PF-114, a Potent and Selective Inhibitor of Native and Mutated BCR/ABL Is Active Against Philadelphia Chromosome-Positive (Ph+) Leukemias Harboring the T315I Mutation

Home , Ponatinib

ORIGINAL ARTICLE PF-114, a potent and selective inhibitor of native and mutated BCR/ABL is active against Philadelphia chromosome-positive (Ph+) leukemias harboring the T315I mutation

AA Mian1,ARaﬁei1, I Haberbosch1, A Zeifman2,3, I Titov2,3, V Stroylov2,3, A Metodieva1, O Stroganov2,3, F Novikov2,3, B Brill4, G Chilov2,3, D Hoelzer1, OG Ottmann1 and M Ruthardt1

Targeting BCR/ABL with tyrosine kinase inhibitors (TKIs) is a proven concept for the treatment of Philadelphia chromosome-positive (Ph+) leukemias. Resistance attributable to either kinase mutations in BCR/ABL or nonmutational mechanisms remains the major clinical challenge. With the exception of ponatinib, all approved TKIs are unable to inhibit the ‘gatekeeper’ mutation T315I. However, a broad spectrum of kinase inhibition increases the off-target effects of TKIs and may be responsible for cardiovascular issues of ponatinib. Thus, there is a need for more selective options for the treatment of resistant Ph+ leukemias. PF-114 is a novel TKI developed with the speciﬁcations of (i) targeting T315I and other resistance mutations in BCR/ABL; (ii) achieving a high selectivity to improve safety; and (iii) overcoming nonmutational resistance in Ph+ leukemias. PF-114 inhibited BCR/ABL and clinically important mutants including T315I at nanomolar concentrations. It suppressed primary Ph+ acute lymphatic leukemia-derived long-term cultures that either displayed nonmutational resistance or harbor the T315I. In BCR/ABL- or BCR/ABL–T315I-driven murine leukemia as well as in xenograft models of primary Ph+ leukemia harboring the T315I, PF-114 signiﬁcantly prolonged survival to a similar extent as ponatinib. Our work supports clinical evaluation of PF-114 for the treatment of resistant Ph+ leukemia.

Leukemia (2015) 29, 1104–1114; doi:10.1038/leu.2014.326

INTRODUCTION potent inhibitors of native BCR/ABL and of most TKD mutants. The Philadelphia chromosome (Ph) is the der(22) of the reciprocal Their clinical use as second-line and more recently first-line 15–20 translocation t(9;22)(q34;q11), which encodes the BCR/ABL, the therapy for all phases of CML and Ph+ ALL has improved oncogenic driver of chronic myeloid leukemia (CML) and 20–25% treatment options and outcome but has not abrogated the of cases of adult acute lymphatic leukemia (ALL). Tyrosine kinase problem of resistance, particularly in advanced disease. Notably, inhibitors (TKI), designed to abrogate the oncogenic function of the pattern of TKD mutations emerging with clinical resistance fi fl BCR/ABL, have greatly improved the overall prognosis of these depends on the speci c TKI administered, and re ects the relative diseases, particularly by altering the natural history of chronic potency of the individual TKIs toward different TKD mutations (for nilotinib: T315, Y253, E255 and F359 residues; dasatinib: T315, phase (CP) CML and preventing the previously inexorable – F317 and V299 residues).21 23 The presence of mutations is progression to terminal blast crisis (BC).1 Most patients with associated with inferior survival in patients resistant to TKI CML-CP treated with imatinib are now anticipated to have a nearly 8,12,24,25 2 highlighting the need for more effective therapy. In normal life expectancy, but its impact on outcome has been less particular, the gatekeeper mutation T315I has emerged as the decisive in patients with CML-BC3 and outcome remains dismal in 4,5 clinically most challenging TKD mutation due to its resistance to patients with CML-BC or Ph+ ALL. This is due to the high rate of all approved second generation TKI. primary and acquired resistance to imatinib in advanced disease Ponatinib is a potent inhibitor of native BCR/ABL and BCR/ABL– stages, but resistance may be observed even in a small but T315I that also inhibits all known BCR/ABL mutants in vitro, as well clinically relevant subset of CML-CP patients. In the pivotal IRIS as in vivo26 and was the first third-generation TKI to be approved trial, ~ 50% of patients displayed either primary resistance based for treatment of CML and Ph+ ALL after showing pronounced on cytogenetic response at 18 months or secondary resistance antileukemic activity in phase I and II studies of patients with after 5-year follow-ups.6,7 multi-TKI-resistant disease or presence of the T315I mutation.27,28 Point mutations in the tyrosine kinase domain (TKD) of BCR/ABL As a multikinase inhibitor, ponatinib also targets kinases have emerged as the predominant cause of acquired resistance. implicated in mutation-independent resistance to TKI, for exam- They are observed in up to 80% of patients with CML-BC and Ph+ ple, SRC. On the other hand, the broad spectrum of kinases – ALL8 12 and ~ 50% of imatinib-resistant CML patients overall.13,14 inhibited by ponatinib, including VEGF receptors, FGF receptors, This led to the development and regulatory approval of second PDGF receptors, EGF receptor, c-KIT, FLT3 and RET kinases, may generation TKI (nilotinib, dasatinib and bosutinib) that are more enhance the potential for adverse effects, including pancreatitis or

1Department of Hematology, Goethe University, Frankfurt, Germany; 2Fusion Pharma, LLC, Moscow, Russian Federation; 3Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Moscow, Russian Federation and 4Institute for Biomedical Research, Georg-Speyer-Haus, Frankfurt, Germany. Correspondence: Dr M Ruthardt, Laboratory for Tumor Stem Cell Biology, Department of Hematology, Clinic of Goethe University Frankfurt, Theodor Stern Kai 7, 60590 Frankfurt, Germany. E-Mail: [email protected] Received 16 July 2014; revised 16 October 2014; accepted 7 November 2014; accepted article preview online 14 November 2014; advance online publication, 12 December 2014 PF-114 against Ph+ leukemias AA Mian et al 1105 myelosuppression.27 This concern has been exacerbated by recent supplemented with 10 ng/ml mIL-3 (Cell Concepts, Umkirch, Germany). Ph clinical data indicating a high rate of cardiac and vascular adverse + ALL PD-LTCs were maintained in a serum-free medium as described events during ponatinib treatment. The development of other previously.45 compounds active against BCR/ABL–T315I, including several aurora kinase inhibitors, has likewise been hampered by issues FLT3-ITD, c-Kit-D814H, BCR/ABL and its mutants fi 29–31 of poor speci city and tolerability. The FLT3-ITD cDNA was kindly provided by Christian Brandts Clinical experience shows that in Ph+ ALL and CML-BC (Goethe University, Frankfurt, Germany). c-Kit-H814D, p185BCR/ABL and its 32,33 responses to all TKIs tested to date are limited and transient, resistant mutants (Y253F, E255K and T315I) were reported previously.46,47 despite administration of TKI considered appropriate based on the The p185-F317LBCR/ABL was created from p185BCR/ABL and the results of mutational analysis.28 Thus, important therapeutic p185–T315I–E255KBCR/ABL from p185–T315IBCR/ABL cDNA by using the obstacles other than tolerability and appearance of resistance, QuikChange1 II Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, TKD mutations are mutation-independent mechanisms of resis- USA) according to the manufacturer’s instructions. The following primers tance that are caused by, as yet, insufficiently characterized cell were used: forward-F317L 5′-GTTCTATATCATCACTGAGCTTATGACCTACGG intrinsic mechanisms.34,35 These include amplification of BCR/ABL, GAACCTCC-3′; reverse-F317L 5′-GGAGGTTCCCGTAGGTCATAAGCTCAGTGA aberrant phosphatase activity, aberrant drug transporter activity TGATATAGAAC-3′ and forward-E255K 5′-GGGGGCCAGTACGGGAAAGTGT ′ ′ and ‘sanctuary sites’ for example, the central nervous system.36,37 ACGAGGGCG-3 ; reverse-E255K 5 -CCCTCGTACACTTTCCCGTACTGGCCCC ′ BCR/ABL Taken together, these data highlight the unmet need of CGCC-3 . After sequencing the cDNAs of FLT3-ITD, p185 and its effective and safe agents available for treatment of patients with mutants were cloned into the pENTR1A Gateway vector for further recombination into the retroviral PINCO Gateway-destination vectors.48 advanced and therapy-resistant Ph+ leukemias, and those harboring BCR/ABL–T315I in particular. Here we report the design and preclinical evaluation of a highly selective, orally available TKI, Western blotting PF-114, which potently inhibits native BCR/ABL, as well as BCR/ Western blotting was performed according to widely established proto- ABL–T315I and other clinically relevant resistance mutations in cols. The following antibodies were used: anti-ABL (α-ABL) (Santa Cruz biochemical, cellular and in vivo survival models. These data Biotechnology, Santa Cruz, CA, USA) and anti-ABL specific for the α – warrant consideration for single-agent clinical testing of PF-114 in phosphorylated Y245 ( -p-ABL Y245) (Upstate-Biotechnology, Lake Placid, patients resistant to available TKI and hold particular promise for NY, USA); anti-CRKL; and anti-phosphorylated CRKL (Cell Signaling, Boston, controlling BCR/ABL–T315I-driven disease. MA, USA), anti-STAT5; and anti-phosphorylated STAT5 (Cell Signaling). Blocking was performed in 5% low-fat dry milk (Roth, Karlsruhe, Germany).

