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Keywords:

  • Cytogenetically normal acute myeloid leukemia model;
  • Histone deacetylase inhibitor;
  • Connectivity Map;
  • HOXA9-MEIS1;
  • Entinostat;
  • Therapeutic

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. SUMMARY
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

The incidence of refractory acute myeloid leukemia (AML) is on the increase due in part to an aging population that fails to respond to traditional therapies. High throughput genomic analysis promises better diagnosis, prognosis, and therapeutic intervention based on improved patient stratification. Relevant preclinical models are urgently required to advance drug development in this area. The collaborating oncogenes, HOXA9 and MEIS1, are frequently co-overexpressed in cytogenetically normal AML (CN-AML), and a conditional transplantation mouse model was developed that demonstrated oncogene dependency and expression levels comparable to CN-AML patients. Integration of gene signatures obtained from the mouse model and a cohort of CN-AML patients using statistically significant connectivity map analysis identified Entinostat as a drug with the potential to alter the leukemic condition toward the normal state. Ex vivo treatment of leukemic cells, but not age-matched normal bone marrow controls, with Entinostat validated the gene signature and resulted in reduced viability in liquid culture, impaired colony formation, and loss of the leukemia initiating cell. Furthermore, in vivo treatment with Entinostat resulted in prolonged survival of leukemic mice. This study demonstrates that the HDAC inhibitor Entinostat inhibits disease maintenance and prolongs survival in a clinically relevant murine model of cytogenetically normal AML. STEM Cells2013;31:1434–1445


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. SUMMARY
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Large-scale analysis of the leukemic genome and transcriptome has unearthed abnormalities in key cellular and molecular pathways. Microarray-based gene expression profiling, in particular, has identified novel disease subclasses at diagnosis [1–5]. The ability to sequence complete genomes at high resolution has resulted in the generation of disease-specific data silos. However, the true value of such technologies to the fundamental understanding of the disease state and integration into clinical practice has yet to be fully realized [6]. Connectivity mapping (cMap) is an experimental bioinformatics approach to identify and map underlying genetic differences in disease states to toxicology profiles of small-molecule therapeutics [7]. A recent adaptation of this method that incorporates a robust statistical significance and perturbation algorithm, termed statistically significant connectivity map (sscMap), provides a platform to interrogate relevant models of disease and integrate expression profiling with treatment response to rationally designed therapeutics for redeployment [8, 9].

Several leukemia-associated gene rearrangements have been recapitulated in genetically engineered mouse models, some of which have been used successfully to monitor response to therapy and provide insights into chemosensitivity and chemoresistance [10]. Similarly, transduction/transplantation studies have been widely used to recapitulate deregulated oncogene expression in the leukemia setting and provide a basis for functional studies. The relatively long disease latencies observed in some models, for example, Mixed-Lineage Leukemia-Eleven Nineteen Leukemia (MLL-ENL) and AML1 suggest the need for additional genetic interactions to obtain robust transplantable models more amenable to therapy-related studies [11, 12]. Transduction and transplantation of hematopoietic stem/progenitor cells with combined oncogenic components such as BCR/ABL and AML1, into syngeneic recipients, results in aggressive and transplantable leukemia [13].

Homeodomain (HD) containing proteins regulate developmental processes including hematopoiesis. A subset with an atypical HD defined by a three-amino-loop-extension motif (TALE) form complexes with proteins encoded by the clustered class I homeobox (HOX) genes. An established body of evidence supports a role for HOX and TALE proteins in leukemogenesis (reviewed by Argiropoulos and Humphries [14]). In particular, altered expression of HOXA9 has been observed in a significant proportion of both human acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL) and reported as the most consistent indicator of poor prognosis in refractory AML [15–17]. The frequent co-overexpression of HOXA9 and MEIS1, particularly in leukemias harboring MLL-rearrangements [18, 19], suggests a vital genetic interaction between the cofactors, which may be required for leukemia maintenance in a context-dependent manner [20–23]. Notably, expression of HOXA9 and MEIS1 is also associated with cytogenetically normal AML (CN-AML) where no major genetic aberrations have been identified [24]. Direct co-overexpression of HOXA9 and MEIS1 results in an aggressive, transplantable, and tractable myeloid leukemia in mouse models with a cytogenetically normal genetic background [25].

A conditional loxP-HOXA9-ires-MEIS1-loxP mouse model of leukemia (A9M-L2) phenotypically comparable to similar reported models [26] and with levels of the oncogenes comparable to CN-AML patient samples was developed. Gene signatures obtained from A9M-L2 and CN-AML patient samples were submitted to the sscMap platform to identify potential connections with small molecule inhibitors. Of particular note, three of five of the top candidate small molecule inhibitors identified were identical for the human and mouse conditions. One of these molecules, Entinostat (MS-275, SNDX275) was selected for further study. Treatment of primary leukemic cells with Entinostat resulted in reduced cell viability, impaired colony forming potential, and loss of leukemia maintenance in the murine model. Furthermore, a single treatment of Entinostat to leukemic mice in vivo resulted in extension in survival compared to vehicle treatment.

