Leukemia (2014) 28, 2155–2164 & 2014 Macmillan Publishers Limited All rights reserved 0887-6924/14 www.nature.com/leu

ORIGINAL ARTICLE Highly effective combination of LSD1 (KDM1A) antagonist and pan- deacetylase inhibitor against human AML cells

W Fiskus1, S Sharma2, B Shah1, BP Portier1, SGT Devaraj1, K Liu1, SP Iyer1, D Bearss3 and KN Bhalla1

The histone LSD1 (KDM1A) demethylates mono- and di-methylated (Me2) (K) 4 on histone H3. High LSD1 expression blocks differentiation and confers a poor prognosis in acute myeloid leukemia (AML). Here, treatment with the novel LSD1 antagonist SP2509 attenuated the binding of LSD1 with the corepressor CoREST, increased the permissive H3K4Me3 mark on the target promoters, and increased the levels of p21, p27 and CCAAT/enhancer binding a in cultured AML cells. In addition, SP2509 treatment or LSD1 shRNA inhibited the colony growth of AML cells. SP2509 also induced morphological features of differentiation in the cultured and primary AML blasts. SP2509 induced more apoptosis of AML cells expressing mutant NPM1 than mixed-lineage leukemia fusion oncoproteins. Treatment with SP2509 alone significantly improved the survival of immune- depleted mice following tail-vein infusion and engraftment of cultured or primary human AML cells. Co-treatment with pan-HDAC inhibitor (HDI) panobinostat (PS) and SP2509 was synergistically lethal against cultured and primary AML blasts. Compared with each agent alone, co-treatment with SP2509 and PS significantly improved the survival of the mice engrafted with the human AML cells, without exhibiting any toxicity. Collectively, these findings show that the combination of LSD1 antagonist and pan-HDI is a promising therapy warranting further testing against AML.

Leukemia (2014) 28, 2155–2164; doi:10.1038/leu.2014.119

INTRODUCTION and SOX2.16 A recent report demonstrated that LSD1 inhibition Following standard chemotherapy, while complete remissions are increased H3K4Me2 levels and induced the expression of myeloid 17 routinely achieved, a majority of patients with acute myeloid differentiation-associated . Co-treatment with the LSD1 leukemia (AML) eventually suffer relapse with treatment-refractory inhibitor (TCP), which also inhibits monoamine disease.1 Consequently, the overall 5-year survival of AML patients oxidase (MAO) A and B, and all-trans-retinoic acid was shown to remains B23%, creating a compelling rationale to develop novel diminish the engraftment of primary AML cells in vivo in the NOD/ 17 therapeutics for AML.2 In the pathogenesis of AML, multiple SCID-IL-2receptor-g-deficient (NSG) mice. Also, LSD1 inhibition mechanisms involving genetic alterations and epigenetic with a TCP analog phenocopied LSD1 knockdown in primary deregulations collaborate to cause aberrant maturation arrest, AML cells expressing mixed-lineage leukemia (MLL) fusion growth and survival of early myeloid progenitor cells.3,4 Among oncoprotein.18 Furthermore, LSD1 was shown to sustain the the deregulated epigenetic mechanisms, in addition to DNA leukemogenic potential of the MLL-AF9 leukemia stem cells.18 methylation and histone (H) deacetylation, alterations in histone Collectively, these reports strongly suggest that targeted H3 lysine (K)-specific methylation are involved in promoting the knockdown of LSD1 levels and activity induces differentiation aberrant gene expression or ‘transcriptome’ in AML cells, which and exerts anti-AML activity. However, in each report, the LSD1 includes the deregulated expression of oncogenes and tumor inhibitor used exhibited severe in vivo toxicity at the concentration suppressor genes.5,6 Although the levels H3K27 trimethylation that inhibited LSD1 activity and reduced the AML burden.17,18 (3Me) and H3K9Me3 are among the repressive chromatin marks, SP2509 is a novel, FAD-binding pocket, non-MAO-A and MAO-B H3K4Me3 is a permissive histone modification that promotes gene inhibitor of LSD1.19 In the present studies, we determined the transcription.3,6 LSD1 (KDM1A) is an FAD-dependent histone chromatin-modifying as well as the in vitro and in vivo anti-AML demethylase, with homology to amine oxidases, which activity of SP2509 against cultured and primary human AML cells. demethylates di- and mono-methylated K4 on histone H3, Recently, treatment with a pan-HDAC inhibitor (HDI) was also reducing the permissive H3K4Me3.7,8 LSD1 is known to interact shown to downregulate LSD1 through Sp1 inhibition.20 In AML with the corepressor complex CoREST, containing RE1-silencing cells, the pan-HDI panobinostat (PS) was also shown to increase transcription factor (REST) and the histone deacetylases (HDAC) 1 H3K4Me3 plus inhibit H3K27Me3 levels, inducing p21 (CDKN1A), and 2, which augments the gene repressor activity of LSD1.9,10 p15 (CDKN2B) and p16 (CDKN2A), as well as inhibiting cell cycle High LSD1 expression has been shown to confer poor prognosis in progression and promoting differentiation and apoptosis in AML cancers.11,12 LSD1 has also been shown to demethylate non- cells.21,22 Therefore, in the present studies, we also determined the histone products most notably p53 and DNMT1, which improves in vitro and in vivo anti-AML activity of co-treatment with SP2509 their stability.13–15 Although the null mutation of LSD1 is and PS. Our findings demonstrate that the combined treatment embryonically lethal,15 LSD1 inhibition has been shown to exerts synergistic in vitro activity against cultured and primary attenuate growth of pluripotent cancer cells by repressing OCT4 AML progenitor/stem cells. In addition, as compared with each

1Cancer Center, Houston Methodist Research Institute, Houston, TX, USA; 2Huntsman Cancer Institute, University of Utah, UT, Salt Lake City, USA and 3Department of Physiology and Developmental Biology, Brigham Young University, Provo, UT, USA. Correspondence: Dr KN Bhalla, Cancer Research, Houston Methodist Research Institute, 6670 Bertner Ave., R9-113 Houston, TX 77030, USA. E-mail: [email protected] Received 10 February 2014; revised 11 March 2014; accepted 24 March 2014; accepted article preview online 4 April 2014; advance online publication, 25 April 2014 LSD1 antagonist and HDAC inhibitor against AML cells W Fiskus et al 2156 agent alone, co-treatment with SP2509 and PS significantly Assessment of percentage nonviable cells improved the survival of immune-depleted mice engrafted with Following designated treatments, cells were washed with 1 Â phosphate- cultured or primary human AML cells. buffered saline (PBS), stained with propidium iodide and analyzed by flow cytometry, as previously described.23,24

