Letters to the Editor 663 are sensitive to NMD in primary AML. Consequently, these AML References cases do not express the predicted truncated WT1 proteins. In these AML cases, mutations in WT1 may result in haploinsuffi- 1 Yang L, Han Y, Suarez Saiz F, Minden MD. A tumor suppressor and ciency rather than expression of truncated WT1 proteins with oncogene: the WT1 story. Leukemia 2007; 21: 868–876. 0 impaired function. Haploinsufficiency and impaired function have 2 King-Underwood L, Pritchard-Jones K. Wilms tumor (WT1) gene been proposed as two possible mechanisms by which mutant WT1 mutations occur mainly in acute myeloid leukemia and may confer drug resistance. Blood 1998; 91: 2961–2968. may affect normal hematopoietic development. Surprisingly, in the 3 Summers K, Stevens J, Kakkas I, Smith M, Smith LL, Macdougall F WT1 mutant AML cases the wild-type WT1 protein is expressed at et al. Wilms’ tumour 1 mutations are associated with FLT3-ITD and relatively high levels. Although, this phenomena has been shown failure of standard induction chemotherapy in patients with normal in sporadic Wilms’ tumors and some related syndromes, it is karyotype AML. Leukemia 2007; 21: 550–551. currently unclear whether the WT1 protein is pathogenic in these 4 Paschka P, Marcucci G, Ruppert AS, Whitman SP, Mrozek K, cases or whether it is related to the etiology of the cell of origin.1 Maharry K et al. Wilms tumor 1 gene mutations independently predict poor outcome in adults with cytogenetically normal acute myeloid leukemia: A Cancer and Leukemia Group B Study. J Clin Oncol 2008; 26: 4595–4602. Conflict of interest 5 Virappane P, Gale R, Hills R, Kakkas I, Summers K, Stevens J et al. Mutation of the Wilms tumor 1 gene is a poor prognostic The authors declare no conflict of interest. factor associated with chemotherapy resistance in normal karyotype acute myeloid leukemia: The United Kingdom Medical Research Council Adult Leukaemia Working Party. J Clin Oncol 2008; 26: 5429–5435. Acknowledgements 6 Gaidzik VI, Schlenk RF, Moschny S, Becker A, Bullinger L, Corbacioglu A et al. Prognostic impact of WT1 mutations in cytogenetically normal Supported by grants from the Dutch Cancer Society (Koningin acute myeloid leukemia (AML): A Study of the German-Austrian AML Wilhelmina Fonds). Study Group (AMLSG). Blood 2009; 113: 4505–4511. 7 Kuzmiak HA, Maquat LE. Applying nonsense-mediated mRNA decay research to the clinic: progress and challenges. Trends Mol S Abbas, CAJ Erpelinck-Verschueren, CS Goudswaard, Med 2006; 12: 306–316. BLo¨wenberg and PJM Valk 8 Wouters BJ, Jorda MA, Keeshan K, Louwers I, Erpelinck-Verschueren Department of Hematology, Erasmus University Medical CA, Tielemans D et al. Distinct gene expression profiles of acute Center, Rotterdam, The Netherlands myeloid/T-lymphoid leukemia with silenced CEBPA and mutations E-mail: [email protected] in NOTCH1. Blood 2007; 110: 3706–3714.