Together these data support a proof-of-principle that integration of focused gene expression profiling with in silico screening using the sscMap platform enables identification of small molecule inhibitors of leukemia maintenance. In addition the HOXA9-ires-MEIS1 murine model recapitulates the human CN-AML phenotype at the molecular level and may provide a basis for proof-of-concept studies to predict future therapeutic approaches to treatment of this leukemia subtype.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. SUMMARY
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Patient Samples and Data

AML samples were obtained at diagnosis. All studies adhered to the tenets of the Declaration of Helsinki, had ethical committee approval, and all samples were collected with informed consent and anonymized. Mononuclear cells were purified using Ficoll-Paque (GE Healthcare BioSciences AB, Uppsala, Sweden, http://www.gelifesciences.com) gradient centrifugation. Expression profiles were generated from human genome expression arrays (HG-U133A or HG-U133 Plus 2.0: Affymetrix, Santa Clara, CA, http://www.affymetrix.com) for nonleukemic (MILE Class 18, n = 74) and intermediate risk (MILE Class 13, n = 351) excluding patients with 11q23 abnormalities as classified (NCBI Gene Expression Omnibus Accession number: GSE13204) [27].

Animals

Congenic donor CD45.1+ (C57Bl6/Pep3b or C57Bl/6-Ly5.1) and recipient CD45.2+ (C57Bl/6J) mice were bred and maintained in specific pathogen-free facilities (IRIC or BRU-QUB) under guidelines of both the Canadian Council on Animal Care (CCAC) and UK Animals (Scientific Procedures) Act 1986. Experimental procedures were approved by the Comité de Déontologie de l'Expérimentation sur les Animaux de l'Université de Montréal and the Ethical Review Committee for Animal Research, Queen's University Belfast.

Retroviral Infection and Transplantation of Hematopoietic Cells

The loxP-HOXA9-ires-MEIS1-loxP-Neo (A9M-L2) vector was constructed by polymerase chain reaction (PCR) cloning (details available on request), and the construct was subcloned into the XhoI/BamHI site of the murine stem cell proviral vector (MSCV). All constructs were verified by DNA sequencing. For MSCV-Cre-GFP, the XhoI/MluI fragment (1,031 bp) of pBluescript-Cre (kindly provided by Dr. Keith Humphries) was blunt ended and subcloned into the HpaI site of the MSCV-GFP vector. Generation of vesicular stomatitis virus-pseudotyped retroviruses, infection of hematopoietic cells, and transplantation into mice were performed as described [28]. Recipient mice were sublethally (650–850 cGy) or lethally (1,300 cGy) irradiated prior to transplantation with bone marrow (BM) or fetal liver (FL) cells isolated at 14.5 dpc. Ex vivo exposure to control GFP or Cre-GFP recombinant retroviruses was limited to 48 hours prior to transplantation.

DNA Analyses

High molecular weight DNA was obtained from hematopoietic tissues, and Southern blot analysis was performed as previously described [29]. The probe used was a SalI and MluI fragment of the neomycin gene (852 bp).

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Figure 1. Experimental design and phenotype of conditional leukemia. (A): Schematic representation of the development and experimentation of a conditional HOXA9-MEIS1 (A9M-L2) mouse model. (B, left panel): Structure of the targeting vector used to generate A9M-L2 leukemias indicating the location of flanking loxP sites, the size of anticipated fragments following Cre recombination, and restriction enzymes used to determine integration and clonality. (B, right panel): A representative Southern blot analysis (NeoR probe) of EcoRI digested genomic DNA obtained from spleen (S), bone marrow (B), or lymph node (L) showing low integration and clonality in three independent A9M-L2 mice. (C): Identification of blast cells in hematopoietic tissues from necropsied A9M-L2 mice compared to age-matched normal controls. (D): Representative dot plots (from n = 5) demonstrating levels of surface markers CD11b/Mac-1, CD48, CD150, and Ter-119 analyzed by flow cytometry in both the A9M-L2 model and age-matched normal control bone marrow. (E): Southern blot analysis (NeoR probe) of NheI digested plasmid (P) or genomic DNA from A9M-L2 mice demonstrating the presence of DNA bands at the sizes expected for successful Cre-recombination of the flanked region at 50% compared to control (pMSCV-GFP) and 95% following selection for GFPHi populations. (F): Kaplan-Meir plot of the percentage survival of recipient mice receiving either MSCV-GFP treated A9M-L2 cells (No Cre), unsorted MSCV-Cre-GFP treated A9M-L2 cells (50%), or GFPHi sorted MSCV-Cre-GFP treated A9M-L2 cells (95%); n = 5 per group. Abbreviations: B, bone marrow; GFP, green fluorescent protein; LDA, limited dilution assay; LTR, long terminal repeat; S, spleen, L, lymph node; Tx, primary transplant.

