Leukemia (2015) 29, 76–85 & 2015 Macmillan Publishers Limited All rights reserved 0887-6924/15 www.nature.com/leu

ORIGINAL ARTICLE Sustained inhibition of STAT5, but not JAK2, is essential for TKI-induced cell death in chronic myeloid leukemia

L Schafranek1,2,3, E Nievergall1,2,3, JA Powell4,5, DK Hiwase3,6, T Leclercq1,3, TP Hughes1,2,3,6,7 and DL White1,2,3,4

Kinase inhibitors block proliferative signals in BCR-ABL1 þ leukemic cells, but their capacity to induce apoptosis is poorly understood. Initial studies suggested that very brief exposure to kinase inhibitors was sufficient to induce apoptosis in chronic myeloid leukemia (CML) cells. However, flaws in this experimental model have subsequently been identified, leading to the conclusion that apoptosis only occurs with sustained low-level kinase inhibition. Thus, the minimum duration of complete kinase inhibition required to commit CML cells to death is unknown. Here we confirm that o1 h is insufficient to induce significant commitment to death in BCR-ABL1 þ cell lines and in primary CD34 þ progenitor cells, and establish that commitment to cell death only occurs if kinase inhibition is maintained for 4 h or more. Remarkably, signal transducer and activator of transcription 5 (STAT5) inhibition in combination with transient (o1 h) tyrosine kinase inhibitor (TKI) exposure proved lethal for CML progenitors, despite the reactivation of Bcr-Abl after 1 h. JAK kinase inhibition did not induce cell death in combination with transient TKI exposure. Thus, STAT5 appears to be a critical determinant of the time-dependent sensitivity of CML progenitor cells to TKI treatment in a Bcr-Abl-dependent, but JAK-independent, manner. We conclude that combining kinase inhibition with STAT5 inhibition represents a promising therapeutic approach in BCR-ABL1 þ leukemias.

Leukemia (2015) 29, 76–85; doi:10.1038/leu.2014.156

13 14 INTRODUCTION the calcium channel and dopamine D2 receptor antagonist Chronic myeloid leukemia (CML) is driven by the oncogenic fusion pimozide was identified as an inhibitor of constitutive STAT5 protein Bcr-Abl, which activates multiple signaling pathways activation in CML, demonstrating specificity for STAT5 over STAT1, promoting leukemia cell survival and proliferation.1 The signal STAT3, MAPK and Src-family kinase and displaying synergy in 15 transducer and activator of transcription 5 (STAT5) is constitutively combination with imatinib and nilotinib. Considering the active in CML cells as a result of Bcr-Abl activation.2 In addition, implication of STAT5 in TKI resistance, together with its essential the STAT5 transcriptional repressor Gfi-1 is down regulated in role in Bcr-Abl-dependent leukemogenesis, it has been proposed CML,3 and we have previously demonstrated that low levels of as an attractive drug target for the treatment of resistant CML. GFI-1 at diagnosis predict progression to blast crisis.4 The therapeutic rationale for TKI therapy was based on the need Conventionally, STAT5 activation occurs in response to cytokine for continuous Bcr-Abl inhibition with imatinib for sustained signaling through JAK kinases. In CML, it is currently unclear disease control, where 41000 ng/ml imatinib trough plasma whether JAK2 is required for the Bcr-Abl-dependent activation of levels strongly correlate with therapeutic responses.16,17 Dasatinib STAT5. JAK kinase inhibition induces little apoptosis in CML cells is a potent second generation TKI and due to a short in vivo and appears to target extrinsic cytokine-mediated survival half-life of 3–5 h18 patients were treated with 50 mg dasatinib signaling rather than Bcr-Abl-dependent signaling,5 however, twice daily. However, patients receiving 100 mg dasatinib once there is some evidence to suggest that JAK2 interacts directly daily achieved similar cytogenetic and molecular responses to with Bcr-Abl6,7 and has a role in the inactivation of the those receiving 50 mg twice daily,19 which resulted in significant tumor suppressor protein phosphatase 2A.8 Moreover, the reduction of adverse effects.20 Plasma levels in CML patients reach absence of JAK2 did not affect leukemia maintenance following 150–200 nM 30 min post administration, but quickly drop over the initial myeloid transformation in a CML-like murine model.9 following 4–24 h with a concomitant reactivation of Bcr-Abl kinase Conversely, STAT5 deletion resulted in failure to maintain activity, within 8 h of dasatinib treatment.21 leukemic hematopoiesis,10 reinforcing the pivotal role for the We and others have previously reported that high dose, short- STAT5 protein in leukemic cell survival. term dasatinib exposure (100 nM for 30 min) induces cell death in Elevated total STAT5 expression has recently been demon- CML cells, despite restoration of Bcr-Abl signaling21–23 2 h after strated as an important mechanism of resistance to tyrosine drug washout.22 However, recently Lipka and colleagues24,25 have kinase inhibitor (TKI) treatment,11 and highlighted as a therapeutic demonstrated that the standard washout procedure established target in CML CD34 þ cells with acquired resistance to imatinib by Shah et al.21 is ineffective at completely removing intracellular where STAT5A protects cells from oxidative stress.12 Recently, TKIs. Residual levels of dasatinib, imatinib and nilotinib were

