Leukemia (2013) 27, 305–314 & 2013 Macmillan Publishers Limited All rights reserved 0887-6924/13 www.nature.com/leu ORIGINAL ARTICLE Receptor kinase profiles identify a rationale for multitarget kinase inhibition in immature T-ALL L Lhermitte1, R Ben Abdelali1, P Villare`se1, N Bedjaoui1, V Guillemot2, A Trinquand1, M Libura1, AS Bedin1, A Petit3, H Dombret4, G Leverger3, N Ifrah5, O Hermine1, E Macintyre1 and V Asnafi1 Constitutively activated FLT3 signaling is common in acute myeloid leukemia, and is currently under evaluation for targeted therapy, whereas little data is available in T-cell acute lymphoblastic leukemia (T-ALL). We analyzed 357 T-ALL cases for FLT3 mutations and transcript expression. FLT3 mutations (3% overall) and overexpression (FLT3 high expresser (FLT3High)) were restricted to immature/TCRgd T-ALLs. In vitro FLT3 inhibition induced apoptosis in only 30% of FLT3High T-ALLs and did not correlate with mutational status. In order to investigate the mechanisms of primary resistance to FLT3 inhibition, a broad quantitative screen for receptor kinome transcript deregulation was performed by Taqman Low Density Array. FLT3 deregulation was associated with overexpression of a network of receptor kinases (RKs), potentially responsible for redundancies and sporadic response to specific FLT3 inhibition. In keeping with this resistance to FLT3 inhibition could be reversed by dual inhibition of FLT3 and KIT with a synergistic effect. We conclude that immature T-ALL may benefit from multitargeted RK inhibition and that exploration of the receptor kinome defines a rational strategy for testing multitarget kinase inhibition in malignant diseases. Leukemia (2013) 27, 305–314; doi:10.1038/leu.2012.177 Keywords: T-ALL; multitarget therapy; receptor kinase; FLT3 INTRODUCTION FLT3 autocrine activation has also been reported in over 70% of T-cell acute lymphoblastic leukemias (T-ALLs) represent expan- AML cases and represents an alternative mechanism for FLT3 15–17 sions of cells arrested at specific stages of thymic development, oncogenic signaling, regardless of FLT3 mutational status. In with the underlying genetic abnormality often determining the AML, FLT3 overexpression associated with autophosphorylation stage of maturation arrest.1 Several recent studies used different and sensitivity to FLT3 inhibition of the receptor and identified 18 approaches but all suggested the existence of an immature an unfavorable prognostic subgroup. Little data is available, subtype (IM) of T-ALL.2–5 IM T-ALLs lack expression of T-cell however, regarding FLT3 expression and receptor tyrosine kinase receptor (TCR) alpha-beta or gamma-delta, cytoplasmic TCR beta profiles in either normal human T-cell maturation or in T-ALL. (cTCRb), and frequently CD4/8 and CD1a markers. In contrast, We assessed oncogenic FLT3 mutation/deregulation in a large expression of CD13 and CD33 myeloid antigens markers, as well as series of T-ALL and identified a concerted activated receptor kinase ‘myeloid’ oncogenic lesions, such as MLL and CALM-AF10 (RK) signature as a mechanism for primary resistance to single-agent rearrangements, are common and relatively specific to this FLT3 inhibition, but sensitivity to dual agent inhibition, thus providing subset of T-ALLs. IM T-ALL, early T-cell precursor leukemia or an original rationale for multi-kinase therapeutic strategies. T/myeloid biphenotypic acute leukemia have been reported to respond poorly to chemotherapy and may therefore benefit from specific targeted therapy.6–9 MATERIALS AND METHODS Tyrosine kinase inhibitors may represent such agents, but Patients efficient evaluation of their potential therapeutic impact requires The study population included 357 unselected patients, consisting of 195 their use in subsets of patients that are most likely to benefit. adults (X15 years) and 162 children (o15 years) with newly diagnosed Constitutive receptor tyrosine kinase activation of the FLT3 T-ALL. Samples were collected centrally for molecular and cellular analysis, pathway by internal tandem duplication (ITD), tyrosine kinase with the support of the adult (GRAALL) and children (FRALLE) protocols. Diagnosis of T-ALL was based on the World Health Organization consensus domain point mutation (D835Y) or an autocrine-loop has been 19 reported in acute myeloid leukemia (AML)10 and MLL rearranged criteria, defined by expression of cytoplasmic CD3, with neither expression of MPO nor strong expression of cytoplasmic CD79a. As such, or hyperdiploid B-cell precursor ALL.11 Activating FLT3 mutations 12,13 mixed phenotype acute leukemias were excluded from the study, in favor have been described in rare cases of T-ALL when they have of pure T-ALL. Maturation stage of arrest was specified further, based been reported to be restricted to the CD117 þ subset in adults or on expression of cTCRbF1 and surface TCR, as previously described.2 associated with a CD4 þ /CD8 À phenotype in pediatric T-ALL, but T-ALL cases were classified as immature (TCR À and cTCRb À ), ab-lineage these data remain controversial.14 (TCRab þ or TCR À /cTCRb þ ) or TCRgd-expressing cases (TCRgd þ ). 