Protein phosphorylation controls the activity of signal transduction pathways regulated by kinases and phosphatases. Little is known, however, about the impact of preanalytical factors, for example, delayed times to tissue fixation, on global phosphoprotein levels in tissues. The aim of this study was to characterize the potential effects of delayed tissue preservation (cold ischemia) on the levels of phosphoproteins using targeted and nontargeted proteomic approaches. Rat and murine liver samples were exposed to different cold ischemic conditions ranging from 10 to 360 min prior to cryopreservation. The phosphoproteome was analyzed using reverse phase protein array (RPPA) technology and phosphoprotein-enriched quantitative tandem mass spectrometry (LC-MS/MS). RPPA analysis of rat liver tissues with long (up to 360 min) cold ischemia times did not reveal statistically significant alterations of specific phosphoproteins even though nonphosphorylated cytokeratin 18 (CK18) showed increased levels after 360 min of delay to freezing. Keeping the samples on ice prior to cryopreservation prevented this effect. LC-MS/MS-based quantification of 1684 phosphorylation sites in rat liver tissues showed broadening of their distribution compared to time point zero, but without reaching statistical significance for individual phosphosites. Similarly, RPPA analysis of mouse liver tissues with short (<60 min) cold ischemia times did not reveal directed or predictable changes of protein and phosphoprotein levels. Using LC-MS/MS and quantification of 791 phosphorylation sites, we found that the distribution of ratios compared to time point zero broadens with prolonged ischemia times, but these were rather undirected and diffuse changes, as we could not detect significant alterations of individual phosphosites. On the basis of our results from RPPA and LC-MS/MS analysis of rat and mouse liver tissues, we conclude that prolonged cold ischemia results in unspecific phosphoproteome changes that can be neither predicted nor assigned to individual proteins. On the other hand, we identified a number of phosphosites which were extraordinarily stable even after 360 min of cold ischemia and, therefore, may be used as general reference markers for future companion diagnostics for kinase inhibitors.
We present a large scale quantitation study of the membrane proteome from Halobacterium salinarum. To overcome problems generally encountered with membrane proteins, we established a membrane preparation protocol that allows the application of most proteomic techniques originally developed for soluble proteins. Proteins were quantified using two complementary approaches. For gel-based quantitation, DIGE labeling was combined with two-dimensional gel electrophoresis on an improved 16-benzyldimethyl-n-hexadecylammonium chloride/SDS system. MS-based quantitation was carried out by combining gel-free separation with the recently developed isotope-coded protein labeling technique. Good correlations between these two independent quantitation strategies were obtained. From computational analysis we conclude that labeling of free amino groups by isotope-coded protein labeling (Lys and free N termini) is better suited for membrane proteins than Cys-based labeling strategies but that quantitation of integral membrane proteins remains cumbersome compared with soluble proteins. Nevertheless we could quantify 155 membrane proteins; 101 of these had transmembrane domains. We compared two growth states that strongly affect the energy supply of the cells: aerobic versus anaerobic/phototrophic conditions. The photosynthetic protein bacteriorhodopsin is the most highly regulated protein. As expected, several other membrane proteins involved in aerobic or anaerobic energy metabolism were found to be regulated, but in total, however, the number of regulated proteins is rather small.
Constitutively activating internal tandem duplication (ITD) alterations of the receptor tyrosine kinase FLT3 (Fms-like tyrosine kinase 3) are common in acute myeloid leukemia (AML) and classifies FLT3 as an attractive therapeutic target. So far, applications of FLT3 small molecule inhibitors have been investigated primarily in FLT3-ITD+ patients. Only recently, a prolonged event-free survival has been observed in AML patients who were treated with the multikinase inhibitor sorafenib in addition to standard therapy. Here, we studied the sorafenib effect on proliferation in a panel of 13 FLT3-ITD− and FLT3-ITD+ AML cell lines. Sorafenib IC50 values ranged from 0.001 to 5.6 μm, whereas FLT3-ITD+ cells (MOLM-13, MV4-11) were found to be more sensitive to sorafenib than FLT3-ITD− cells. However, we identified two FLT3-ITD− cell lines (MONO-MAC-1 and OCI-AML-2) which