1Cancer Theme, South Australian Health and Medical Research Institute (SAHMRI), Adelaide, South Australia, Australia; 2Centre for Personalised Cancer Medicine (CPCM), Faculty of Health Science, University of Adelaide, Adelaide, South Australia, Australia; 3School of Medicine, University of Adelaide, Adelaide, South Australia, Australia; 4Discipline of Paediatrics, Faculty of Health Science, University of Adelaide, Adelaide, South Australia, Australia; 5Department of Immunology, SA Pathology, Adelaide, South Australia, Australia; 6Department of Haematology, SA Pathology, Adelaide, South Australia, Australia and 7Centre for Cancer Biology, University of South Australia, Adelaide, South Australia, Australia. Correspondence: Professor DL White, Cancer Theme, South Australian Health and Medical Research Institute SAHMRI, Adelaide, South Australia 5000, Australia. E-mail: [email protected] Received 27 November 2013; revised 14 April 2014; accepted 25 April 2014; accepted article preview online 12 May 2014; advance online publication, 27 June 2014 STAT5 inactivation is critical for sensitivity to TKI treatment L Schafranek et al 77 detected using HPLC in K562 and Ba/F3-BCR-ABL cells following Colony-forming assays 24,25 standard washout (STD wash). However, equilibration of cells Following a 72-h cell viability assay, viable CD34 þ cells were plated for in drug-free media between washes (optimal wash) resulted in colony-forming unit-granulocyte and macrophage assay in MethoCult-H4230 complete removal of residual TKI. Following the effective removal (StemCell Technologies) together with five growth factors (20 ng/ml of of dasatinib, transient TKI exposure was no longer capable of granulocyte macrophage-colony stimulating factor, interleukin-3, interleukin-6 inducing apoptosis in CML cells. This has subsequently been and fms-like tyrosine kinase-3 ligand, and 50 ng/ml of stem cell factor) confirmed by other groups postulating that reduction of pSTAT5 is in all culture conditions. Colony-forming unit-granulocyte and macrophage colonies were counted after 14 days incubation at 37 1C/5% CO . a sensitive marker for the presence of residual/low concentrations 2 of TKI, rather than reduced CrkL phosphorylation.26,27 Western blot analysis In this study, we have used the optimal washout (OPT wash) 5 method as a model of the effect of TKI availability on Bcr-Abl Following drug treatments, 2 Â 10 cells/ml were cultured over 72 h at 37 1C/5% CO2. Cells were lysed with Laemmli buffer and probed as activation and evaluated the critical factors involved in commit- previously described.29 Primary antibodies phospho-c-Abl (Y245), c-Abl, ment to TKI-induced cell death. Accordingly, the duration of phospho-STAT5 (Y694), STAT5, phospho-Erk1/2 (Thr202/Tyr204), Erk1/2, potent TKI exposure required to commit BCR-ABL1 þ cells to b-actin, Bcl-2, Bcl-xL, Mcl-1, Bim and cleaved poly (ADP-ribose) polymerase death was investigated in the setting of transient Bcr-Abl kinase (PARP) were obtained from Cell Signaling Technologies and alkaline- inhibition. We hypothesized that continuous inhibition of STAT5 phosphatase-conjugated anti-rabbit immunoglobulin was purchased from phosphorylation, independent of JAK2, is critical for the commit- Santa Cruz (Santa Cruz, CA, USA). Bound antibodies were detected with ment of CML cells to death in the setting of transient Bcr-Abl enhanced chemifluorescence substrate (Amersham Pharmacia, Castle Hill, kinase inhibition. NSW, Australia) and analyzed on a Typhoon FLA 9000 scanner (GE Healthcare, Buckinghamshire, UK). MATERIALS AND METHODS Cell lines RESULTS BCR-ABL1-expressing cell lines KU812, Meg01 and K562, obtained from the Bcr-Abl-driven STAT5 activation is especially sensitive to low levels American Type Culture Collection (Manassas, VA, USA), were cultured in of dasatinib RPMI1640 (Sigma, Castle Hill, NSW, Australia) supplemented by 10% fetal calf Recent reports suggest that low concentrations of TKIs remain in serum (SAFC Bioscience, Lenexa, KS, USA), L-glutamine (SAFC Bioscience) cells following the ‘standard’ washout of 100 nM dasatinib or 32.5 mM and penicillin/streptomycin (Sigma Life Sciences, St Louis, MO, USA). imatinib,24–26 with Simara et al.26 reporting that residual intracellular concentrations of dasatinib after 72 h were equivalent Patient samples to a 1 nM continuous dasatinib treatment. We therefore analyzed Blood was collected from newly diagnosed chronic phase (CP) CML patients the signaling profile of cells exposed to 30 min 100 nM dasatinib with informed consent, before TKI therapy. Mononuclear cells were isolated 1 using Lymphoprep (Axis-Shield PoCAs, Oslo, Norway) density gradient followed by three washes at 37 C (standard, STD, wash), compared centrifugation. CD34 þ progenitor cells were isolated by magnetic-assisted with 1 nM continuous dasatinib in the BCR-ABL1 þ cell line KU812. cell sorting (Miltenyi Biotech, Aurburn, CA, USA), and the purity was checked Sensitive inhibition of Erk and STAT5 phosphorylation in with anti-CD34-PE (BD Biosciences, San Jose, CA, USA). comparison to pBcr-Abl was observed for both treatments, suggesting residual dasatinib may remain following STD wash TKIs (Supplementary Figure 1). This loss of Bcr-Abl survival signals resulted in the inhibition of Mcl-1, Bcl-x and Bcl-2 and the Dasatinib and imatinib were purchased from Symansis (Shanghai, China). L The MEK inhibitor U0126 was purchased from Cell Signaling Technology induction of Bim and cleaved PARP (Supplementary Figure 1). (Beverly, MA, USA). The JAK1/2 inhibitor ruxolitinib was purchased from Active Biochemicals (Wan Chai, HongKong). Pimozide and chloroquine Removal of residual TKI following 30 min 100 nM dasatinib were purchased from Sigma-Aldrich (St Louis, MO, USA). The STAT5 prevents cell death inhibitor N’-((4-Oxo-4 H-chromen-3-yl)methylene)nicotinohydrazide (STAT5i) We confirm that a complete OPT wash of dasatinib, by was purchased from Merck (Darmstadt, Germany). equilibration of cells for an hour at 37 1C/5% CO2 in drug-free media between washes prevented cell death in KU812 (82.6%, Washout protocols viable OPT wash vs 16.1% viable STD wash) and Meg01 (78.7% Cells were cultured with either 100 nM dasatinib or 32.5 mM imatinib for viable OPT wash vs 8.5% viable STD wash) BCR-ABL1 þ cells 30 min to 8 h followed by standard or optimal wash protocols. In the (Supplementary Figure 2) as previously observed.24–26 standard wash protocol, cells were centrifuged at 1400 r.p.m. for 5 min following incubation with TKI. The supernatant was aspirated and the cells were washed with 37 1C phosphate-buffered saline three consecutive Transient exposure to TKI exceeding 4 h induces cell death despite times before being suspended in TKI-free media for the remainder of the complete removal of TKI 72 h at 37 1C/5% CO2. In the OPT wash protocol, cells were allowed to Original standard washout experiments assessed TKI exposures of equilibrate for 1 h at 37 1C/5% CO2 in drug-free media between each wash 20 min to 24 h before washout, however, recent reports have only (1400 r.p.m. for 5 min for three times). investigated exposures of p2 h. Peak plasma levels of dasatinib occur up to 6 h following administration18 and can be available for 24 h.21 Cell viability We determined that continuous treatment with 100 nM dasatinib Cell death was assessed by Annexin V/7AAD staining as previously reduced the percentage of viable cells to 4.5% in KU812 (Figure 1a) described.28 Briefly, following drug treatments, cells (2 Â 105/ml) were and 5.3% in Meg01 cells (Figure 1b). Prolonging 100 nM dasatinib cultured for 72 h at 37 1C/5% CO2 in RPMI/10% fetal calf serum for cell lines exposures to 4 and 8 h before OPT wash resulted in a significant or in serum-deprived medium, Iscove’s Modification of Dulbecco Medium decrease in cell viability in KU812 (53%, and 14% viable, respectively; (Sigma-Aldrich, Castle Hill, NSW, Australia) supplemented with 1% bovine Figure 1a) and Meg01 cells (42% and 19% viable, respectively; serum albumin (Sigma Aldrich), 1 U/ml insulin (Actrapid, Novo Nordisk Figure 1b). Importantly, apoptosis was also achieved with transient Pharmaceuticals Pty Ltd, Bagsuaerd, Denmark), 200 mg/ml transferrin exposures to potent imatinib treatments of 32.5 mM (Supplementary (StemCell Technologies, Vancouver, BC, Canada), 10 mg/ml low-density Figure 3), indicating that this discovery is not TKI specific. lipoproteins (Sigma) and 0.1 mM 2-b-mercaptoethanol (Sigma). Cells were subsequently washed and double stained with Annexin V-PE Subsequently, we determine the relevance of these findings in (BD Biosciences) and 7AAD (Invitrogen, Carlsbad, CA, USA), followed by primary CML progenitors. In comparison to the STD wash (55.8% immediate analysis on a FC500 flow cytometer (Beckman Coulter, Miami, colony-forming units (CFUs) normalized to control), application of FL, USA). the OPT wash following a 30-min exposure to 100 nM dasatinib