MATERIALS AND METHODS Cytotoxicity/proliferation and apoptosis Compounds, ponatinib and PF-114 Cytotoxicity/proliferation was assessed by using the XTT Proliferation Kit ’ PF-114 was synthesized as described in Supplementary Figure S1. (Roche, Mannheim, Germany) according to the manufacturer s instructions. Ponatinib was purchased from Selleck Chemicals (Houston, TX, USA). The Cell growth was assessed by dye exclusion using Trypan-blue and 46 compounds were diluted in dimethyl sulfoxide to a stock solution 1000 × , apoptosis by 7-AAD staining as described before. then diluted to working concentrations. For the in vivo experiments, the compounds were diluted to working concentration in 0.5% methyl- Isolation of Sca1+ hematopoietic stem and progenitor cells cellulose (Methocel 65HG, Fluka/Sigma, Taufkirchen, Germany). Sca1+ cells were isolated from the bone marrow (BM) of 8- to 12-week-old female C57BL/6N mice (Janvier St. Berthevin, France) using the Sca1+ Kinase inhibition assay Enrichment Kit according to the manufacturer’s instructions (Miltenyi, Kinase inhibition was measured at Reaction Biology Corp. (San Diego, CA, Bergisch Gladbach, Germany). USA), using its proprietary ‘Kinase Hot Spot’ assay. For IC50 measurements of PF-114 and ponatinib on a selected set of kinases, 10 concentrations Retroviral infection were tested in duplicates starting from 1 μM with threefold serial dilution. fi Ecotropic Phoenix packaging cells were transfected with the PINCO Staurosporine was used as a positive control. For kinase selectivity pro ling 46 + PF-114, ponatinib, dasatinib and nilotinib were tested in a single vectors. The retroviral supernatant was collected after 36 h. Sca1 cells fi concentration of 100 nM in duplicate against a panel of 337 kinases. prestimulated for 2 days in Dulbecco's modi ed Eagle's medium, 10% FCS, mIL-3 (20 ng/ml), mIL-6 (20 ng/ml) and mSCF (100 ng/ml; Cell Concepts) or Ba/F3 cells were plated onto retronectin-coated (Takara-Shuzo, Shiga, Molecular modeling Japan) nontissue culture 24-well plates and exposed to the retroviral 38 The initial coordinates of ABL WT (PDB ID: 3OXZ), ABL T315I (PDB ID: supernatant for 3 h at 37 °C. 3IK3),26 B-RAF (PDB ID: 3Q96)39 and VEGFR2 (PDB ID: 3VHE)40 kinases were obtained from the Protein Data Bank. Polar hydrogen atoms and missing protein residues were added using Build model program.41 Binding poses Syngeneic transduction/transplantation model of CML of PF-114 and ponatinib were first hypothesized using Lead Finder docking C57BL/6N mice (8–12 weeks) were sublethally irradiated with 4.5 Gy. software42 and then refined according to the following molecular Retrovirally infected donor cells (105) were inoculated via tail vein. dynamics protocol. Models of protein–ligand complexes were solvated Mice were killed at the first appearance of morbidity (loss of weight 410%, with water molecules using the genbox utility of the GROMACS 4.5 neurological abnormalities and failure to thrive or diarrhea). simulation package43 (crystallographic water molecules were retained). Molecular mechanics parameterization of ligands was performed with ACPYPE.44 OPLS-AA force field and TIP4P water model were used in further Syngeneic BCR/ABL-induced ALL 4 molecular dynamics simulations, which included energy minimization step, Cryopreserved BM cells (4 × 10 ) from C57BL/6N mice with BCR/ABL-driven 100-ns NVT equilibration and 2-ns NPT production run. ALL were injected via tail vein into sublethally irradiated recipient. Mice were killed at the first appearance of morbidity. Cell lines and patient-derived long-term cultures (PD-LTCs) All cell lines were obtained from the German Collection of Microorganisms K562 xenograft in nude mice 6 and Cell Cultures (DSMZ, Braunschweig, Germany). K562, KCL-22, SupB15, K562 cells (5 × 10 ) were implanted subcutaneously into the right flank of Tom-1, BV-173 and Jurkat cells were maintained in RPMI 1640 female BALB/cAnNRj-Foxn1nu mice (Charles River, Sulzfeld, Germany). Mice supplemented with 10% fetal calf serum (FCS; Gibco/Invitrogen, Karslruhe, were randomized to treatment groups when the average tumor volume Germany). The packaging cell line Phoenix was cultured in dimethyl (tumor volume = L × W2 × 0.5) reached about 500 mm3. Mean tumor sulfoxide with 10% FCS. Ba/F3 cells were grown in RPMI+10% FCS volume was measured every 3 days.

