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Letters to the Editor 1684 12 Sun L, Wang D, Liang J, Zhang H, Feng X, Wang H et al. Umbilical cord activated to secrete the anti-inflammatory protein TSG-6. Cell Stem Cell 2009; 2: mesenchymal stem cell transplantation in severe and refractory systemic 54 --63. erythematosus. Arthritis Rheum 2010; 62: 2467 --2475. 14 Sato K, Ozaki K, Mori M, Muroi K, Ozawa K. Mesenchymal stromal cells for graft- 13 Lee RH, Pulin AA, Seo MJ, Kota DJ, Ylostalo J, Larson B et al. Intravenous hMSC versus-host disease: basic aspects and clinical outcomes. J Clin Exp Hematop 2010; improve myocardial infarction in mice because cells embolized in lung are 50:79--89.

Supplementary Information accompanies the paper on the Leukemia website (http://www.nature.com/leu) A role for in chronic lymphocytic leukemia

Leukemia (2012) 26, 1684--1686; doi:10.1038/leu.2011.386; progressive Rai Stage II CLL, diagnosed 6 years earlier and published online 13 January 2012 characterized by the 11q deletion with high CD38 expression (44%), was being considered for first-line chemotherapy on the The clinical management of chronic lymphocytic leukemia (CLL) basis of increased fatigue and lymphocyte doubling time patients with fludarabine-resistant disease or tumor cells with 17p (Figure 1a). The patient also had episodal from an deletions remains problematic.1 High-dose (HDGCs) acquired C1 esterase inhibitor deficiency related to CLL.4 Prior to a are among the most effective treatment approaches for such dental procedure, her allergist prescribed Danazol 300 mg p.o. patients, as well as elderly patients who cannot tolerate conven- b.i.d. for 1 week to raise C1-inhibitor levels in order to prevent tional chemotherapy. However, HDGCs are not without significant possible airway obstruction from oral angioedema.5 Coinciden- side effects and are only palliative in nature.2 Identification of tally, she was seen in the CLL clinic a week later where her white nontoxic agents that could improve the therapeutic efficacy of blood cell (WBC) count had dropped from 76 to 42 106 cells/ml HDGCs would help significantly in managing these patients. (Figure 1a) and she reported improvements in subjective symptoms. We report a case that suggests the weak Danazol3 Another dental procedure was scheduled for the following month may be such an agent. A 61-year-old female with slowly and the patient consented to give blood before and after the

Danazol Danazol 12 80 70 10 60 8 /ml) 50 -6 40 6 30 4

Cells (x10 20 10 2

0 Danazol-Specific Death (%) 0 0 2 4 6 8 10 12 0 0.3 1 3 10 30 Months -2 Danazol (μM)

None

60 p > 0.05

50

30 μM Dex

40 7AAD

10 μM Dan 30

20 Danazol-Specific Death (%)

30 μM Dax + 10 10 μM Dan

0 0 200 400 600 800 Females Males 1000 FSC (n=6) (n=5) Figure 1. Effect of Danazol on CLL cells in vivo and in vitro.(a) WBC counts 412 months for the patient described in the text. One week of Danazol caused a drop in the WBC count, which was confirmed by the response to a second treatment. (b) CLL cells obtained from consenting patients who were untreated for at least 3 months, and with local Research Ethics Board (REB) approval, were purified by negative selection12 and cultured in serum-free media in different concentrations of Danazol (Sigma, St Louis, MO, USA). Exclusion of 7-amino-actinomycin D (7AAD) by flow cytometry was used to measure cell viability after 48 h. Danazol-specific death is the difference þ in %7AAD cells between Danazol-treated cells and control cells. Subsequent experiments used 10 mM of Danazol. (c) Example of flow cytometric analysis. Numbers in the dot-plots represent percentages of viable 7AAD cells. (d) Danazol-specific death after 48 h for six female and five male patient samples. The P-value for the difference between sample means was calculated using a two-sample t-test.

Leukemia (2012) 1681 --1729 & 2012 Macmillan Publishers Limited Letters to the Editor 1685 prophylactic course of Danazol. Remarkably, the WBC count, which although it was weaker than Dex (Figure 2a). However, Danazol- had increased in the time off Danazol, decreased significantly mediated killing was not blocked by the GR antagonist again in response to Danazol, although it increased again in the ,6 in contrast to Dex (not shown). Consistent with a following weeks (Figure 1a). Similar declines in circulating WBC GR-independent mechanism, Danazol had additive effects on Dex- counts have been observed in two male patients treated with induced cytotoxicity (Figure 2b). Danazol (400 mg p.o. b.i.d.) for 2 months (not shown). We then examined the effects of Danazol specifically on CLL This apparent clinical activity of Danazol prompted laboratory cells with 17p deletions (Figure 2c, bottom left panel). At a dose of studies of its effects on CLL cells. Danazol was found to be toxic to 10 mM, Danazol remained capable of killing such cells, especially in CLL cells above 3 mM (Figure 1b), although it was generally less combination with Dex. Compared with CLL cells without this toxic than the synthetic (Dex) cytotogenetic abnormality, 17p tumor cells appeared less (Figure 1c). Danazol killed both male and female CLL cells, sensitive to Danazol, with or without Dex, although the differences although the former appeared somewhat more sensitive were not statistically significant (Figure 2c, right panel). Note the (Figure 1d). We considered that Danazol might have off-target results for Figure 2c were obtained with cryopreserved cells and effects on the glucocorticoid (GR) to explain its cytotoxic the higher levels of spontaneous death following thawing account activity against CLL cells. Indeed, Danazol induced phosphoryla- for the lower specific death levels in comparison with Figure 2b, tion of the GR (indicating activation of the receptor) in CLL cells, which were obtained with fresh tumor cells.

