FOXO1 forkhead domain mutants in B-cell lymphoma lack transcriptional activity.

Somatic point mutations of the FOXO1 transcription factor were reported in non-Hodgkin lymphoma including diffuse large B-cell lymphoma, follicular lymphoma and Burkitt lymphoma. These alterations were associated with a poor prognosis and resistance to therapy. Nearly all amino acid substitutions are localized in two major clusters, affecting either the N-terminal region (Nt mutations) or the forkhead DNA-binding domain (DBD mutations). While recent studies have focused on Nt mutations, we characterized FOXO1 DBD mutants. We analyzed their transcriptional activity, DNA binding, phosphorylation and protein-protein interaction. The majority of DBD mutants showed a decrease in activity and DNA binding, while preserving AKT phosphorylation and interaction with the cytoplasmic ATG7 protein. In addition, we investigated the importance of conserved residues of the α-helix 3 of the DBD. Amino acids I213, R214, H215 and L217 appeared to be crucial for FOXO1 activity. Our data underlined the key role of multiple amino-acid residues of the forkhead domain in FOXO1 transcriptional activity and revealed a new type of FOXO1 loss-of-function mutations in B-cell lymphoma.

Atomistic mechanism of the constitutive activation of PDGFRA via its transmembrane domain.

Single-point mutations in the transmembrane (TM) region of receptor tyrosine kinases (RTKs) can lead to abnormal ligand-independent activation. We use a combination of computational modeling, NMR spectroscopy and cell experiments to analyze in detail the mechanism of how TM domains contribute to the activation of wild-type (WT) PDGFRA and its oncogenic V536E mutant. Using a computational framework, we scan all positions in PDGFRA TM helix for identification of potential functional mutations for the WT and the mutant and reveal the relationship between the receptor activity and TM dimerization via different interfaces. This strategy also allows us design a novel activating mutation in the WT (I537D) and a compensatory mutation in the V536E background eliminating its constitutive activity (S541G). We show both computationally and experimentally that single-point mutations in the TM region reshape the TM dimer ensemble and delineate the structural and dynamic determinants of spontaneous activation of PDGFRA via its TM domain. Our atomistic picture of the coupling between TM dimerization and PDGFRA activation corroborates the data obtained for other RTKs and provides a foundation for developing novel modulators of the pathological activity of PDGFRA.

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

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.

PDGF-induced fibroblast growth requires monounsaturated fatty acid production by stearoyl-CoA desaturase.

Stearoyl-coenzyme A desaturase (SCD) catalyzes the Δ9-cis desaturation of saturated fatty acids (SFA) to generate monounsaturated fatty acids (MUFA). This enzyme is highly up-regulated by platelet-derived growth factor (PDGF) in human fibroblasts. Accordingly, the analysis of cellular fatty acids by gas chromatography showed that PDGF significantly increased the proportion of MUFA, particularly palmitoleate, in cellular lipids. To further analyze the role of SCD in fibroblasts, we used small hairpin RNA targeting SCD (shSCD), which decreased the MUFA content. SCD down-regulation blunted the proliferation of fibroblasts in response to PDGF. This was confirmed using a pharmacological inhibitor of SCD. In addition, proliferation was blocked by palmitate and stearate (two SCD substrates) but not by palmitoleate and oleate (two SCD products). In the presence of an equal amount of oleate, palmitate had no effect on cell proliferation. SCD inhibition or down-regulation did not decrease PDGF receptor activity or signaling. However, by measuring plasma membrane lipid lateral diffusion by fluorescence recovery after photobleaching, we showed that the modulation of the MUFA/SFA ratio by PDGF and SCD inhibitor was related to modifications of membrane fluidity. Altogether, our data suggest that SCD is required for the response of normal fibroblasts to growth factors.

Idiopathic basal ganglia calcification-associated PDGFRB mutations impair the receptor signalling.

Platelet-derived growth factors (PDGF) bind to two related receptor tyrosine kinases, which are encoded by the PDGFRA and PDGFRB genes. Recently, heterozygous PDGFRB mutations have been described in patients diagnosed with idiopathic basal ganglia calcification (IBGC or Fahr disease), a rare inherited neurological disorder. The goal of the present study was to determine whether these mutations had a positive or negative impact on the PDGFRB activity. We first showed that the E1071V mutant behaved like wild-type PDGFRB and may represent a polymorphism unrelated to IBGC. In contrast, the L658P mutant had no kinase activity and failed to activate any of the pathways normally stimulated by PDGF. The R987W mutant activated Akt and MAP kinases but did not induce the phosphorylation of signal transducer and activator of transcription 3 (STAT3) after PDGF stimulation. Phosphorylation of phospholipase Cγ was also decreased. Finally, we showed that the R987W mutant was more rapidly degraded upon PDGF binding compared to wild-type PDGFRB. In conclusion, PDGFRB mutations associated with IBGC impair the receptor signalling. PDGFRB loss of function in IBGC is consistent with recently described inactivating mutations in the PDGF-B ligand. These results raise concerns about the long-term safety of PDGF receptor inhibition by drugs such as imatinib.

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 bortezomib 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.