were also sorafenib sensitive. Phosphoproteome analyses revealed that the affected pathways differed in sorafenib sensitive FLT3-ITD− and FLT3-ITD+ cells. In MV4-11 cells sorafenib suppressed mTOR signaling by direct inhibition of FLT3. In MONO-MAC-1 cells sorafenib inhibited the MEK/ERK pathway. These data suggest that the FLT3 status in AML patients might not be the only factor predicting response to treatment with sorafenib. Constitutively activating internal tandem duplication (ITD) alterations of the receptor tyrosine kinase FLT3 (Fms-like tyrosine kinase 3) are common in acute myeloid leukemia (AML) and classifies FLT3 as an attractive therapeutic target. So far, applications of FLT3 small molecule inhibitors have been investigated primarily in FLT3-ITD+ patients. Only recently, a prolonged event-free survival has been observed in AML patients who were treated with the multikinase inhibitor sorafenib in addition to standard therapy. Here, we studied the sorafenib effect on proliferation in a panel of 13 FLT3-ITD− and FLT3-ITD+ AML cell lines. Sorafenib IC50 values ranged from 0.001 to 5.6 μm, whereas FLT3-ITD+ cells (MOLM-13, MV4-11) were found to be more sensitive to sorafenib than FLT3-ITD− cells. However, we identified two FLT3-ITD− cell lines (MONO-MAC-1 and OCI-AML-2) which were also sorafenib sensitive. Phosphoproteome analyses revealed that the affected pathways differed in sorafenib sensitive FLT3-ITD− and FLT3-ITD+ cells. In MV4-11 cells sorafenib suppressed mTOR signaling by direct inhibition of FLT3. In MONO-MAC-1 cells sorafenib inhibited the MEK/ERK pathway. These data suggest that the FLT3 status in AML patients might not be the only factor predicting response to treatment with sorafenib. In acute myeloid leukemia (AML) 1The abbreviations used are: AML, acute myeloid leukemia; FLT3, Fms-like tyrosine kinase 3; ITD, internal tandem duplications; MSA, multistage activation; PEP, posterior error probability.1The abbreviations used are: AML, acute myeloid leukemia; FLT3, Fms-like tyrosine kinase 3; ITD, internal tandem duplications; MSA, multistage activation; PEP, posterior error probability. the Fms like tyrosine kinase 3 (FLT3) gene is frequently altered by the insertion of internal tandem duplications (ITD) in the juxtamembrane domain or by point mutations in the tyrosine kinase domain (TKD). These genetic alterations lead to an aberrant activation of downstream signaling proteins and promote cell proliferation of AML cells (1.Meshinchi S. Appelbaum F.R. Structural and functional alterations of FLT3 in acute myeloid leukemia.Clin. Cancer Res. 2009; 15: 4263-4269Crossref PubMed Scopus (169) Google Scholar). Deregulated kinases are promising targets in the treatment of cancer. Numerous FLT3 kinase inhibitors such as lestaurtinib (CEP-701) (2.Levis M. Ravandi F. Wang E.S. Baer M.R. Perl A. Coutre S. Erba H. Stuart R.K. Baccarani M. Cripe L.D. Tallman M.S. Meloni G. Godley L.A. Langston A.A. Amadori S. Lewis I.D. Nagler A. Stone R. Yee K. Advani A. Douer D. Wiktor-Jedrzejczak W. Juliusson G. Litzow M.R. Petersdorf S. Sanz M. Kantarjian H.M. Sato T. Tremmel L. Bensen-Kennedy D.M. Small D. Smith B.D. Results from a randomized trial of salvage chemotherapy followed by lestaurtinib for patients with FLT3 mutant AML in first relapse.Blood. 2011; 117: 3294-3301Crossref PubMed Scopus (314) Google Scholar), midostaurin (PKC412) (3.Fischer T. Stone R.M. Deangelo D.J. Galinsky I. Estey E. Lanza C. Fox E. Ehninger G. Feldman E.J. Schiller G.J. Klimek V.M. Nimer S.D. Gilliland D.G. Dutreix C. Huntsman-Labed A. Virkus J. Giles F.J. Phase IIB trial of oral Midostaurin (PKC412), the FMS-like tyrosine kinase 3 receptor (FLT3) and multi-targeted kinase inhibitor, in patients with acute myeloid leukemia and high-risk myelodysplastic syndrome with either wild-type or mutated FLT3.J Clin Oncol. 2010; 28: 4339-4345Crossref PubMed Scopus (403) Google Scholar), and quizartinib (AC220) (4.Cortes J.E. Kantarjian H. Foran J.M. Ghirdaladze D. Zodelava M. Borthakur G. Gammon G. Trone D. Armstrong R.C. James J. Levis M. Phase I study of quizartinib administered daily to patients with relapsed or refractory acute myeloid leukemia irrespective of FMS-like tyrosine kinase 3-internal tandem duplication status.J Clin Oncol. 2013; 31: 3681-3687Crossref PubMed Scopus (285) Google Scholar) have been developed and evaluated either in clinical trials as monotherapy or in combination with standard chemotherapeutic protocols in the last years. Sorafenib is a multikinase inhibitor targeting different receptor tyrosine kinases including FLT3, vascular endothelial growth factor receptor (VEGFR), Kit and RET, which play an important role during myeloid cell differentiation (5.Wilhelm S. Carter C. Lynch M. Lowinger T. Dumas J. Smith R.A. Schwartz B. Simantov R. Kelley S. Discovery and development of sorafenib: a multikinase inhibitor for treating cancer.Nat. Rev. Drug Discov. 