& 2015 Macmillan Publishers Limited Leukemia (2015) 76 – 85 STAT5 inactivation is critical for sensitivity to TKI treatment L Schafranek et al 78

Figure 1. Extension of dasatinib exposure before OPT washouts induce cell death in CML cells. (a)KU812and(b) Meg01 cells were incubated with 100 nM dasatinib for 0–8 h, followed by the STD or OPT wash and then cultured for 72 h in drug-free media. Cells were then analyzed by Annexin V/ 7-aminoactinomycin D staining (data are mean þ s.e.m., n ¼ 3). *Po0.05, **Po0.01, ***Po0.001 compared with 30 min OPT washout. CD34 þ cells isolated from newly diagnosed CP-CML patients were treated with (c) 100 nM dasatinib or (d) 32.5 mM imatinib for 0–8 h, followed by the STD or OPT wash and then cultured for 72 h in drug-free media. Cells were then analyzed for clonogenic potential using the colony-forming unit-granulocyte and macrophage assay at 2 weeks (data are mean±s.e.m., n ¼ 3). *Po0.05, **Po0.01, ***Po0.001 compared with 30 min OPT washout.

resulted in significantly more colony-forming cells (84.8% CFUs, Bcr-Abl phosphorylation recovered following the OPT wash and P ¼ 0.03; Figure 1c). In addition, colony-forming ability was was fully restored within 8 h (Figure 2a). Notably, there was also restored in cells treated with 32.5 mM imatinib following OPT wash reactivation of both STAT5 and Erk, with no effect on anti- (82.9% CFUs) opposed to STD wash (46.8% CFUs, P ¼ 0.03; apoptotic Mcl-1, Bcl-xL and Bcl-2 and subsequently no significant Figure 1d). However, exposure to dasatinib or imatinib increase in Bim, or cleaved PARP to indicate induction of apoptosis (Supplementary Figure S4) for 2–8 h before OPT wash induced (Figure 2a). apoptosis and resulted in significant reduction in CFUs Extension of the initial 100 nM dasatinib exposure to 8 h before compared with 30 min treatment before OPT wash, in dasatinib- OPT wash lead to reduction in pBcr-Abl and complete inhibition of and imatinib-treated cells, respectively, at 2 h (51.3%, pSTAT5 and pErk upon washout (Figure 2b), in contrast to the P ¼ 0.004 and 41.2%, P ¼ 0.01), 4 h (39.6%, P ¼ 0.002 and 24.4%, 30-min OPT wash where Bcr-Abl activity resumed upon washout P ¼ 0.0006) and 8 h (30.1%, P ¼ 0.002 and 14.4%, P ¼ 0.0003). (Figure 2a). This extended treatment induced a pro-apoptotic state as indicated by loss of the STAT5 targets Mcl-1 and Bcl-xL, but not Bcl-2 protein expression (associated with the Ras/Raf/MEK/Erk The loss of STAT5 activity is critical for irreversible induction of cell pathway), indicating that loss of STAT5 activation was an death important factor. The induction of apoptosis markers Bim and To determine the critical factors required for TKI-induced cell cleaved PARP were also observed (Figure 2b). death, signaling dynamics were characterized in KU812 cells To confirm that exposures of greater than 30 min to 100 nM exposed to 100 nM dasatinib for 30 min followed by the OPT wash. dasatinib followed by OPT wash maintain inhibition of STAT5

Leukemia (2015) 76 – 85 & 2015 Macmillan Publishers Limited STAT5 inactivation is critical for sensitivity to TKI treatment L Schafranek et al 79

Figure 2. Extension of Bcr-Abl inhibition, and therefore inhibition of pSTAT5, before OPT washouts induces apoptosis despite reactivation of Bcr-Abl signaling. KU812 cells were transiently exposed to 100 nM dasatinib (Das) for either (a)30minor(b) 8 h followed by the OPT washout, then cultured for 0–48 h in Das-free media. Protein lysates were analyzed by western blotting for Bcr-Abl signaling (left) and apoptotic signaling (right). (c)CP-CMLCD34þ cells were transiently exposed to 100 nM Das for 0–8 h followed by either the STD or OPT washout. Cells were then analyzed by pSTAT5 (Y694) intracellular phosphoflow. Left panel: graph of mean fluorescence intensity, MFI (data are mean þ s.e.m., n ¼ 3). *Po0.05, **Po0.01, compared with 30 min OPT washout. Right panel, histogram of one representative sample. Ctrl, control. inhibition occurs in primary cells, pSTAT5 was assessed in de novo treatment. STAT5 inhibition with pimozide (Figure 3a) and the CP-CML CD34 þ cells by flow cytometry (Figure 2c). In CML cells, specific STAT5 inhibitor N0-((4-oxo-4H-chromen-3-yl)methylene) STAT5 is constitutively activated by Bcr-Abl, and as such, there was nicotinohydrazide (from now on referred to as STAT5i; Figure 3b) a decrease in the mean fluorescence intensity (MFI) of pSTAT5 had little effect. However, only 30 min exposure to 100 nM from 8.3 in untreated cells to 3.9 in cells treated with 100 nM dasatinib (OPT wash) in combination with either pimozide or dasatinib for 30 min (Figure 2c). In cells treated for 30 min with STAT5i effectively induced cell death in both KU812 cells (2.3 and 100 nM dasatinib followed by the STD wash, STAT5 was 21% viable; Figures 3a and b), Meg01 (24.4 and 29.8% viable) and inhibited (2.8 MFI); however, upon OPT wash, pSTAT5 was K562 (16.6 and 26.1% viable) cells (Supplementary Figure 5). restored (8.5 MFI). In contrast, increasing the initial dasatinib The mechanism of STAT5 activation by Bcr-Abl is unclear, and it exposure to 4 or 8 h before OPT wash demonstrated an inhibition has been reported that JAK2 is involved.6 Intriguingly, a of pSTAT5 measured at 30 min following the OPT wash (18% and combination of the pan JAK kinase inhibitor ruxolitinib with 3.5%, respectively). 30 min 100 nM dasatinib OPT wash (69.5% viable) was unable to reduce viable cells compared with treatment with ruxolitinib alone in KU812 cells (73.9%, P ¼ 0.41; Figure 3c). Surprisingly, Erk STAT5 inhibition sensitizes BCR-ABL1 þ cells to Bcr-Abl inhibition inhibition had no additional effect on cell death following the Inhibitors of STAT5 were utilized to assess the role of STAT5 in 30 OPT wash (69.2% viable) compared with U0126 alone (61.1% the commitment of BCR-ABL1 þ cells to death following TKI viable, P ¼ 0.73; Figure 3d).