MATERIALS AND METHODS Assessment of differentiation and CD86 expression Reagents and antibodies Following designated treatments, cells were washed with 1 Â PBS, and then incubated in 0.2% bovine serum albumin/PBS with IgG isotype LSD1 antagonist, SP2509 and its inactive enantiomer, SP2513, were kindly control, anti-CD11b, anti-CD14 or anti-CD86 antibodies in the dark at 4 1C provided by Salarius Pharmaceuticals (Holladay, UT, USA). PS was provided for 30 min. Cells were washed with 0.2% bovine serum albumin/PBS and by Novartis Pharmaceuticals Inc. (East Hanover, NJ, USA). Anti-H3K4Me3, the percentage of CD11b, CD14 or CD86-positive cells was determined by H3K9Me2 and H3K27Me3 antibodies for chromatin immunoprecipitation flow cytometry.23,26 For determination of CD68 expression, cells were fixed were obtained from Millipore (Billerica, MA, USA). Anti-LSD1, cleaved poly with paraformaldehyde, permeabilized with methanol and incubated in (ADP-ribose) polymerase, anti-c-MYC and anti-BIM antibodies were 0.2% bovine serum albumin/PBS with IgG isotype control or anti-CD68 obtained from Cell Signaling (Danvers, MA, USA). Anti-p21WAF antibody antibody in the dark at 4 1C for 30 min. Cells were washed with 0.2% was obtained from Neomarkers (Fremont, CA, USA). Anti-p27KIP antibody bovine serum albumin/PBS and the percentage of CD68-positive cells was was obtained from BD Biosciences (San Jose, CA, USA). Anti-CoREST determined by flow cytometry. For assessment of morphological antibody was obtained from Abcam (Cambridge, MA, USA) Anti-b-actin differentiation of cultured and primary AML cells, cells were cytospun antibody and lentiviral short hairpin RNAs (shRNAs) targeting LSD1 or onto glass slides for 5 min. Cells were stained with hematoxylin and eosin nontargeting shRNA were obtained from Sigma-Aldrich (St Louis, MO, USA). and the cells were observed by light microscopy. The percentage of morphological differentiation was determined by counting 100–200 cells on the slides.23 SP2509 activity assays The LSD1 screening biochemical assay kit was purchased from Cayman Colony growth assay Chemical (Ann Arbor, MI, USA). Test compounds were diluted to 20 Â the desired test concentration in 100% dimethyl sulfoxide and 2.5 ml of the Cultured AML cells were treated with SP2509 and/or PS for 48 h. At the end diluted drug sample was added to a black 384-well plate. The LSD1 of treatment, cells were washed free of the drugs and 500 cells per condition were plated in methylcellulose and incubated at 37 1C. Colony enzyme stock was diluted 17-fold with assay buffer and 40 ml of the diluted 23,24 LSD1 enzyme was added to the appropriate wells. Substrate, consisting of formation was measured 7–10 days after plating. horseradish peroxidase, dimethyl K4 peptide corresponding to the first 21 amino acids of the N-terminal tail of histone H3, and 10-acetyl-3,7- Chromatin immunoprecipitation and real-time PCR dihydroxyphenoxazine was then added to wells. Resorufin was analyzed OCI-AML3 and MOLM13 cells were treated with SP2509 for 16 h. Following on an Envision plate reader (Perkin Elmer, Waltham, MA, USA) with an drug exposure, cross-linking, cell lysis, sonication and chromatin immuno- excitation wavelength of 530 nm and an emission wavelength of 595 nm. precipitation for 3MeK4 Histone H3, 2MeK9 Histone H3 or 3MeK27 Histone The activity of SP2509 on the other oxidases was determined by using H3 was performed according to the manufacturer’s protocol (Millipore). For commercially available kits. For determining the glucose oxidase activity quantitative assessment of p57KIP, KLF4 and p21 promoter DNA in the (which also noncovalently binds FAD in an elongate conformation), the chromatin immunoprecipitates, a SYBR Green Mastermix from Applied glucose oxidase kit used was procured from Life Technologies (Carlsbad, Biosystems (Foster City, CA, USA) was used. Relative enrichment of the CA, USA; catalog number A22189). The MAO assays were performed using promoter DNA in the chromatin immunoprecipitates was normalized the MAO-glo kit (Promega V1401, Madison, WI, USA) with MAO-A from against the amount of p57KIP, KLF4 and p21 promoter DNA in the input Promega (V1452) and MAO-B from Sigma (M7441-1VL, St Louis, MO, USA). samples.22,27

Cell culture RNA isolation and quantitative PCR OCI-AML3 and MOLM13 cells were obtained from the DSMZ (Braunsch- Following the designated treatments with SP2509, total RNA was isolated weig, Germany) and cultured in the recommended media. MV4-11 cells with a High Pure RNA isolation kit (Roche Diagnostics, Indianapolis, IN, were obtained from the American Type Culture Collection (Manassas, VA, USA) and reverse transcribed. Quantitative real-time PCR analysis for the expression of p57KIP, KLF4 and p21 was performed on complementary USA) and cultured in the recommended media. All experiments with cell 22,23 lines were performed within 6 months after thawing or obtaining from the DNA using TaqMan probes from Applied Biosystems. Relative American Type Culture Collection or DSMZ. Cell line authentication was mRNA expression was normalized to the expression of glyceraldehyde performed by the American Type Culture Collection or DSMZ. The 3-phosphate dehydrogenase. American Type Culture Collection and DSMZ use short tandem repeat profiling for characterization and authentication of cell lines. shRNA to LSD1 Lentiviral shRNAs targeting LSD1 or nontargeting shRNA were procured from Sigma-Aldrich and transduced into OCI-AML3 cells, as previously Primary normal progenitor and AML blast progenitor cells described.23 Forty-eight hours post transduction, the cells were washed Primary peripheral blood and/or bone marrow aspirate AML samples were with complete media and plated with or without PS for 48 h for assessing obtained and prepared for the studies below, as previously described.22,23 apoptosis, cell proliferation or colony growth. Banked, de-linked and de-identified, normal or AML CD34 þ or AML CD34 þ CD38 À LIN À bone marrow progenitor/stem cells were purified, Cell lysis, protein quantification and immunoblot analyses as previously described.21,22 Untreated or drug-treated cells were centrifuged, and the cell pellets were lysed and the protein quantification and immunoblot analyses were 22–24 Assessment of apoptosis by Annexin V staining performed, as previously described. Immunoblot analyses were performed at least twice and representative immunoblots are shown. Untreated or drug-treated cells were stained with Annexin V (Pharmingen, San Diego, CA, USA) and TO-PRO-3 iodide and the percentages of apoptotic cells were determined by flow cytometry, as previously In vivo model of AML described.23,24 The combination index for each drug combination and All in vivo studies were approved by, and conducted in accordance with the evaluation of the synergistic interactions were calculated by median the guidelines of the Institutional Animal Care and Use Committees at the dose effect analyses (assuming mutual exclusivity) utilizing the Houston Methodist Research Institute. Female NOD/SCID mice were commercially available software Calcusyn (Biosoft, Ferguson, MO, USA).25 exposed to 2.5 Gy of radiation. The following day, 5 million OCI-AML3 Combination index values of less than 1.0 represent a synergistic cells were injected into the lateral tail vein of the mice and the mice were interaction of the two drugs in the combination. monitored for 7 days. Following treatments were administered in cohorts

