Leukemia (2013) 27, 1567–1614 & 2013 Macmillan Publishers Limited All rights reserved 0887-6924/13 www.nature.com/leu

LETTERS TO THE EDITOR Nilotinib shows prolonged intracellular accumulation upon pulse-exposure: a novel mechanism for induction of apoptosis in CML cells

Leukemia (2013) 27, 1567–1570; doi:10.1038/leu.2012.364 resulted in decreased levels of apoptosis as compared with nilotinib pulse-treated K562 wild-type controls. The difference reached statistical difference for K562 cells overexpressing ABCG2 During the last decade, tyrosine kinase inhibitors (TKI) have (Figure 1d). This observation is in line with publications showing fundamentally changed the landscape of cancer treatment.1–6 that ABC-transporter expression reduces responsiveness of Based upon preclinical data, it was believed that continuous BCR-ABL-positive cells to TKI treatment.14,15 Based on these kinase inhibition is a prerequisite for successful TKI treatment: It findings, activity of important signaling pathways was assessed has been shown in a chronic myeloid leukemia (CML) mouse by western blot: While P-CRKL showed marked rephosphorylation model that prolonged inhibition of BCR-ABL is necessary to after the first drug wash out, P-STAT5 and BCR-ABL (Y412) eradicate the disease.7 In line with this, clinical trials identified a remained dephosphorylated (‘1x’) and showed only incomplete close relationship between imatinib serum trough-levels and recovery of phosphorylation (‘2x’, ‘3x’) as compared with the clinical effectiveness.8,9 untreated control (‘M’) even after the third round of washing In 2008, Shah et al.10 introduced the concept that transient (Figure 2a). In contrast, phosphorylation of BCR-ABL (Y177) potent BCR-ABL inhibition irreversibly commits CML cells to seemed to be largely unaffected even upon continuous apoptosis. Consecutively, other groups published similar data on nilotinib exposure for 2 and 10 h. Western blot analysis of nilotinib dasatinib and nilotinib.11,12 Very recently, we have performed a pulse-treated K562 cells revealed similar results (Figure 2b). comprehensive investigation on the molecular mechanisms Results of propidium iodide staining and western blotting involved in apoptosis induced upon transient kinase inhibition.13 suggested that nilotinib might be enriched intracellularly upon Interestingly, cellular TKI accumulation and retention was pulse-exposure. Furthermore, slow release into the cell culture identified as the underlying molecular mechanism of induction media has been described for other TKI upon HD-TKI pulse.13 To of apoptosis upon high-dose TKI (HD-TKI) pulse-exposure using quantify a potential nilotinib release into the media after drug either imatinib or dasatinib.13 wash out, a novel liquid chromatography (LC)/mass spectrometry Based upon these findings, we here investigated the behavior (MS)/MS assay was used.16 BCR-ABL þ murine and human cell of nilotinib when applied as high-dose pulse using an established lines, as well as primary human cells from CML patients and from in vitro model of BCR-ABL transformation. For this purpose, Ba/ healthy donors were treated as described above. LC/MS/MS- F3-BCR-ABL cells were treated for 2 h with 1 mM nilotinib, followed measurements of cell culture supernatants after drug wash out by thorough repetitive drug wash out with phosphate-buffered revealed a time-dependent increase of nilotinib, reaching 17 saline, each consisting of two rounds of media exchange (‘1x’). concentrations far above the in vitro IC50 (Figure 2c). Using the This procedure was repeated 2 and 4 h after reseeding of cells in same assay, we measured nilotinib in lysates of BCR-ABL þ cells TKI free cell culture media (‘2x’, ‘3x’). Before washing, cell culture after nilotinib incubation. Based on the cellular volume that supernatants were transferred to previously untreated cells (‘S1’, was derived by calculation from cell diameter measurements ‘S2’, ‘S3’) (Figure 1a). Apoptosis as assessed by propidium iodide (total cellular volume for 0.2 Â 106 cells was calculated to be staining after 24 h showed high levels of apoptotic cell death 0.19 ml for Ba/F3-BCR-ABL cells and 0.5 ml for K562 cells), we following nilotinib pulse-treatment (Figure 1a, ‘1x’), which is in line then calculated the intracellular TKI concentration (for details, with published data.10–13 Upon repeated washing, we observed a please see Lipka et al.13). Thus, we determined the intracellular partial rescue from apoptosis. However, in contrast to our previous nilotinib peak concentration upon 2 h exposure to 1 mM nilotinib to findings employing imatinib and dasatinib, this rescue was be about 960 mM for Ba/F3-BCR-ABL cells and 261 mM for K562 cells. incomplete (Figure 1a ‘2x’, ‘3x’). Transferred supernatants showed This demonstrates that nilotinib dramatically accumulates TKI activity by inducing cell death in previously untreated cells, intracellularly. Taken together, our data indicate intracellular TKI indicating the presence of biologically relevant nilotinib concen- retention upon nilotinib pulse-exposure, which translates into trations (‘S2’, ‘S3’). Employing two other apoptosis assays (staining prolonged inhibition of BCR-ABL kinase, resulting in induction of for AnnexinV and intracellular cleaved caspase3) we were able to apoptosis. These results are in line with recently reported data on confirm these data (Figures 1b and c). As a control, Ba/F3 parental imatinib and dasatinib.13 Previous publications already had cells did not undergo apoptosis after nilotinib pulse-treatment, suggested that transient but potent kinase inhibition can indicating the absence of cytotoxic off-target effects at the TKI efficiently induce apoptosis.10–12 However, our data presented concentrations used (data not shown). We also tested the effects here and in Lipka et al.13 add a novel view to this concept: we of nilotinib pulse-exposure on the human CML cell line K562 and provide evidence that transient kinase inhibition is not sufficient similarly, we observed a gradual decrease in the percentage of to induce apoptosis. Instead, nilotinib (but also imatinib and apoptotic cells upon repetitive washing (data not shown). Then dasatinib13) dramatically accumulates intracellularly upon HD-TKI we investigated the effects of ABC-transporter expression on pulse-exposure. This then results in prolonged intracellular TKI apoptosis upon HD-TKI pulse-exposure. Therefore, we used K562 exposure even after drug removal in the surrounding media. cells overexpressing either ABCB1 or ABCG2. Nilotinib pulse- In the case of nilotinib, we here observed relatively high treatment of cells overexpressing either transporter protein levels of apoptosis even after three rounds of drug wash out