& 2015 Macmillan Publishers Limited Leukemia (2015) 76 – 85 STAT5 inactivation is critical for sensitivity to TKI treatment L Schafranek et al 80

Figure 3. Combination of STAT5 inhibition, but not JAK1/2, autophagy or MEK inhibition, with 30 min dasatinib exposure induces cell death in CML cells. (a–d) KU812 cells were transiently exposed to 100 nM dasatinib for 30 min followed by either the STD wash or the OPT wash in the presence or absence of the inhibitors (a)5mM or 10 mM of the STAT5 inhibitor pimozide and (b)50mM of the STAT5 inhibitor N0-((4-oxo-4H- chromen-3-yl)methylene) nicotinohydrazide (STAT5i), (c)1mM of the JAK inhibitor ruxolitinib, (d)10mM of the autophagy inhibitor chloroquine (CQ), (e)5mM of the MEK inhibitor U0126 and then cultured for 72 h in dasatinib-free media. Cells were then analyzed by Annexin V/7-aminoactinomycin D staining (data are mean þ s.e.m., n ¼ 3). (e, f) CP-CML CD34 þ cells were treated with 100 nM dasatinib for 30 min, followed by the OPT wash in the presence or absence of the STAT5 inhibitors (e) pimozide or (f) STAT5i and then cultured for 72 h in dasatinib- free media containing that STAT5 inhibitor. Cells were then analyzed for clonogenic potential using the colony-forming unit-granulocyte and macrophage assay at 2 weeks (data are mean þ s.e.m., n ¼ 3). Ctrl, control.

In de novo CP-CML CD34 þ cells, the combination of 30 min In CP-CML CD34 þ cells, treatment with 10 mM pimozide alone 100 nM dasatinib OPT wash with continuous exposure to pimozide caused a decreased pSTAT5 MFI (3.3) compared with untreated (12.8% CFUs, Po0.0001; Figure 3e) or STAT5i (17.9% CFUs, control (8.3 MFI; Figure 4b). In cells treated with 10 mM pimozide in Po0.01; Figure 3f) significantly reduced colony formation combination with 30 min 100 nM dasatinib OPT wash, pSTAT5 compared to 30 min 100 nM dasatinib OPT wash alone (86.1% remained inhibited (3.4 MFI) compared with the dasatinib alone CFUs). This reduction was also observed with a 30-min 32.5 mM OPT wash treatment (8.6 MFI). The combination of STAT5i with imatinib OPT wash alone (85.7% CFUs) compared with combina- 30 min 100 nM OPT wash also resulted in sustained inhibition of tion with pimozide (8.9% CFUs, Po0.001) or STAT5i (16.9% CFUs, pSTAT5 (Supplementary Figure 7). These data confirm the Po0.005; Supplementary Figure 6). Pimozide alone also induced a importance of inhibition of pSTAT5 following the transient reduction in colonies (56.4%); however, this may be attributed to exposure to dasatinib. off-target effects of pimozide, as STAT5i alone had little effect on the inhibition of colony formation (97.2%, P ¼ 0.24). JAK kinase inhibition prevents cytokine activation of STAT5, but not Bcr-Abl activation of STAT5 Loss of STAT5 activation results in loss of Bcl-xL and Mcl-1, and Chemical inhibition of STAT5, but not ruxolitinib inhibition of JAK induces apoptotic markers despite the reactivation of Bcr-Abl kinases, in combination with 30 min TKI treatment induced cell Analysis of Bcr-Abl and apoptotic signaling was assessed following death, suggesting JAK signaling is not significantly involved in this 30 min OPT treatment in combination with STAT5 inhibition by setting (Figures 3a–c). To assess the contribution of the JAK-STAT pimozide. Inhibition of pBcr-Abl and pErk was completely restored pathway to Bcr-Abl survival signaling, the effect of 1000 nM by 2 h following the removal of dasatinib (Figure 4a). However, we ruxolitinib on the cytokine activation of STAT5 was assessed. observed sustained loss of pSTAT5 following the OPT wash, which STAT5 activation was assessed in the presence and absence of a confirmed efficient blockade of STAT5 activation by pimozide. growth factor mix (5GF) in CP-CML CD34 þ cells by flow Furthermore, we observed increased expression of Bim and cytometry. In the absence of 5GF, ruxolitinib alone had little cleaved PARP signaling the irreversible induction of apoptosis, effect on pSTAT5, however, 100 nM dasatinib inhibited pSTAT5 accompanied by decreased STAT5 partners Bcl-xL and Mcl-1, but whether alone or in combination with ruxolitinib (Figure 5a). The not Bcl-2 (Figure 4a). addition of 5GF stimulated further activation of STAT5 (Figure 5a).

Leukemia (2015) 76 – 85 & 2015 Macmillan Publishers Limited STAT5 inactivation is critical for sensitivity to TKI treatment L Schafranek et al 81

Figure 4. Combination of STAT5 inhibition, with 30 min dasatinib exposure on Bcr-Abl signaling in CML cells. (a) KU812 cells were transiently exposed to 100 nM dasatinib and 10 mM of the STAT5 inhibitor pimozide, followed by the OPT wash and then cultured for 0–48 h in dasatinib- free media containing pimozide. Protein lysates were analyzed by western blotting for Bcr-Abl signaling (left) and apoptotic signaling (right). (a) CP-CML CD34 þ cells were transiently exposed to 100 nM dasatinib and 10 mM of pimozide for 30 min, followed by the OPT wash and then cultured in dasatinib-free media containing pimozide. Cells were then analyzed by pSTAT5 (Y694) intracellular phosphoflow. Cells were then analyzed by pSTAT5 (Y694) intracellular phosphoflow. Left panel: graph of mean fluorescence intensity, MFI (data are mean þ s.e.m., n ¼ 3). *Po0.05 compared with 30 min OPT washout. Right panel: histogram of one representative sample.

