Eight-channel iTRAQ Enables Comparison of the Activity of Six Leukemogenic Tyrosine Kinases

Abstract

There are a number of leukemogenic protein-tyrosine kinases (PTKs) associated with leukemic transformation. Although each is linked with a specific disease their functional activity poses the question whether they have a degree of commonality in their effects upon target cells. Exon array analysis of the effects of six leukemogenic PTKs (BCR/ABL, TEL/PDGFRβ, FIP1/PDGFRα, D816V KIT, NPM/ALK, and FLT3ITD) revealed few common effects on the transcriptome. It is apparent, however, that proteome changes are not directly governed by transcriptome changes. Therefore, we assessed and used a new generation of iTRAQ tagging, enabling eight-channel relative quantification discovery proteomics, to analyze the effects of these six leukemogenic PTKs. Again these were found to have disparate effects on the proteome with few common targets. BCR/ABL had the greatest effect on the proteome and had more effects in common with FIP1/PDGFRα. The proteomic effects of the four type III receptor kinases were relatively remotely related. The only protein commonly affected was eosinophil-associated ribonuclease 7. Five of six PTKs affected the motility-related proteins CAPG and vimentin, although this did not correspond to changes in motility. However, correlation of the proteomics data with that from the exon microarray not only showed poor levels of correlation between transcript and protein levels but also revealed alternative patterns of regulation of the CAPG protein by different oncogenes, illustrating the utility of such a combined approach. There are a number of leukemogenic protein-tyrosine kinases (PTKs) associated with leukemic transformation. Although each is linked with a specific disease their functional activity poses the question whether they have a degree of commonality in their effects upon target cells. Exon array analysis of the effects of six leukemogenic PTKs (BCR/ABL, TEL/PDGFRβ, FIP1/PDGFRα, D816V KIT, NPM/ALK, and FLT3ITD) revealed few common effects on the transcriptome. It is apparent, however, that proteome changes are not directly governed by transcriptome changes. Therefore, we assessed and used a new generation of iTRAQ tagging, enabling eight-channel relative quantification discovery proteomics, to analyze the effects of these six leukemogenic PTKs. Again these were found to have disparate effects on the proteome with few common targets. BCR/ABL had the greatest effect on the proteome and had more effects in common with FIP1/PDGFRα. The proteomic effects of the four type III receptor kinases were relatively remotely related. The only protein commonly affected was eosinophil-associated ribonuclease 7. Five of six PTKs affected the motility-related proteins CAPG and vimentin, although this did not correspond to changes in motility. However, correlation of the proteomics data with that from the exon microarray not only showed poor levels of correlation between transcript and protein levels but also revealed alternative patterns of regulation of the CAPG protein by different oncogenes, illustrating the utility of such a combined approach. Human leukemias are often associated with chromosome translocations that generate a fusion transcript that can be implicated in disease pathogenesis (for a review, see Ref. 1Mitelman F. Johansson B. Mertens F. The impact of translocations and gene fusions on cancer causation.Nat. Rev. Cancer. 2007; 7: 233-245Crossref PubMed Scopus (995) Google Scholar). Many of these translocation gene products encode for constitutively activated protein-tyrosine kinases (PTKs). 1The abbreviations used are: PTK, protein-tyrosine kinase; PDGFR, platelet-derived growth factor receptor; iTRAQ, isobaric tags for relative and absolute quantitation; ITD, internal tandem duplication; MSCV, murine stem cell virus; SCX, strong cation exchange; HMGB2, high mobility group protein B2. 