Accepted article preview online 12 December 2012; advance online publication, 11 January 2013 Letters to the Editor 1568

Figure 1. Repetitive washing or overexpression of ABC-transporters prevent apoptosis upon nilotinib pulse-exposure in BCR-ABL-positive 4 cells. Apoptosis assays in Ba/F3 cells: Ba/F3-BCR-ABL cells (5 Â 10 cells/ml) were exposed for 2 h to 1 mM nilotinib and subsequently underwent repetitive wash out procedures as described earlier.13 Cells exposed to 0.1% dimethyl sulfoxide served as controls (‘M’). ‘1x’, ‘2x’, ‘3x’: Cells were treated with HD-TKI for 2 h and drug wash out was performed in 2 h intervals. Furthermore, cell culture supernatants were transferred to previously untreated cells before each washing (‘S1’, ‘S2’, ‘S3’). Percentage of cells in subG1 phase at 24 h was determined by propidium iodide staining. Experiments were performed in triplicate and are shown þ s.e.m. (a) As additional apoptosis assays we determined levels of annexinV-staining (b) and caspase3-cleavage using flow-cytometry (c). One representative experiment is shown in (b) and (c). Impact of ABC- transporter overexpression on HD-TKI-induced cell death: (d) K562, K562-ABCB1 and K562-ABCG2 cells were treated with 1 mM nilotinib for 2 h followed by single drug wash out procedure. Percentage of cells in subG1 phase at 24 h was determined by propidium iodide staining. Experiments were performed in triplicate and are shown þ s.e.m. The asterisk (*) indicates Po0.05 in a two-tailed t-test.