Although dasatinib completely inhibited pSTAT5 in the absence of STAT5 inhibition and JAK inhibition offers no further benefit 5GF, this could be partially restored by 5GF stimulation. Ruxolitinib except in the presence of cytokines, with STAT5 common to both removed 5GF-stimulated pSTAT5 both in the presence and pathways. absence of dasatinib, suggesting that the role of JAK2 activation of STAT5 in BCR-ABL1 þ cells may predominantly occur through Cytokine protection from TKI-induced cell death is prevented by cytokine signaling and not via Bcr-Abl. STAT5 inhibition As cytokine activation of JAK2 activates STAT proteins other that 32 Ruxolitinib targets cytokine protection of CP-CML CD34 þ cells STAT5, the ability of STAT5i to abrogate cytokine protection from TKI-induced cell death was assessed. Exposure to 1 nM Ruxolitinib specifically inhibits JAK1 (IC50 2.7 nM), JAK2 (IC50 dasatinib significantly reduced cell viability compared with 4.5 nM) and JAK3 (IC50 322 nM) and is commonly used in vitro at 30,31 untreated cells (8.3% viable, P 0.0001), with restoration 300–1000 nM, thus we assessed optimal ruxolitinib dose in o observed in the presence of 5GF (63.7% viable, P 0.003; BCR-ABL1 þ cells. In the absence of cytokines, ruxolitinib had little ¼ effect on cell viability; however, in the presence of 5GF, ruxolitinib Figure 6a). In the absence of cytokines, STAT5i alone (85.9% resulted in a dose-dependent reduction in live cells (Figure 5b). viable) had a minimal effect on cell viability and was not Ruxolitinib enhanced dasatinib-induced cell death only in the significantly different from ruxolitinib alone (89.4% viable, presence of 5GF, providing no further benefit to dasatinib P ¼ 0.39; Figure 6a). However, in the presence of 5GF (107.6% treatment alone in the absence of cytokines. This suggests that viable), STAT5i significantly reduced the proportion of viable cells JAK inhibition may only be effective in the context of cytokine in the absence (90.8% viable, P ¼ 0.007) or presence of dasatinib stimulation, irrespective of Bcr-Abl activity. No additional effect of (22% viable, Po0.0001; Figure 6a). Interestingly, the amount of cell death induced by STAT5i was not significantly different from that 3000 nM ruxolitinib was observed, thus concentrations of 300 and induced by ruxolitinib in the presence of dasatinib with 5GF 1000 nM were selected. (12.7% viable, P ¼ 0.1; Figure 6a). In the absence of cytokines, 10 nM dasatinib alone significantly reduced CP-CML CD34 þ colony formation (28.5% CFUs of untreated control, P ¼ 0.01); however, 300 nM ruxolitinib alone JAK inhibition, but not STAT5 inhibition, reduced normal did not significantly reduce CFUs (87.7% CFUs, P ¼ 0.15) and the CD34 þ CFUs combination of dasatinib with 300 nM ruxolitinib (23.5%, P ¼ 0.82) Previous observations have demonstrated that JAK2 inhibition is did not further reduce CFUs compared with dasatinib alone toxic to normal hematopoietic cells,5 thus, to determine any toxic (Figure 5c). The addition of 5GF significantly increased colony effects of STAT5 or JAK inhibition, the effect of ruxolitinib and formation (125.4% CFUs), which was significantly reduced by STAT5i on CD34 þ cells from normal bone marrow donors was 10 nM dasatinib (74% P ¼ 0.03) or 300 nM ruxolitinib (78.7%, assessed. Exposure to dasatinib alone resulted in minimal P ¼ 0.01; Figure 5d). The combination of dasatinib with 300 nM reduction of colonies (93.3% CFUs of untreated control; ruxolitinib resulted in a further decrease in colonies (27.1% CFUs, Figure 6b). Based on the in vivo reports30,33 and our in vitro P ¼ 0.01). Again, suggesting a specific role for JAK inhibition is in observations in CP-CML CD34 þ cells (Figure 5), concentrations of cytokine blockade. Therefore, dasatinib may result in effective 300 and 1000 nM ruxolitinib were assessed. Ruxolitinib decreased

& 2015 Macmillan Publishers Limited Leukemia (2015) 76 – 85 STAT5 inactivation is critical for sensitivity to TKI treatment L Schafranek et al 82

Figure 5. Bcr-Abl activation of STAT5 is not via JAK-2. (a) CP-CML CD34 þ cells were treated without (top) or with (bottom) a 5 growth factor mix (5GF) in the presence or absence of ruxolitinib (JAKi) and/or dasatinib (Das). Cells were then analyzed by pSTAT5 (Y694) intracellular phosphoflow. Histograms are representative of at least two independent experiments. (b) CP-CML CD34 þ cells were treated with increasing concentrations of ruxolitinib in the absence (top) or the presence (bottom) of a 5GF mix, ±10 nM dasatinib. Cells were then analyzed for Annexin V/7-aminoactinomycin D at 72 h (n ¼ 1). (c) CP-CML CD34 þ cells were treated with 300 nM or 1000 nM ruxolitinib in the presence or absence of 5GF, with or without 10 nM dasatinib for 72 h. Cells were then analyzed for clonogenic potential using the colony-forming unit-granulocyte and y yy macrophage assay at 2 weeks (data are mean þ s.e.m., n ¼ 3). *Po0.05 compared with no GF untreated control. Po0.05, Po0.01 compared with 5GF untreated control.