© 2015 Macmillan Publishers Limited Leukemia (2015) 1104 – 1114 PF-114 against Ph+ leukemias AA Mian et al 1106 T315I-positive Ph+ ALL xenograft model Statistical analysis BCR/ABL-T315I-positive PD-LTC (KÖ) cells (4 × 106) were inoculated via tail Student's t-tests or Mantel-Cox test for the determination of statistical vein into sublethally irradiated (2.5 Gy) NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ significance of survival curves were performed using the GraphPad Prism (NSG) mice. These mice were bred at the animal facility of the Georg- (GraphPad Software, San Diego, CA, USA). Speyer Haus, Frankfurt, Germany, under specific pathogen-free condi- tions. Mice were killed at the first appearance of morbidity. RESULTS Design of PF-114 for interaction with ABL-T315I and improved Retrovirus-based mutagenesis screen kinase selectivity For a modified retrovirus-based mutagenesis screen,37 Ba/F3 cells were For the design of an inhibitor with the same potency but a better retrovirally transduced with either p185BCR/ABL or its resistance mutants selectivity profile two main structural hypotheses were consid- and selected by interleukin (IL)-3 withdrawal. A perfectly balanced pool of ered: (i) to render repulsion with the main chain carbonyl oxygen, 107 cells was cultured with increasing concentrations of PF-114 (0, 50, 100 present in the ATP-binding site of many off-target kinases (for and 500 nM). After 28 days clones were obtained by limiting dilution in example, main chain carbonyl of residue E917 in VEGFR2; 96-well plates. Genomic DNA for sequencing the BCR ABL kinase domain Supplementary Figure S2, Supplementary Table S1), a partially was extracted using QIAamp DNA Mini Kit (Qiagen, Düsseldorf, Germany). negatively charged nitrogen atom was placed into corresponding For amplification the following primers were used: ALL-TB 5′-GCAAGACC GGGCAGATCT-3′ and R-ABL-A 5′-GTTGCACTCCCTCAGGTAGTC-3′.10 PCR position of the ponatinib scaffold (Figure 1a); (ii) to disrupt products were sequenced by Seqlab (Göttingen, Germany) using the AN4 potential hydrogen bond between the water molecule present in 5′-TGGTTCATCATCATTCAACGGTGG-3′.49 The sequence data were analyzed the active site of some off-target kinases (for example, in B-RAF for mutations with Clone Manager Professional (Sci ED Software, Morrison, kinase active site, Supplementary Figure S1 and S2) and piridazine, NC, USA). nitrogen atom of ponatinib was replaced with a carbon atom

Figure 1. Suggestive molecular interactions of PF-114 to some off-target. (a) Binding of ponatinib to some off-target kinases (CH…O bond with main chain carbonyl oxygen example of VEGFR2, H-bond with active site water molecule example of B-Raf). Disfavor off-target interactions of PF-114 to these off-target kinases. (b) Modeled binding mode of PF-114 in the ABL active site and in the ABL-T315I active site. (c) Kinase inhibition proﬁles of PF-114, ponatinib, dasatinib and nilotinib. Cell-free enzymatic assay was performed with a panel of 337 kinases. Selectivity of PF-114 was assessed in direct comparison with that of nilotinib, dasatinib and ponatinib all at 100 nM concentration. Highlighted are individual kinases with residual activity o1%, 1–10% and 10–50%. (d) The effect of PF-114 and ponatinib on the factor-independent growth of Ba/F3 expressing either FLT3-ITD or c-Kit-D814H was performed on cells selected by IL-3 withdrawal. These cells were exposed to increasing concentrations of PF-114 or ponatinib as indicated. Cell proliferation and viability was assessed by XTT assays.

Leukemia (2015) 1104 – 1114 © 2015 Macmillan Publishers Limited PF-114 against Ph+ leukemias AA Mian et al 1107 (Figure 1a). The favorable binding of PF-114 in the active site of Table 1. PF-114 inhibits ABL kinase and its clinically relevant mutants ABL and its T315I-mutant was confirmed by molecular modeling (Figure 1b; for ponatinib see Supplementary Figure S3). Enzyme PF-114 lC50 nM The inhibitory effects of PF-114 on the ABL kinase and ABL ABL 0.49 harboring the clinically relevant mutations were confirmed in a ABL(T315l) 0.78 cell-free kinase inhibition assays (Table 1). The activity range of ABL(E255k) 9.5 26,50 PF-114 was similar to that of ponatinib, whereas IC50s for ABL(F3171) 2.0 51 ABL(G250E) 7.4 nilotinib were higher. Selectivity of PF-114 was investigated in ABL(H396P) 1.0 direct comparison with ponatinib, dasatinib and nilotinib by the ABL(T351T) 2.8 determination of the inhibition profile of 337 human kinases. We ABL(Q252H) 12 used a concentration of 100 nM, which corresponds to the ABL(Y253F) 4.1 clinically feasible plasma concentrations of ponatinib and

Figure 2. PF-114 inhibits kinase activity of BCR/ABL and BCR/ABL-T315I and factor-independent growth of Ba/F3 cells mediated by BCR/ABL and its resistance mutants. (a) Western blot analysis of lysates of Ba/F3 cells expressing BCR/ABL and BCR/ABL-T315I using antibodies directed against: c-ABL; ABL–Y245 (α-p-ABL); Crkl; phosphorylated Crkl (α-p-Crkl); STAT5; phosphorylated STAT5 (α-p-STAT5); and β-tubulin (anti-β- tubulin). Molecular mass references (kDa) are presented and c-ABL and β-Tubulin were used as a loading control. (b) The effect of PF-114 on the factor-independent growth of Ba/F3 expressing BCR/ABL was performed on cells selected by IL-3 withdrawal. These cells were exposed to increasing concentrations of PF-114. Ponatinib was used as a control. Cell proliferation and viability was assessed by XTT assays. Cytotoxic effect was conﬁrmed on empty vector-transduced Ba/F3 cells in the presence of IL-3 at concentrations at least until 2 μM.(c) The effect of PF-114 on the factor-independent growth of Ba/F3 expressing BCR/ABL resistance mutations was assessed by XTT assay. (d) The apoptosis rate of Ba/F3 cells expressing BCR/ABL and BCR/ABL–T315I upon exposure to increasing concentration of PF-114 was determined by staining with 7-AAD. Ponatinib was used as a control. The data represent the mean of three independent experiments ± s.d.

© 2015 Macmillan Publishers Limited Leukemia (2015) 1104 – 1114 PF-114 against Ph+ leukemias AA Mian et al 1108 dasatinib, respectively.52 As a threshold, a minimum of 90% of on constitutively activated FLT3 and c-KIT in a cellular model. We inhibited kinase activity was selected. PF-114 potently inhibited 11 used FLT3-ITD or c-Kit-D814H, the murine analogue of c-KIT- kinases, whereas ponatinib inhibited 47, dasatinib 36 and nilotinib D816H and retrovirally expressed them in factor-dependent Ba/F3 4 kinases, respectively (Figure 1c, Supplementary Table S2). cells. After selection by factor withdrawal the cells were exposed Extrapolating the kinase inhibition of nilotinib to its clinically to increasing concentrations of PF-114 and ponatinib. Whereas relevant concentration of 4 μM the number of inhibited kinases PF-114 did not show any activity below a concentration of 500 nM, reached 21 (Supplementary Table S2). ponatinib inhibited both FLT3-ITD and c-Kit-D814H in a low Like ponatinib and dasatinib, PF-114 potently inhibited ABL2, nanomolar range (Figure 1d). DDR1, DDR2, FMS, FRK, LCK, LYN and PDGFR kinases, whereas it Whether the different target profiles influence toxicity was did not inhibit c-SRC, CSK or c-KIT. Furthermore as compared with investigated on primary Sca1+/lin- murine hematopoietic stem ponatinib it spared EPHRIN, FLT3, FGFR, VEGFR and B-RAF. To and progenitor cells in a colony assay in semisolid medium. confirm differences between PF-114 and ponatinib regarding their Whereas PF-114 did not reduce the colony number even at 5 μM, target profile, we compared the effects of PF-114 and ponatinib ponatinib began to reduce colonies starting from 500 nM

Figure 3. PF-14 inhibits human Ph+ patient-derived cell lines. XTT assays were performed on human Ph+ cell lines derived from ALL or CML patients. (a) K562 and KCL-22, both expressing p210BCR/ABL (myeloid CML-BCs). Jurkat and Nalm-6 cells were used as negative controls. (b) SupB15 and Tom-1 (Ph+ ALL) expressing p185BCR/ABL or BV-173 expressing p210BCR/ABL (lymphatic CML-BC) were used as Ph+ lymphatic cell lines. The means ± s.d. of triplicates of one representative experiment out of three performed are given. (c) Western blot analysis of lysates of SupB15 and K562 using antibodies directed against: c-ABL; ABL–Y245; and Tubulin (anti-β-Tubulin) was performed. Molecular mass references (kDa) are presented and c-ABL and Tubulin were used as a loading control.