Leukemia (2014) 2155 – 2164 & 2014 Macmillan Publishers Limited LSD1 antagonist and HDAC inhibitor against AML cells W Fiskus et al 2157 of eight mice for each treatment: vehicle alone, 25 mg/kg SP2509, 5 mg/kg the maximum increase observed for the mRNA of p21, following PS and SP2509 plus PS. Treatments were initiated on day 7 for OCI-AML3 exposure to SP2509 for 16 h (Figure 2c). Treatment with SP2509 cells. SP2509 (formulated in solubilization buffer (20% Cremaphor, 20% exerted similar effects on the chromatin alterations and the mRNA dimethyl sulfoxide, 60% sterile water)) was administered twice per week levels in MOLM13 cells (Supplementary Figures 3A and B). We also (Tuesday and Thursday) intraperitoneally for 3 weeks, and then determined whether treatment with SP2509 affected the associa- discontinued. PS (formulated in 5% dimethyl sulfoxide/95% normal saline) was administered by intraperitoneally injection 3 days per week (Monday, tion of LSD1 with the corepressor CoREST in the cultured AML Wednesday, Friday) for 3 weeks and discontinued. The survival of mice is cells. As shown in Figure 3a, SP2509 treatment disrupted the represented by a Kaplan–Meier survival plot.22,23 The doses of PS used in association of LSD1 with CoREST in OCI-AML3 and MOLM13 cells, these studies were determined to be safe and effective through previously without affecting the levels of LSD1. Concomitantly, treatment reported studies.22 A separate in vivo experiment was conducted using with SP2509 also induced the protein levels of p53 and p21, as NSG mice and primary AML cells.28,29 Following engraftment of the AML well as of the myeloid differentiation-associated transcription cells (presence of greater than 1% CD45 þ cells in the peripheral blood), factor CCAAT/enhancer binding protein a (C/EBPa) in the AML mice were treated with SP2509 and/or PS, as described above, for 3 cells (Figure 3b). weeks.9,29 The survival of mice is represented by a Kaplan–Meier plot.