colonies at both 300 nM (68.4% CFUs) and 1000 nM (49.4% CFUs), signaling was observed during continuous 1 nM dasatinib whereas STAT5i demonstrated minimal toxicity to normal CD34 þ treatment that was comparable to a 30 min treatment with cells (79.8% CFUs). 100 nM dasatinib following the standard wash protocol. Using an OPT wash procedure reported to prevent induction of apoptosis due to the complete removal of residual TKI,24,25 we demonstrate DISCUSSION that Bcr-Abl, STAT5 and Erk signaling are immediately restored Bcr-Abl protects cells from apoptosis through the activation of cell following removal of 100 nM dasatinib, suggesting that no residual survival signaling involving both STAT5 and Erk, and the dasatinib remains to induce apoptotic signaling. expression of anti-apoptotic proteins to promote cell survival. Pharmacokinetic studies in humans demonstrate that a Progenitor and mature BCR-ABL1 þ cells are dependent on Bcr- significant amount of dasatinib remains available to cells for 4 Abl signaling and therefore exposure to Abl TKIs commits cells to and 8 h after drug ingestion, at doses equating to approximately apoptosis. The treatment paradigm established with the clinical 100 and 50 ng/ml, respectively,34 and that Bcr-Abl activation is development of imatinib was based on the requirement for inhibited for at least 7 h.35 For the first time, we demonstrate that continuous TKI exposure, and therefore continuous Bcr-Abl kinase commitment of BCR-ABL1 þ cells to death requires at least 4 h inhibition, for induction of apoptosis in BCR-ABL1 þ cells. This fails exposure to dasatinib in vitro. This is despite complete removal of to account for the clinical efficacy of once-daily dasatinib therapy dasatinib and reactivation of Bcr-Abl, and subsequent reactivation in CML, which, because of its short half-life in humans, only of STAT5 and Erk. We also observed induction of cell death transiently inhibits Bcr-Abl in vivo. following transient exposures of greater than 2 h to potent Recent reports suggest that a low level of TKI, similar to 1 nM imatinib treatments of 32.5 mM, indicating that this phenomenon is continuous dasatinib for 72 h,26 remains following standard not TKI specific. washout (three consecutive washes) of TKI after a 30-min Importantly, distinct phospho-signaling dynamics were clearly exposure.24–26 Here, we provide new evidence verifying the established between 30 min and 8 h of transient dasatinib hypothesis of Lipka et al.24 that low levels of dasatinib may remain exposure correlating with commitment to apoptosis. This offers following standard washout. Sensitive inhibition of STAT5 and Erk a rationale for the success of once daily dasatinib in the clinical

Leukemia (2015) 76 – 85 & 2015 Macmillan Publishers Limited STAT5 inactivation is critical for sensitivity to TKI treatment L Schafranek et al 83

Figure 7. Proposed mechanism of STAT5 signaling in CML. We propose that JAK-2 may only phosphorylate STAT5 following cytokine receptor activation and that Bcr-Abl-dependent activation of STAT5 occurs despite JAK inhibition and therefore may be JAK-2 independent.

Figure 6. STAT5 inhibition removes protection of TKI-induced cell death signaling in BCR-ABL1 þ cells with minimal effect on non- increased the sensitivity of BCR-ABL1 þ cells to dasatinib and malignant cells. (a) KU812 cells were treated with 50 mM STAT5i, imatinib, whereas combined JAK or MEK/Erk inhibition had no 1000 nM ruxolitinib (Rux) or 1 nM dasatinib (Das) in the presence or additional effect. absence of 5GF, for 72 h. Cells were then analyzed for Annexin V Notably, cell death was achieved despite the reactivation of 7-aminoactinomycin D at 72 h (data are mean þ s.e.m., n ¼ 3). Bcr-Abl. This indicates that STAT5 inhibition is an important factor ****P 0.0001 compared with Das þ GF. (b) CD34 þ cells from o in commitment to cell death, without impairing cell viability or normal bone marrow donors were treated with 50 mM STAT5i, 300 nM or 1000 nM ruxolitinib (Rux) in the presence or absence of 5GF, with clonogenic potential as a sole agent. Bcr-Abl activation of STAT5 or without 10 nM dasatinib (Das) for 72 h. Cells were then analyzed has previously been implicated in the protection of cells from DNA for clonogenic potential using the colony-forming unit-granulocyte damage-induced apoptosis,37 and in protection of BCR-ABL1 þ and macrophage assay at 2 weeks (data are mean þ s.e.m., n ¼ 2). cells from stress (that is, from the loss of Bcr-Abl-dependent survival signals).12 Attenuation of STAT5A with RNAi did not induce apoptosis, but resulted in increased ROS and subsequently setting. STAT5 mediates expression of anti-apoptotic proteins activation of the p53/Chk2 pathway.12 In addition, high expression 10 Bcl-xL and Mcl-1, and is required for maintenance of leukemia. of STAT5 was found to facilitate imatinib resistance, independent Comparisons between the 30 min and 8 h signaling profiles of JAK2, significantly reducing sensitivity to TKI-induced 11 highlighted STAT5 and its downstream targets, Bcl-xL and Mcl-1 apoptosis and elevated STAT5 mRNA correlates to increased as the key determinants of survival or death. Recent experiments mutation rates in CML patients,38 thus supporting our findings that differentially knockdown STAT5A and STAT5B have that direct inhibition of STAT5 may be a more promising demonstrated that genetic loss of STAT5 is not necessarily lethal therapeutic goal than JAK inhibition. to cells, rather response to cell stress and DNA damage is Therefore to clearly define the role of JAK in CML, we observed impaired.12 We therefore targeted signaling pathways associated the combination of dasatinib and ruxolitinib in the presence and with Bcr-Abl to overcome prevention of apoptosis using the OPT absence of cytokines, as cytokine activation of JAK2 has previously washout. been described to have a protective effect on CML cells.28,39 In the STAT5 is a critical participant in TKI resistance11,12 and the setting of a transient exposure to dasatinib (30 min OPT wash), we clinically available dopamine reuptake inhibitor pimozide has did not observe any benefit of the addition of ruxolitinib to been demonstrated to inhibit STAT5 activation in CML cells, achieving induction of cell death, as observed with combined subsequently enhancing TKI-induced apoptosis.3,15 We establish STAT5 inhibition. In a non-pathological setting, activation of STAT5 that treatment with STAT5i, which targets the SH2 domain of occurs by cytokine and growth factor signaling through JAK STAT5,36 had little effect as a sole agent in cell lines or primary CP- kinases. In CML, STAT5 is constitutively active because of the CML CD34 þ cells. Inhibition of STAT5 with pimozide or STAT5i in presence of the Bcr-Abl oncoprotein, with some reports that JAK2 combination with only 30 min exposure to TKI, induced death may phosphorylate or complex with Bcr-Abl.6,7,40 Neviani et al.8 in both BCR-ABL1 þ cell lines and primitive CP-CML CD34 þ cells recently advocated inactivation of the tumor suppressor protein despite complete removal of dasatinib or imatinib after. Thus, phosphatase 2A through a JAK2/b-catenin pathway for the demonstrating STAT5 as a viable target rather than simply a treatment of TKI-refractory CML; however, the involvement of marker of sensitive inhibition of Bcr-Abl as previously proposed.24 STAT5 was not assessed. In this study, we demonstrated a minimal Pimozide alone induced cell death and decreased colonies in reduction of pSTAT5 by ruxolitinib in the absence of cytokines. primary CP-CML CD34 þ cells, however, this may be attributed to Observations of ruxolitinib alone in CD34 þ CP-CML cells did not nonspecific inhibition of other targets by pimozide, which is also significantly reduce viable and colony-forming cells unless in the reported to inhibit proliferation of breast cancer cells by blocking presence of a growth factor cocktail. Furthermore, JAK inhibition T-type calcium channels.13,36 Importantly, both STAT5 inhibitors was only able to remove the live cell population stimulated by