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Leukemia morphology, Immunophenotype, and Flow Cytometry

Tissue infiltration, morphology, and flow cytometry analyses were performed as described [28]. Allophycocyanin (APC)-conjugated CD11b (BD Pharmingen, San Jose, CA, San Diego, http://www.bdbiosciences.com/index[lowen]us.shtml), Fluorescein isothiocyanate (FITC)-conjugated TER119, Phycoerythrin (PE)-conjugated CD150, and APC-conjugated CD48 (eBiosciences, San Diego, CA) antibodies were used for immunophenotyping. APC-conjugated CD45.1 antibody and PE-conjugated CD45.2 antibody (eBiosciences, http://www.ebioscience.com) were used for repopulation and PE-conjugated CD45.1 antibody (Southern Biotech, Alabama) for homing assays. All data were acquired and analyzed using an LSRII cytometer and FACSDiva 6.1.2 (BD Pharmingen) or FlowJo 7.6.5 software (TreeStar Inc., Ashland, OR, http://www.treestar.com). Tissues were treated with Modified Wright's stain and images were captured at 20× objective. Images were captured using an inverted microscope (CKX41) with attached digital camera (E620) both Olympus, Southend-on-Sea, UK.

Gene Expression Profiling

Total BM was collected by flushing femurs and tibias of age-matched control or A9M-L2 mice, and total RNA was isolated and cDNAs were generated as previously described [30]. For stem cell pluripotency and immune response profiling, cDNA samples were prepared with TaqMan Universal PCR mix and (200 ng/100 μL) loaded into each of the eight ports on the predesigned 384 well cards of the TaqMan Gene Expression Array. Quantitative reverse transcriptase PCR (qRT-PCR) was performed as per the manufacturer protocol and analyzed using the 7900 HT Sequence Detection System, all ABI (Applied Biosystems, Foster City, CA, Foster City, CA, http://www.appliedbiosystems.com). 18S rRNA was used as the endogenous control calibrator for subsequent analysis. For analysis by the Biotrove transcription factor panel array, RNA was converted to cDNA with High capacity cDNA RT kit (ABI #4368813) and used at 108 ng/μL of cDNA for 32 cycles of qRT-PCR. Expression data were filtered by Ct confidence [mt]300 and presence or absence of a symmetrical peak in a melting curve analysis. An arbitrary Ct value of 26 was used as the cut-off between expressed and nonexpressed genes. The combined expression of peptidylprolyl isomerase A (Ppia) and β-Actin was used as the endogenous control calibrators for subsequent analysis.

A subset of candidate genes identified from the array platforms were used for validation by individual SYBR Green 1 based qRT-PCR assays. For these assays, the original experimental cDNA was used and validated primer sets were obtained from PrimerBank (http://pga.mgh.harvard.edu/primerbank/) using the relevant gene accession numbers.

Bioinformatics and cMap

Connections between drug-induced gene expression profiles and gene signatures representing the AML disease or normal BM (NBM) state were obtained by sscMap [8] to 1,309 reference profiles obtained from the BROAD Institute cMap database. For the mouse model, a list of all genes with a minimum ± fourfold change in expression between AML and NBM (n = 5 per group) were ranked and converted to Affymetrix HG-U133A probeset IDs where possible. Differentially expressed genes were identified using a t test. A gene signature with 47 Affymetrix probeset IDs, representing 30 individual genes, returned significant connections to drugs at 1% false discovery rate (FDR = 0.01). For the MILE study, the Affymetrix MAS5 value on the log scale was used as a measure of gene expression level. Class 13 (CN-AML plus fewer than three structural abnormalities, excluding 11q23, n = 351) versus Class 18 (nonleukemia and healthy BM, n = 74) comparison was carried out using two-sample t test to identify significantly differentially expressed genes. Following the same FDR criterion as above, a gene signature with 24 Affymetrix Probeset IDs, representing 21 individual genes, was constructed. The two gene lists were submitted to sscMap for connectivity mapping. A perturbation stability score of robustness was calculated for each significant connection (1 = robust, 0 = weak). Candidate drugs were prioritized, based on combined rational application of the connection, statistical significance and perturbation score.

Ex Vivo Entinostat Treatment

Primary AML samples were rapidly thawed and cultured for up to 72 hours in roswell park memorial institute (RPMI) 10% fetal calf serum (FCS) (Life Technologies, Paisley, U.K. http://www.lifetech.com) containing Entinostat, or the pan-histone deacetylase inhibitor Panobinostat (both Selleckchem, Munich, Germany, http://www.selleckchem.com). To reduce the potential for nonspecific effects, the inhibitors were used at the lowest IC50 doses reported to result in effective histone deacetylase (HDAC)-1 and pan-HDAC inhibition, [31, 32] or Dimethyl Sulfoxide (DMSO; vehicle control). Cell viability was quantified by evaluation of ATP levels using CellTiter-Glo (Promega, Madison, WI, http://www.promega.com). Primary A9M-L2 or mouse NBM cells were rapidly thawed and allowed to recover in expansion media, as previously described [26]. Recovered mouse cells or primary AML samples were treated in culture with Entinostat or Panobinostat at the stated concentrations or DMSO (vehicle control, final concentration 0.01%) for 24 hours, then plated in MethoCult GF M3434 media (Stem Cell Technologies, Vancouver, BC, Canada, http://www.stemcell.com) or directly transplanted into irradiated recipient mice at the stated concentration and dosage. Methylcellulose cultures were maintained in a humidified incubator at 37°C with 5% CO2 for up to 10 days when colony formation, colony counts, and photo images were captured and analyzed. Colony staining with 1 mg/mL p-iodonitrotetrazolium violet (INT, Sigma-Aldrich, Gillingham, U.K., http://www.sigmaaldrich.com) for 16 hours enabled visualization of colonies and determination of metabolic activity. Images were captured at the stated magnification using an inverted microscope (CKX41) with attached digital camera (E620) both Olympus. Recipient mice were followed until disease development or up to 80 days at which point necropsy was performed and hematopoietic tissue was examined.