2006; 5: 835-844Crossref PubMed Scopus (1406) Google Scholar). Several preclinical studies have demonstrated that AML cells with activating FLT3 receptor mutations are sensitive against sorafenib (6.Auclair D. Miller D. Yatsula V. Pickett W. Carter C. Chang Y. Zhang X. Wilkie D. Burd A. Shi H. Rocks S. Gedrich R. Abriola L. Vasavada H. Lynch M. Dumas J. Trail P. Wilhelm S.M. Antitumor activity of sorafenib in FLT3-driven leukemic cells.Leukemia. 2007; 21: 439-445Crossref PubMed Scopus (144) Google Scholar, 7.Zhang W. Konopleva M. Shi Y. Mcqueen T. Harris D. Ling X. Estrov Z. Quintás-cardama A. Small D. Cortes J. Andreeff M. Mutant FLT3: A direct target of sorafenib in acute myelogenous leukemia.J. Nat. Cancer Inst. 2008; 6: 184-198Crossref Scopus (302) Google Scholar, 8.Pratz K.W. Sato T. Murphy K.M. Stine A. Rajkhowa T. Levis M. FLT3-mutant allelic burden and clinical status are predictive of response to FLT3 inhibitors in AML.Blood. 2010; 115: 1425-1432Crossref PubMed Scopus (191) Google Scholar). Recently, sorafenib has been studied as monotherapy (9.Borthakur G. Kantarjian H. Ravandi F. Zhang W. Konopleva M. Wright J.J. Faderl S. Verstovsek S. Mathews S. Andreeff M. Cortes J.E. Phase I study of sorafenib in patients with refractory or relapsed acute leukemias.Haematologica. 2011; 96: 62-68Crossref PubMed Scopus (156) Google Scholar) or in combination with chemotherapeutics in clinical trials (10.Ravandi F. Arana Yi C. Cortes J.E. Levis M. Faderl S. Garcia-Manero G. Jabbour E. Konopleva M. O'Brien S. Estrov Z. Borthakur G. Thomas D. Pierce S. Brandt M. Pratz K. Luthra R. Andreeff M. Kantarjian H. Final report of phase II study of sorafenib, cytarabine and idarubicin for initial therapy in younger patients with acute myeloid leukemia.Leukemia. 2014; 28: 1543-1545Crossref PubMed Scopus (68) Google Scholar, 11.Macdonald D.A. Assouline S.E. Brandwein J. Kamel-Reid S. Eisenhauer E.A. Couban S. Caplan S. Foo A. Walsh W. Leber B. A phase I/II study of sorafenib in combination with low dose cytarabine in elderly patients with acute myeloid leukemia or high-risk myelodysplastic syndrome from the National Cancer Institute of Canada Clinical Trials Group: trial IND.186.Leuk. Lymphoma. 2013; 54: 760-766Crossref PubMed Scopus (37) Google Scholar, 12.Serve H. Krug U. Wagner R. Sauerland M.C. Heinecke A. Brunnberg U. Schaich M. Ottmann O. Duyster J. Wandt H. Fischer T. Giagounidis A. Neubauer A. Reichle A. Aulitzky W. Noppeney R. Blau I. Kunzmann V. Stuhlmann R. Krämer A. Kreuzer K.-A. Brandts C. Steffen B. Thiede C. Müller-Tidow C. Ehninger G. Berdel W.E. Sorafenib in combination with intensive chemotherapy in elderly patients with acute myeloid leukemia: results from a randomized, placebo-controlled trial.J. Clin. Oncol. 2013; 31: 3110-3118Crossref PubMed Scopus (257) Google Scholar). Indeed, response rates for sorafenib in patients with FLT3-ITD+ are often higher than in patients without FLT3 alterations, but significant differences in overall survival have not been observed (10.Ravandi F. Arana Yi C. Cortes J.E. Levis M. Faderl S. Garcia-Manero G. Jabbour E. Konopleva M. O'Brien S. Estrov Z. Borthakur G. Thomas D. Pierce S. Brandt M. Pratz K. Luthra R. Andreeff M. Kantarjian H. Final report of phase II study of sorafenib, cytarabine and idarubicin for initial therapy in younger patients with acute myeloid leukemia.Leukemia. 2014; 28: 1543-1545Crossref PubMed Scopus (68) Google Scholar). Especially, elderly patients did not benefit from a sorafenib therapy (12.Serve H. Krug U. Wagner R. Sauerland M.C. Heinecke A. Brunnberg U. Schaich M. Ottmann O. Duyster J. Wandt H. Fischer T. Giagounidis A. Neubauer A. Reichle A. Aulitzky W. Noppeney R. Blau I. Kunzmann V. Stuhlmann R. Krämer A. Kreuzer K.-A. Brandts C. Steffen B. Thiede C. Müller-Tidow C. Ehninger G. Berdel W.E. Sorafenib in combination with intensive chemotherapy in elderly patients with acute myeloid leukemia: results from a randomized, placebo-controlled trial.J. Clin. Oncol. 2013; 31: 3110-3118Crossref PubMed Scopus (257) Google Scholar). However, results of the randomized SORAML study showed a prolonged event-free survival in AML patients (< 60 years), who were treated with sorafenib in addition to standard induction and consolidation therapy (13.Röllig C. Serve H. Hüttmann A. Noppeney R. Müller-Tidow C. Krug U. Baldus C.D. Brandts C.H. Kunzmann V. Einsele H. Krämer A. Schäfer-Eckart K. Neubauer A. Burchert A. Giagounidis A. Krause S.W. Mackensen A. Aulitzky W. Herbst R. Hänel M. Kiani A. Frickhofen N. Kullmer J. Kaiser U. Link H. Geer T. Reichle A. Junghanß C. Repp R. Heits F. Dürk H. Hase J. Klut I.