& 2015 Macmillan Publishers Limited Leukemia (2015) 76 – 85 STAT5 inactivation is critical for sensitivity to TKI treatment L Schafranek et al 84 cytokines, and did not further enhance dasatinib-mediated cell 12 Casetti L, Martin-Lannere´e S, Najjar I, Plo I, Auge´ S, Roy L et al. Differential killing. These findings support previous reports that cytokines contributions of STAT5A and STAT5B to stress protection and tyrosine kinase protect cells from TKI-induced apoptosis by Bcr-Abl-independent inhibitor resistance of chronic myeloid leukemia stem/progenitor cells. Cancer Res activation of JAK2/STAT5 pathway.39 2013; 73: 2052–2058. Our data are also supported by in vivo evidence, which suggests 13 Bertolesi GE, Shi C, Elbaum L, Jollimore C, Rozenberg G, Barnes S et al. The Ca(2 þ ) that JAK kinase signaling is only relevant in Bcr-Abl-independent, channel antagonists mibefradil and pimozide inhibit cell growth via different extrinsic activation of STAT5 as JAK2 inhibition had no effect on cytotoxic mechanisms. Mol Pharmacol 2002; 62: 210–219. Bcr-Abl-driven STAT5 signaling or cell death as a sole agent in the 14 Tecott LH, Kwong LL, Uhr S, Peroutka SJ. Differential modulation of dopamine D2 5,9 receptors by chronic haloperidol, nitrendipine, and pimozide. Biol Psychiatry 1986; absence of cytokines. As expected, ruxolitinib significantly 21: 1114–1122. reduced viable cells in the presence of 5GF, but intriguingly 15 Nelson EA, Walker SR, Weisberg E, Bar-Natan M, Barrett R, Gashin LB et al. The STAT5i also significantly reduced viability in the presence of 5GF STAT5 inhibitor pimozide decreases survival of chronic myelogenous leukemia to the same extent as ruxolitinib. Therefore, we propose that cells resistant to kinase inhibitors. Blood 2011; 117: 3421–3429. STAT5 activation as a result of active Bcr-Abl does not 16 Larson RA, Druker BJ, Guilhot F, O’Brien SG, Riviere GJ, Krahnke T et al. Imatinib occur through JAK2, but instead is in close correlation to pharmacokinetics and its correlation with response and safety in chronic-phase phosphorylation at the kinase site of Abl (Figure 7). chronic myeloid leukemia: a subanalysis of the IRIS study. Blood 2008; 111: This study demonstrates that STAT5 inhibition together with 4022–4028. transient TKI treatment also results in marked cell death in primary 17 Picard S, Titier K, Etienne G, Teilhet E, Ducint D, Bernard MA et al. Trough imatinib plasma levels are associated with both cytogenetic and molecular responses to CP-CML CD34 þ cells, whereas JAK inhibition only removes standard-dose imatinib in chronic myeloid leukemia. Blood 2007; 109: 3496–3499. cytokine-induced growth and survival. For the first time, we 18 Brave M, Goodman V, Kaminskas E, Farrell A, Timmer W, Pope S et al. Sprycel provide evidence that continuous inhibition of Bcr-Abl is not for chronic myeloid leukemia and Philadelphia chromosome-positive acute required to induce cell death in CML cells, rather that the lymphoblastic leukemia resistant to or intolerant of imatinib mesylate. Clin Cancer continuous inhibition of its downstream partner STAT5 appears Res 2008; 14: 352–359. essential. Our findings suggest STAT5 is the critical downstream 19 Talpaz M, Shah NP, Kantarjian H, Donato N, Nicoll J, Paquette R et al. Dasatinib in partner of Bcr-Abl and provide a tenable therapeutic target for imatinib-resistant Philadelphia chromosome-positive leukemias. N Engl J Med combination therapy approaches for the treatment of CML. 2006; 354: 2531–2541. 20 Kantarjian H, Cortes J, Kim DW, Dorlhiac-Llacer P, Pasquini R, DiPersio J et al. Phase 3 study of dasatinib 140 mg once daily versus 70 mg twice daily in patients with chronic myeloid leukemia in accelerated phase resistant or intolerant to CONFLICT OF INTEREST imatinib: 15-month median follow-up. Blood 2009; 113: 6322–6329. Nievergall and Hiwase: CSL, research funding, White: Novartis Oncology, honoraria 21 Shah NP, Kasap C, Weier C, Balbas M, Nicoll JM, Bleickardt E et al. Transient and research funding; BMS, honoraria and research funding; CSL, research funding; potent BCR-ABL inhibition is sufficient to commit chronic myeloid leukemia cells Ariad, research funding, Hughes: Novartis Oncology, honoraria, membership on irreversibly to apoptosis. Cancer Cell 2008; 14: 485–493. an entity’s Board of Directors or advisory committees and research funding; 22 Hiwase DK, White DL, Saunders VA, Kumar S, Melo JV, Hughes TP. Short-term BMS, honoraria, membership on an entity’s Board of Directors or advisory committees intense Bcr-Abl kinase inhibition with nilotinib is adequate to trigger cell death in and research funding; Ariad, honoraria, membership on an entity’s Board of Directors BCR-ABL( þ ) cells. Leukemia 2009; 23: 1205–1206. or advisory committees. 23 Snead JL, O’Hare T, Adrian LT, Eide CA, Lange T, Druker BJ et al. Acute dasatinib exposure commits Bcr-Abl-dependent cells to apoptosis. Blood 2009; 114: 3459–3463. 24 Lipka DB, Wagner MC, Dziadosz M, Schnoder T, Heidel F, Schemionek M et al. REFERENCES Intracellular retention of ABL kinase inhibitors determines commitment to 1 Deininger MWN, Goldman JM, Melo JV. The molecular biology of chronic myeloid apoptosis in CML cells. PLoS One 2012; 7: e40853. leukemia. Blood 2000; 96: 3343–3356. 25 Wagner MC, Dziadosz M, Melo JV, Heidel F, Fischer T, Lipka DB. Nilotinib shows 2 Ilaria Jr RL, Van Etten RA. P210 and P190(BCR/ABL) induce the tyrosine phos- prolonged intracellular accumulation upon pulse-exposure: a novel mechanism phorylation and DNA binding activity of multiple specific STAT family members. for induction of apoptosis in CML cells. Leukemia 2012; 27: 1567–1570. J Biol Chem 1996; 271: 31704–31710. 26 Simara P, Stejskal S, Koutna I, Potesil D, Tesarova L, Potesilova M et al. 3 Soliera AR, Mariani SA, Audia A, Lidonnici MR, Addya S, Ferrari-Amorotti G et al. Apoptosis in chronic myeloid leukemia cells transiently treated with imatinib or Gfi-1 inhibits proliferation and colony formation of p210BCR/ABL-expressing dasatinib is caused by residual BCR-ABL kinase inhibition. Am J Hematol 2013; 88: cells via transcriptional repression of STAT 5 and Mcl-1. Leukemia 2012; 26: 385–393. 1555–1563. 27 O’Hare T, Eide CA, Agarwal A, Adrian LT, Zabriskie MS, Mackenzie RJ et al. 4 Kok CH, Watkins DB, Leclercq T, D’Andrea RJ, Hughes TP, White DL. Low GFI1 Threshold levels of ABL tyrosine kinase inhibitors retained in chronic myeloid expression in white blood cells of CP-CML patients at diagnosis is strongly leukemia cells define commitment to apoptosis. Cancer Res 2013; 73: 3356–3370. associated with subsequent blastic transformation. Leukemia 2014; 27: 1427–1430. 28 Hiwase DK, White DL, Powell JA, Saunders VA, Zrim SA, Frede AK et al. Blocking 5 Traer E, MacKenzie R, Snead J, Agarwal A, Eiring AM, O’Hare T et al. Blockade of cytokine signaling along with intense Bcr-Abl kinase inhibition induces apoptosis JAK2-mediated extrinsic survival signals restores sensitivity of CML cells to ABL in primary CML progenitors. Leukemia 2010; 24: 771–778. inhibitors. Leukemia 2011; 26: 1140–1143. 29 White D, Saunders V, Lyons AB, Branford S, Grigg A, To LB et al. In vitro sensitivity 6 Samanta A, Perazzona B, Chakraborty S, Sun X, Modi H, Bhatia R et al. Janus kinase to imatinib-induced inhibition of ABL kinase activity is predictive of molecular 2 regulates Bcr-Abl signaling in chronic myeloid leukemia. Leukemia 2011; 25: response in patients with de novo CML. Blood 2005; 106: 2520–2526. 463–472. 30 Shi JG, Chen X, McGee RF, Landman RR, Emm T, Lo Y et al. The pharmacokinetics, 7 Yamada O, Ozaki K, Furukawa T, Machida M, Wang YH, Motoji T et al. Activation of pharmacodynamics, and safety of orally dosed INCB018424 phosphate in healthy STAT5 confers imatinib resistance on leukemic cells through the transcription of volunteers. J Clin Pharmacol 2011; 51: 1644–1654. TERT and MDR1. Cell Signal 2011; 23: 1119–1127. 31 Quinta´s-Cardama A, Vaddi K, Lui P, Manshouri T, Li J, Scherle PA et al. Preclinical 8 Neviani P, Harb JG, Oaks JJ, Santhanam R, Walker CJ, Ellis JJ et al. PP2A-activating characterization of the selective JAK1/2 inhibitor INCB018424: therapeutic impli- drugs selectively eradicate TKI-resistant chronic myeloid leukemic stem cells. cations for the treatment of myeloproliferative neoplasms. Blood 2010; 115: J Clin Invest 2013; 123: 4144–4157. 3109–3117. 9 Hantschel O, Warsch W, Eckelhart E, Kaupe I, Grebien F, Wagner K-U et al. BCR-ABL 32 DaSilva L, Rui H, Erwin RA, Howard OM, Kirken RA, Malabarba MG et al. Prolactin uncouples canonical JAK2-STAT5 signaling in chronic myeloid leukemia. Nat recruits STAT1, STAT3 and STAT5 independent of conserved receptor tyrosines Chem Biol 2012; 8: 285–293. TYR402, TYR479, TYR515 and TYR580. Mol Cell Endocrinol 1996; 117: 131–140. 10 Hoelbl A, Schuster C, Kovacic B, Zhu B, Wickre M, Hoelzl MA et al. Stat5 is 33 Verstovsek S, Kantarjian H, Mesa RA, Pardanani AD, Cortes-Franco J, Thomas DA et al. indispensable for the maintenance of Bcr/Abl-positive leukaemia. EMBO Mol Med Safety and efficacy of INCB018424, a JAK1 and JAK2 inhibitor, in myelofibrosis. 2010; 2: 98–110. N Engl J Med 2010; 363: 1117–1127. 11 Warsch W, Kollmann K, Eckelhart E, Fajmann S, Cerny-Reiterer S, Holbl A et al. High 34 Christopher LJ, Cui D, Wu C, Luo R, Manning JA, Bonacorsi SJ et al. Metabolism STAT5 levels mediate imatinib resistance and indicate disease progression in and disposition of dasatinib after oral administration to humans. Drug Metab chronic myeloid leukemia. Blood 2011; 117: 3409–3420. Dispos 2008; 36: 1357–1364.