(Figures 1a–c, ‘3x’), which might be due to even more prolonged Taken together, our data presented here underline our intracellular TKI retention of nilotinib as compared with imatinib previous findings that HD-TKI pulse-exposure confers apoptotic and dasatinib. Data presented by Manley et al.18,19 showing cell death by prolonged intracellular TKI activity. Our data indicate increased residence time of nilotinib further support this that monitoring not only plasma but also intracellular drug hypothesis. Nevertheless, further investigations involving a direct levels of TKI may have the potential to optimize dosing schedules. comparison of different TKI are needed to define differential Furthermore, our data indicate that nilotinib might exhibit cellular retention profiles. As published recently for imatinib and more prolonged intracellular retention as compared with imatinib dasatinib,13 we would like to point out that phosphorylation and dasatinib. This interesting point has to be clarified in dynamics of signaling pathways upon repetitive drug wash out further studies directly comparing different TKI for their differ for BCR-ABL(Y412) in comparison with BCR-ABL(Y177), and capacity of intracellular accumulation and retention. for STAT5 in comparison with CRKL. In our view, this explains why These data might then provide a rationale for clinical trials testing in previous work relevant residual TKI levels were considered to be once daily or discontinuous TKI dosing for chronic myeloid ruled out.10–12 leukemia.

Leukemia (2013) 1567 – 1614 & 2013 Macmillan Publishers Limited Letters to the Editor 1569

Figure 2. Intracellular signaling events and nilotinib measurements demonstrate intracellular nilotinib retention upon pulse-exposure. Ba/F3-BCR-ABL (a) and K562 (b) cells were pulse-treated with nilotinib. Drug wash out was performed as described above (a) or according to the protocol described by Shah et al.10 (b). Lysates for western blotting were prepared at each time-point and immunoblotting was performed using antibodies as indicated. Quantification of signals was performed using the public domain open source software ImageJ 1.46 (http:// rsbweb.nih.gov/ij/download.html). Signals were normalized to ‘media’ (M) and further to the respective total protein levels. (c) Nilotinib concentrations were measured in cell culture supernatants using LC/MS/MS as described.16 Ba/F3-BCR-ABL cells (I), K562 cells (II), primary þ human CML patient mononuclear cells (MNCs) (III) and normal CD34 enriched cells (IV) were pulse-exposed to 1 mM nilotinib for 2 h followed by drug wash out. The red lines represent the IC50 for nilotinib in vitro. Supernatant measured at the end of exposure (‘EOE’) served as a positive control. One representative experiment is shown.

CONFLICT OF INTEREST REFERENCES DBL received research funding from Novartis and honoraria from Novartis and BMS; 1 Druker BJ, Guilhot F, O’Brien SG, Gathmann I, Kantarjian H, Gattermann N et al. FH and TF received honoraria from Novartis. All other authors declare no conflicts of Five-year follow-up of patients receiving imatinib for chronic myeloid leukemia. interest. N Engl J Med 2006; 355: 2408–2417. 2 Johnson JR, Cohen M, Sridhara R, Chen YF, Williams GM, Duan J et al. Approval summary for erlotinib for treatment of patients with locally advanced or meta- ACKNOWLEDGEMENTS static non-small cell lung cancer after failure of at least one prior chemotherapy regimen. Clin Cancer Res 2005; 11: 6414–6421. This work was supported by research funds granted by Novartis Pharma GmbH 3 Goodman VL, Rock EP, Dagher R, Ramchandani RP, Abraham S, Gobburu JV. (to DBL), by the ‘Wilhelm-Sander-Stiftung’ (No 2011.079.1 to FH, DBL, and TF) and Approval summary: sunitinib for the treatment of imatinib refractory or intolerant by the ‘Deutsche Forschungsgemeinschaft’ (GRK-1167, P17-2 to TF and SFB-854/1, gastrointestinal stromal tumors and advanced renal cell carcinoma. Clin Cancer TP20 to TF). Res 2007; 13: 1367–1373. 4 Kantarjian H, Giles F, Wunderle L, Bhalla K, O’Brien S, Wassmann B et al. Nilotinib in imatinib-resistant CML and Philadelphia chromosome-positive ALL. M-C Wagner1, M Dziadosz2, JV Melo3, F Heidel1, N Engl J Med 2006; 354: 2542–2551. T Fischer1 and DB Lipka1 5 Talpaz M, Shah NP, Kantarjian H, Donato N, Nicoll J, Paquette R et al. Dasatinib in 1Department of Hematology and Oncology, University Medical imatinib-resistant Philadelphia chromosome-positive leukemias. N Engl J Med Center, Otto-von-Guericke-University, Magdeburg, Germany; 2006; 354: 2531–2541. 2Institute of Forensic Medicine, University Medical Center, 6 Flaherty KT, Puzanov I, Kim KB, Ribas A, McArthur GA, Sosman JA et al. Inhibition of mutated, activated BRAF in metastatic melanoma. N Engl J Med 2010; 363: Otto-von-Guericke-University, Magdeburg, Germany and 3 809–819. Department of Haematology, Centre of Cancer Biology–IMVS, 7 le Coutre P, Mologni L, Cleris L, Marchesi E, Buchdunger E, Giardini R et al. In vivo Adelaide, South Australia, Australia; eradication of human BCR/ABL-positive leukemia cells with an ABL kinase E-mail: [email protected] inhibitor. J Natl Cancer Inst 1999; 91: 163–168.