Leukemia (2015) 1104 – 1114 © 2015 Macmillan Publishers Limited PF-114 against Ph+ leukemias AA Mian et al 1109 (Supplementary Figure S4). For further preclinical toxicity data and empty vector-transduced Ba/F3 cells in the presence of IL-3 at pharmacokinetics of PF-114 see Supplementary information and concentrations at least until 2 μM (Figure 3b). Supplementary Tables S3-S5). Next we extended our investigations on the clinically most Taken together, these data show that PF-114 is a potent TKI important resistance mutants of BCR/ABL like Y253F, E255K with a more restricted selectivity proﬁle as compared with (imatinib resistance), F317L (dasatinib resistance) and T315I ponatinib or dasatinib. (global resistance). PF-114 effectively inhibited the growth of Ba/F3 cells expressing these mutants with an IC50 of 25–100 nM. – PF-114 inhibits the autophosphorylation of BCR/ABL and BCR/ The BCR/ABL F317L, responded starting from 100 nM of PF-114 ABL–T315I and abolishes factor-independent growth of Ba/F3 cells (Figure 2c). Growth inhibition by both PF-114 and ponatinib, at mediated by BCR/ABL and its resistance mutants low dosages, could not be entirely attributed to an induction of apoptosis, as assessed by 7-AAD staining as depicted exemplarily The activity of a kinase inhibitor in a cell context can strongly for native BCR/ABL and BCR/ABL–T315I (Figure 2d). differ from that in cell-free systems. Therefore we studied the effects of PF-114 on the autophosphorylation of BCR/ABL and BCR/ABL-T315I in Ba/F3 cells, as a read out of ABL kinase activity. PF-114 potently inhibits Ph+ patient-derived cell lines We retrovirally transduced Ba/F3 cells with either native BCR/ To conﬁrm the effects of PF-14 on human Ph+ patient-derived cell ABL or BCR/ABL-T315I and exposed them to increasing concen- lines, we performed XTT assays on human Ph+ leukemia cell lines trations of PF-114. Ponatinib (100 nM) was used as a positive derived from ALL or CML patients. We used K562 and KCL-22 BCR/ABL control. Autophosphorylation of BCR/ABL was addressed by an (both myeloid CML-BC expressing p210 ) and the Ph+ antibody directed against the phosphorylated Y245 residue in lymphatic cell lines SupB15, Tom-1 (both Ph+ ALL expressing BCR/ABL BCR/ABL ABL. The IL-3-dependent Ba/F3 cells are factor independent upon p185 ) and BV-173 (lymphatic CML-BC expressing p210 ). expression of BCR/ABL. To avoid bias of stress-induced signaling As a BCR/ABL negative control we used the Jurkat and Nalm-6 by factor withdrawal, we performed these experiments in the cells. Here we show that PF-114 inhibited proliferation of K562 and presence of IL-3. PF-114 inhibited the autophosphorylation of KCL-22 with an IC50 of 8 and 9 nM, respectively. Jurkat and Nalm-6 BCR/ABL and BCR/ABL-T315I in a dose-dependent manner similar cells were not affected in their growth (Figure 3a). PF-114 to ponatinib (Figure 2a). PF-114 also inhibited substrate phos- inhibited proliferation of BV-173, Tom-1 (IC50 of 5 nM) and SupB15 phorylation as shown by the reduced Crkl-phosphorylation and (IC50 of 50 nM) cells (Figure 3b). downstream activation of Stat5 by BCR/ABL, as well as by BCR/ Western blotting on lysates of SupB15 and K562 revealed a ABL-T315I (Figure 2a). dose-dependent inhibition of autophosphorylation of BCR/ABL by The effect of PF-114 on the factor-independent growth of Ba/F3 PF-114 (Figure 3c). expressing BCR/ABL was performed on cells selected by IL-3 withdrawal. Cell proliferation and viability was assessed by XTT PF-114 abolishes tumor growth in a K562 nude mouse xenograft assays. As shown in Figure 2b, PF-114 potently inhibited model proliferation of Ba/F3 cells expressing native BCR/ABL in a dose- To further validate the antitumor activity of PF-114 in a cell line- dependent manner with IC50 of 5–10 nM. No effect was seen on based model, we used a xenograft model in which K562 cells were

Figure 4. PF-114 abolishes K562 cells-driven tumor growth in nude mouse xenograft model. K562 cells (5 × 106) were subcutaneously inoculated into nude mice (BALB/cAnNRj-Foxn1nu). Tumor bearing animals (volume of 500 mm3) were treated once daily by oral gavage with 25 or 40 mg/kg of PF-114 or 25 mg/kg of ponatinib for 14 consecutive days (treatment). Control group was kept untreated. The mean tumor volume ± s.d. is shown. PF-114 treatment group was compared to the untreated group using Student's t-tests (n = 3/group).

© 2015 Macmillan Publishers Limited Leukemia (2015) 1104 – 1114 PF-114 against Ph+ leukemias AA Mian et al 1110 injected subcutaneously into nude mice. Treatment was started at primary cells from Ph − and Ph+ ALL patients or lymphatic CML- the moment the tumors reached a volume of about 500 mm3. BC patients, which remain genetically as well as immunopheno- PF-114 was administered at 25 and 40 mg/kg/day and ponatinib typically stable for at least 6 months, without entering senescence at 25 mg/kg/day. Tumor growth was completely inhibited by or passing the typical crisis of cell lines. Of 7 Ph+ PD-LTCs 40 mg PF-14 as compared with untreated mice within 10 days available, two expressed p210BCR/ABL (VB and CM) and three (Figure 4). At this dosage PF-114 caused a 100% reduction in mean p185BCR/ABL (PH, DW and KW).45,53 Also the nonmutational TKI- tumor volume at the ﬁnal measurement (P o0.001) compared resistant BV and the T315I-positive KÖ PD-LTCs harbored the with the start of treatment and tumors did not recur even after p185BCR/ABL. To avoid the bias of unspeciﬁc toxicity of PF-114, the 30 weeks from stopping the treatment (Figure 4). In contrast at HP, a Ph − PD-LTC, was used as a negative control. Cytotoxicity/ 25 mg/kg both PF-114 and ponatinib were able to cause a 100% proliferation was assessed by XTT. PF-114 inhibited the prolifera- reduction of the mean tumor volume within 4 and 2 weeks, tion of all TKI-responsive PD-LTCs, with IC50 values between 6 nM respectively, but were unable to avoid recurrence of the tumor and a maximum of 20 nM and did not affect the proliferation of growth (Figure 4). Ph − PD-LTC HP (Figure 5a). The TKI-resistant BV and KÖ PD-LTC were inhibited by PF-114 at an IC50 of 10 and 150 nM, respectively PF-114 suppresses growth of Ph+ PD-LTC with nonmutational (Figure 5b). The response of PD-LTCs toward PF-114 was related to resistance as well as T315I mutation the inhibition of BCR/ABL kinase activity as shown by the Ph+ ALL in adults is not fully represented by cell lines. We took reduction of the BCR/ABL autophosphorylation in PH, BV and KÖ advantage of a recently established unique culture system for cells (Figure 5c).