Statistical analysis Treatment with SP2509 induces differentiation of cultured and Significant differences between values obtained in a population of AML primary AML cells cells treated with different experimental conditions were determined using Previous reports have demonstrated that treatment with the a two-tailed, paired t-test or a one-way analysis of variance within an nonspecific LSD1 antagonist TCP or a biguanide polyamine analog analysis package of Microsoft Excel 2010 software or using GraphPad Prism induces myelo-monocytic differentiation-associated markers and (GraphPad Software Inc., CA, USA). P-values of less than 0.05 were assigned induces the morphologic features of differentiation in AML cells.17 significance. Statistical differences in the survival of the mice treated with On the basis of this and prompted by the observation that SP2509 SP2509, PS or SP2509 þ PS were determined by log-rank (Mantel–Cox) test. P-values of less than 0.05 were assigned significance. treatment induced p21 levels and increased the expression of C/EBPa, we determined the ability of SP2509 to induce differentiation in AML cells. Treatment with SP2509 increased the RESULTS expression of the myelo-monocytic differentiation marker CD11b, with only a minimal increase in the expression of CD68 in OCI-AML3 SP2509 inhibits LSD1 activity, reduces colony growth and induces apoptosis of cultured AML cells cells (Figure 4a). In contrast, exposure to SP2509 increased the expression of CD14 and CD68 in MOLM13 cells (Figure 4b). Utilizing a cell-free in vitro assay, we first determined the activity of MOLM13 cells do not express CD11b, and SP2509 treatment did not SP2509 against a variety of oxidases including the MAOs A and B induce its expression in MOLM13 cells. Unlike what has been (MAO-A and MAO-B) (Figures 1a and b). SP2509 was found to previously reported, SP2509 treatment did not increase the selectively inhibit LSD1 at low nanomolar concentrations expression of CD86 in OCI-AML3 or MOLM13 cells (Figure 1b). The 50% inhibitory concentration of SP2509 against (Supplementary Figure 4).26 Treatment with SP2509 also increased LSD1 was determined to be 13 nM. SP2509 was inactive against the expression of CD11b and CD14 in four separate samples of MAO-A, MAO-B, lactate dehydrogenase and glucose oxidase. We primary AML blasts expressing NPM1c þ and/or FLT3-ITD next determined the ability of SP2509 to induce apoptosis in (Figure 4c). Importantly, SP2509 treatment also induced the cultured AML cells. As shown in Figure 1c, treatment with SP2509 morphological features of differentiation in OCI-AML3 and MOLM13 for 48 h dose dependently induced modest levels of apoptosis in cells, as exemplified in Figure 4d. Exposure to SP2509 also dose AML cells (Figure 1c). However, OCI-AML3 cells, which are known 23 dependently induced morphological features of differentiation in to express mutant NPM1 and DNMT3A, were significantly more the primary AML blasts (Figure 4e), as also exemplified and sensitive than the other AML cell types (Po0.05). As determined presented in Figure 4f. Induction of the morphological features of by propidium iodide uptake and flow cytometry, following 96 h of differentiation is regarded as the ‘gold standard’ of evidence for exposure to SP2509, OCI-AML3 cells also exhibited the highest loss differentiation in the myeloid lineage AML cells. of viability compared with the other AML cell types (Figure 1d). Treatment with SP2509 also dose dependently depleted the percentage of cells positive for Ki67 expression and inhibited the Targeted knockdown of LSD1 mimics the anti-AML activity of colony growth of OCI-AML3 significantly more than that of the SP2509 other AML cell types studied (Po0.01), showing greater than 90% To confirm that the effects of SP2509 on the AML cells was due to loss of clonogenic survival of OCI-AML3 cells (Figure 1e and SP2509-mediated inhibition of LSD1, we also determined the Supplementary Figure 1). effect of the shRNA-mediated genetic knockdown of LSD1 in OCI- AML3 cells. As compared with the nontargeted (NT) control SP2509 treatment inhibits the association of LSD1 with CoREST, shRNA, the LSD1-targeted shRNA knocked down the expression of increases promoter-specific H3K4Me3 and induces p53, p21 and LSD1 by B50% (Figure 5a). Consistent with the previous reports C/EBPa in AML cells documenting that LSD1 stabilizes DNMT1 levels,15 targeted knock Turning to the chromatin effects of SP2509, using chromatin down of LSD1 also depleted DNMT1 in OCI-AML3 cells (Figure 5a). immunoprecipitation analysis with anti-H3K4Me3 antibody, we Attenuation of LSD1 was associated with increase in the levels of determined the effect of SP2509 treatment on the levels of global histone H3K4Me2, H3K4Me3 and H3K9Me2, but depletion H3K4Me3 that was associated with the chromatin of the known in the levels of c-Myc protein (Figure 5a). Lentiviral transduction of LSD1 target gene promoters, for example, those of p57KIP, KLF4 LSD1 shRNA in OCI-AML3 cells resulted in decline in the and p21. Treatment with SP2509 increased the level of H3K4Me3 suspension culture growth over 96 h (Figure 5b) and colony from 1.7-fold (p57KIP and KLF4) to 3.5-fold (CDKN1 A, p21) culture growth in semisolid medium over 10 days (Figure 5c). It is associated with the chromatin of these gene promoters in OCI- noteworthy that, compared with OCI-AML3 cells transduced by AML3 (Figure 2a). SP2509 treatment also increased the H3K9Me2 the NT shRNA, SP2509 treatment-induced expression of CD11b levels (Supplementary Figure 2) but did not alter the H3K27Me3 and the loss of viability was significantly abrogated in OCI-AML3 levels associated with the promoters of these genes (Figure 2b). cells transduced by the shRNA to LSD1 (Figures 5d and e). This Consistent with this, SP2509 treatment also increased the mRNA indicated that the differentiation and loss of viability mediated by levels of p57KIP, KLF4 and p21 in a dose-dependent manner, with SP2509 was at least partly mediated through inhibition of LSD1.

& 2014 Macmillan Publishers Limited Leukemia (2014) 2155 – 2164 LSD1 antagonist and HDAC inhibitor against AML cells W Fiskus et al 2158 SP2509

OH O O O 40 N N S OCI-AML3 CI N 35 H O MV4-11 30 MOLM13 25 150 LSD1 20

MAO A 15 100 % apoptosis MAO B 10 D-LDH 50 GO 5

% Activity IC50 for LSD1 = 13 nM 0 0 Control 250 500 1000 -8 -6 -4 -2 nM, SP2509, 48 h log[SP-2509] M -50

80 110 OCI-AML3 100 MOLM13 70 MV4-11 90 MV4-11 60 OCI-AML3 MOLM13 * 80 50 70 60 40 50 30 40

% colony growth % colony 30 20 (relative to control) 20 10 * % non-viable cells (PI positive) % non-viable 10 0 0 Control 250 500 1000 Control 250 500 1000 nM, SP2509, 96 h nM, SP2509, 96 h Figure 1. Treatment with the LSD1 antagonist, SP2509, inhibits LSD1 activity, depletes colony growth and induces apoptosis and cell death of cultured human AML cells. (a) Chemical structure of SP2509. (b) In vitro assay demonstrating the low nanomolar activity of SP2509 for LSD1 compared with other MAOs (MAO-A and MAO-B), lactate dehydrogenase (D-LDH) and glucose oxidase (GO). (c) OCI-AML3, MV4-11 and MOLM13 cells were treated with the indicated concentrations of SP2509 for 48 h. At the end of treatment, cells were stained with Annexin V and TO-PRO-3 iodide and the percentage of Annexin V-positive, apoptotic cells was determined by flow cytometry. Columns, mean of three experiments; bars, s.e.m. (d) OCI-AML3, MV4-11 and MOLM13 cells were treated with the indicated concentrations of SP2509 for 96 h. Then, cells were stained with propidium iodide and the percentage of nonviable cells was determined by flow cytometry. Columns, mean of three experiments; bars, s.e.m. * Indicates values significantly greater in OCI-AML3 cells compared with MOLM13 and MV4-11 cells (Po0.01). (e) OCI-AML3, MV4-11 and MOLM13 cells were treated with the indicated concentrations of SP2509 for 96 h. At the end of treatment, live cells were plated in methylcellulose and incubated at 37 1C for 7–10 days. The percentage of colony growth is reported relative to the untreated cells. Columns, mean of three experiments; bars, s.e.m. * Indicates values significantly less in OCI-AML3 cells compared with MOLM13 and MV4-11 cells (Po0.01).