& 2013 Macmillan Publishers Limited Leukemia (2013) 1567 – 1614 Letters to the Editor 1570 8 Larson RA, Druker BJ, Guilhot F, O’Brien SG, Riviere GJ, Krahnke T et al. Imatinib altered anti-cancer effects and pharmacological properties. Br J Pharmacol 2009; pharmacokinetics and its correlation with response and safety in chronic-phase chronic 158: 1153–1164. myeloid leukemia: a subanalysis of the IRIS study. Blood 2008; 111: 4022–4028. 15 Dohse M, Scharenberg C, Shukla S, Robey RW, Volkmann T, Deeken JF et al. 9 Picard S, Titier K, Etienne G, Teilhet E, Ducint D, Bernard MA et al. Trough Comparison of ATP-binding cassette transporter interactions with the tyrosine imatinib plasma levels are associated with both cytogenetic and molecular kinase inhibitors imatinib, nilotinib, and dasatinib. Drug Metab Disposition 2010; responses to standard-dose imatinib in chronic myeloid leukemia. Blood 2007; 38: 1371–1380. 109: 3496–3499. 16 Dziadosz M, Wagner M-C, Lipka DB, Fischer T, Bartels H. High-performance liquid 10 Shah NP, Kasap C, Weier C, Balbas M, Nicoll JM, Bleickardt E et al. Transient chromatography with ultraviolet detection and protein precipitation as a way of potent BCR-ABL inhibition is sufficient to commit chronic myeloid leukemia cells quantitative determination of nilotinib with and without internal standard. JLiq irreversibly to apoptosis. Cancer Cell 2008; 14: 485–493. Chromatogr Relat Techno 2012. 11 Snead JL, O’Hare T, Adrian LT, Eide CA, Lange T, Druker BJ et al. Acute dasatinib 17 Weisberg E, Manley PW, Breitenstein W, Bruggen J, Cowan-Jacob SW, Ray A et al. exposure commits Bcr-Abl-dependent cells to apoptosis. Blood 2009; 114: 3459–3463. Characterization of AMN107, a selective inhibitor of native and mutant Bcr-Abl. 12 Hiwase DK, White DL, Saunders VA, Kumar S, Melo JV, Hughes TP. Short-term Cancer Cell 2005; 7: 129–141. intense Bcr-Abl kinase inhibition with nilotinib is adequate to trigger cell death in 18 Manley PW, Cowan-Jacob S, Fendrich G, Liebetanz J, Mestan J, Martin N, Fabbro D. BCR-ABL( þ ) cells. Leukemia 2009; 23: 1205–1206. 100th Annual Meeting of the American Association for Cancer Research; 13 Lipka DB, Wagner M-C, Dziadosz M, Schno¨der T, Heidel F, Schemionek M et al. 18–22 April, 2009; Abstract no. 3715. Intracellular retention of ABL kinase inhibitors determines commitment to 19 Manley PW, Cowan-Jacob SW, Fendrich G, Jahnke W, Fabbro D. Nilotinib, in apoptosis in CML cells. PLoS One 2012; 7: e40853. comparison to both dasatinib and imatinib, possesses a greatly prolonged resi- 14 Hegedu+sC,O¨ zvegy-Laczka C, Apa´ti A´ , Mago´csi M, Ne´met K, Orfi+ L et al. Interaction dence time when bound to the BCR-ABL kinase SH1 domain. Blood (ASH Annu of nilotinib, dasatinib and bosutinib with ABCB1 and ABCG2: implications for Meet Abstr) 2011; 118: 1674.