Figure 5. PF-114 suppresses growth of Ph+ PD-LTC with nonmutational resistance, as well as T315I mutation. (a) XTT assay on TKI-sensitive PD-LTCs expressing p185BCR/ABL or p210BCR/ABL cells upon exposure to increasing concentration of PF-114. Ponatinib was used as control. HP (Ph − ) was used as BCR/ABL negative control PT-DLTCs. (b) XTT assay on TKI-resistant PD-LTCs BV and KÖ (p185–T315IBCR/ABL) upon exposure to increasing concentration of PF-114. The means ± s.d. of triplicates from one representative experiment out of three performed are given. (c) Western blot analysis of lysates of PH, BV and KÖ using antibodies directed against: c-ABL; ABL–Y245; and Tubulin (anti-Tubulin) was performed. Molecular mass references are presented and c-ABL and Tubulin were used as a loading control.

Leukemia (2015) 1104 – 1114 © 2015 Macmillan Publishers Limited PF-114 against Ph+ leukemias AA Mian et al 1111 PF-114 prolongs the survival of mice with both BCR/ABL- and BCR/ efficacy of PF-114 in Ph+ ALL cells harboring the T315I we ABL-T315I-driven CML-like disease inoculated 4 × 106 KÖ into sublethally irradiated (2.5 Gy) NSG mice. To evaluate the in vivo efficacy of PF-114 on native p185BCR/ABL Treatment for 14 days with either PF-114 (50 mg/kg) or ponatinib and p185-T315IBCR/ABL, we used a syngeneic mouse model for (25 mg/kg) significantly prolonged survival from a median of CML.54 Sublethally irradiated C57BL/6N mice were transplanted about 116 to 130 days (P = 0.0001) (Figure 6d) suggesting a clinical with 105 Sca1+ BM cells expressing p185BCR/ABL or p185-T315IBCR/ABL. significance of PF-114 in advanced Ph+ leukemia even in the The mice were treated with PF-114 (50 mg/kg) or ponatinib presence of T315I. (25 mg/kg) once daily for 20 days. Both PF-114 and ponatinib extended median survival significantly from 28 days to 39 and PF-114 selects a BCR/ABL–T315I–E255K compound mutation 60 days, respectively (Figure 6a). Clinically relevant mutations can be predicted by mutagenesis – BCR/ABL In mice transplanted with p185 T315I -expressing cells screens. Two methods are actually used: (i) the retrovirus-based daily oral treatment with PF-114 and ponatinib for 20 days screen that plays on the natural mutation rate of the reverse fi prolonged signi cantly median survival from 68 to 132 and transcriptase during retroviral infection of Ba/F3 cells37 or by 124 days, respectively (P = 0.006 for PF-114 and P = 0.04 for pharmacologically induced point mutations using a mutagen, ponatinib) (Figure 6b). such as ethyl-nitrosurea.26,56 To confirm the resistance profile at the level of single mutations and to extend it to compound PF-114 prolongs the survival of mice with syngeneic BCR/ABL- mutations, defined as two TKD mutations in the same molecule as driven ALL or harboring a xenograft of T315I-positive Ph+ ALL a reason for resistance,23,26 we here used a modified reverse The BCR/ABL-driven CML-like disease in the mouse represents a transcriptase mutagenesis screen as schematically described in model of CML-CP. To study the effect of PF-114 in an advanced Ph+ Figure 7a. This method was used because mutations are limited to leukemia we took advantage from the fact that in our transduc- the provirus and do not involve the entire genome as in mutagen- tion/transplantation CML-like disease model in very rare cases treated cells. At 50 nM, PF-114 suppressed all mutants with the (o1%; data not shown) we obtained BCR/ABL-driven ALLs. These exception of E255K or T315I or a combination of the two ALLs are dependent on BCR/ABL activity (data not shown). Thus, (Figure 7b). The combination of T315I and T315L was about a spleen cells from ALL mice were inoculated into sublethally clone with the integration of two provirus, one the original T315I (4.5 Gy) irradiated recipients. The mice were treated either with and another mutated to T315L. At 100 nM, PF-114 suppressed all PF-114 (50 mg/kg) or ponatinib (25 mg/kg) for 20 days. PF-114 and resistance mutants and allowed the outgrowth only of a ponatinib extended significantly the median survival from 31 to 41 combined T315I–E255K mutant (Figure 7b). This mutant took and 46 days, respectively (Figure 6c). origin from an additional mutation as confirmed by the fact that We recently established a PD-LTC (KÖ) from a Ph+ ALL patient the triplet encoding the E255K did not correspond to the original harboring the T315I,45,55 which gives origin to a full blown cDNA sequence of the BCR/ABL–E255K clones (data not shown). leukemia in NSG mice within about 100 days. To investigate the At 500 nM, PF-114 suppressed all clones (data not shown).

Figure 6. The efﬁcacy of PF-114 in vivo in models of Ph+ leukemia. (a and b) For the induction of CML-like disease sublethally irradiated C57BL/ 6N mice were transplanted intravenously with 1 × 105 Sca1+-positive BM cells expressing p185BCR/ABL or p185–T315IBCR/ABL. Eight mice per group were treated orally either with PF-114 (50 mg/kg) or ponatinib (25 mg/kg) once daily for 20 days (treatment). (c) PF-114 prolongs the 4 survival of mice with BCR/ABL-derived ALL. Spleen cells (5 × 10 ) from ALL mice (frozen stock in liquid N2) were transplanted into sublethally (4.5 Gy) irradiated recipients. The mice were treated with PF-114 (50 mg/kg) or ponatinib (25 mg/kg) by gavage for 20 days. (d) Cells (4 × 106) from the PD-LTC KÖ (expressing BCR/ABL-T315I) were transplanted into sublethally irradiated (2.5 Gy) NSG recipient mice. Eight mice per group were treated with PF-114 (50 mg/kg) or ponatinib (25 mg/kg) by gavage for 14 days (treatment).

Figure 7. Compound mutations selected by PF-114. (a) Schematic representation of a modiﬁed reverse transcriptase-based mutagenesis screen. Ba/F3 cells infected with BCR/ABL and its indicated resistance mutants were selected to a 100% positive cell population for each construct by factor withdrawal. These populations were pooled in an exact proportional ratio and exposed to 50 and 100 nM (and 500 nM—not shown) of PF-114. PF-114 was freshly added with each medium change. After 28 days, the resulting bulk populations were subject to limiting dilution. Genomic DNA of resulting clones were then sequenced for the detection of primary mutations, as well as of additional compound mutations. (b) Resistant clones recovered from the pool of Ba/F3 cells infected with BCR/ABL and its mutants upon of PF-114 at 50 and 100 nM. Pre-existing mutations: outgrowth of clones from one of the mutant population in the pool (no changes in the sequence as compared with the provirus used for infection); new: additional mutations occurring in a provirus harboring one of the resistance mutations. (c) Resistance of the compound mutation BCR/ABL–T315I–E255K against PF-114 and ponatinib The effect of PF-114 on the factor-independent growth of Ba/F3 expressing BCR/ABL or BCR/ABL–T315I–E255K was performed on cells selected by IL-3 withdrawal. These cells were exposed to the indicated concentrations of PF-114 and ponatinib. Cell proliferation and viability was assessed by XTT assays.