Dasatinib promotes ATRA-induced differentiation of AML cells

Leukemia (2010) 24, 663–665; doi:10.1038/leu.2009.267; characterized by defective myeloid differentiation and the published online 24 December 2009 accumulation of proliferative blasts. One subtype of AML, acute promyelocytic leukemia (APL), undergoes differentiation in Dasatinib is a US Food and Drug Administration-approved response to treatment with all-trans retinoic acid (ATRA), and compound that was developed as an inhibitor of ABL and Src the combination of ATRA and chemotherapy results in a high family kinases (SFKs).1 Dasatinib is substantially more potent cure rate in this disease. However, the other subtypes of AML, than imatinib against wild-type BCR–ABL. Moreover, with the representing the majority of AML cases, are largely unresponsive exception of the T315I mutant, dasatinib retains activity against to ATRA differentiation therapy. We have previously shown that most imatinib-resistant BCR–ABL mutants, helping to explain SFKs can negatively regulate myeloid differentiation.8 In this the effectiveness of dasatinib in all stages of imatinib-resistant report, we sought to determine whether treatment of AML cell CML. The efficacy of dasatinib in CML is furthered by the ability lines and primary cells with dasatinib might restore or enhance of this compound to inhibit the SFK member LYN, which is ATRA-induced differentiation. persistently hyperactivated in imatinib-resistant disease.2 To determine the impact of dasatinib treatment on myeloid Recent studies have revealed dasatinib effects on hemato- differentiation, HL-60 and NB4 cells were incubated with poietic cells other than CML, and have suggested additional dasatinib in the presence or absence of ATRA, followed by flow therapeutic opportunities, as well as adverse toxicities. Dasati- cytometric analysis of the myeloid differentiation marker CD11b nib inhibits the production of osteoclasts and tumor-associated (Figures 1a and b). HL-60 cells were derived from a patient with macrophages, as well as the functional activities of osteoclasts, AML (FAB M2), and NB4 from a patient with APL (FAB M3). platelets, T cells and natural killer cells.3–7 As these cell types do Both undergo granulocytic differentiation when cultured not express BCR–ABL, inhibition of SFK members such as LYN, in ATRA. To determine whether dasatinib would enhance or other kinase targets, is likely responsible for the effects of ATRA-induced differentiation in these cell-line models, we used dasatinib on these cells. ATRA doses that yielded only low levels of differentiation in a Overexpression and/or hyperactivation of SFKs is common in 72-h assay (1 mM for HL-60; 0.01 mM for NB4; Figures 1a and b, acute myeloid leukemia (AML), but the impact of dasatinib on data not shown). Dasatinib was used at doses (100, 500 nM and this disease is largely unknown. AML is a diverse malignancy 1.0 mM) that did not alter cell viability (not shown). Figures 1a

Leukemia Letters to the Editor 664 and b show that treatment with dasatinib alone did not promote lysates of HL-60 and NB4 cells (Figures 2a and c), as well as differentiation. By contrast, co-treatment with dasatinib and primary AML cells (Supplementary Figure 1). In conjunction, ATRA for 72 h resulted in marked induction of CD11b phosphorylated/activated enzymes were detected only in the expression, relative to that seen with ATRA or dasatinib alone. LYN immunoprecipitate pellet (Figures 2b and d). These results Enhancement of ATRA-induced differentiation was seen using indicate that LYN is the predominant activated SFK in the dasatinib concentrations as low as 100 nM. Qualitatively and HL-60, NB4 and primary AML cells studied, and are consistent quantitatively similar results were seen when differentiation was with the recent observation by Dos Santos et al.14 pointing to scored by cytochemical staining with nitroblue tetrazolium the predominant activation of LYN in AML cells. These findings (Supplementary Table 1). also raise the possibility that selective targeting of LYN To investigate whether dasatinib could be used to restore may be useful for optimizing ATRA-induced differentiation, ATRA-induced differentiation in primary AML cells representing providing further refinement and impact to our discovery that a non-APL subtype, we treated leukemic blasts from a patient treatment with dasatinib can enhance ATRA-stimulated differ- diagnosed with AML FAB M5 subtype (Figure 1c). As shown, co- entiation in AML. treatment with dasatinib and ATRA resulted in enhanced differentiation relative to treatment with either agent alone. These findings suggest that FDA-approved dasatinib in combi- nation with ATRA may provide therapeutic benefit in the p < 0.01 treatment of non-APL AML, wherein current chemotherapy 80 p < 0.05 regimens are associated with considerable adverse toxicities and only modest improvement in survival outcomes. 70 Our findings are consistent with recent findings that 60 p > 0.05 the tyrosine-kinase inhibitors gefitinib and erlotinib can be 50 9–11 used to promote myeloid differentiation. Impressively, 40 treatment of NSCLC patients harboring concomitant AML with 30 the EGFR inhibitor erlotinib has been shown to result in 20 complete remission of the AML.12,13 The effects of gefitinib 10

