Federica Toffalini

Transcription factor regulation can be accurately predicted from the presence of target gene signatures in microarray gene expression data

Deciphering transcription factor networks from microarray data remains difficult. This study presents a simple method to infer the regulation of transcription factors from microarray data based on well-characterized target genes. We generated a catalog containing transcription factors associated with 2720 target genes and 6401 experimentally validated regulations. When it was available, a distinction between transcriptional activation and inhibition was included for each regulation. Next, we built a tool (www.tfacts.org) that compares submitted gene lists with target genes in the catalog to detect regulated transcription factors. TFactS was validated with published lists of regulated genes in various models and compared to tools based on in silico promoter analysis. We next analyzed the NCI60 cancer microarray data set and showed the regulation of SOX10, MITF and JUN in melanomas. We then performed microarray experiments comparing gene expression response of human fibroblasts stimulated by different growth factors. TFactS predicted the specific activation of Signal transducer and activator of transcription factors by PDGF-BB, which was confirmed experimentally. Our results show that the expression levels of transcription factor target genes constitute a robust signature for transcription factor regulation, and can be efficiently used for microarray data mining.

New insights into the mechanisms of hematopoietic cell transformation by activated receptor tyrosine kinases

A large number of alterations in genes encoding receptor tyrosine kinase (RTK), namely FLT3, c-KIT, platelet-derived growth factor (PDGF) receptors, fibroblast growth factor (FGF) receptors, and the anaplastic large cell lymphoma kinase (ALK), have been found in hematopoietic malignancies. They have drawn much attention after the development of tyrosine kinase inhibitors. RTK gene alterations include point mutations and gene fusions that result from chromosomal rearrangements. In both cases, they activate the kinase domain in the absence of ligand, producing a permanent signal for cell proliferation. Recently, this simple model has been refined. First, by contrast to wild-type RTK, many mutated RTK do not seem to signal from the plasma membrane, but from various locations inside the cell. Second, their signal transduction properties are altered: the pathways that are crucial for cell transformation, such as signal transducer and activator of transcription (STAT) factors, do not necessarily contribute to the physiologic functions of these receptors. Finally, different mechanisms prevent the termination of the signal, which normally occurs through receptor ubiquitination and degradation. Several mutations inactivating CBL, a key RTK E3 ubiquitin ligase, have been recently described. In this review, we discuss the possible links among RTK trafficking, signaling, and degradation in leukemic cells.

KANK1, a candidate tumor suppressor gene, is fused to PDGFRB in an imatinib-responsive myeloid neoplasm with severe thrombocythemia

KANK1 , a candidate tumor suppressor gene, is fused to PDGFRB in an imatinib-responsive myeloid neoplasm with severe thrombocythemia

Critical Role of the Platelet-derived Growth Factor Receptor (PDGFR) β Transmembrane Domain in the TEL-PDGFRβ Cytosolic Oncoprotein

The fusion of TEL with platelet-derived growth factor receptor (PDGFR) β (TPβ) is found in a subset of patients with atypical myeloid neoplasms associated with eosinophilia and is the archetype of a larger group of hybrid receptors that are produced by rearrangements of PDGFR genes. TPβ is activated by oligomerization mediated by the pointed domain of TEL/ETV6, leading to constitutive activation of the PDGFRβ kinase domain. The receptor transmembrane (TM) domain is retained in TPβ and in most of the described PDGFRβ hybrids. Deletion of the TM domain (ΔTM-TPβ) strongly impaired the ability of TPβ to sustain growth factor-independent cell proliferation. We confirmed that TPβ resides in the cytosol, indicating that the PDGFRβ TM domain does not act as a transmembrane domain in the context of the hybrid receptor but has a completely different function. The ΔTM-TPβ protein was expressed at a lower level because of increased degradation. It could form oligomers, was phosphorylated at a slightly higher level, co-immunoprecipitated with the p85 adaptor protein, but showed a much reduced capacity to activate STAT5 and ERK1/2 in Ba/F3 cells, compared with TPβ. In an in vitro kinase assay, ΔTM-TPβ was more active than TPβ and less sensitive to imatinib, a PDGFR inhibitor. In conclusion, we show that the TM domain is required for TPβ-mediated signaling and proliferation, suggesting that the activation of the PDGFRβ kinase domain is not enough for cell transformation.

The fusion proteins TEL-PDGFRβ and FIP1L1-PDGFRα escape ubiquitination and degradation

Upon growth factor-induced activation, receptor tyrosine kinases such as the PDGF and FGF receptors are targeted for lysosomal degradation via a mechanism that involves ubiquitination of receptor lysines. In this study, it is shown that constitutively active oncogenic fusion proteins that contain PDGF or FGF receptor moieties, caused by specific chromosomal translocations in chronic myeloid neoplasms, escape this negative regulatory mechanism. Background Chimeric oncogenes encoding constitutively active protein tyrosine kinases are associated with chronic myeloid neoplasms. TEL-PDGFRβ (TPβ, also called ETV6-PDGFRB) is a hybrid protein produced by the t(5;12) translocation, FIP1L1-PDGFRα (FPα) results from a deletion on chromosome 4q12 and ZNF198-FGFR1 is created by the t(8;13) translocation. These fusion proteins are found in patients with myeloid neoplasms associated with eosinophilia. Wild-type receptor tyrosine kinases are efficiently targeted for degradation upon activation, in a process that requires Cbl-mediated monoubiquitination of receptor lysines. Since protein degradation pathways have been identified as useful targets for cancer therapy, the aim of this study was to compare the degradation of hybrid and wild-type receptor tyrosine kinases. Design and Methods We used Ba/F3 as a model cell line, as well as leukocytes from two patients, to analyze hybrid protein degradation. Results In contrast to the corresponding wild-type receptors, which are quickly degraded upon activation, we observed that TPβ, FPα and the ZNF198-FGFR1 hybrids escaped down-regulation in Ba/F3 cells. The high stability of TPβ and FPα hybrid proteins was confirmed in leukocytes from leukemia patients. Ubiquitination of TPβ and FPα was much reduced compared to that of wild-type receptors, despite marked Cbl phosphorylation in cells expressing hybrid receptors. The fusion of a destabilizing domain to TPβ induced protein degradation. Instability was reverted by adding the destabilizing domain ligand, Shield1. The destabilization of this modified TPβ reduced cell transformation and STAT5 activation. Conclusions We have shown that chimeric receptor tyrosine kinases escape ubiquitination and down-regulation and that their stabilization is critical to efficient stimulation of cell proliferation.

