Leukemia (2009) 23, 477–485 & 2009 Macmillan Publishers Limited All rights reserved 0887-6924/09 $32.00 www.nature.com/leu ORIGINAL ARTICLE Global target profile of the kinase inhibitor bosutinib in primary chronic myeloid leukemia cells LL Remsing Rix1, U Rix1, J Colinge1, O Hantschel1, KL Bennett1, T Stranzl1,AMu¨ller1, C Baumgartner2, P Valent2, M Augustin3, JH Till3 and G Superti-Furga1 1Center for Molecular Medicine of the Austrian Academy of Sciences (CeMM), Vienna, Austria; 2Division of Hematology and Hemostaseology, Department of Internal Medicine I, Medical University of Vienna, Vienna, Austria and 3Millipore UK Ltd, Gemini Crescent, Dundee Technology Park, Dundee, UK The detailed molecular mechanism of action of second- AMN107) are two such inhibitors that have been approved for generation BCR–ABL tyrosine kinase inhibitors, including the treatment of patients who are resistant or intolerant to perturbed targets and pathways, should contribute to rationa- 5,6 lized therapy in chronic myeloid leukemia (CML) or in other imatinib. An up-and-coming next-generation BCR–ABL affected diseases. Here, we characterized the target profile of inhibitor is bosutinib (SKI-606), a dual Sarcoma/Abelson (SRC/ the dual SRC/ABL inhibitor bosutinib employing a two-tiered ABL) kinase inhibitor that has recently been introduced in approach using chemical proteomics to identify natural binders clinical trials against CML (phase III) and breast cancer (phase in whole cell lysates of primary CML and K562 cells in parallel to II).7–9 Bosutinib has been shown to target several imatinib in vitro kinase assays against a large recombinant kinase panel. resistance-causing BCR–ABL mutants, except the T315I mutant, The combined strategy resulted in a global survey of bosutinib targets comprised of over 45 novel tyrosine and serine/ and is unique in that it does not inhibit either the mast/stem cell threonine kinases. We have found clear differences in the (KIT) or platelet-derived (PDGFR) growth factor receptors, 7,8 target patterns of bosutinib in primary CML cells versus the possibly explaining its higher therapeutic index. Furthermore, K562 cell line. A comparison of bosutinib with dasatinib across bosutinib results in a delayed compensatory upregulation in the whole kinase panel revealed overlapping, but distinct, mitogen-activated protein kinase (MAPK) activity as compared inhibition profiles. Common among those were the SRC, with imatinib, suggesting further differences in their target ABL and TEC family kinases. Bosutinib did not inhibit KIT 10 or platelet-derived growth factor receptor, but prominently profile. targeted the apoptosis-linked STE20 kinases. Although in vivo Recent reports have demonstrated the success of large-scale bosutinib is inactive against ABL T315I, we found this clinically profiling of kinase inhibitors to determine their full spectrum of important mutant to be enzymatically inhibited in the mid- target proteins in, for example, cell lines representing CML nanomolar range. Finally, bosutinib is the first kinase inhibitor (K562) and breast cancer (Hs578T).11–15 In our laboratory, we shown to target CAMK2G, recently implicated in myeloid emphasize identifying drug targets in their clinically relevant leukemia cell proliferation. Leukemia (2009) 23, 477–485; doi:10.1038/leu.2008.334; patient cell populations. Thus, we previously used a chemical published online 27 November 2008 proteomic approach in which we immobilized coupleable Keywords: chronic myeloid leukemia; bosutinib; dasatinib; kinase analogs of imatinib, dasatinib and nilotinib and exposed them profiling; chemical proteomics to total cell lysates not only of the BCR–ABL-positive K562 cell line, but also to extracts of primary CML patient cells, thereby identifying and subsequently validating both novel kinase (BTK, DDR1) and nonkinase (NQO2) targets.14,16 Here, we used a combined chemical proteomic and in vitro profiling method to evaluate (i) the natural target profile of Introduction bosutinib in primary CML and K562 cells and (ii) the activity of bosutinib versus dasatinib against over 270 recombinant Chronic myeloid leukemia (CML) is a three-phase hemato- kinases, thereby maximizing the cross-section of the kinome poietic disease originating in the stem cell compartment. under investigation. This combined approach revealed that A reciprocal translocation between chromosomes 9 and 22 bosutinib, similar to dasatinib, is also a broad specificity kinase results in the expression of the BCR–ABL fusion oncoprotein, a inhibitor, targeting not only SRC and ABL kinases, but also TEC deregulated tyrosine kinase that activates several proliferative 1 family kinases, including BTK, which is known to have host and antiapoptotic signaling pathways. The BCR–ABL inhibitor