and erlotinib on AML seem to be due to off-target effects on % CD11b-positive cells other tyrosine kinases, possibly SFKs, because AML cells do not 0 M M M express EGF receptors. We propose that similar therapeutic μ μ μM μM μ

activity against AML might be achieved using dasatinib/ATRA DMSO combination. Das 1 ATRA 1 SFKs are key targets of dasatinib and are overexpressed and/or Das 100nMDas 500nM hyperactivated in a majority of AML specimens. Immunoblotting for total protein levels revealed that HL-60 and NB4 cells Das 1nM + ATRA 1 expressed the SFK members LCK, LYN, FYN, HCK and c-SRC, Das 100nMDas + ATRA 500nM 1 + ATRA 1 whereas primary AML (FAB M5) cells expressed LCK, LYN and HCK (data not shown). To determine which of these SFKs were present in active form, we performed immunodepletion studies 80 p < 0.001 in which specific SFKs were immunoprecipitated from cell 70 lysates, followed by immunoblotting with an antibody 60 (a-phosphoSRC Y418) that recognizes the phosphorylated/ 50 p < 0.001 activated form of multiple SFK members. These experiments 40 showed that immunodepletion of LYN resulted in nearly 30 p > 0.05 complete removal of the phosphorylated/activated SFKs from 20 10 % CD11b-positive cells 0

Figure 1 Dasatinib promotes ATRA-induced differentiation of AML μM μM μM μM μM cell lines and primary non-APL AML cells. HL-60 (a) and NB4 (b) cells DMSO 0.01 were treated for 72 h with 0.1% DMSO, ATRA alone (1 mM for HL-60 Das 1 and 0.01 mM for NB4), dasatinib alone (Das; 100, 500 nM or 1 mM), or Das 100nMDas 500nM the combination of ATRA and varying doses of dasatinib. After ATRA 0.01 ATRA 0.01ATRA 0.01 treatment, cells were incubated with anti-CD11b-PE antibody or isotype-matched antibody, followed by flow cytometric analysis of CD11b expression. Columns represent the mean of three independent Das 1nM + ATRA experiments, and bars the s.e.. Statistical significance was determined Das 100nMDas + 500nM + by one-way analysis of variance, followed by Tukey’s multiple comparison test. (c) Leukemic blasts were collected from the Increase in % CD11b-positive cells peripheral blood of a patient with FAB M5 AML after obtaining relative to DMSO-treated control informed consent in accordance with institutional guidelines, then isolated using Ficoll–Paque Plus. The isolated blasts were treated for Dasatinib 13.8 % 72 h with 0.1% DMSO alone, 1 mM dasatinib alone, 2 mM ATRA alone, or the combination of dasatinib and ATRA. After treatment, the ATRA 2.3 % percentage of CD11b-positive cells was determined by flow cyto- metric analysis. The data are plotted as the increase in percentage of CD11b-positive cells relative to DMSO-treated control. The experi- Dasatinib + ATRA 31.5 % ment was performed twice with similar results.

Leukemia Letters to the Editor

665

[ phospho-SFK [ phospho-SFK

lgG [ phospho-SFK phospho-SFK lgG

Figure 2 LYN is the predominant activated SFK in AML cells. Whole cell lysates from HL-60 (a, b) or NB4 (c, d) cells were subjected to immunoprecipitation with control IgG antibody, or antibodies directed against total LCK, LYN, FYN, HCK or c-SRC proteins. After immunoprecipitation of different SFKs, the immunodepleted cell lysates (a, c) and immunoprecipitate pellets (b, d) were subjected to immunoblotting with anti-phospho-SFK antibody (a-phosphoSRC Y418). Input (untreated) lysates were loaded in the left lanes as controls, and blots of immunodepleted lysates were stripped and reprobed with anti-b-actin to show equal protein input. Immunodepletion of LYN resulted in removal of activated SFKs from lysates and appearance of activated SFKs in the LYN immunoprecipitate pellets. The experiments were performed three times, with similar results each time.