-M. Illmer T. Bornhäuser M. Schaich M. Parmentier S. Görner M. Thiede C. von Bonin M. Schetelig J. Kramer M. Berdel W.E. Ehninger G. Addition of sorafenib versus placebo to standard therapy in patients aged 60 years or younger with newly diagnosed acute myeloid leukaemia (SORAML): a multicentre, phase 2, randomised controlled trial.Lancet Oncol. 2015; 16: 1691-1699Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar). Of interest, only 17% of all patients in this study had the status of FLT3-ITD+. Therefore, FLT-3 ITD alone may not be sufficient to predict sorafenib response and the addition of other biomarkers may be required to improve the prediction accuracy. Here, we hypothesized, that the activation of other protein kinases beside FLT3 might predict the AML cell responsiveness to sorafenib. Advances in sample processing, mass spectrometry, and computer algorithms have enabled the application of mass spectrometry-based proteomics to monitoring phosphorylation events on a global scale, allowing the identification and quantification of thousands of phosphorylation sites in a single experiment (14.Olsen J.V. Blagoev B. Gnad F. Macek B. Kumar C. Mortensen P. et al.Global, in vivo, and site-specific phosphorylation dynamics in signaling networks.Cell. 2006; 127: 635-648Abstract Full Text Full Text PDF PubMed Scopus (2825) Google Scholar, 15.Schaab C. Analysis of phosphoproteomics data.Methods Mol. Biol. 2011; 696: 41-57Crossref PubMed Scopus (13) Google Scholar, 16.Sharma K. D'Souza R.C.J. Tyanova S. Schaab C. Wiœniewski J.R. Cox J. Mann M. Ultradeep human phosphoproteome reveals a distinct regulatory nature of tyr and ser/thr-based signaling.Cell Rep. 2014; 8: 1583-1594Abstract Full Text Full Text PDF PubMed Scopus (639) Google Scholar, 17.Riley N.M. Coon J.J. Phosphoproteomics in the age of rapid and deep proteome profiling.Anal. Chem. 2016; 88: 74-94Crossref PubMed Scopus (166) Google Scholar). When applied to cells treated with small molecules or antibodies, these methods allow the unbiased analysis of the mode of action of these agents (18.Conradt L. Godl K. Schaab C. Tebbe A. Eser S. Diersch S. Michalski C.W. Kleeff J. Schnieke A. Schmid R.M. Saur D. Schneider G. Disclosure of erlotinib as a multikinase inhibitor in pancreatic ductal adenocarcinoma.Neoplasia. 2011; 13: 1026-1034Crossref PubMed Scopus (40) Google Scholar, 19.Weigand S. Herting F. Maisel D. Nopora A. Voss E. Schaab C. Klammer M. Tebbe A. Global quantitative phosphoproteome analysis of human tumor xenografts treated with a CD44 antagonist.Cancer Res. 2012; 72: 4329-4339Crossref PubMed Scopus (31) Google Scholar, 20.Pan C. Olsen J.V. Daub H. Mann M. Global effects of kinase inhibitors on signaling networks revealed by quantitative phosphoproteomics.Mol. Cell. Proteomics. 2009; 8: 2796-2808Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). Recently, phosphoproteomics was also applied in the context of AML to study the mode of action of kinase inhibitors (21.Alcolea M.P. Casado P. Rodriguez-Prados J.C. Vanhaesebroeck B. Cutillas P.R. Phosphoproteomic analysis of leukemia cells under basal and drug-treated conditions identifies markers of kinase pathway activation and mechanisms of resistance.Mol. Cell. Proteomics. 2014; 11: 453-466Abstract Full Text Full Text PDF Scopus (58) Google Scholar, 22.Bertacchini J. Guida M. Accordi B. Mediani L. Martelli A.M. Barozzi P. Petricoin E. Liotta L. Milani G. Giordan M. Luppi M. Forghieri F. De Pol A. Cocco L. Basso G. Marmiroli S. Feedbacks and adaptive capabilities of the PI3K/Akt/mTOR axis in Acute Myeloid Leukemia revealed by pathway selective inhibition and phosphoproteome analysis.Leukemia. 2014; 28: 2197-2205Crossref PubMed Scopus (54) Google Scholar) or to discover predictive biomarker candidates for kinase inhibitors (23.Schaab C. Oppermann F.S. Klammer M. Pfeifer H. Tebbe A. Oellerich T. Krauter J. Levis M. Perl A.E. Daub H. Steffen B. Godl K. Serve H. Global phosphoproteome analysis of human bone marrow reveals predictive phosphorylation markers for the treatment of acute myeloid leukemia with quizartinib.Leukemia. 