Co-treatment with SP2509 and PS exerts synergistic lethal activity AML3, MOLM13 and MV4-11 cells, with combination indices below against cultured and primary AML cells 1.0 as determined by isobologram analysis (Figure 6c and Next, on the basis of the observation that pan-HDIs induce Supplementary Figures 5A–C). As compared with treatment with differentiation and apoptosis, as well as deplete LSD1 levels in each agent alone, co-treatment with PS and SP2509 was also AML cells,20,22 we determined the effects of co-treatment with PS associated with greater increase in the levels of p21, p27, BIM and on the anti-AML activity of SP2509. In these studies, we used cleaved poly (ADP-ribose) polymerase (Figure 6d). We also deter- relatively lower concentrations of PS that are well tolerated mined whether exposure to PS and SP2509 more than each agent 30,31 (5–15 nM), but biologically effective as epigenetic modifiers. alone induced biomarkers associated with induction of myeloid Figure 6a demonstrates that co-treatment with 10 nM of PS differentiation in the cultured AML cells. As shown in Supplementary significantly enhanced SP2509-induced but not SP2513 (the inactive Figures 6A and B, compared with treatment with each agent alone, enantiomer of SP2509)-induced apoptosis, in OCI-AML3 and MOLM13 co-treatment with SP2509 and PS for 96 h induced higher levels of cells. Consistent with this observation, treatment with low concentra- p21, p16 and C/EBPa, as well as induced higher levels of CD11b in tions of PS induced more apoptosis in OCI-AML3 cells transduced OCI-AML3 cells (Po0.05). We next determined the lethal effects of with shRNA to LSD1, as compared with the OCI-AML3 cells SP2509 and/or PS against patient-derived, CD34 þ primary AML transduced with the NT shRNA (Figure 6b). Importantly, combined versus normal bone marrow progenitor cells. Figure 7a demonstrates therapy with PS and SP2509 synergistically induced apoptosis of OCI- that treatment with either SP2509 or PS induced more loss of cell

Leukemia (2014) 2155 – 2164 & 2014 Macmillan Publishers Limited LSD1 antagonist and HDAC inhibitor against AML cells W Fiskus et al 2159 2.5 4 OCI-AML3 H3K27Me3 chIP OCI-AML3 H3K4Me3 chIP 3.5 p57KIP p57KIP 2 3 KLF4 KLF4 2.5 1.5 p21 2 p21 1

1.5 fold enrichment (relative to untreated)

fold enrichment 1 0.5

(relative to untreated) 0.5 0 0 Control 1000 Control 1000 nM, SP2509, 16 h nM, SP2509, 16 h

7 OCI-AML3 6 p21

5 p57KIP1

4 KLF4

3

2

Relative mRNA expression 1

0 Control 250 500 1000 nM, SP2509, 16 h Figure 2. Treatment with SP2509 increases H3K4Me3 on the promoters of p57Kip, KLF4 and p21, and induces mRNA expression of p57Kip, KLF4 and p21 in AML cells. (a, b) OCI-AML3 cells were treated with the indicated concentrations of SP2509 for 16 h. At the end of treatment, chromatin was cross-linked, sonicated and chromatin immunoprecipitation (chIP) was performed for H3K4Me3 and H3K27Me3. The chIPed DNA was used as template for quantitative PCR. The fold enrichment was calculated using the Ct value of the chIPed DNA versus the Ct for the input DNA. (c) OCI-AML3 cells were treated with the indicated concentrations of SP2509 for 16 h. Then, total RNA was isolated and reverse transcribed. The resulting complementary DNA was used for quantitative PCR of p57Kip, KLF4 and p21 using TaqMan probes. The relative mRNA expression was normalized against glyceraldehyde 3-phosphate dehydrogenase.

OCI-AML3 0 1000 nM, SP2509 MOLM13

input IgG control IB: OCI-AML3 (MLL-AF9) CoREST 0 250 1000 0 250 1000 nM SP2509, 24 h IP:LSD1 LSD1 p53 p21

MOLM13 C/EBPα p42

0 1000 nM, SP2509 C/EBPα p30 IB: β-Actin CoREST IP:LSD1 LSD1

Figure 3. Treatment with SP2509 inhibits binding of LSD1 with CoREST and induces p53, p21 and CCAAT/enhancer binding protein a (C/EBPa) in AML cells. (a) OCI-AML3 and MOLM13 cells were treated with the indicated concentration of SP2509 for 16 h. At the end of treatment, total cell lysates were prepared and LSD1 was immunoprecipitated. After thorough washing, SDS–polyacrylamide gel electrophoresis and immunoblot analyses were conducted for CoREST and LSD1 in the immunoprecipitates. (b) OCI-AML3 and MOLM13 cells were treated with the indicated concentration of SP2509 for 24 h. Following this, cells were collected, total cell lysates were prepared and immunoblot analyses were conducted for the expression levels of p53, p21, C/EBPa and b-actin in the lysates. viability of the primary AML versus normal progenitor cells. but induction of p21, p27 and BIM in the AML progenitor cells Furthermore, co-treatment with the combination was significantly (Figure 7b). We next determined whether the combination was also more lethal against AML versus normal progenitor cells (Po0.01; highly active against the purified CD34 þ CD38 À LIN À primary AML Figure 7a). In addition, compared with each agent alone, co-treatment stem/progenitor cells. As shown in Figure 7c and Supplementary with SP2509 and PS caused more loss of viability of the primary AML Figure 5D, co-treatment with SP2509 and PS was synergistically lethal progenitor cells. This was associated with greater depletion of c-Myc against the AML stem/progenitor cells.

& 2014 Macmillan Publishers Limited Leukemia (2014) 2155 – 2164 LSD1 antagonist and HDAC inhibitor against AML cells W Fiskus et al 2160 30 30 40 Primary AML cells (n=4) OCI-AML3 MOLM13 35 25 25 CD11b CD14 CD11b 30 CD68 CD68 20 20 CD14 25 15 20 15 15 10 10 % positive cells positive % % positive cells % % positive cells positive % 10 5 5 5 0 0 0 Control 250 1000 Control 250 1000 Control 250 1000 nM, SP2509, 96 h nM, SP2509, 96 h nM, SP2509, 96 h