* * 70 CLL Patient 4 CLL Patient 5CLL Patient 6 60 Dan --+ + - - + + - - ++ Dex ---+++ + - + --+ 50

pGR 40

30

β-actin Specific Death (%) 20

10 * 0 * Dex Dan Dex+Dan 50 40 Non 17p- 17p- (n=5) 30 (n=6) 50 Non 17p- (n=6) 20 p>0.05 40 10 0 ** 30 p>0.05 p>0.05 30 **

Specific Death (%) 20 20

17p- 10 Average % Specific Death 10 (n=5) 0 Dex Dan Dex+Dan 0 Dex Dan Dex+Dan

HDGC 200

160 HDGC+Danazol

/ml) 120 HDGC+Danazol -6

80

Cells (x10 40

0 024681210 Months Figure 2. Effect of Danazol and glucocorticoids on CLL cells in vitro and in vivo.(a) Purified CLL cells from three different patients were cultured 12 alone, with Dexamethasone (Dex) (Sigma) (30 mM), Danazol (Dan) (10 mM) or both. After 4 h, cell lysates were collected and examined by immunoblotting with antibodies to the phosphorylated glucorticoid receptor (pGR) (Cell Signaling, Beverley, MA, USA), using b-actin as a loading control. The results indicate that Dan can weakly phosphorylate and activate the GR in CLL cells. Similar results were seen with four other samples. (b) Summary of cell viability results, measured with 7AAD-staining and flow cytometry after 48 h of culture, for 13 different patient samples. (c) Cryopreserved cells from 11 additional patients, with (n ¼ 5) or without (n ¼ 6) 17p deletions determined by fluorescence in situ hybridization, were thawed, rested overnight and examined for cell viability, following treatment with Dex, Dan or both. Specific deaths for individual patient samples are shown in the left panels and summarized in the right graph. P-values for the differences between sample means were calculated using a single factor analysis of variance test. *Po0.01; **Po0.02. (d) Changes in the WBC count of the patient described in the text, indicating that addition of Dan to HDGCs can potentially enhance efficacy without increased toxicity.