2014; 28: 716-719Crossref PubMed Scopus (23) Google Scholar). In the present study, we analyzed the effects of the multityrosine-kinase sorafenib on a panel of AML cell lines with different FLT3 status. Moreover, a phosphoproteome analysis was performed to understand the different modes of action in sorafenib sensitive and less sensitive AML cell lines. Our results show that subsets of both FLT3 wild-type and ITD mutated cell lines respond to treatment with sorafenib. However, the responses in these cell lines are mediated through different modes of action. Thus, beside the FLT3 status, the activation of certain signaling pathways might be used to predict the responsiveness of AML cells to treatment with sorafenib. Furthermore, we used a chemical proteomics approach to identify protein binding partners of sorafenib with differential binding affinity or differential expression in FLT3-ITD and FLT3 wild-type cell lines. Our data identified novel target pathways of sorafenib in AML cells. A panel of 13 AML cell lines with different morphology and FLT3 status (Table I) was used to study the effect of sorafenib treatment. All cell lines were purchased from Deutsche Sammlung von Mikroorganismen und Zellkultur (DSMZ, Braunschweig, Germany) and cultured according to the supplier's protocols. Stocks of cryopreserved cells were applied quickly after receipt from DSMZ and cells were passaged for less than 6 month. Cell lines were carefully checked for immune phenotype using flow cytometry and morphologic consistency by microscope. In addition, cell cultures were checked for mycoplasma contamination using Mycoalert Detection Kit (Lonza, Basel, Switzerland).Table ICharacteristics of AML cell lines. The columns list the FAB classification, the FLT3-ITD and FLT3-TKD status and the cell growth IC50sCell linesFABFLT3-ITDFLT3-TKDIC50 (72h)MOLM-13M5a+−0.001 μmMV4-11M5a+−0.001 μmMONO-MAC-1M5a−−0.02 μmOCI-AML-2M4−−0.08 μmSH-2M2−−0.4 μmKG-1M6a−−0.5 μmNB-4M3−−1.1 μmHL-60M2−−1.3 μmOCI-M1M6−−1.7 μmNOMO-1M5a−−1.9 μmSKM-1M5a−−2.8 μmPL-21M3+−4.1 μmM-07eM7−−5.6 μm Open table in a new tab NB-4, HL-60, MOLM-13, MV4-11, NOMO-1, and KG-1 cell lines were cultured Roswell Park Memorial Institute (RPMI) 1640 medium (Biochrom AG, Berlin, Germany), supplemented with 10% fetal bovine serum (FBS) (PAA Laboratories GmbH, Pasching, Austria) and 100 μg/ml penicillin and streptomycin (P/S) (PAA Laboratories GmbH). MONO-MAC-1 cell line was cultured in RPMI 1640, supplemented with 10% FBS, 2 mm l-Glutamine (Biochrom AG), 1 mm Na-Pyruvate (Biochrom AG) and 100 μg/ml P/S. PL-21 and SKM-1 cell lines were cultured in RPMI 1640, supplemented with 20% FBS and100 μg/ml P/S. OCI-AML-2 cell line was cultured in alpha Minimum Essential Eagle's medium supplemented with 10% FBS and 100 μg/ml P/S. M-07e cells were cultured in RPMI 1640, supplemented with 20% FBS, 10 ng/ml GM-CSF (Bayer Healthcare Pharmaceuticals, LLC Seattle, WA) and and100 μg/m P/S. OCI-M1 and SH-2 cell lines were cultured in Iscove's Modified Dulbecco's Medium supplemented with 20% FBS and 100 μg/mg P/S. Cultures were maintained in a 5% CO2 humidified atmosphere at 37°C. Genomic DNA was extracted from all cell lines using NucleoBond CB 100 (Macherey-Nagel, Dueren, Germany) and screened for FLT3-ITD and FLT3-TKD mutations. The presence of an ITD was confirmed by PCR amplification of the juxtamembrane domain from FLT3 exons 14 to 15 with a fluorescently labeled primer followed by fragment analysis using ABI Prism 3100 Avant Genetic Analyzer (Applied Biosystems, Darmstadt, Germany). Point mutations at codon 835 and 836 were analyzed by amplification of FLT3 exon 20 followed by restriction fragment length polymorphism with EcoRV and gel electrophoretic analysis of cleavage products as previously reported (24.Kiyoi H. Naoe T. Yokota S. Nakao M. Minami S. Kuriyama K. Takeshita A. Saito K. Hasegawa S. Shimodaira S. Tamura J. Shimazaki C. Matsue K. Kobayashi H. Arima N. Suzuki R. Morishita H. Saito H. Ueda R. Ohno R. Internal tandem duplication of FLT3 associated with leukocytosis in acute promyelocytic leukemia.. 1997; 11: 1447-1452Google Scholar). Sorafenib was purchased from LC Laboratories (Woburn, MA). Linifanib and vandetanib were obtained from Hycultec GmbH (Beutelsbach, Germany). All inhibitors were dissolved in dimethyl sulfoxide (DMSO). Stock solutions were prepared with a concentration of 10 mm and stored at −20 °C. Working solutions were prepared freshly by diluting stock solution with media. Cells (3.33 × 105/ml) were seeded in 24-well or 96-well plates (Nunc, Langenselbold, Germany) and incubated with increasing concentrations of sorafenib, linifanib, or vandetanib (0.001–10 μm) for up to 72 h. Control cells were cultured in a medium containing the same concentration of DMSO as the treated cells. After treatment, cell counts were determined using trypan blue staining. Metabolic activity was determined using tetrazolium compound WST-1 (Roche Applied Science, Mannheim, Germany) as previously described (25.Schult C. Dahlhaus M. Ruck S. Sawitzky M. Amoroso F. Lange S. Etro D. Glass A. Fuellen G. Boldt S. Wolkenhauer O. Neri L.M. Freund M. Junghanss C. The multikinase inhibitor Sorafenib displays significant antiproliferative effects and induces apoptosis via caspase 3, 7 and PARP in B- and T-lymphoblastic cells.BMC Cancer. 