nM SP2509, 96 h 25 0 250 1000 Primary AML (n=5) 20 nM SP2509, 96 h 0 250 1000

AML3 15 - AML

OCI 10 % differentiation % morphology) (by 5 Primary

0 MOLM13 Control 250 1000 nM, SP2509, 96 h Figure 4. Treatment with SP2509 induces features of morphological differentiation of cultured and primary AML cells (a, b) OCI-AML3 and MOLM13 cells were treated with the indicated concentrations of SP2509 for 96 h. At the end of treatment, cells were stained with CD11b, CD14 or CD68 antibodies and the percentage of cells positive for these markers was determined by flow cytometry. Columns represent the mean of three independent experiments ±s.e.m. (c) CD34 þ progenitor cells purified from AML blasts (n ¼ 4) were treated with the indicated concentrations of SP2509 for 96 h. Following this, cells were stained with CD11b or CD14 antibodies and the percentage of cells positive for these markers was determined by flow cytometry. Columns represent the mean percent of CD11b or CD14-positive cell ±s.e.m. (d) OCI-AML3 and MOLM13 cells were treated with the indicated concentrations of SP2509 for 96 h. Following this, cells were cytospun onto glass slides and stained with hematoxylin and eosin. Cells were observed at  40 magnification using a light microscope. Images of differentiated cells were obtained using a charge-coupled device (CCD) camera. Representative cells are shown. (e) CD34 þ progenitor cells purified from AML blasts (n ¼ 5) were treated with the indicated concentrations of SP2509 for 96 h. Then, cells were cytospun onto glass slides and stained with hematoxylin and eosin. The percentages of differentiated cells were determined by light microscopy. Columns, mean morphological differentiation of the AML samples; bars, s.e.m. (f) CD34 þ progenitor cells purified from AML blasts were treated with the indicated concentrations of SP2509 for 96 h. Following this, cells were cytospun onto glass slides and stained with hematoxylin and eosin. Cells were observed at  40 magnification using a light microscope. Images of differentiated AML cells were obtained using a CCD camera.

Combined treatment with SP2509 and PS exerts superior in vivo of SP2509 and/or PS on the survival of NSG mice engrafted with activity against AML xenografts and ‘primagrafts’ the primary AML blasts co-expressing FLT3-ITD and NPM1c þ .As We next determined the in vivo anti-AML activity of SP2509 and/or shown, treatment with SP2509 or PS alone for 3 weeks PS against cultured (OCI-AML3) in NOD/SCID mice, as well as significantly improved the median survival of the mice, as against primary AML blasts engrafted in the bone marrow, that is, compared with the treatment with vehicle control (Po0.01). ‘primagrafts’ in the NSG mice.29 Following the tail vein infusion Combined therapy with SP2509 and PS was superior to the and engraftment of OCI-AML3 cells, the effect of the treatment treatment with SP2509 or PS alone in improving the median with the vehicle control, PS or SP2509 alone, or the combination survival of the mice (95 versus 80 and 56 days, respectively; of SP2509 and PS, for 3 weeks on the survival of the NOD/SCID Po0.01), with 50% of the mice surviving more than 100 days after mice was determined. The Kaplan–Meier plot depicting the the infusion of the AML blasts in the tail veins of the mice. This survival of the mice demonstrates that, as compared with plateau in the survival curve suggests a potentially curative impact treatment with the vehicle alone, treatment with either SP2509 of the combination on the survival of the mice (Figure 7e). We also or PS significantly improved the median survival of the mice determined the ex vivo sensitivity of the primary AML sample infused with OCI-AML3 cells (37 and 36.5 days, respectively, versus utilized as the primagraft in the NSG mice. As shown in 19.5 days for vehicle control) (Po0.05; Figure 7d). Notably, Supplementary Figure 7A, co-treatment with SP2509 and PS combined treatment with PS and SP2509 further improved the induced more apoptosis than either agent alone. Ex vivo co- median survival of the mice (44.5 days), as compared with treatment with SP2509 and PS also synergistically induced treatment with SP2509 or PS alone (Po0.01). One-third of the apoptosis of the primary AML cells with combination indices mice co-treated with SP2509 and PS were alive 80 days following o1.0, as determined by median dose effect and isobologram the infusion of the AML cells. Figure 7e demonstrates the effects analysis (Supplementary Figure 7B).

Leukemia (2014) 2155 – 2164 & 2014 Macmillan Publishers Limited LSD1 antagonist and HDAC inhibitor against AML cells W Fiskus et al 2161

1.2 110 OCI-AML3 OCI-AML3 100 OCI-AML3 1 90 + - NT shRNA, 72 h NT shRNA 80 - + LSD1 shRNA #72, 72 h 0.8 LSD1 shRNA 70 LSD1 60 0.6 1.0 0.49 50 DNMT1 40 0.4 *

% colony growth colony % 30

1.0 0.62 cell/mL (in millions) 20 0.2 10 H3K4Me3 0 0 NT-shRNA LSD1 shRNA H3K4Me2 48 72 96 120 144 hours, post transduction H3K9Me2 25 45 OCI -AML3 OCI-AML3 c-Myc 40 NT shRNA NT shRNA 1.0 0.53 20 35 LSD1 shRNA LSD1 shRNA β-Actin 30 15 25 20 10 15

% non-viable cells non-viable % 10 5

% CD11b positive cells positive CD11b % 5 0 0 Control 500 1000 Control 500 1000 nM, SP2509, 96 h nM, SP2509, 96 h Figure 5. shRNA-mediated knockdown of LSD1 inhibits cell proliferation, colony growth and significantly enhances pan-HDI PS-induced apoptosis of AML cells. (a) OCI-AML3 cells were transduced with nontargeting (NT) shRNA or LSD1-specific shRNA for 72 h. Following this, cells were collected and total cell lysates were prepared. Immunoblot analyses were conducted for the expression levels of LSD1, DNMT1, c-MYC, H3K4Me3, H3K4Me2, H3K9Me2 and b-actin in the cell lysates. (b) OCI-AML3 cells were transduced with NT and LSD1 shRNA for 48 h. Then, cells were counted and 50 000 cells were plated in duplicate. Cell counts were taken every 24 h for 144 h post transduction. Values represent the mean of three experiments ±s.e.m. (c) OCI-AML3 cells were transduced with NT and LSD1 shRNA for 48 h. Following this, cells were plated in methylcellulose and incubated for 7–10 days. Total number of colonies was counted and the percentage of colony growth is calculated relative to the NT shRNA-transduced cells. * Indicates colony growth values significantly less in LSD1 shRNA-transduced cells compared with NT shRNA-transduced cells (Po0.001). (d, e) OCI-AML3 cells were transduced with NT and LSD1 shRNA for 48 h. Then, cells were treated with the indicated concentrations of SP2509 for 96 h and the percentage of CD11b-positive cells (d) and nonviable cells (e) was determined by flow cytometry. Columns, mean of three experiments; bars, s.e.m.