1Department of Hematology, Hoˆpital Necker-Enfants-Malades APHP, CNRS UMR 8147, Universite´ Paris-5 Descartes, Paris, France; 2Laboratoire d’Exploration fonctionnelle des Ge´nomes, CEA, Ge´nopole, Evry, France; 3Department of Hematology, Hoˆpital Armand Trousseau, Paris, France; 4Department of Hematology, Hoˆpital St-Louis, Paris, France and 5PRES LUNAM, CHU Angers service des Maladies du Sang et INSERM U 892, Angers, France. Correspondence: Dr V Asnafi, Laboratoire d’He´matologie, Tour Pasteur, Hoˆ pital Necker, 149–161, rue de Se`vres, 75743 Paris cedex 15, France. E-mail: vahid.asnafi@nck.aphp.fr Received 17 September 2011; revised 13 June 2012; accepted 26 June 2012; accepted article preview online 3 July 2012; advance online publication, 27 July 2012 A rationale for multitarget kinase inhibition L Lhermitte et al 306 Normal thymus samples were obtained from children undergoing CD3 expression. Stained samples were analyzed with a FACS Canto II cardiac surgery at the Necker-Enfants-Malades Hospital, with informed (BD Biosciences). Thymic cells were sorted with a combination of CD3, CD4, consent from the parents. CD8, CD34, CD1a and CD45 fluorochrome-labeled antibodies using a FACS Aria II cell-sorter (BD Biosciences) (Supplementary Figure S3). Western Molecular analysis (real-time quantitative PCR, FLT3 mutational blotting of FLT3 was performed using anti-FLT3 antibody (clone S18; Santa status and Taqman Low Density Array (TLDA)) Cruz Biotechnology, Santa Cruz, CA, USA) and anti-phosphotyrosine (clone 4G10) as previously described.24 Phosphorylation of KIT was analyzed An unselected series of 357 T-ALLs with available cDNA were screened for 20 using anti-phospho-c-Kit (Tyr719) (Cell Signaling Technology, Berverly, MA, FLT3 ITD and D835Y mutations, as previously described. FLT3 transcript USA) as previously reported.25 expression was quantified by real-time quantitative PCR for 301 cases. Flow-cytometry monitoring of phosphoepitopes of FLT3 and down- Molecular oncogenetic analyses (TLX1, TLX3, SIL-TAL and CALM-AF10) 2,3 stream signaling molecules was performed using a polyclonal anti- were performed centrally, as previously reported. Results of phospho-FLT3 (Y591) Alexa Fluor 488 (Cell Signaling Technology), transcriptional analysis were expressed as target/reference transcript monoclonal anti-phospho-STAT5 (Y694) Alexa Fluor 647 (Clone 47; BD ratios. ABL and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) Biosciences) and monoclonal anti-phospho-ERK1/2 (T202/Y204) Alexa Fluor were shown to be highly correlated, including for cases harboring the 647 (Clone 20a, BD Biosciences). Leukemic samples were incubated in NUP-ABL abnormality, and as such were used as reference genes culture medium for 3 h and then secondarily subjected to different (Supplementary Figure S1). Quantification of RK expression was performed combinations of activators and/or inhibitors; FL, CEP701 and AC220. on quality-controlled cDNA using predesigned TaqMan Low Density Monitoring of phospho-protein state was performed after 30 min of microfluidic cards (TLDA Human Protein Kinase Array; Applied Biosystems). exposure for FLT3, and 1 h for ERK and STAT5 molecules. Cells were Real-time measurements were performed using the ABI Prism 7900HT immediately fixed by addition of BD CytoFix fixation buffer (BD Biosciences) (Applied Biosystems, Foster City, CA, USA). Samples with GAPDH cycle into the culture well and then incubated for 15 min at 37 1C. After washing, thresholds superior to 21 were excluded from analysis. Each probe value cells were permeabilized using BD PhosFlow Perm Buffer III (BD Biosciences) was normalized with respect to GAPDH copy number and analyzed with 13 according to the manufacturer’s instructions. Samples were then stained Dcycle threshold threshold to discard background noise. Reproducibility of using the above-mentioned reagents added with a backbone combination measurements and correlation with classical real-time quantitative PCR of markers including CD45 V500 (HI30) and CD3 PE (UCHT1) for gating of experiments were validated (Supplementary Figure S2). blast cells. Samples were analyzed using a FACS Canto II (BD Biosciences). Data were processed using Cytobank software.26 Changes in phosphory- Statistical analysis lation of signaling proteins following activator and/or inhibitor exposure All monoparametric measurement comparisons were determined using were calculated using the log2 ratio