The fusion proteins TEL-PDGFRbeta and FIP1L1-PDGFRalpha escape ubiquitination and degradation

Upon growth factor-induced activation, receptor tyrosine kinases such as the PDGF and FGF receptors are targeted for lysosomal degradation via a mechanism that involves ubiquitination of receptor lysines. In this study, it is shown that constitutively active oncogenic fusion proteins that contain PDGF or FGF receptor moieties, caused by specific chromosomal translocations in chronic myeloid neoplasms, escape this negative regulatory mechanism. Background Chimeric oncogenes encoding constitutively active protein tyrosine kinases are associated with chronic myeloid neoplasms. TEL-PDGFRβ (TPβ, also called ETV6-PDGFRB) is a hybrid protein produced by the t(5;12) translocation, FIP1L1-PDGFRα (FPα) results from a deletion on chromosome 4q12 and ZNF198-FGFR1 is created by the t(8;13) translocation. These fusion proteins are found in patients with myeloid neoplasms associated with eosinophilia. Wild-type receptor tyrosine kinases are efficiently targeted for degradation upon activation, in a process that requires Cbl-mediated monoubiquitination of receptor lysines. Since protein degradation pathways have been identified as useful targets for cancer therapy, the aim of this study was to compare the degradation of hybrid and wild-type receptor tyrosine kinases. Design and Methods We used Ba/F3 as a model cell line, as well as leukocytes from two patients, to analyze hybrid protein degradation. Results In contrast to the corresponding wild-type receptors, which are quickly degraded upon activation, we observed that TPβ, FPα and the ZNF198-FGFR1 hybrids escaped down-regulation in Ba/F3 cells. The high stability of TPβ and FPα hybrid proteins was confirmed in leukocytes from leukemia patients. Ubiquitination of TPβ and FPα was much reduced compared to that of wild-type receptors, despite marked Cbl phosphorylation in cells expressing hybrid receptors. The fusion of a destabilizing domain to TPβ induced protein degradation. Instability was reverted by adding the destabilizing domain ligand, Shield1. The destabilization of this modified TPβ reduced cell transformation and STAT5 activation. Conclusions We have shown that chimeric receptor tyrosine kinases escape ubiquitination and down-regulation and that their stabilization is critical to efficient stimulation of cell proliferation.

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.

The fusion proteins TEL-PDGFR and FIP1L1-PDGFR escape ubiquitination and degradation

Upon growth factor-induced activation, receptor tyrosine kinases such as the PDGF and FGF receptors are targeted for lysosomal degradation via a mechanism that involves ubiquitination of receptor lysines. In this study, it is shown that constitutively active oncogenic fusion proteins that contain PDGF or FGF receptor moieties, caused by specific chromosomal translocations in chronic myeloid neoplasms, escape this negative regulatory mechanism. Background Chimeric oncogenes encoding constitutively active protein tyrosine kinases are associated with chronic myeloid neoplasms. TEL-PDGFRβ (TPβ, also called ETV6-PDGFRB) is a hybrid protein produced by the t(5;12) translocation, FIP1L1-PDGFRα (FPα) results from a deletion on chromosome 4q12 and ZNF198-FGFR1 is created by the t(8;13) translocation. These fusion proteins are found in patients with myeloid neoplasms associated with eosinophilia. Wild-type receptor tyrosine kinases are efficiently targeted for degradation upon activation, in a process that requires Cbl-mediated monoubiquitination of receptor lysines. Since protein degradation pathways have been identified as useful targets for cancer therapy, the aim of this study was to compare the degradation of hybrid and wild-type receptor tyrosine kinases. Design and Methods We used Ba/F3 as a model cell line, as well as leukocytes from two patients, to analyze hybrid protein degradation. Results In contrast to the corresponding wild-type receptors, which are quickly degraded upon activation, we observed that TPβ, FPα and the ZNF198-FGFR1 hybrids escaped down-regulation in Ba/F3 cells. The high stability of TPβ and FPα hybrid proteins was confirmed in leukocytes from leukemia patients. Ubiquitination of TPβ and FPα was much reduced compared to that of wild-type receptors, despite marked Cbl phosphorylation in cells expressing hybrid receptors. The fusion of a destabilizing domain to TPβ induced protein degradation. Instability was reverted by adding the destabilizing domain ligand, Shield1. The destabilization of this modified TPβ reduced cell transformation and STAT5 activation. Conclusions We have shown that chimeric receptor tyrosine kinases escape ubiquitination and down-regulation and that their stabilization is critical to efficient stimulation of cell proliferation.