DISCUSSION AML cells at the micromolar level. This is similar to what has 19 In the present studies, we demonstrate for the first time that a been previously reported. Unlike the situation in the novel, potent LSD1 antagonist SP2509, induces growth inhibition, cell-free enzyme assay, the lower intracellular potency of SP2509 differentiation and apoptosis in human AML cells. Importantly, is likely due to the presence and activity of LSD1 in the regulatory unlike the reported experience with the TCP analogs, exposure to protein complexes in the intracellular setting, and the SP2509 did not induce toxicity in immune-depleted mice potential regulation of the biological outcome of LSD1 inhibition 9,10,19 engrafted with AML at doses that exerted anti-AML activity. by other intracellular factors. For example, the demethylase Consistent with the previous report demonstrating that LSD1 is activity of LSD1 at the nucleosomes is enhanced by its association essential for the survival of MLL-AF9-expressing leukemia stem with the corepressor complexes such as Co-REST and NURD.9,19,32 cells, our findings show that inhibition of LSD1 is lethal against Our studies also show that the binding of LSD1 and AML expressing MLL fusion oncoprotein. Moreover, our studies CoREST is partially disrupted by SP2509 treatment. Inhibition of also demonstrate that SP2509 is relatively more active in inducing LSD1 by SP2509 resulted in an increase in the promoter-specific, loss of viability of OCI-AML3 cells expressing mutant NPM1, which permissive H3K4Me3 modification, as well as induced p53, p21 had not been previously reported. SP2509 treatment also and C/EBPa levels. Whether this was due to transcriptional exhibited in vivo anti-AML activity in immune-depleted mice and/or post-transcriptional effects was not determined here; engrafted with OCI-AML3 cells or primary AML blasts expressing however, these alterations due to SP2509 treatment were clearly mutant NPM1 plus FLT3-ITD, without inducing any toxicity in the associated with growth inhibition, differentiation and the mice, which was noted with the use of TCP or its analogs as the loss of viability of the AML cells. An inverse correlation of LSD1 LSD1 antagonist.17,18 levels with differentiation has also been reported in SP2509 is a small molecule reversible inhibitor of LSD1.19 It neuroblastoma cells.33 In a separate report, alteration in the showed activity at low nanomolar levels against LSD1 in the cell- expression of the Myc ‘core module’ genes was identified in free in vitro enzyme assay, but exerted the biological activity in the the leukemia stem cells in which LSD1 was knocked down.18

& 2014 Macmillan Publishers Limited Leukemia (2014) 2155 – 2164 LSD1 antagonist and HDAC inhibitor against AML cells W Fiskus et al 2162

80 80 MOLM13 OCI -AML3 80 70 70 0 nM SP 0 nM SP OCI-AML3 1000 nM SP2513 70 60 1000 nM SP2513 60 NT shRNA 1000 nM SP2509 1000 nM SP2509 60 50 50 LSD1 shRNA 50 40 40 40 30 30 30 % apoptosis % % apoptosis % % apoptosis % 20 20 20 10 10 10 0 0 0 010 010 Control 2.5 5 10 nM, PS, 48 h nM, PS, 48 h nM, PS, 48 h

OCI-AML3 MOLM13 + 15 nM PS + 15 nM PS 0 1000 0 1000 0 1000 0 1000 nM, SP2509 p21 1.5 1.5 OCI-AML3 MOLM13 SP2509 + PS p27 SP2509 + PS 1.0 1.0 BIMEL CI CI 0.5 0.5 BIML BIMS 0 0 Cl PARP 0 0.2 0.4 0.6 0.8 1.0 0 0.2 0.4 0.6 0.8 1.0 Fractional Effect Fractional Effect β-Actin

Figure 6. Combined treatment with SP2509, but not its inactive enantiomer SP2513, and PS synergistically induces apoptosis of cultured AML cells. (a) OCI-AML3 and MOLM13 were treated with the indicated concentrations of SP2509, SP2513 and/or PS for 48 h. At the end of treatment, cells were stained with Annexin V and TO-PRO-3 iodide. The percentage of Annexin V-positive, apoptotic cells was determined by flow cytometry. Columns, mean of three experiments; bars, s.e.m. * Indicates values significantly greater in SP2509 and PS-treated cells compared with those treated with SP2513 and PS (Po0.05). (b) OCI-AML3 cells were transduced with NT and LSD1 shRNA for 48 h. Then, cells were treated for an additional 48 h with the indicated concentrations of PS and the percentage of Annexin V-positive, apoptotic cells were determined by flow cytometry. Columns, mean of three experiments; bars, s.e.m. * Indicates values significantly greater in LSD1 shRNA- transduced cells treated with PS (Po0.05). (c) OCI-AML3 and MOLM13 cells were treated with SP2509 (dose range: 500–1000 nM) and/or PS (dose range: 5–20 nM) at a constant ratio for 48 h. Following this, cells were stained with Annexin V and TO-PRO-3 iodide and the percentage of Annexin V-positive, apoptotic cells was determined by flow cytometry. Median dose effect and isobologram analyses were performed using calcusyn. Combination index (CI) values less than 1.0 indicate a synergistic interaction of the two agents in the combination. (d) OCI-AML3 and MOLM13 cells were treated with the indicated concentrations of SP2509 and/or PS for 24 h. At the end of treatment, cell lysates were prepared and immunoblot analyses were performed as indicated. The expression levels of b-actin in the lysates served as a loading control.