The resistance toward PF-114 was confirmed by the exposure of responsible for a variety of drug-related adverse events that Ba/F3 retrovirally transduced with p185–T315I–E255KBCR/ABL to compromise both short-term and long-term tolerability and PF-114 and ponatinib (Figure 7c). safety, such as pleural effusions or pulmonary arterial hypertension by dasatinib,18,57 thrombocytopenia, arterial thrombosis rash, dry skin and abdominal pain by ponatinib28 or liver function DISCUSSION abnormalities and thrombocytopenia by INNO-406.58 By direct In developing PF-114, we focused on three prerequisites for a comprehensive comparison of the kinase inhibition profiles of novel ABL-targeting kinase inhibitor: (i) inhibition of native BCR/ PF-114, nilotinib, dasatinib and ponatinib using the same ABL at nanomolar concentrations; (ii) potent inhibition of BCR/ biochemical assay, we demonstrate a significantly greater ABL-T315I, and of other major TKD mutants; and (iii) the highest selectivity of PF-114, with fewer TKs inhibited by 490%. This possible degree of selectivity in terms of inhibiting kinase targets. was achieved by a rational molecular design of triazolopyridin Utilizing the same scaffold as for imatinib, nilotinib and ponatinib, moiety that preferably binds in the ATP-binding site of ABL our structure-based chemical modifications ensured high-affinity compared with other kinases. binding to native BCR/ABL in the low nanomolar range with nearly PF-114 is an orally available TKI, which is active in a range of the same potency as ponatinib. Furthermore no difference was 10–150 nM within it suppress all tested mutants. Its high tumor- seen between p210BCR/ABL and p185BCR/ABL, suggesting a similar specific activity was confirmed by the lack of any toxicity up to degree of BCR/ABL kinase inhibition in CML and Ph+ ALL. 5 μM in cellular assays using primary murine hematopoietic stem Although off-target effects of ABL-directed multikinase inhbitors and progenitor cells, Ba/F3 cells, Ph − PD-LTCs and Ph − cell lines. have been credited with potential therapeutic value in CML, for Together with these data, the higher selectivity of PF-114 as example, the inhibition of SRC family kinases, they are also compared with dasatinib and ponatinib suggests that PF-114 may

