Engineering and Functional Characterization of Fusion Genes Identifies Novel Oncogenic Drivers of Cancer
Hengyu LuNicole VillafañeTurgut DogrulukCaitlin L. GrzeskowiakKathleen KongYiu Huen TsangOksana ZagorodnaAngeliki PantaziLixing YangNicholas J. NeillYoung Won KimChad J. CreightonRoel G.W. VerhaakGordon B. MillsPeter J. ParkRaju KucherlapatiKenneth L. Scott
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Abstract Oncogenic gene fusions drive many human cancers, but tools to more quickly unravel their functional contributions are needed. Here we describe methodology permitting fusion gene construction for functional evaluation. Using this strategy, we engineered the known fusion oncogenes, BCR-ABL1, EML4-ALK, and ETV6-NTRK3, as well as 20 previously uncharacterized fusion genes identified in The Cancer Genome Atlas datasets. In addition to confirming oncogenic activity of the known fusion oncogenes engineered by our construction strategy, we validated five novel fusion genes involving MET, NTRK2, and BRAF kinases that exhibited potent transforming activity and conferred sensitivity to FDA-approved kinase inhibitors. Our fusion construction strategy also enabled domain-function studies of BRAF fusion genes. Our results confirmed other reports that the transforming activity of BRAF fusions results from truncation-mediated loss of inhibitory domains within the N-terminus of the BRAF protein. BRAF mutations residing within this inhibitory region may provide a means for BRAF activation in cancer, therefore we leveraged the modular design of our fusion gene construction methodology to screen N-terminal domain mutations discovered in tumors that are wild-type at the BRAF mutation hotspot, V600. We identified an oncogenic mutation, F247L, whose expression robustly activated the MAPK pathway and sensitized cells to BRAF and MEK inhibitors. When applied broadly, these tools will facilitate rapid fusion gene construction for subsequent functional characterization and translation into personalized treatment strategies. Cancer Res; 77(13); 3502–12. ©2017 AACR.AIM To construct a plasmid expressing fusion protein,and to study the antiviral activity of the proteins.METHODS Artifically modified hIFNα1 cDNA was fused precisely to the reading frame of CB cDNA and further cloned into expression vector pBV220. The plasmid was transformed into E. coli and expressed under thermal induction. The fusion protein was purified by preparetive SDS-PAGE.RESULTS Expression analysis revealed accounts for 2.8% of total cellular proteins. Antiviral activity of this fusion protein was 2.62×10 6 IU/mg by CPE. CONCLUSION We succeeded in constructing the recombinant plasmid expressing hIFNα1-CB fusion proteins. Factors such as non-native conformation and β-amidation were responsible for the low activity of hIFNα1-CB fusion molecule.
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Oncogene Proteins
MEN1
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Objective:To obtain human S100A2-GST fusion protein for further research on human S100A2(hS100A2)protein function and its interactions with other proteins and systems.Methods:hS100A2 gene from pHAHA-hS100A2 was subcloned into an expression vector pGST-moluc to construct pGST-moluc-hS100A2.The new plasmid was identified by digestion with XhoⅠand EcoRⅠ.The recombinant BL21 was induced with IPTG to express recombinant fusion protein hS100A2-GST,which was purified by Glutathion-Sepharose 4B ball beads,identified by SDS-PAGE and Western Blot,and quantified by Bradford method.Results:After the digestion,the recombinant plasmid was cutted into two fragments,300 bp and 5kb.After treated with IPTG,the recombinant BL21 strain expressed a protein,which was about 36 kD and was recognized specifically by an-ti-hS100A2.Its yield was 5 mg/L bacterial culture.After isolated by Glutathion-Sepharose 4B ball beads,its purity was 92%.Conclusion:hS100A2-GST expression plasmid pGST-moluc-hS100A2 was constructed successfully.hS100A2-GST fusion pro-tein could be expressed in Escherichia coli with higher yield and isolated with higher purity,which lays the foundation for the follow-up of the hS100A2 research.
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Objective To obtain high-level expression and purification of fusion protein of VEGF121 and amphoteric molecules KLAK in E.coli BL21(DE3) and laid foundations for studying its effection resistence to neoplastic angiogenesis. Methods The genes of VEGF121 and KLAK by RT-PCR method was ligated and was subcloned into pET-28a which was then transformed into competent E.coli BL21(DE3). Induced by IPTG VEGF121-KLAK fusion protein was expressed in recombinant E.coli BL21(DE3) and was detected with SDS-PAGE and Western blot. Results: The genes of VEGF121 and KLAK were obtained and the recombinant expressing vector was constructed. SDS-PAGE and Western blot analysis indicated that the molecular weight of fusion protein was right. Conclusion: The recombinant expressing vector constructed could produce VEGF121-KLAK fusion protein and the high purified fusion protein was obtained.