& 2012 Macmillan Publishers Limited Leukemia (2012) 1681 --1729 Letters to the Editor 1686 Although Danazol and low doses of glucocorticoids could ACKNOWLEDGEMENTS potentially compete for the GR, the possibility of combining This study was supported by the Canadian Institutes of Health Research (CIHR) and Danazol with HDGCs is supported by the example of an 85-year- the Leukemia and Lymphoma Society of Canada (to DS) and a QEII-SST graduate old female with symptomatic Rai Stage IV CLL and trisomy 12 scholarship from the University of Toronto (to ST). tumor cells. Fludarabine- and alkylator-based regimens were felt to be contraindicated because of the patient’s congestive heart S Tung1,2 and DE Spaner1,2,3,4 failure and she was treated with HDGCs (1500 mg PO 1Division of Molecular and Cellular Biology, 5 days),7 but suffered side effects of fluid retention and Sunnybrook Research Institute, Toronto, Canada; profound fatigue. On the basis of previous use of Danazol as a 2Department of Medical Biophysics, University of Toronto, -sparing agent in autoimmune disorders,8 the prednisone Toronto, Canada; dose was lowered to 1000 mg p.o. daily for 4 days and Danazol 3Sunnybrook Odette Cancer Center, Toronto, Canada and (200 mg p.o. b.i.d.) was added to the next two treatment cycles. 4Department of Medicine, University of Toronto, The treatments were better tolerated and the clinical response Toronto, Canada was maintained and possibly even enhanced (Figure 2d). E-mail: [email protected] These observations suggest that Danazol has clinical activity in CLL and can be used safely with HDGCs. Danazol has been REFERENCES prescribed for almost 40 years3 and has a well-known toxicity profile. It causes in young women with , 1 Gribben JG, O’Brien S. Update on therapy of CLL. J Clin Oncol 2011; 29: which is not necessarily a problem for older male or post- 544 --550. 2 Castro JE, James DF, Sandoval-Sus JD, Jain S, Bole J, Rassenti L et al. Rituximab menopausal female CLL patients. Danazol is otherwise relatively in combination with high-dose for the treatment of CLL. safe but patients must be monitored for thrombosis and lipid and Leukemia 2009; 23: 1779 --1789. 9 4,8 abnormalities. Alongwithitsuseinautoimmunedisorders, 3 Fontana V, Dudkiewicz P, Ahn ER, Horstman L, Ahn YS. Danazol therapy combined 3 Danazol is thought to have activity in myelodysplastic syndromes with intermittent application of chemotherapy induces lasting remission in and can prevent interferon-induced thrombocytopenia.10 Over 2500 myeloproliferative disorder (MPD). Hematology 2011; 16:90--94. papers on Danazol are listed in PubMED but, to our knowledge, 4 Cicardi M, Zingale LC, Pappalardo E, Folcioni A, Agostoni A. Autoantibodies and its activity in CLL has not been reported before. lymphoproliferative diseases in acquired C1-inhibitor deficiencies. Medicine The of Danazol in CLL is not clear. Blood levels (Baltimore) 2003; 82: 274 --281. of CD23 were lowered by Danazol in endometriosis,11 suggesting a 5 Morcavallo PS, Leonida A, Rossi G, Mingardi M, Martini M, Monguzzi R et al. in oral surgery. J Oral Maxillofac Surg 2010; 68: direct effect on activated B cells, which are considered the normal 12 2307--2311. counterparts of CLL cells. However, we could not detect an effect of 6 Johanssen S, Allolio B. Mifepristone (RU 486) in Cushing’s syndrome. Eur J Danazol on CD23 expression by CLL cells in vitro (not shown). Danazol Endocrinol 2007; 157: 561 --569. has been reported to decrease the production of inflammatory 7 Spaner DE. Oral high-dose glucocorticoids and ofatumumab in flurarabine- cytokines, including interleukin-6 and tumor necrosis factor-a,which resistant chronic lymphocytic leukemia. Leukemia 2011; e-pub ahead of print may limit their ability to cause proliferation of CLL cells in vivo.13 8 November 2011; doi:10.1038/leu.2011.329. Danazol can also intercalate into cell membranes,14 which may 8 Boruchov DM, Gururangan S, Driscoll MC, Bussel JB. Multiagent induction and dysrupt growth-promoting signaling processes and account for a maintenance therapy for patients with refractory ITP. Blood 2007; 110: 3526--3531. mechanism of action apparently independent of glucocorticoid, 9 Shepherd J. Danazol and plasma lipoprotein . Int J Gynaecol Obstet 1995; 50(Suppl 1): S23 --S26. and androgen receptors (Figure 1d). 10 Alvarez G, Go´ mez-Galicia D, Rodr´ıguez-Fragoso L, Marina V, Dorantes L, Sa´nchez- The observations reported in this letter suggest clinical trials to Alema´nMet al. Danazol improves thrombocytopenia in HCV patients treated determine whether the anti-CLL effect of Danazol can be with peginterferon and ribavirin. Ann Hepatol 2011; 10: 458 --468. exploited to improve the clinical efficacy of HDGCs. Conventional 11 Matalliotakis I, Neonaki M, Koumantaki Y, Goumenou A, Kyriakou D, Koumantakis oral doses of Danazol have been reported to achieve erythrocyte E. A randomized comparison of danazol and leuprolide acetate membrane levels that approach the mM levels that appear to be suppression of serum-soluble CD23 levels in endometriosis. Obstet Gynecol needed for cytotoxicity (Figures 1 and 2). Initial clinical studies will 2000; 95: 810 --813. focus on determining whether optimal dosing of Danazol in CLL 12 Tomic J, Lichty B, Spaner DE. Aberrant interferon-signaling is associated with patients can be achieved through the oral route,7 or whether i.v. aggressive CLL. Blood 2011; 117: 2668 --2680. 13 Tanaka T. Danazol regulates the functions of normal human endometrial stromal administration will be required. cell subpopulations by modifying endometrial cytokine networks. Int J Mol Med 2009; 23: 421 --428. 14 Horstman L, Jy W, Arce M, Ahn YS. Danazol distribution in plasma and cell CONFLICT OF INTEREST membranes as related to altered cell properties: implications for mechanism. The authors declare no conflict of interest. Am J Hematol 1995; 50: 179 --187. Phase I and pharmacokinetic study of elacytarabine, a novel 50-elaidic acid derivative of cytarabine, in adults with refractory hematological malignancies

Leukemia (2012) 26, 1686--1689; doi:10.1038/leu.2012.1 Reduced hENT1 expression and/or activity is associated with adverse therapeutic outcomes in patients with acute myeloid leukemia (AML) treated with ara-C.3,4 Elacytarabine (CP-4055) Cytarabine (ara-C) enters cells primarily as a false substrate (Figure 1), the lipophilic 50-elaidic acid ester of ara-C, enters cells through specialized nucleoside transporter proteins, predomi- independently of hENT1 and is active in both ara-C-resistant cell nantly hENT1 (the human equilibrative nucleoside transporter).1,2 lines5--7 and animal tumor xenograft models.8,9 In patients with

Accepted article preview online 6 January 2012; advance online publication, 27 January 2012

Leukemia (2012) 1681 --1729 & 2012 Macmillan Publishers Limited