multicentric form of the disease and in 1 out of 8 patients with isolated myofibroma. Two patients had the same mutation in multiple separated lesions. By contrast, a third patient had three different PDGFRB mutations in the three nodules analyzed. Mutations were located in the transmembrane, juxtamembrane and kinase domains of the receptor. We showed that these mutations activated receptor signaling in the absence of ligand and transformed fibroblasts. In one case, a weakly‐activating germline variant was associated with a stronger somatic mutation, suggesting a two‐hit model for familial myofibromatosis. Furthermore, the mutant receptors were sensitive to the tyrosine kinase inhibitor imatinib, except D850V, which was inhibited by dasatinib and ponatinib, suggesting a targeted therapy for severe myofibromatosis. In conclusion, we identified gain‐of‐function PDGFRB mutations in the majority of multifocal infantile myofibromatosis cases, shedding light on the mechanism of disease development, which is reminiscent of multifocal venous malformations induced by TIE2 mutations. Our results provide a genetic test to facilitate diagnosis, and preclinical data for development of molecular therapies.

t(5;9)(q32;p24) KANK1/PDGFRB

The fusion protein contains the first 741 residues of KANK1 fused to the last 692 residues of PDGFRB (Medves et al., 2010). This includes several coiled-coil domains of KANK1 and the fifth immunoglobulin-like domain, the transmembrane domain and the kinase domain of PDGFRB. The deletion of the immunoglobulin-like domain does not affect the oncogenic activity of the fusion (Medves et al., 2010). By contrast, deletion of the coiled-coil domains prevents cell transformation and signaling via STAT5 and MAP kinases (Medves et al., 2011). Our data suggest that KANK1-PDGFRB adopts a trimeric and multimeric conformation (Medves et al., 2011).

O40 De nouvelles cibles pour contrôler l’inflammation dans le muscle squelettique: les microARNs régulés par l’adiponectine

Introduction L’adiponectine (ApN) est une hormone diminuee dans le syndrome metabolique qui possede des proprietes antidiabetiques et anti-inflammatoires. Dans le muscle squelettique, l’ApN previent l’inflammation et le stress oxydatif causes par l’obesite. Les microARNs controlent l’expression genique en induisant notamment la degradation des ARNm. Dans ce travail, nous avons caracterise les microARNs qui pourraient contribuer a l’action anti-inflammatoire de l’ApN sur le muscle squelettique. Materiels et methodes Le muscle tibialis anterior gauche des souris deficientes en ApN (ApN-KO) a ete injecte, puis electropore, avec un plasmide codant pour le gene de l’ApN tandis que la patte controlaterale a recu un plasmide controle. Les souris ont ensuite ete soumises a une injection ip de lipopolysaccharides (LPS) afin d’induire un etat inflammatoire. L’expression de marqueurs de l’inflammation (TNFα, IL-1β) et du stress oxydatif (peroxiredonine-3 ; PRDX3) a ete quantifiee. Un microRNA array (Exiqon) a ete realise. Des myotubes murins (C2C12) ont ete traites avec de l’ApN recombinante ou transfectes par un miR-mimic, avant une inflammation causee par le LPS. Resultats L’ApN exercait, comme attendu, son action anti-inflammatoire: l’expression du TNFα, de l’IL-1β et du PRDX3 etaient diminues (– 50 %) dans le muscle electropore avec l’ApN compare au controlateral. Le microRNA array a revele que l’ApN augmentait (~ +150 %) l’expression du miR-711, donnees validees par RT-qPCR. L’analyse in silico a montre que ce microARN peut reprimer l’expression de genes impliques dans differentes voies inflammatoires et notamment la voie Toll-like receptor 4, activee par le LPS. Dans les cellules C2C12, l’ApN attenuait egalement l’induction du TNFα causee par le LPS. La transfection du miR-711 mimic reproduisait l’effet de l’ApN. Conclusion Le miR-711 semble etre implique dans l’action anti-inflammatoire de l’ApN, ce qui peut ouvrir de nouvelles perspectives therapeutiques pour controler l’inflammation musculaire. Declaration d’interet Les auteurs declarent ne pas avoir d’interet direct ou indirect (financier ou en nature) avec un organisme prive, industriel ou commercial en relation avec le sujet presente.