Leukemia (2015) 1104 – 1114 © 2015 Macmillan Publishers Limited PF-114 against Ph+ leukemias AA Mian et al 1113 have an improved risk profile as compared with these two 4 Giles FJ, Kantarjian HM, le Coutre PD, Baccarani M, Mahon FX, Blakesley RE et al. compounds, which was confirmed on our toxicity studies in rats Nilotinib is effective in imatinib-resistant or -intolerant patients with chronic and dogs (see supplementary information). In particular, the lack myeloid leukemia in blastic phase. Leukemia 2012; 26:959–962. of inhibition of members of the VEGFR-family might contribute to 5 Ottmann OG, Larson RA, Kantarjian HM, le Coutre PD, Baccarani M, Hochhaus A less cardiac or vascular toxicity, although the precise mechanisms et al. Phase II study of nilotinib in patients with relapsed or refractory Philadelphia chromosome--positive acute lymphoblastic leukemia. Leukemia of these adverse events have not yet been elucidated. In fact, also 2013; 27: 1411–1413. nilotinib presents an increased cardiovascular risk profile although 59 6 Druker BJ, Guilhot F, O'Brien SG, Gathmann I, Kantarjian H, Gattermann N et al. unable to inhibit members of the VEGFR-family. Even if our Five-year follow-up of patients receiving imatinib for chronic myeloid leukemia. short-term toxicity studies in large animals suggest a good N Engl J Med 2006; 355: 2408–2417. tolerance for PF-114, the risk profile of PF-114 has to be 7 O'Brien SG, Guilhot F, Larson RA, Gathmann I, Baccarani M, Cervantes F et al. investigated in further toxicity studies and mainly in phase I Imatinib compared with interferon and low-dose cytarabine for newly diagnosed studies. Regarding the potential selection of compound mutations chronic-phase chronic myeloid leukemia. N Engl J Med 2003; 348: 994–1004. in patients, PF-114, seems to select the E255 site in addition to 8 Soverini S, Iacobucci I, Baccarani M, Martinelli G. Targeted therapy and the T315I 92 – T315I, similar to ponatinib.26 mutation in Philadelphia-positive leukemias. Haematologica 2007; :437 439. 9 Branford S, Hughes TP. Mutational analysis in chronic myeloid leukemia: when Here we focused our experimental approach on the therapeutic and what to do? Curr Opin Hematol 2011; 18:111–116. potential of PF-114 for Ph+ lymphoid leukemias using in vitro and 10 Pfeifer H, Wassmann B, Pavlova A, Wunderle L, Oldenburg J, Binckebanck A et al. in vivo models that closely represent human Ph+ ALL, as this type Kinase domain mutations of BCR-ABL frequently precede imatinib-based therapy of leukemia is clinically particularly aggressive and susceptible to and give rise to relapse in patients with de novo Philadelphia-positive acute clonal evolution. In the PD-LTCs, PF-114 was highly effective not lymphoblastic leukemia (Ph+ ALL). Blood 2007; 110: 727–734. only for cells harboring the BCR/ABL–T315I, but also for patient- 11 Shah NP, Nicoll JM, Nagar B, Gorre ME, Paquette RL, Kuriyan J et al. Multiple derived samples displaying nonmutational resistance. Even BCR-ABL kinase domain mutations confer polyclonal resistance to the tyrosine though the resistance mechanisms in these cells are not known, kinase inhibitor imatinib (STI571) in chronic phase and blast crisis chronic myeloid 2 – these data further strengthen the rationale for use of PF-114 as leukemia. Cancer Cell 2002; :117 125. 12 Soverini S, Colarossi S, Gnani A, Rosti G, Castagnetti F, Poerio A et al. Contribution treatment for advanced, therapy-refractory Ph+ leukemia. of ABL kinase domain mutations to imatinib resistance in different subsets of Our pharmacokinetic studies, together (Supplementary Table S3) Philadelphia-positive patients: by the GIMEMA Working Party on Chronic Myeloid with the excellent response in our xenograft models of high-risk Leukemia. Clin Cancer Res 2006; 12: 7374–7379. Ph+ ALL in NSG mice, with and without BCR/ABL–T315I, clearly 13 Hughes T, Saglio G, Branford S, Soverini S, Kim DW, Muller MC et al. Impact of show that PF-114 has sufficient oral availability as to exert a potent baseline BCR-ABL mutations on response to nilotinib in patients with chronic antileukemic effect not restricted to CML-CP, but also on myeloid leukemia in chronic phase. J Clin Oncol 2009; 27: 4204–4210. aggressive human Ph+ ALL. 14 Muller MC, Cortes JE, Kim DW, Druker BJ, Erben P, Pasquini R et al. Dasatinib The F317L mutation that is implicated in clinical resistance to treatment of chronic-phase chronic myeloid leukemia: analysis of responses 114 – dasatinib21,23,60 was the only mutation for which our cellular according to preexisting BCR-ABL mutations. Blood 2009; : 4944 4953. – 15 Cortes JE, Jones D, O'Brien S, Jabbour E, Konopleva M, Ferrajoli A et al. Nilotinib as assays showed an IC50 beyond the 10 100 nM range, at 150 nM, front-line treatment for patients with chronic myeloid leukemia in early and in direct comparison was less potent than ponatinib. This chronic phase. J Clin Oncol 2010; 28: 392–397. concentration of 150 nM is likely to be clinically achievable based 16 Cortes JE, Jones D, O'Brien S, Jabbour E, Ravandi F, Koller C et al. Results of on our toxicity data, making it likely that PF-114 will have clinical dasatinib therapy in patients with early chronic-phase chronic myeloid leukemia. efficacy even in patients with BCR/ABL–F317L as also confirmed J Clin Oncol 2010; 28:398–404. by its inhibition of ABL–F317I in the cell-free kinase assay and by 17 Cortes JE, Kim DW, Kantarjian HM, Brummendorf TH, Dyagil I, Griskevicius L et al. the fact that in the resistance screen no BCR/ABL–F317L-positive Bosutinib versus imatinib in newly diagnosed chronic-phase chronic myeloid 30 – clone was detected. leukemia: results from the BELA trial. J Clin Oncol 2012; : 3486 3492. In conclusion, PF-114 is a highly potent, yet selective, TKI in 18 Kantarjian H, Shah NP, Hochhaus A, Cortes J, Shah S, Ayala M et al. Dasatinib versus imatinib in newly diagnosed chronic-phase chronic myeloid leukemia. biochemical cellular and in vivo assays with a very good toxicity 362 – fi fi N Engl J Med 2010; : 2260 2270. pro le, suggesting signi cant therapeutic potential in CML and 19 Rosti G, Palandri F, Castagnetti F, Breccia M, Levato L, Gugliotta G et al. Nilotinib high-risk advanced Ph+ leukemia. Its ability to overcome TKI for the frontline treatment of Ph(+) chronic myeloid leukemia. Blood 2009; 114: resistance induced by either BCR/ABL–T315I or other major 4933–4938. resistance mutations, or mutation-independent mechanisms 20 Saglio G, Kim DW, Issaragrisil S, le Coutre P, Etienne G, Lobo C et al. Nilotinib makes PF-114 a promising candidate for the treatment of CML versus imatinib for newly diagnosed chronic myeloid leukemia. N Engl J Med and Ph+ ALL patients resistant or refractory to all currently 2010; 362: 2251–2259. available TKIs. 21 Cortes J, Jabbour E, Kantarjian H, Yin CC, Shan J, O'Brien S et al. Dynamics of BCR- ABL kinase domain mutations in chronic myeloid leukemia after sequential treatment with multiple tyrosine kinase inhibitors. Blood 2007; 110: 4005–4011. CONFLICT OF INTEREST 22 Kantarjian HM, Giles F, Gattermann N, Bhalla K, Alimena G, Palandri F et al. Nilotinib (formerly AMN107), a highly selective BCR-ABL tyrosine kinase inhibitor, fi The entire study was nanced by Fusion Pharma LLC, Moscow, Russia. AZ, IT, VS, OS is effective in patients with Philadelphia chromosome-positive chronic fl and GC are employees of Fusion Pharma. The remaining authors declare no con ict myelogenous leukemia in chronic phase following imatinib resistance and of interest. intolerance. Blood 2007; 110: 3540–3546. 23 Shah NP, Skaggs BJ, Branford S, Hughes TP, Nicoll JM, Paquette RL et al. Sequential ABL kinase inhibitor therapy selects for compound drug-resistant BCR- REFERENCES ABL mutations with altered oncogenic potency. J Clin Invest 2007; 117: 1 Hughes TP, Hochhaus A, Branford S, Muller MC, Kaeda JS, Foroni L et al. Long-term 2562–2569. prognostic significance of early molecular response to imatinib in newly 24 Nicolini FE, Corm S, Le QH, Sorel N, Hayette S, Bories D et al. Mutation status diagnosed chronic myeloid leukemia: an analysis from the International and clinical outcome of 89 imatinib mesylate-resistant chronic myelogenous Randomized Study of Interferon and STI571 (IRIS). Blood 2010; 116:3758–3765. leukemia patients: a retrospective analysis from the French intergroup of CML 2 Hoglund M, Sandin F, Hellstrom K, Bjoreman M, Bjorkholm M, Brune M et al. Tyrosine (Fi(phi)-LMC GROUP). Leukemia 2006; 20: 1061–1066. kinase inhibitor usage, treatment outcome, and prognostic scores in CML: report 25 Nicolini FE, Hayette S, Corm S, Bachy E, Bories D, Tulliez M et al. Clinical outcome from the population-based Swedish CML registry. Blood 2013; 122:1284–1292. of 27 imatinib mesylate-resistant chronic myelogenous leukemia patients 3 Talpaz M, Silver RT, Druker BJ, Goldman JM, Gambacorti-Passerini C, Guilhot F harboring a T315I BCR-ABL mutation. Haematologica 2007; 92:1238–1241. et al. Imatinib induces durable hematologic and cytogenetic responses in patients 26 O'Hare T, Shakespeare WC, Zhu X, Eide CA, Rivera VM, Wang F et al. AP24534, a with accelerated phase chronic myeloid leukemia: results of a phase 2 study. pan-BCR-ABL inhibitor for chronic myeloid leukemia, potently inhibits the T315I Blood 2002; 99: 1928–1937. mutant and overcomes mutation-based resistance. Cancer Cell 2009; 16:401–412.