2014; 10: 560Crossref Scopus (33) Google Scholar). The IC50 value, at which 50% of the cell growth is inhibited compared with DMSO control, was calculated with probit analysis using the SPSS software, version 22 (IBM, Ehningen, Germany). For examination of cell morphology, cells were fixed on glass slides in 200 μl cell suspension (based on DMSO treated cells) using a Shandon 3 Cytospin centrifuge (Shandon, Frankfurt/Main, Germany). After centrifugation, glass slides were air-dried and incubated with May Gruenwald and Giemsa solution (Merck, Darmstadt, Germany) as previously reported (26.Kretzschmar C. Roolf C. Langhammer T.-S. Sekora A. Pews-Davtyan A. Beller M. Frech M.J. Eisenlöffel C. Rolfs A. Junghanss C. The novel arylindolylmaleimide PDA-66 displays pronounced antiproliferative effects in acute lymphoblastic leukemia cells.BMC Cancer. 2014; 14: 71Crossref PubMed Scopus (14) Google Scholar). Cell morphology was examined and visualized with Evos XL Core Imaging System (Life technologies, Darmstadt, Germany). For analysis of apoptosis, cells were stained with Annexin V-Fluorescein isothiocyanate (FITC) (BD Biosciences, Heidelberg, Germany) and propidium iodide (PI) (Sigma Aldrich, St. Louis, MO) and analyzed by flow cytometry as previously described (25.Schult C. Dahlhaus M. Ruck S. Sawitzky M. Amoroso F. Lange S. Etro D. Glass A. Fuellen G. Boldt S. Wolkenhauer O. Neri L.M. Freund M. Junghanss C. The multikinase inhibitor Sorafenib displays significant antiproliferative effects and induces apoptosis via caspase 3, 7 and PARP in B- and T-lymphoblastic cells.BMC Cancer. 2014; 10: 560Crossref Scopus (33) Google Scholar). Measurement of early apoptotic (Annexin V-FITC+ and PI−) and late apoptotic cells (Annexin V-FITC+ and PI+) was performed using FACSCalibur (Becton and Dickinson, Heidelberg, Germany). For phosphoproteomics MV4-11, MONO-MAC-1, and SKM-1 cells were cultured in SILAC media containing 60.6 mg/ml Arg0 and 50 mg/ml Lys0 (light label), 62.3 mg/ml Arg6 and 61.1 mg/ml Lys4 (medium label), and 63.4 mg/ml Arg10 and 62.2 mg/ml Lys8 (heavy label). SILAC media were supplemented with 10% dialyzed fetal bovine serum (Invitrogen, Carlsbad, CA), 1% penicillin and streptomycin (Biochrom AG, Berlin, Germany), 1% glutamine (PAA, Pasching, Austria) and 1% sodium pyruvate (PAA). Cells were cultured for at least seven cell doublings to reach > 95% of incorporation of amino acids arginine and lysine, before treatment for 2 hours with DMSO, 0.01 or 0.2 μm sorafenib, respectively. Cells were lysed in ice-cold lysis buffer (8 M urea, 75 mm NaCl, 50 mm Tris pH 8.2, 5 mm EDTA, 5 mm EGTA, 10 mm sodium pyrophosphate, 10 mm NaF, 10 mm beta-glycerophosphate, 2 mm Na3VO4, phosphatase inhibitor mixture 1 and 2 (Sigma-Aldrich) and protease inhibitor mixture tablet (Roche Applied Science, Mannheim, Germany)) and sonicated three times for 1 min on ice. Lysates from the differently SILAC-encoded cells were pooled, reduced with dithiothreitol, alkylated with iodoacetamide and digested in-solution with endoproteinase Lys-C (Wako, Neuss, Germany) and trypsin (Promega, Mannheim, Germany) as described before (27.Daub H. Olsen J.V. Bairlein M. Gnad F. Oppermann F.S. Körner R. Greff Z. Kéri G. Stemmann O. Mann M. Kinase-selective enrichment enables quantitative phosphoproteomics of the kinome across the cell cycle.Mol. Cell. 2008; 31: 438-448Abstract Full Text Full Text PDF PubMed Scopus (491) Google Scholar). Lys-C and trypsin were used in excess to the protease inhibitors. Tryptic peptides were desalted using reversed-phase 500 mg C18 SepPak cartridges (Waters, Eschborn, Germany) and fractionated by SCX chromatography (250 × 9.4 mm polySULFOETHYL A column, 200 Å pore size, 5 μm particle size (PolyLC)) operated with ÄKTA explorer system (GE Healthcare, München, Germany) as described previously (28.Villén J. Gygi S.P. The SCX/IMAC enrichment approach for global phosphorylation analysis by mass spectrometry.Nat. Protoc. 2008; 3: 1630-1638Crossref PubMed Scopus (500) Google Scholar). The resulting twelve peptide samples were desalted using reversed-phase 100 mg C18 SepPak cartridges (Waters) before phosphopeptide enrichment with immobilized metal affinity chromatography (IMAC) in two successive enrichment steps essentially as described before (28.Villén J. Gygi S.P. The SCX/IMAC enrichment approach for global phosphorylation analysis by mass spectrometry.Nat. Protoc. 2008; 3: 1630-1638Crossref PubMed Scopus (500) Google Scholar, 29.Rappsilber J. Mann M. Ishihama Y. Protocol for micro purification, enrichment, pre fractionation and storage of peptides for proteomics using StageTips.Nat. Protoc. 2007; 2: 1896-1906Crossref PubMed Scopus (2590) Google Scholar, 30.Dephoure N. Zhou C. Villén J. Beausoleil S.A. Bakalarski C.E. Elledge S.J. Gygi S.P. A quantitative atlas of mitotic phosphorylation.Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 10762-10767Crossref PubMed Scopus (1255) Google Scholar). The resulting 24 samples per cell line were subjected to LC-MS/MS analysis. Phosphopeptide-enriched samples were loaded onto a reverse phase analytical column (packed in-house with C18 beads), resolved by an acetonitrile gradient using an Easy nLC system (Thermo Fisher Scientific, Waltham, MA) and directly electrosprayed via a nanoelectrospray ion source into an LTQ Orbitrap Velos mass spectrometer (Thermo Fisher Scientific) (27.Daub H. Olsen J.V. Bairlein M. Gnad F. Oppermann F.S. Körner R. Greff Z. Kéri G. Stemmann O. Mann M. Kinase-selective enrichment enables quantitative phosphoproteomics of the kinome across the cell cycle.Mol. Cell. 2008; 31: 438-448Abstract Full Text Full Text PDF PubMed Scopus (491) Google Scholar, 31.Olsen J.V. Schwartz J.C. Griep-Raming J. Nielsen M.L. Damoc E. Denisov E. Lange O. Remes P. Taylor D. Splendore M. Wouters E.R. Senko M. Makarov A. Mann M. Horning S. A dual pressure linear ion trap Orbitrap instrument with very high sequencing speed.Mol. Cell. Proteomics. 2009; 8: 2759-2769Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar). The Orbitrap mass spectrometer was operated in a data-dependent acquisition mode to automatically switch between full scans in the orbitrap mass analyzer (resolution r = 60.000) and the acquisition of CID fragmentation spectra (MS/MS mode) of the fifteen most abundant peptide ions in the linear ion trap (LTQ). For optimal phosphopeptide dissociation the multistage activation (MSA) modus was selected. Identification and quantification of phosphorylation sites was performed using the MaxQuant software (version 1.4.0.6) (32.Cox J. Mann M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification.Nat Biotechnol. 2008; 26: 1367-1372Crossref PubMed Scopus (9223) Google Scholar) using the human Swissprot database including isoform sequences (version 07.2013 with 39,268 entries). Carbamidomethylation of cysteine was set as a fixed modification. Oxidation of methionine, N-terminal acetylation and phosphorylation on serine, threonine and tyrosine were set as variable modifications. The minimum required peptide length was seven amino acids, the minimum ratio count was set to two, the maximum number of missed cleavages was set to two, the main search tolerance was set to 4.5 ppm, and the MS/MS tolerance was set to 10 ppm. In-silico peptides were generated with "Trypsin/P," which cleaves after lysine and arginine also if a proline follows. A false discovery rate (FDR) of 0.01 was selected for both protein and peptide identifications and a posterior error probability (PEP) below or equal to 0.1 for each peptide-to-spectral match was required. The match between runs option was enabled for a time window of 1 min. Phosphorylation sites were quantified using the SILAC ratios. In case multiple peptides were detected for the same site, the one with the least modifications was used for quantification. The mass-spectrometry raw data and the MaxQuant output tables have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository with the data set identifier PXD004442. Intensity ratios were transformed to reflect sorafenib versus DMSO control. Phosphorylation sites of low confidence (non-Class-I), reverse hits, or potential contaminants were removed. Significantly regulated phosphorylation sites were identified using the parametric MeanRank test (33.Klammer M. Dybowski J.N. Hoffmann D. Schaab C. Identification of significant features by the global mean rank test.PLoS ONE. 2014; 9: e104504Crossref PubMed Scopus (15) Google Scholar). Significance testing was performed separately for all three cell models, using sites that were quantified in at least two of the three replicates. The MeanRank test intrinsically handles missing values and automatically corrects for multiple hypothesis testing. Phosphorylation sites were considered significantly regulated at an FDR of 0.05. Enrichment analyses of gene ontology (GO) cellular component categories and KEGG pathways were performed for phosphoproteins regulated upon treatment with sorafenib compared with all phosphoproteins harboring quantified phosphorylation sites by non-conditional hypergeometric testing using Fisher's Exact Test. Enrichment of kinase substrates among significantly regulated phosphorylation sites was performed analogously. To correct for multiple hypothesis testing, FDR correction according to Benjamini-Hochberg was performed (FDR 0.05) (34.Benjamini Y. Hochberg Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing.J. R. Stat. Soc. 1995; 57: 289-300Google Scholar). Kinase selectivity of sorafenib in MV4-11 and MONO-MAC-1 cells was determined using KinAffinity as described (35.Sharma K. Weber C. Bairlein M. Greff Z. Kéri G. Cox J. Olsen J.V. Daub H. Proteomics strategy for quantitative protein interaction profiling in cell extracts.Nat. Methods. 2014; 6: 741-744Crossref Scopus (116) G