Leukemia (2015) 76 – 85 & 2015 Macmillan Publishers Limited STAT5 inactivation is critical for sensitivity to TKI treatment L Schafranek et al 85 35 Luo FR, Yang Z, Camuso A, Smykla R, McGlinchey K, Fager K et al. Dasatinib 38 Warsch W, Grundschober E, Berger A, Gille L, Cerny-Reiterer S, Tigan AS et al. (BMS-354825) Pharmacokinetics and pharmacodynamic biomarkers in STAT5 triggers BCR-ABL1 mutation by mediating ROS production in chronic animal models predict optimal clinical exposure. Clin Cancer Res 2006; 12: myeloid leukaemia. Oncotarget 2012; 3: 1669–1687. 7180–7186. 39 Wang Y, Cai D, Brendel C, Barett C, Erben P, Manley PW et al. Adaptive secretion of 36 Strobl JS, Kirkwood KL, Lantz TK, Lewine MA, Peterson VA, Worley 3rd JF. granulocyte-macrophage colony-stimulating factor (GM-CSF) mediates imatinib Inhibition of human breast cancer cell proliferation in tissue culture by the and nilotinib resistance in BCR/ABL þ progenitors via JAK-2/STAT-5 pathway neuroleptic agents pimozide and thioridazine. Cancer Res 1990; 50: 5399–5405. activation. Blood 2007; 109: 2147–2155. 37 Hoover RR, Gerlach MJ, Koh EY, Daley GQ. Cooperative and redundant effects of 40 Chen M, Gallipoli P, DeGeer D, Sloma I, Forrest DL, Chan M et al. Targeting STAT5 and Ras signaling in BCR/ABL transformed hematopoietic cells. Oncogene primitive chronic myeloid leukemia cells by effective inhibition of a new 2001; 20: 5826–5835. AHI-1-BCR-ABL-JAK2 complex. J Natl Cancer Inst 2013; 105: 405–423.

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

& 2015 Macmillan Publishers Limited Leukemia (2015) 76 – 85