© 2015 Macmillan Publishers Limited Leukemia (2015) 1104 – 1114 PF-114 against Ph+ leukemias AA Mian et al 1114 27 Cortes JE, Kantarjian H, Shah NP, Bixby D, Mauro MJ, Flinn I et al. Ponatinib in 44 Sousa da Silva AW, Vranken WF. ACPYPE - AnteChamber PYthon Parser interfacE. refractory Philadelphia chromosome-positive leukemias. N Engl J Med 2012; 367: BMC Res Notes 2012; 5: 367. 2075–2088. 45 Badura S, Tesanovic T, Pfeifer H, Wystub S, Nijmeijer BA, Liebermann M et al. 28 Cortes JE, Kim DW, Pinilla-Ibarz J, le Coutre P, Paquette R, Chuah C et al. A phase 2 Differential effects of selective inhibitors targeting the PI3K/AKT/mTOR pathway trial of ponatinib in Philadelphia chromosome-positive leukemias. N Engl J Med in acute lymphoblastic leukemia. PLoS One 2013; 8: e80070. 2013; 369: 1783–1796. 46 Mian AA, Oancea C, Zhao Z, Ottmann OG, Ruthardt M. Oligomerization inhibition, 29 Giles FJ, Cortes J, Jones D, Bergstrom D, Kantarjian H, Freedman SJ. MK-0457, combined with allosteric inhibition, abrogates the transformation potential of a novel kinase inhibitor, is active in patients with chronic myeloid leukemia or T315I-positive BCR/ABL. Leukemia 2009; 23:2242–2247. acute lymphocytic leukemia with the T315I BCR-ABL mutation. Blood 2007; 109: 47 Zheng X, Oancea C, Henschler R, Ruthardt M. Cooperation between constitutively 500–502. activated c-Kit signaling and leukemogenic transcription factors in the 30 Giles FJ, le Coutre PD, Pinilla-Ibarz J, Larson RA, Gattermann N, Ottmann OG et al. determination of the leukemic phenotype in murine hematopoietic stem cells. Nilotinib in imatinib-resistant or imatinib-intolerant patients with chronic myeloid Int J Oncol 2009; 34: 1521–1531. leukemia in chronic phase: 48-month follow-up results of a phase II study. Leu- 48 Beissert T, Puccetti E, Bianchini A, Guller S, Boehrer S, Hoelzer D et al. Targeting kemia 2013; 27: 107–112. of the N-terminal coiled coil oligomerization interface of BCR interferes with 31 Tanaka R, Squires MS, Kimura S, Yokota A, Nagao R, Yamauchi T et al. Activity of the transformation potential of BCR-ABL and increases sensitivity to STI571. the multitargeted kinase inhibitor, AT9283, in imatinib-resistant BCR-ABL-positive Blood 2003; 102: 2985–2993. leukemic cells. Blood 2010; 116: 2089–2095. 49 Hochhaus A, Kreil S, Corbin AS, La Rosee P, Muller MC, Lahaye T et al. Molecular 32 Melo JV, Barnes DJ. Chronic myeloid leukaemia as a model of disease evolution in and chromosomal mechanisms of resistance to imatinib (STI571) therapy. human cancer. Nat Rev Cancer 2007; 7:441–453. Leukemia 2002; 16: 2190–2196. 33 Ottmann OG, Wassmann B. Treatment of Philadelphia chromosome-positive 50 Shah NP, Tran C, Lee FY, Chen P, Norris D, Sawyers CL. Overriding imatinib acute lymphoblastic leukemia. Hematology Am Soc Hematol Educ Program 2005: resistance with a novel ABL kinase inhibitor. Science 2004; 305:399–401. 118–122. 51 Manley PW, Drueckes P, Fendrich G, Furet P, Liebetanz J, Martiny-Baron G et al. 34 O'Hare T, Deininger MW, Eide CA, Clackson T, Druker BJ. Targeting the BCR-ABL Extended kinase profile and properties of the protein kinase inhibitor nilotinib. signaling pathway in therapy-resistant Philadelphia chromosome-positive Biochim Biophys Acta 2010; 1804: 445–453. leukemia. Clin Cancer Res 2011; 17:212–221. 52 Baccarani M, Deininger MW, Rosti G, Hochhaus A, Soverini S, Apperley JF et al. 35 Pfeifer H, Lange T, Wystub S, Wassmann B, Maier J, Binckebanck A et al. European LeukemiaNet recommendations for the management of chronic Prevalence and dynamics of bcr-abl kinase domain mutations during imatinib myeloid leukemia: 2013. Blood 2013; 122:872–884. treatment differ in patients with newly diagnosed and recurrent bcr-abl positive 53 Nijmeijer BA, Szuhai K, Goselink HM, van Schie ML, van der Burg M, de Jong D acute lymphoblastic leukemia. Leukemia 2012; 26: 1475–1481. et al. Long-term culture of primary human lymphoblastic leukemia cells in the 36 Kimura S, Naito H, Segawa H, Kuroda J, Yuasa T, Sato K et al. NS-187, a potent absence of serum or hematopoietic growth factors. Exp Hematol 2009; 37: and selective dual Bcr-Abl/Lyn tyrosine kinase inhibitor, is a novel agent for 376–385. imatinib-resistant leukemia. Blood 2005; 106: 3948–3954. 54 Li S, Ilaria Jr. RL, Million RP, Daley GQ, Van Etten RA. The P190, P210, and P230 37 von Bubnoff N, Barwisch S, Speicher MR, Peschel C, Duyster J. A cell-based forms of the BCR/ABL oncogene induce a similar chronic myeloid leukemia-like screening strategy that predicts mutations in oncogenic tyrosine kinases: impli- syndrome in mice but have different lymphoid leukemogenic activity. J Exp Med cations for clinical resistance in targeted cancer treatment. Cell Cycle 2005; 4: 1999; 189:1399–1412. 400–406. 55 Mian AA, Metodieva A, Badura S, Khateb M, Ruimi N, Najajreh Y et al. Allosteric 38 Zhou T, Commodore L, Huang WS, Wang Y, Thomas M, Keats J et al. Structural inhibition enhances the efficacy of ABL kinase inhibitors to target unmutated mechanism of the Pan-BCR-ABL inhibitor ponatinib (AP24534): lessons for BCR-ABL and BCR-ABL-T315I. BMC Cancer 2012; 12: 411. overcoming kinase inhibitor resistance. Chem Biol Drug Des 2011; 77:1–11. 56 Bradeen HA, Eide CA, O'Hare T, Johnson KJ, Willis SG, Lee FY et al. Comparison 39 Gould AE, Adams R, Adhikari S, Aertgeerts K, Afroze R, Blackburn C et al. Design of imatinib mesylate, dasatinib (BMS-354825), and nilotinib (AMN107) in an and optimization of potent and orally bioavailable tetrahydronaphthalene Raf N-ethyl-N-nitrosourea (ENU)-based mutagenesis screen: high efficacy of drug inhibitors. J Med Chem 2011; 54: 1836–1846. combinations. Blood 2006; 108:2332–2338. 40 Oguro Y, Miyamoto N, Okada K, Takagi T, Iwata H, Awazu Y et al. Design, synthesis, 57 Montani D, Bergot E, Gunther S, Savale L, Bergeron A, Bourdin A et al. Pulmonary and evaluation of 5-methyl-4-phenoxy-5H-pyrrolo[3,2-d]pyrimidine derivatives: arterial hypertension in patients treated by dasatinib. Circulation 2012; 125: novel VEGFR2 kinase inhibitors binding to inactive kinase conformation. Bioorg 2128–2137. Med Chem 2010; 18: 7260–7273. 58 Kantarjian H, le Coutre P, Cortes J, Pinilla-Ibarz J, Nagler A, Hochhaus A et al. Phase 41 Stroganov OV, Novikov FN, Zeifman AA, Stroylov VS, Chilov GG. TSAR, a new 1 study of INNO-406, a dual Abl/Lyn kinase inhibitor, in Philadelphia graph-theoretical approach to computational modeling of protein side-chain chromosome-positive leukemias after imatinib resistance or intolerance. Cancer flexibility: modeling of ionization properties of proteins. Proteins 2011; 79: 2693–2710. 2010; 116:2665–2672. 42 Stroganov OV, Novikov FN, Stroylov VS, Kulkov V, Chilov GG. Lead finder: an 59 Le Coutre P, Rea D, Abruzzese E, Dombret H, Trawinska MM, Herndlhofer S et al. approach to improve accuracy of protein-ligand docking, binding energy esti- Severe peripheral arterial disease during nilotinib therapy. J Natl Cancer Inst 2011; mation, and virtual screening. J Chem Inf Model 2008; 48: 2371–2385. 103:1347–1348. 43 Pronk S, Pall S, Schulz R, Larsson P, Bjelkmar P, Apostolov R et al. GROMACS 4.5: 60 Burgess MR, Skaggs BJ, Shah NP, Lee FY, Sawyers CL. Comparative analysis of two a high-throughput and highly parallel open source molecular simulation toolkit. clinically active BCR-ABL kinase inhibitors reveals the role of conformation-specific Bioinformatics 2013; 29:845–854. binding in resistance. Proc Natl Acad Sci USA 2005; 102: 3395–3400.

Supplementary Information accompanies this paper on the Leukemia website (http://www.nature.com/leu)