A placebo-controlled phase 3 trial demonstrated that the epidermal growth factor receptor (EGFR) inhibitor erlotinib in combination with gemcitabine was especially efficient in a pancreatic ductal adenocarcinoma (PDAC) subgroup of patients developing skin toxicity. However, EGFR expression was not predictive for response, and markers to characterize an erlotinib-responding PDAC group are currently missing. In this work, we observed high erlotinib IC50 values in a panel of human and murine PDAC cell lines. Using EGFR small interfering RNA, we detected that the erlotinib response was marginally influenced by EGFR. To find novel EGFR targets, we used an unbiased chemical proteomics approach for target identification and quality-controlled target affinity determination combined with quantitative mass spectrometry based on stable isotope labeling by amino acids in cell culture. In contrast to gefitinib, we observed a broad target profile of erlotinib in PDAC cells by quantitative proteomics. Six protein kinases bind to erlotinib with similar or higher affinity (Kd = 0.09-0.358 μM) than the EGFR (Kd 0.434 μM). We provide evidence that one of the novel erlotinib targets, ARG, contributes in part to the erlotinib response in a PDAC cell line. Our data show that erlotinib is a multikinase inhibitor, which can act independent of EGFR in PDAC. These findings may help to monitor future erlotinib trials in the clinic.
Human protein biomarker discovery relies heavily on pre-clinical models, in particular established cell lines and patient-derived xenografts, but confirmation studies in primary tissue are essential to demonstrate clinical relevance. We describe in this study the process that was followed to clinically translate a 5-protein response signature predictive for the activity of an anti-HER3 monoclonal antibody (lumretuzumab) originally measured in fresh frozen xenograft tissue. We detail the development, qualification, and validation of the multiplexed targeted mass spectrometry assay used to assess the signature performance in formalin-fixed, paraffin-embedded human clinical samples collected in a phase Ib trial designed to evaluate lumretuzumab in patients with metastatic breast cancer. We believe that the strategy delineated here provides a path forward to avoid the time- and cost-consuming step of having to develop immunological reagents against unproven targets. We expect that mass spectrometry-based platforms may become part of a rational process to rapidly test and qualify large number of candidate biomarkers to identify the few that stand a chance for further development and validation.
Abstract Since targeted drugs selectively block molecular pathways that are typically over-activated in tumour cells, they are more precise and show fewer adverse effects than traditional chemotherapeutic agents. At the same time the proportion of patients that benefit from targeted drugs is smaller. Therefore, predictive molecular markers are needed to confidently predict the patient's response to a specific therapy. Such markers would facilitate therapy personalization, where the selected therapy is based on the molecular profile of the patient. We sought to identify a signature of protein phosphorylations that predicts the response to dasatinb in non-small cell lung cancer cell lines. A panel of cell lines were profiled in a global, unbiased, phosphoproteomics study yielding quantitative information for roughly 25,000 phosphorylation sites. The identical cell lines were tested for their response to dasatinib. From the phosphoproteome profiles, we identified a signature of twelve phosphorylation sites that can accurately predict dasatinib sensitivity. Four of the phosphorylation sites belong to integrin α4, a protein that mediates cell-matrix or cell-cell adhesion. We evaluated the performance of this signature in a cross-validation set-up and investigated the robustness of the selected predictive features. Finally, we confirmed the predictive power of the signature in an independent set of breast cancer cell lines. We showed that the phosphorylations of integrin β4 as well as eight further proteins are candidate biomarkers for predicting response to dasatinib in solid tumours. Furthermore, we demonstrated that identifying predictive phosphorylation signatures from global, quantitative phosphoproteomic data is possible, and opens a new path to discovering molecular markers for response prediction. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4808. doi:1538-7445.AM2012-4808