In Vivo Entinostat Treatment of Leukemic Mice

Sublethally irradiated (850 cGy) recipient mice (CD45.2+) were transplanted with A9M-L2-derived BM cells (1 × 106) to regenerate leukemia. After 14 days of transplantation, leukemia harboring mice were treated intravenously with vehicle control or a bolus of Entinostat (30 mg/kg) previously shown to be within the maximal tolerated dose range [33, 34]. Mice were monitored until disease development, necropsied and hematopoietic tissue was examined.

Protein Analysis

Total cell lysates were prepared after incubation of leukemic cells (primary A9M-L2 or A9M cell line) in vehicle (DMSO) or histone deacetylase class I inhibitor (HDACi) containing media at the indicated dosage and time, using an SDS extraction protocol (Cell Signaling Technology, Danvers, MA, http://www.cellsignal.com). Proteins were separated by SDS-PAGE and probed with the following antibodies: caspase 3 (1:1,000; Cell Signaling Technology); p21CIP/WAF (1:200; Santa Cruz Biotechnology, Santa Cruz, CA, http://www.scbt.com); Acetyl-Histone H3 (Lys9; C5B11), Histone H3 (D1H2), Acetyl-Histone H4 (Lys8), Histone H4 (L64C1) (1:1,000; Acetyl-Histone Antibody Sampler Kit #9933, Cell Signaling Technology). Equal loading was assessed using a mouse monoclonal β-actin primary antibody (1:5,000; Sigma-Aldrich, St Louis, MO). Blots were developed with a chemiluminescence detection system (Immobilon Western Chemiluminescent horseradish peroxidase (HRP) Substrate; MerkMillipore, Billerica, MA, http://www.millipore.com), exposed to X-ray film (SLS/MOL 7016, Analab, Lisburn, U.K.) for up to 20 minutes, developed, and images were obtained using an Auto-Chemi Imaging system (UVP, Upland, CA). Densitometry was performed using an Auto-Chemi Imaging system (UVP, Upland, CA) and LabWorks software (LabWorks software, Version 4.6, UVP, Upland, CA).

Chromatin Immunoprecipitation and qRT-PCR Analysis

A9M or A9M-L2 cells were cultured at a density of 0.625 × 106 per mL in maintenance media supplemented with 300 nM Entinostat or vehicle control. Cells were crosslinked for 10 minutes at 37°C with 1.5% (w/v) formaldehyde after the appropriate treatment time. Chromatin was isolated, sonicated, and immunoprecipited as per manufacturer protocols using the Acetyl-Histone H4 Immunoprecipitation (ChIP) Assay Kit (Millipore, Billerica, MA). qRT-PCR for ChIP enrichment was as per the manufacturer protocol using SYBR Green PCR mastermix (Roche Diagnostics Limited, West Sussex, U.K., http://www.roche-applied-science.com) and analyzed by the ABI PRISM 7500 system (Applied Biosystems). Control immunoprecipitations were performed by substituting Acetyl-Histone H4 antibody with negative control rabbit immunoglobulins (Dako, Cambridgeshire, U.K., http://www.dako.com). Fold enrichment was determined by normalizing threshold cycle values of ChIP samples against sonicated whole cell DNA extract, set at a value of 1. Primer sequences for the p21CIP/WAF ChIP assays were (5′-TCAAAACGACCTGAATGCCTA-3′ and 5′-GTACAGTTAGAGC TGAGTGAGT-3′). The A9M cell line and A9M-L2 primary leukemia cells were also treated with vehicle (DMSO) or 300 nM Entinostat for 8, 16, or 24 hours. Gene expression was assessed by qRT-PCR using SYBR Green PCR mastermix and p21CIP/WAF primers (5′-CACAGGCACCATGTCCA ATC-3′, 5′-GAA ATCTGTCAGGCTGGTCT-3′).

Statistical Analysis

ANOVA of microarray experiments was implemented using Partek Genomics Suite and ANOVA or Student's t tests were performed by GraphPad Prism software (GraphPad software, Version 5.0, LA Jolla, CA) or SPSS software package (IBM, Portsmouth, U.K.).

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. SUMMARY
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Generation and Functional Validation of the A9M-L2 CN-AML Model