immunomodulatory activity.17 In contrast to dasatinib, bosutinib imatinib (Gleevec, STI-571) entered the market in 2001 as the 2 does not inhibit KIT or PDGFR, but rather displays a striking first targeted therapeutic against CML; however, it soon affinity toward the STE family of kinases, in particular the STE20 became clear that both primary and secondary, or acquired, subfamily. CAMK2G, a Ca2 þ /calmodulin-dependent protein resistance toward imatinib can occur and thus necessitated the 3,4 kinase recently shown to be intricately involved in regulating development of second-generation BCR–ABL inhibitors. signaling networks controlling the proliferation of myeloid Dasatinib (Sprycel, BMS-354825) and nilotinib (Tasigna, leukemia cells,18 was also identified as a novel kinase target inhibited by bosutinib, but not dasatinib. Furthermore, using Correspondence: Dr G Superti-Furga, Center for Molecular Medicine chemical proteomics, clear differences were observed between of the Austrian Academy of Sciences (CeMM), Lazarettgasse 19, the bosutinib target profiles within primary CML cells and the Vienna 1090, Austria. E-mail: [email protected] K562 cell line. In addition to the correlation found between Received 25 August 2008; revised 26 September 2008; accepted 14 inhibitor affinity purification and inhibition of kinase activity, October 2008; published online 27 November 2008 this study exemplifies the complementary use of such kinase Bosutinib target profile in CML LL Remsing Rix et al 478 panels together with chemical proteomics for (near) kinome- were obtained from two CML patients with accelerated-phase wide identification of possible targets that could be responsible disease upon clinical admission. Upon taking the blood for clinically observed (side) effects or represent secondary samples, these patients had previously only received hydro- therapeutic points of intervention. xyurea, but no kinase inhibitor therapy (additional patient information, for example, blood makeup, is provided in Supplementary Table S1). In addition to the patient samples, Materials and methods K562 cells were used to allow for comparisons between the bosutinib profile obtained here and the profiles of bosutinib and Biological material other BCR–ABL inhibitors generated in this cell line.11,14 The list K562 cell pellets were generated by Cilbiotech (Mons, Belgium). of identified proteins (Supplementary Tables S2–S4) obtained Unfractionated peripheral blood leukocytes (buffy coat) were from cell lysates of the two CML patient samples and K562 obtained from patients in accelerated-phase CML. Peripheral following incubation with the bosutinib affinity matrix was blood mononuclear cells were obtained using Ficoll. Studies filtered against a ‘core proteome’ data set (comprised of the most were approved by the institutional review board (Medical prevalent proteins in the total cell lysate of K562, for example, University of Vienna). Written informed consent in accordance DNAPK, GCN1L1, HSPs, actin, tubulin) and a list of ‘frequent with the Declaration of Helsinki was obtained before blood hitters’ (that is, nonspecific proteins, such as mTOR or COG6, donation. identified in purifications using seven unrelated drugs).14 Table 1 lists the protein kinases present in this filtered list (see also Supplementary Table S5). Compounds and immobilization In parallel to the chemical proteomic investigation, bosutinib Bosutinib was a gift from Oridis Biomed (Graz, Austria). and c-bosutinib were assayed at 1 and 10 mM against 272 c-Bosutinib was synthesized by Vichem Chemie Ltd (Budapest, recombinant kinases, including several clinically relevant kinase Hungary) and acetylated or immobilized on NHS-activated mutants (Supplementary Table S6). This analysis revealed a Sepharose 4 Fast Flow (GE Healthcare Bio-Sciences AB, strong correlation (Po2.2E-16 for 1 mM and Po2E-16 for 10 mM) Uppsala, Sweden) through its amino-functionality as described 14 between inhibition by bosutinib and c-bosutinib further validat- previously. Dasatinib was synthesized by WuXi PharmaTech ing the use of c-bosutinib as a probe for detecting bosutinib- (Shanghai, China). binding proteins in physiological samples (Figure 1b). A correlation was also found between the two profiling Kinase inhibition analysis techniques in that the stronger a kinase was purified using the chemical proteomic approach (that is, higher sequence coverage), Bosutinib, c-bosutinib and its N-acetyl derivative were the stronger it was found to be inhibited (that is, lower IC )inthe assayed in vitro for inhibition of recombinant full-length 50 kinase panel (Figure 1c). Indeed, over half of the overlapping c-ABL (Millipore, Dundee, UK). Phosphorylation of 5FAM- kinases were completely inhibited (less than 5% remaining