Conflict of interest 4 Brownlow N, Mol C, Hayford C, Ghaem-Maghami S, Dibb NJ. Dasatinib is a potent inhibitor of tumour-associated The authors declare no conflict of interest. macrophages, osteoclasts and the FMS receptor. Leukemia 2009; 23: 590–594. 5 Gratacap MP, Martin V, Valera MC, Allart S, Garcia C, Sie P et al. Acknowledgements The new tyrosine-kinase inhibitor and anti-cancer drug dasatinib reversibly affects platelet activation in vitro and in vivo. Blood 2009; 114: 1884–1892. We thank Bristol-Myers Squibb (New York, NY) for providing 6 Schade AE, Schieven GL, Townsend R, Jankowska AM, Susulic V, dasatinib. This work was supported by National Institutes of Zhang R. Dasatinib, a small-molecule protein tyrosine kinase Health Grant R01 CA108904. inhibitor, inhibits T-cell activation and proliferation. Blood 2008; 111: 1366–1377. 1 1 1 1 1,2 PL Kropf , L Wang , Y Zang , RL Redner and DE Johnson 7 Blake SJ, Bruce Lyons A, Fraser CK, Hayball JD, Hughes TP. 1 Department of Medicine, University of Pittsburgh Dasatinib suppresses in vitro natural killer cell cytotoxicity. Blood and the University of Pittsburgh Cancer Institute, 2008; 111: 4415–4416. Pittsburgh, PA, USA and 8 Miranda MB, Redner RL, Johnson DE. Inhibition of Src family 2Department of Pharmacology, University of Pittsburgh, kinases enhances retinoic acid induced gene expression and Pittsburgh, PA, USA myeloid differentiation. Mol Cancer Ther 2007; 6: 3081–3090. E-mail: [email protected] 9 Stegmaier K, Corsello SM, Ross KN, Wong JS, Deangelo DJ, Golub TR. Gefitinib induces myeloid differentiation of acute myeloid leukemia. Blood 2005; 106: 2841–2848. References 10 Miranda MB, Duan R, Thomas SM, Grandis JR, Redner RL, Jones JE et al. Gefitinib potentiates myeloid cell differentiation by ATRA. 1 Lombardo LJ, Lee FY, Chen P, Norris D, Barrish JC, Behnia K et al. Leukemia 2008; 22: 1624–1627. Discovery of N-(2-chloro-6-methyl-phenyl)-2-(6-(4-(2-hydroxy- 11 Boehrer S, Ades L, Braun T, Galluzzi L, Grosjean J, Fabre C et al. ethyl)-piperazin-1-yl)-2-methylpyrimidin-4-ylamino)thiazole-5- Erlotinib exhibits antineoplastic off-target effects in AML and MDS: carboxamide (BMS-354825), a dual Src/Abl kinase inhibitor with a preclinical study. Blood 2008; 111: 2170–2180. potent antitumor activity in preclinical assays. J Med Chem 2004; 12 Chan G, Pilichowska M. Complete remission in a patient with 47: 6658–6661. acute myelogenous leukemia treated with erlotinib for non small- 2 Wu J, Meng F, Kong LY, Peng Z, Ying Y, Bornmann WG et al. cell lung cancer. Blood 2007; 110: 1079–1080. Association between imatinib-resistant BCR–ABL mutation-nega- 13 Pitini V, Arrigo C, Altavilla G. Erlotinib in a patient with acute tive leukemia and persistent activation of LYN kinase. J Natl myelogenous leukemia and concomitant non-small-cell lung Cancer Inst 2008; 100: 926–939. cancer. J Clin Oncol 2008; 26: 3645–3646. 3 Vandyke K, Dewar AL, Farrugia AN, Fitter S, To LB, Hughes TP 14 Dos Santos C, Demur C, Bardet V, Prade-Houdellier N, et al. Therapeutic concentrations of dasatinib inhibit in vitro Payrastre B, Recher C. A critical role for Lyn in acute myeloid osteoclastogenesis. Leukemia 2009; 23: 994–997. leukemia. Blood 2008; 111: 2269–2279.

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

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