Retroviral transduction of HOXA9-ires-MEIS1 (A9M) into BM or FL hematopoietic cells has previously been demonstrated to give rise to aggressive leukemias in mice [26]. To investigate the requirement of these collaborating oncogenes in leukemia maintenance, we exploited the Cre-Lox system to generate a conditional clonal A9M leukemia (A9M-L2). A serial limiting dilution transplantation model was designed (Fig. 1A) and Southern blot analysis of digested genomic DNA from hematopoietic tissue indicated low integration and clonality as demonstrated by a small number of EcoRI digested fragments and equivalent banding patterns in all mice and all tissues (Fig. 1B). The leukemia latency, gross morphology (Fig. 1C), immunophenotype (Fig. 1D; supporting information Fig. S1), and tissue infiltration were consistent with previous studies [26]. Retroviral transduction of the A9M-L2 leukemia cells with Cre-GFP followed by cell sorting, to isolate the transduced (Cre+GFP+) cells, resulted in a population of cells (Crehi/GFPhi) in which the HOXA9-ires-MEIS1 provirus was deleted (∼95%) as determined by Southern blot analysis (Fig. 1E), and a population of cells in which the level of exposure to Cre was reduced (Crelo/GFPlo) resulting in 50% retention of the transgenes (Fig. 1E; supporting information Fig. S2). The latency of leukemia development in recipients of control GFP+ or Crelo/GFPlo A9M-L2 cells was comparable (30 ± 3 days, Fig. 1F) suggesting that the remaining A9M-L2 cells were sufficient to regenerate leukemia. In contrast, recipients of Crehi/GFPhi A9M-L2 cells in which the transduced HOXA9 and MEIS1 transgenes were effectively deleted, resulting in reduced activity of clonogenic progenitors, altered cell growth and gene expression (supporting information Fig. S2), remained healthy, without any overt signs of disease, during the 150-day observation period (Fig. 1F), indicating that expansion of the leukemic cell population depended on continuous overexpression of HOXA9 and MEIS1.

qRT-PCR analysis demonstrated that the clonal mouse leukemia expressed HOXA9 and MEIS1 above the levels determined for either normal nonmanipulated mice or human AML with favorable prognostic outcome, but within the range determined for AML with intermediate or adverse prognosis (supporting information Fig. S3). Therefore, combined low viral titers, in vivo limiting dilution, and clonal selection generated a mouse model of induced leukemia characterized by expression levels of HOXA9 and MEIS1 comparable to those detected in primary human leukemias. This represented an appropriate model to search for inhibitors of HOXA9/MEIS1-associated leukemia maintenance and/or expansion.

A9M-L2 Model Is Associated with Reduced Gene Expression

Overexpression of HOXA9 and MEIS1 results in an AML phenotype. A focus on the associated biology underlying the disease was taken with respect to quantifying molecular signatures. Since HOX and TALE proteins are transcription factors that work within networks and affect progenitor/stem cell function, the Biotrove transcription factor panel and the ABI TaqMan pluripotent stem cell gene expression array were used to quantify gene expression levels. In addition, a TaqMan immune response array was used to quantify expression levels and contribution of immunoregulatory gene networks to the general AML-associated gene signature. In total, 817 individual qRT-PCR assays were examined in duplicate for biological replicates (up to n = 5) from age-matched normal control or A9M-L2 leukemia BM. A stringent fourfold difference in expression (equivalent to ΔΔCt = ±2) was used as a cut-off to identify robustly increased or decreased expression of candidate genes. Using this criterion, 14% (114/817) of the genes examined demonstrated differential expression between the normal and leukemic states (supporting information Table S1). Of the differentially expressed genes, 83% (95/114) demonstrated reduced expression and only 17% (19/114) demonstrated increased expression in the leukemic state compared to normal controls (Fig. 2A–2C). A cohort of candidate genes from the array datasets was validated by individual qRT-PCR assays, 87% of which showed similar trends in differential expression (supporting information Fig. S4). Perhaps not surprisingly, HOXA9 and MEIS1 showed the most significant fold change in expression in A9M-L2 cells (161- and 248-fold, respectively, p [lt] .05) when compared to NBM, further validating both the model and the array platform approach.

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Figure 2. Differential gene expression in A9M-L2 leukemia. Waterfall plots from quantitative reverse transcriptase polymerase chain reaction analysis of an array of genes obtained from bone marrow of A9M-L2 mice compared to age-matched normal bone marrow controls (n = 5 per group in duplicate). Relative changes in expression are displayed as ΔΔCT values corrected for endogenous controls. Only consistent changes in ΔΔCT values of greater than ±2 are presented. Mean values ± S.E.M are plotted from (A) stem cell pluripotency, (B) immune response, and (C) transcription factor array platforms. *A subset of array genes were further validated by individual assays (supporting information Fig. S3).

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A9M-L2 Candidate Genes Associate with Key Molecular Pathways

Submission of the A9M-L2 signature (n = 114 genes) to the gene ontology (GO) platform GOEAST and DAVID databases identified cell proliferation and transcription factor activities among the highest ranking biological processes significantly affected in the mouse model of leukemia development. In addition to transcriptional regulation and immune responses, submission of the gene list to either the DAVID analysis platform (supporting information Table S2a, upper panel) or the GOEAST platform (supporting information Table S3) identified enrichment for genes associated with cell proliferation, activation, and apoptosis. Convincingly, both platforms identified similar subsets of genes including cytokines and chemokines, E2F transcription families, and cluster of differentiation markers as being associated with key processes (supporting information Table S2b). While GOEAST was able to identify similar ontologies to DAVID bioinformatics analysis, it was unable to assess pathway perturbation. The leukemic gene signature was therefore compared to the Kyoto Encyclopedia of Genes and Genomes using the DAVID software database. Key biological pathways involving cytokine signaling and adhesion were significantly enriched within the gene signature, and “Hematopoietic Lineage” and “Pathways in Cancer” were highlighted as major associated processes (supporting information Table S2a, lower panel).