1The abbreviations used are: PTK, protein-tyrosine kinase; PDGFR, platelet-derived growth factor receptor; iTRAQ, isobaric tags for relative and absolute quantitation; ITD, internal tandem duplication; MSCV, murine stem cell virus; SCX, strong cation exchange; HMGB2, high mobility group protein B2. These include BCR/ABL (2Konopka J.B. Watanabe S.M. Witte O.N. An alteration of the human c-Abl protein in K562 leukemia-cells unmasks associated tyrosine kinase-activity.Cell. 1984; 37: 1035-1042Abstract Full Text PDF PubMed Scopus (678) Google Scholar), a PTK associated with chronic myeloid leukemia, and TEL/PDGFRβ (3Golub T.R. Barker G.F. Lovett M. Gilliland D.G. Fusion of PDGF receptor-β to a novel Ets-like gene, Tel, in chronic myelomonocytic leukemia with t(5;12) chromosomal translocation.Cell. 1994; 77: 307-316Abstract Full Text PDF PubMed Scopus (1081) Google Scholar) observed in some cases of chronic myelomonocytic leukemia. FIP1/PDGFRα fusion gene is associated with the pathogenesis of about 50% of patients with the hypereosinophilic syndrome (4Cools J. DeAngelo D.J. Gotlib J. Stover E.H. Legare R.D. Cortes J. Kutok J. Clark J. Galinsky I. Griffin J.D. Cross N.C.P. Tefferi A. Malone J. Alam R. Schrier S.L. Schmid J. Rose M. Vandenberghe P. Verhoef G. Boogaerts M. Wlodarska I. Kantarjian H. Marynen P. Coutre S.E. Stone R. Gilliland D.G. A tyrosine kinase created by fusion of the PDGFRA and FIP1L1 genes as a therapeutic target of imatinib in idiopathic hypereosinophilic syndrome.N. Engl. J. Med. 2003; 348: 1201-1214Crossref PubMed Scopus (1492) Google Scholar). A t(2, 5) translocation results in the fusion kinase NPM/ALK found in cases of anaplastic large cell lymphoma (5Morris S.W. Kirstein M.N. Valentine M.B. Dittmer K.G. Shapiro D.N. Saltman D.L. Look A.T. Fusion of a kinase gene, ALK, to a nucleolar protein gene, NPM, in non-Hodgkin's lymphoma.Science. 1994; 263: 1281-1284Crossref PubMed Scopus (1943) Google Scholar). Recently other forms of PTK dysregulation have been observed in myeloproliferative disorders and the leukemias: the KIT receptor PTK undergoes activating mutations (D816V) in acute myeloid leukemia (6Nagata H. Worobec A.S. Oh C.K. Chowdhury B.A. Tannenbaum S. Suzuki Y. Metcalfe D.D. Identification of a point mutation in the catalytic domain of the protooncogene c-Kit in peripheral blood mononuclear cells of patients who have mastocytosis with an associated hematologic disorder.Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10560-10564Crossref PubMed Scopus (794) Google Scholar) and in solid tumors; internal tandem duplication at the juxtamembrane region of the FLT3 gene is a somatic change detected in 20% of cases of acute myeloid leukemia that leads to enhanced ligand-independent PTK activity in the FLT3 internal tandem duplication (ITD) form of the protein (7Nakao M. Yokota S. Iwai T. Kaneko H. Horiike S. Kashima K. Sonoda Y. Fujimoto T. Misawa S. Internal tandem duplication of the flt3 gene found in acute myeloid leukemia.Leukemia. 1996; 10: 1911-1918PubMed Google Scholar). Targeted inhibition of the PTKs (such as KIT, BCR/ABL, and TEL/PDGFRβ) via inhibitors that bind the ATP pocket of the kinase domain has been a successful strategy to achieve hematologic or molecular remission (8Baselga J. Targeting tyrosine kinases in cancer: the second wave.Science. 2006; 312: 1175-1178Crossref PubMed Scopus (397) Google Scholar). In chronic myeloid leukemia the use of imatinib, a BCR/ABL kinase inhibitor, has profoundly altered approaches to treatment (9Deininger M. Buchdunger E. Druker B.J. The development of imatinib as a therapeutic agent for chronic myeloid leukemia.Blood. 2005; 105: 2640-2653Crossref PubMed Scopus (1058) Google Scholar). However, after 5 years a significant proportion of patients develop a resistance to the drug, although other molecular therapies remain effective (10Hochhaus A. Kantarjian H.M. Baccarani M. Lipton J.H. Apperley J.F. Druker B.J. Facon T. Goldberg S.L. Cervantes F. Niederwieser D. Silver Stone R. hematologic and in chronic myeloid leukemia after of imatinib 2007; PubMed Scopus Google Scholar). The for in treatment of this and other leukemias is apparent, and of for tyrosine leukemic is The development of new for the of effects of genes in cells from patients with leukemia. of protein levels in such cells is in changes in the levels of not as a for changes in the proteome R.D. D.L. D. E. K. A. E. proteomics as a factor in stem 2006; PubMed Scopus Google Scholar). The effects of the TEL/PDGFRβ on a cell proteome have been to be by altered regulation of protein levels R.D. A. proteomic analysis isobaric protein tags of changes in transcript and protein levels in 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). However, protein analysis has been of or be quantification proteomics the to and the effects of leukemogenic PTKs. an isobaric for relative quantification to and tandem the of protein levels four can be for their common effects B. K. S. S. S. S. 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