In this report, the knockdown and inhibition of LSD1 was also A previous study demonstrated that co-treatment with LSD1 shown to inhibit growth and induce differentiation of AML antagonist enhanced the differentiation inducing activity of all- progenitor cells expressing MLL fusion oncoprotein.18 We also trans-retinoic acid against AML cells.17 LSD1-associated CoREST determined that the biological effects of SP2509 treatment were and NURD complexes also contain class I HDAC-associated HDAC also phenocopied by shRNA-mediated knockdown of LSD1 in AML activity.9,10,19,32 Consistent with this, and with our previously cells, suggesting that the effects of SP2509 were mediated by reported findings that treatment with pan-HDI PS induces LSD1 inhibition. Recent reports have highlighted that the NPM1 differentiation in AML blast progenitor cells,22 our present mutation is the founder mutation in the AML stem/progenitor in vitro findings confirm that co-treatment with SP2509 or cells where acquisition of FLT3-ITD mutation results in the knockdown of LSD1 by shRNA enhanced the lethal effects of emergence of an aggressive AML phenotype.4,34 In our studies, relatively low and clinically achievable concentrations of PS in SP2509 exerted a relatively higher level of activity against AML AML cells.30,31 We also demonstrate here that co-treatment with cells expressing NPM1 mutation. Although the precise mechanism SP2509 and PS was associated with greater induction of the underlying this was not further probed here, a clear role of LSD1- markers of myeloid differentiation in AML cells, which could dependent demethylase activity has been demonstrated in potentially contribute to the superior anti-AML activity of the normal differentiation and in regulating cell death upon combination. Moreover, we observed synergistic apoptotic differentiation of stem/progenitor cells.14,35,36 In addition, LSD1- effects of SP2509 and PS against cultured and primary AML associated corepressor complexes have been implicated in TAL1 cells. This may be explained by greater induction of the levels of and Notch signaling, which are also involved in regulating the pro-apoptotic p27 and BIM in AML cells exposed to co- biology of hematopoietic stem/progenitor cells.37–39 treatment with SP2509 and PS, as compared with each drug Consistent with this, our findings show that SP2509 exerted alone.40,41 Previous reports have identified that LSD1-mediated in vitro and in vivo anti-AML activity especially against histone demethylation triggers Myc-induced transcription, and the leukemia-initiating primary AML progenitor/stem cells the Myc ‘core module’ gene expression was altered in the co-expressing NPM1 and FLT3 mutations. leukemia stem cells in which LSD1 was knocked down.18,42

Leukemia (2014) 2155 – 2164 & 2014 Macmillan Publishers Limited LSD1 antagonist and HDAC inhibitor against AML cells W Fiskus et al 2163 Primary AML 70 Primary CD34+ Primary CD34+ AML cells (n=6) + 15 nM PS 60 CD38-LIN- AML cells 1.0 Normal CD34+ cells (n=6) 0 1000 0 1000 nM, SP2509 50 c-Myc 0.8 40 p21 0.6 CI 30 0.4 p27 0.2 20 SP2509 + PS % non-viable cells non-viable % 10 BIMEL 0 0 0.2 0.4 0.6 0.8 1.0

0 BIML Fractional Effect

BIMS β-Actin

OCI-AML3 xenografts Primary AML xenografts 150 Vehicle Vehicle 25 mg/kg SP2509 150 5 mg/kg PS 5 mg/kg PS 25 mg/kg SP2509 + 5 mg/kg PS 15 mg/kg SP2509 100 15 mg/kg SP2509 + 5 mg/kg PS 100

50 SP2509 50 SP2509 (T/Th) Percent survival

Percent survival (T/Th) PS (M, W, F) PS (M, W, F) 3 weeks p< 0.01 3 weeks p< 0.01 0 0 0 20406080100 050100150 Days Post Implantation Days Post Implantation

Median Survival (Days) Median Survival (Days) Vehicle= 19.5 Vehicle= 40 5 mg/kg PS= 36.5 5 mg/kg PS= 56 25 mg/kg SP2509= 37 15 mg/kg SP2509= 80 SP2509+ PS = 44.5 SP2509 + PS = 95

Figure 7. Treatment with SP2509 and/or PS significantly enhances PS-mediated loss of viability of CD34 þ primary AML cells and improves the survival of mice bearing AML xenografts and primagrafts. (a) CD34 þ progenitor cells purified from AML blasts and normal CD34 þ cells were treated with the indicated concentrations of SP2509 and/or PS for 48 h. At the end of treatment, cells were stained with propidium iodide. The percentage of nonviable cells was determined by flow cytometry. * Indicates values significantly greater in CD34 þ AML cells treated with the w combination of SP2509 and PS, compared with treatment with either agent alone (Po0.01). Indicates values significantly less in normal CD34 þ cells treated with the combination of SP2509 and PS compared with CD34 þ AML cells (Po0.05). (b) Primary AML cells were treated with SP2509 and/or PS as indicated for 24 h. Total cell lysates were prepared and immunoblot analyses were conducted for the expression levels of c-Myc, p21, p27, BIM and b-actin in the lysates. (c) CD34 þ CD38 À Lin À progenitor cells purified from AML blasts were treated with SP2509 and/or PS for 48 h. Then, cells were stained with propidium iodide and the percentage of nonviable cells was determined by flow cytometry. Median dose effect and isobologram analyses were conducted using Calcusyn. Combination index (CI) values less than 1.0 indicate a synergistic interaction of the two agents in the combination. (d) NOD/SCID mice that had been exposed to 2.5 Gy of gamma irradiation were injected with 5 million OCI- AML3 cells via the lateral tail vein. Seven days after implantation, mice were treated with 25 mg/kg of SP2509 and/or 5 mg/kg of PS for 3 weeks. Survival of the mice is represented by a Kaplan–Meier plot. (e) NSG mice that had been preconditioned with 2.5 Gy of gamma irradiation were injected with 10 million primary AML cells. Treatment was initiated when mice demonstrated 1% CD45 þ cells in the peripheral blood. Mice were treated with SP2509 and/or PS, as indicated, for 3 weeks. Survival of the mice is represented by a Kaplan–Meier plot.

Here, we also noted that synergistic activity of co-treatment with strongly support the rationale for further in vivo testing of SP2509 and PS was associated with a greater depletion of c-Myc SP2509-based combination with pan-HDI such as PS against expression in the primary AML blast progenitor cells expressing AML, especially those expressing NPM1 mutation. mutant NPM1. Although other reports have shown that combined inhibition of LSD1 and HDACs achieves superior antitumor activity against glioblastoma and breast cancer CONFLICT OF INTEREST 43,44 cells, in these reports, the LSD1 antagonists employed are DB is founder and Chief Scientific Officer of Salarius Pharmaceuticals; SS is Chief known to be either irreversible LSD1 inhibitors45 or exhibit Medical Officer of Salarius Pharmaceuticals. The remaining authors declare no conflict intolerable in vivo activity in the mouse models of AML where of interest. these inhibitors were studied.17,18 In contrast, our in vivo studies demonstrate that co-treatment with SP2509 and PS yields significantly better survival without inducing toxicity, and AUTHOR CONTRIBUTIONS suggests a potential for cure of the immune-depleted mice WF, BS, BPP, SGTD and KL performed in vitro experiments in cultured and engrafted with AML cells expressing NPM1 mutation irrespective primary AML cells and analyzed the data. WF performed the in vivo studies in of the co-expression of FLT3-ITD. Collectively, these findings the NOD/SCID and NSG mice. SS and DJB provided a critical new reagent for the

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