Leukemia Gene Signature sscMap Analysis

To obtain a list of small molecule inhibitors with the potential to affect the leukemic state, differentially expressed gene signatures (supporting information Table S4) from the mouse model (n = 47 probesets representing 30 genes) or MILE dataset (n = 24 probesets representing 21 genes) were submitted for sscMap analysis. In total 133 of 1,309 and 130 of 1,309 (A9M-L2) connections were shown to be statistically significant, that is, −log10 p [mt] 3.15 for the MILE and A9M-L2 datasets, respectively (Fig. 3A). The expected number of false connections = 1 giving an estimated FDR of 0.0075 for the MILE dataset and 0.0077 for the A9M-L2 model dataset, respectively. Application of a perturbation algorithm to the data whereby each gene identified from the normal set (condition 1) versus leukemia set (condition 2) was individually removed to test the strength of the biological connections resulted in reducing the number of candidate small molecule inhibitors for functional studies (Table 1). A perturbation score of 1 is indicative of an extremely strong connection between a particular drug and the related gene signature, a perturbation score of 0 is indicative of a weak connection.

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Figure 3. sscMap validation, acetylation of histones, and induction of p21CIP/WAF in Entinostat-treated A9M-L2 cells. (A): A scattergram volcano plot depicting statistically significant connections (SSC) above the 3.15 threshold for –log10 (p value) between 24 MILE data probesets (upper panel) and 47 A9M-L2 data probesets (lower panel). (B): A bar graph of quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) values obtained from A9M-L2 leukemias following short-term treatment (24 hours) with Entinostat (300 nM) or vehicle (DMSO) control (n = 3 per group). Relative expression is displayed as log2 estimated copy number values for HOXA9 and MEIS1 (where Ct 35 = 5 copies). A subset of the genelist (n = 10) used in the statistically significant connectivity map analysis was examined for differential expression. (C): Western blot and densitometry analysis of A9M cells showing the effect of treatment on levels of proteins implicated in cell survival and activity. Representative data from three independent experiments normalized to β-actin are shown, drug dosages in nM are presented. (D): Bar graph demonstrating a transient increase in AcH4 binding at the p21CIP/WAF promoter following Entinostat treatment of A9M and A9M-L2 primary cells as determined by ChIP-qRT-PCR. (E): A bar graph demonstrating transient upregulation of p21CIP/WAF mRNA expression following Entinostat treatment in A9M and A9M-L2 cells as measured by qRT-PCR analysis. For both (B), (D), and (E) mean values of duplicates ± SEM are plotted (n = 3 per group). Significant differences obtained from Student' t tests denoted by *, p ≤ .05; **, p ≤ .01; ***, p ≤ .001. Abbreviations: AML, acute myeloid leukemia; DMSO, Dimethyl Sulfoxide; NBM, normal bone marrow; A9M, HOXA9 plus Meis1 primary cell line; LBM leukemic bone marrow cells from A9M-L2 transplanted mice; ENTO, Entinostat.

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Table 1. Candidate small molecule inhibitors (statistically significant connectivity map)
  1. Abbreviations: CN-AML, cytogenetically normal acute myeloid leukemia; NBM, normal bone marrow.

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A shortlist of five potential therapeutics with high normalized connectivity scores (−3.8 to −4.4) and a perturbation score of 1 (p [lt] .00015) was identified from the MILE signature. Similar analysis of the A9M-L2 signature demonstrated connectivity scores of −3.2 to −4.1 (p [lt] .0015). Comparative analysis identified overlap of three candidate drugs from the mouse model and patient samples. Exemestane, a steroidal aromatase inhibitor, registered a robust perturbation stability of 1 for the MILE but a lower score (0.85) for the A9M-L2 gene signature. Two drugs, namely; TTNPB (tetrahydro-tetramethyl-naphthalenyl-propenyl benzoic acid) and Entinostat had a strong biological connection and perturbation score of 1.0 for both signatures. TTNPB is a retinoic acid agonist shown previously to exhibit in vivo toxicity in mouse [35]. Entinostat, a well-tolerated HDACi [33, 34], was therefore selected as a candidate drug for further analysis. Another HDACi, Panobinostat, not in the 1,309 BROAD Institute cMap Reference Profiles database, was included for comparative analysis.

Validation of sscMap and Entinostat Activity in A9M-L2 Cells

Short-term Entinostat treatment (24 hours) of A9M-L2 cells resulted in increased expression of a subset of genes (9/10), previously identified as being decreased in A9M-L2 cells compared to controls, and part of the signature originally used to obtain the sscMap (Fig. 3B). These data validated the extension of the sscMap from cell lines to the primary leukemia cells. The reported cellular effects of Entinostat, most notably antiproliferative responses, were functionally validated by exposing A9M-L2 cells to the drug for up to 48 hours. This treatment resulted in transient hyperacetylation of Histones H3 and H4 and increased p21CIP/WAF levels. However, no significant decrease in caspase 3 levels or accumulation of cleaved Poly (ADP-ribose) polymerase (PARP) or caspase 3 products could be detected by Western blot analysis (Fig. 3C and data not shown). The transient increase in p21CIP/WAF protein levels following Entinostat treatment was mimicked by a transient increase in AcH4 occupancy at the p21CIP/WAF promoter locus (Fig. 3D) and upregulation of p21CIP/WAF mRNA expression in both A9M and A9M-L2 primary cells (Fig. 3E). The transient effects may be due to low metabolic stability of Entinostat or high background of HDAC activity in A9M leukemic cells.