OPEN Initially disadvantaged, TEL-AML1 cells expand and initiate leukemia in response to irradiation and cooperating mutations

Leukemia (2013) 27, 1570–1573; doi:10.1038/leu.2013.15 control. We initially surveyed 5-week-old mice for induction of EGFP in various hematopoietic tissues (Figure 1b). We did not observe any cell type in which TEL-AML1 provided a selective Although TEL-AML1 is present in one-fourth of childhood advantage. There was a clear selective disadvantage of TEL-AML1 precursor B-cell acute lymphoblastic leukemia (pre-B ALL), the in B-cells and in T-cells. TEL-AML1 had a modest negative impact expression of the ETV6-RUNX1 gene encoding this fusion protein on the development of bone marrow myeloid cells and, has been a poor initiator of leukemia in multiple mouse models.1–8 interestingly, was somewhat tolerated by peritoneal B-cells. To gain insight into why this common lesion does not readily We also took advantage of mice that were part of our survival initiate disease, we took advantage of a novel mouse model, in study (see below) to assess how TEL-AML1 impacted hematopoi- which TEL-AML1 expression was linked to a fluorescence marker esis in aging mice. TEL-AML1 did not impact the fitness of and could be induced in a subset of hematopoietic cells during hematopoietic stem and most progenitor cells, but was selected embryonic development. The laboratory of Corrinne Lobe had against in megakaryocyte-erythroid progenitors (MEP, Figure 1c). generated a reporter mouse in which enhanced green fluorescent Results in these aged mice confirmed a selective disadvantage in protein (EGFP) was expressed following Cre-mediated excision maturing lymphocytes, as well as a sizeable disadvantage during (designated herein as ZEG mice), and subsequently generated an granulopoiesis in older mice (Figure 1d). The substantially analogous mouse, in which a human TEL-AML1 encoding impaired contribution to granulopoiesis in older mice might complementary DNA and an internal ribosomal entry site were reflect an interaction of TEL-AML1 with the shifting hematopoietic stem/early progenitor numbers and lineage output that occur in placed upstream of EGFP (designated herein as TA1 mice). 9,10 We crossed the inducible ZEG and TA1 mice with mice aged mice. However, as shown in Supplementary Figure S1, expressing Cre under the control of the Tie2(Tek) promoter, TEL-AML1 expression had no impact on survival. Low-dose radiation can cause lymphoid tumors in mice which is expressed in endothelial cells as well as during early 11 hematopoietic development. These crosses resulted in mice (Gross et al. and references therein). We irradiated Cre/ZEG carrying either the Tie2-Cre and inducible ZEG transgenes and Cre/TA1 mice at 5 and 6 weeks of age with 1.7 Gy. We (Cre/ZEG mice) or the Tie2-Cre and inducible TA1 transgenes observed the impact of this irradiation on the percentage of cells (Cre/TA1 mice). Mice carrying the inducible TA1 transgene but in the peripheral blood that expressed EGFP and on survival. lacking Cre were also generated (Cre-/TA1 þ control mice). Cre/ZEG mice had a high percentage of EGFP expressing The Tie2-Cre cassette resulted in EGFP expression in most, but cells in their peripheral blood whether or not they were not all, mature hematopoietic cells of Cre/ZEG mice (Figures 1a irradiated (Figure 1e). This high percentage of EGFP was seen in and b). The fact that Tie2-Cre resulted in variegated EGFP cells with low side scatter (primarily lymphocytes) and cells with expression enabled us to determine whether TEL-AML1 expres- higher side scatter (primarily granulocytes). In contrast, Cre/TA1 sion had a selective advantage or disadvantage on hematopoietic mice had low levels of EGFP in the blood before irradiation development. If TEL-AML1 provided a selective advantage in Cre/ (greater expression of EGFP was observed in granulocytes than in TA1 mice, we expected the percentage of EGFP expressing cells to lymphocytes), and the percentage of GFP increased greatly in the be increased relative to that observed in the Cre/ZEG mice. blood (in both the low and higher side scatter blood cells) when However, if TEL-AML1 provided a selective disadvantage, EGFP assessed 5–14 months after irradiation. Irradiation curtailed expressing cells would be decreased relative to the Cre/ZEG survival and induced tumors in both the Cre/ZEG and Cre/TA1

Accepted article preview online 16 January 2013; advance online publication, 26 February 2013

Leukemia (2013) 1567 – 1614 & 2013 Macmillan Publishers Limited