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Fusion gene detection is widely used in the diagnosis and treatment of leukemia. This study developed a rapid detection method of eight common pediatric leukemia fusion genes.In this study, one step multiplex RT-PCR assay was developed for the simultaneous detection of eight common leukemia fusion genes, including BCR-ABL, ETV6-RUNX1, MLL-AF4, E2A-PBX1, AML1-ETO, PML-RARα, CBFβ-MYH11 and SIL-TAL1. The single step RT-PCR approach is mediated by universal primers after obtaining total RNA from bone marrow specimens. The size of the amplified fragments were analyzed by capillary electrophoresis assay. A total of 122 patients with positive leukemia fusion genes were tested by real-time PCR.Respectively, 21 cases were detected as CBRB-MYH11 fusion gene, 13 cases were detected as SIL-TAL1 fusion gene, 16 cases were detected as ETV6-RUNX1 fusion gene, 16 cases were detected as E2A-PBX1 fusion gene, 15 cases were detected as PML-RARα fusion gene, 14 cases were detected as AML1-ETO fusion gene, 13 cases were detected as MLL-AF4 fusion gene, except for 1 case where no fusion gene was detected.This method has a high accuracy and detection rate. Therefore, one step multiplex RT-PCR combined with a capillary electrophoresis analysis system can be used as an important tool for the clinical diagnosis, treatment and prognosis of pediatric leukemia.
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Oncogene Proteins
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Objective:To study the detection and clinical significance of the rearrangements of MLL gene and its fusion gene in leukemia.Method:Rearrangements of MLL gene of 70 leukemia patients were detected by fluorescence in situ hybridization(FISH) and 6 common MLL fusion genes were detected by nested RT-PCR.Result:9 of 70 leukemic patients were found with rearrangements of MLL gene,the incidence of which was 12.86%.5 of 9 patients were diagnosed as B-ALL.3 patients were diagnosed as AML-M5,1 patient was diagnosed as JMML.Among 5 B-ALL patients,2 patients were confirmed to express MLL/ENL fusion gene,1 patient was confirmed to express MLL/AF4 fusion gene,the other 2 patients did not express the fusion genes.Among 3 AML-M5 patients,2 patients were confirmed to express MLL/MF9 fusion gene,the other 1 patient did not express the fusion gene.The fusion gene of 1 JMML patient with rearrangements of MLL gene was MLL/ENL.Conclusion:Nested RT-PCR is a convenient and effective method to detect the fusion genes resulting from rearrangements of MLL gene.The rearrangements of MLL gene were found in B-ALL,AML-M5 and JMML.It indicates a poor prognosis.
Chimeric gene
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Lineage (genetic)
Gene rearrangement
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To construct a vector containing protein transduction domain (PTD) and bcr/abl fusion gene of chronic myelogenous leukemia and express PTD-bcr/abl fusion protein in E. Coli.DNA fragment encoding PTD was synthesized and fused to PCR-amplified bcr/abl gene fragment, then inserted into plasmid pET-16b to get the expression vector pEPb containing PTD-bcr/abl fusion gene, which was transfected and expressed in E. Coli LB21. PTD-bcr/abl fusion protein was purified by affinity chromatography.523 bp bcr/abl fusion gene was effectively amplified. The PTD-bcr/abl gene sequencing showed the same sequence as scheduled. The fusion peptide was successfully expressed in E. Coli and purified.The results may provide a new PTD-bcr/abl fusion peptide for the immunotherapy of CML.
ABL
breakpoint cluster region
Chronic myelogenous leukemia
Transduction (biophysics)
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Objective:To obtain human S100A6-GST fusion protein as material of the study of function of hS100A6.Methods:The hS100A6 gene from pHAHA-hS100A6 was subcloned into the prokaryotic GST fusion protein expression plasmid,pGST-moluc,to form pGST-moluc-hS100A6. The recombinant plasmid was transformed to E.coli BL2l and the expression of hS100A6-GST fusion protein was induced with IPTG. The protein was detected by SDS-PAGE and purified with Glutathion-Sepharose 4B beads,then identified by western blot assays. Results:A 36 000 Dalton protein,as expected,was obtained evidently.The concentration of the purified fusion protein was 3mg/L bacterial culture,and the purity was about 92%. Conclusion:A prokaryotic system expressing hS100A6-GST fusion protein was successfully constructed.hS100A6-GST fusion protein with high purity could be obtained after it was efficiently expressed in E.coli BL21 and purified with Glutathion-Sepharose 4B beads. This will facilitate our study of the function of hS100A6.
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