Three drugs with neutral connectivity scores ([lt] ±0.15) and perturbation values of 0 namely; tamoxifen, thiamine, and ascorbic acid were examined as negative controls for the sscMap analysis. None of the treatments resulted in significant increase in the sscMap gene signature or affected colony formation of A9M-L2 cells (supporting information Fig. S5).

Entinostat Reduces CN-AML Viability and Colony Formation of A9M-L2

Primary CN-AML patient samples (n = 6) cultured in Panobinostat (7 nM) or Entinostat (300 nM) for up to 72 hours showed a significant decrease in viability compared to DMSO (vehicle) controls in liquid culture (Fig. 4A). Methylcellulose culture demonstrated reduced clonogenic potential and cellularity of colonies produced from Entinostat-treated primary CN-AML cells compared to Panobinostat or vehicle treated controls (Fig. 4B), even though treatment of A9M-L2 cells with 300 nM Entinostat or 7 nM Panobinostat resulted in comparable increased histone acetylation (supporting information Fig. S6). Entinostat treatment of A9M-L2 primary leukemia cells resulted in a similar marked reduction in clonogenic potential and decreased cellularity compared to Panobinostat, or vehicle and untreated controls, as demonstrated by INT staining (Fig. 4C). The observation that Entinostat treatment did not result in a significant decrease in colony formation of NBM cells (Fig. 4D) suggests the leukemic cells are particularly Entinostat sensitive.

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Figure 4. Entinostat reduces cytogenetically normal acute myeloid leukemia (CN-AML) viability and reduces colony formation of A9M-L2 cells. (A): Bar graph demonstrating viability of primary CN-AML samples following Entinostat (300 nM) or Panobinostat (7 nM) treatment (n = 6 per group). Cell viability was assessed as relative fluorescence units obtained from the measurement of ATP concentration at the specified time. (B): Representative colony images and the total colony forming cell (CFC) content (lower right panel) of the primary CN-AML cell populations treated with vehicle (DMSO), Panobinostat (7 nM), or Entinostat (300 nM). (C): Colony formation of A9M-L2 cells (n = 3 per group) following control, DMSO, Entinostat (300 nM), or Panobinostat (7 nM) treatment. Left panel, total CFC content of the treated cell populations (mean ± SD, n = 3, two experiments); right panel, representative images of colonies. (D): Colony formation of normal bone marrow samples (n = 3 per group) following control, DMSO, or Entinostat (300 nM) treatment. Left panel, total CFC content (mean ± SD, n = 3, two experiments); right panel, representative images of colonies. Asterisks denote significant differences between the treatments and controls (Student's t tests *, p ≤ .05; ** p ≤ .01; *** p ≤ .001). Abbreviation: DMSO, Dimethyl Sulfoxide.

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Entinostat Treatment Prevents A9M-L2 Leukemia Maintenance and Extends Survival of Leukemic Mice

The sensitivity of A9M-L2 cells to Entinostat was further demonstrated in parallel transplantation assays. Aliquots of the ex vivo-treated and control A9M-L2 cells used for the colony assays were directly transplanted into recipient mice. A dramatic decrease in the number of leukemia initiating cells in the Entinostat treated cohorts was demonstrated by a complete absence of overt leukemia during the 80-day observation period (Fig. 5A left panel), and no leukemic cell infiltrates (Fig. 5A right panel). In contrast, recipients of untreated, DMSO-treated, or Panobinostat-treated A9M-L2 cells succumbed to leukemia within approximately 32 days after transplantation. Entinostat treatment of NBM cells was well tolerated by recipient mice following transplantation (Fig. 5A left panel) and homing to the spleen or short-term repopulation in spleen and bone marrow were not markedly affected compared to controls (supporting information Figs. S7, S8). Interestingly, both NBM and A9M-L2 cells treated with Entinostat demonstrated a slight reduction in homing to the bone marrow (supporting information Fig. S7), which may warrant further examination. The low dose of HDACis used in the ex vivo model treatments, sufficient to activate p21CIP/WAF, did not induce apoptosis as measured by Annexin V/Propidium Iodide staining. This phenomenon was, however, observed when drug concentrations were increased to 1 μM and 25 nM for Entinostat and Panobinastat, respectively (supporting information Fig. S9).

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Figure 5. Entinostat treatment prevents A9M-L2 leukemia maintenance and results in prolonged survival in vivo. Kaplan-Meier plots demonstrating correlation between treatment regimens and survival in leukemic mice. (A, left panel): A9M-L2 cells were incubated ex vivo for 24 hours in culture with control media, or in media supplemented with vehicle, or Panobinostat (7 nM), or Entinostat (300 nM) then transplanted (5 × 105 cells) into sublethally irradiated (850 cGy) mice, which were closely monitored and sacrificed at the first sign of disease. Normal bone marrow cells treated with Entinostat (300 nM) were transplanted in a control mouse cohort. (A, right panel): Blood smears and tissue touchpreps stained with Wright-Giemsa showing leukemia infiltration of peripheral blood and solid tissues. (B, left panel): Kaplan-Meier plots demonstrating prolonged survival in leukemic (A9M-L2) mice, given an i.v. bolus of Entinostat (30 mg/kg) at day 14 following transplantation, compared to DMSO controls (n = 5 per group). A control mouse cohort (NBM) was similarly treated and demonstrated no morbidity within the observation time frame. Significant difference between the treatment and control (Log-rank Mantel-Cox Test; p value = .014). Scale bars = 20 μM. Abbreviations: DMSO, Dimethyl Sulfoxide; NBM, normal bone marrow.

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To determine whether Entinostat was capable of suppressing leukemia development in vivo, recipients of A9M-L2 cells were treated with a single bolus of Entinostat or vehicle control on day 14 after leukemia initiation by transplantation and assessed daily for development of AML. Entinostat treatment prolonged the survival time of treated leukemic mice by up to 8 days (p = .0140) compared to controls (Fig. 5B left panel), suggesting that in vivo Entinostat treatment noticeably suppressed and delayed expansion of leukemic cell populations. Mice that succumbed to leukemia following the single Entinostat treatment presented with a similar phenotype, including tissue infiltration (Fig. 5B right panel) as vehicle controls.

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. SUMMARY
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

The frequent co-overexpression of HOXA9 and MEIS1 in AML, particularly in the absence of an underlying genetic rearrangement, [36, 37] suggests an important role for this axis above and beyond the well-established link with 11q23 translocations (reviewed by Muntean and Hess [38]). Co-overexpression of HOXA9 and MEIS1 results in an aggressive and tractable leukemia in mouse which makes it an attractive model for molecular and genetic studies [26]. In this work, we explored the usefulness of a conditional HOXA9/MEIS1-dependent mouse model to investigate and challenge leukemia maintenance. A combination of low viral titer and transplantation with limiting numbers of leukemia initiating cells resulted in a robust clonal disease that expressed the collaborating oncogenes at levels representative of CN-AML patient samples. Deletion of the collaborating oncogenes following exposure to Cre-recombinase resulted in altered colony and cell growth and loss of leukemia maintenance, defining a potential therapeutic window. However, therapeutic targeted gene deletion is currently far removed from the clinical setting and for that reason a drug redeployment strategy was adopted for candidate drug discovery.

Gene expression analyses of the A9M-L2 leukemia identified reduced expression (below fourfold) of an array of transcription factor, immune responsive and pluripotent stem cell associated genes compared to age-matched normal controls. Only a minor fraction of differentially expressed genes (17%) demonstrated increased expression above fourfold compared to NBM controls. The alteration in gene expression obtained from leukemic bone marrow of A9M-L2 mice reflects the biology of end-stage disease and is thus unlikely to represent direct targets of the collaborating oncogenes. However, some of the genes within the signature, including CD34 and CD28, have been previously identified as potential targets of HOXA9/MEIS1 [39].

GO and pathway analysis of the gene signature obtained from the A9M-L2 model reflected the pathways interrogated by the specific arrays but furthermore identified regulation of proliferation, apoptosis, hematopoietic lineage, and cancer pathways (among others) as key processes associated with the differentially expressed genes. Submission of the CN-AML and A9M-L2 signatures to sscMap analysis identified robust connectivity between five drugs, in both the clinical and mouse model samples, associated with promoting condition 1 (normal bone marrow) over condition 2 (leukemia), thereby indicating therapeutic potential. Furthermore, three of the drugs identified (Entinostat, TTNPB, and Exemestane) showed direct overlap between the CN-AML patients and A9M-L2 model of which two (Entinostat and TTNPB) demonstrated statistically robust perturbation scores of 1. One of the compounds (TTNPB) had previously been shown to have in vivo toxicity in mouse models [35] and, therefore, Entinostat, a HDACi shown to be well-tolerated in vivo [33, 34], was selected for further analysis.

Short-term exposure of A9M-L2 cells to low-dose Entinostat (300 nM) resulted in acetylation of Histones H3 and H4, upregulation of the cell cycle inhibitor p21CIP/WAF, and lack of induction of apoptosis in agreement with previous findings [40]. Low-dose Entinostat or Panobinostat treatments significantly decreased cell viability of primary CN-AML cells in liquid culture. Additionally, A9M-L2 leukemia cells treated with Entinostat demonstrated reduced numbers, size, and metabolism of clonogenic progenitors compared to controls or Panobinostat-treated cells. The differential cellular effects of two HDACis used at concentrations that attain comparable levels of Histone acetylation emphasizes the complexity of epigenetic regulation and warrants further study.

The decrease in colony formation following Entinostat treatment correlated with a dramatic reduction in leukemia initiating cells compared to Panobinostat, control, or vehicle-treated A9M-L2 cells. This clearly demonstrates that A9M-L2 leukemia cells are sensitive to short-term exposure of Entinostat that results in significant depletion and potential purging of the leukemia-initiating cell possibly by a mechanism of reduced proliferation/self-renewal and increased differentiation, in part due to the increased activity of p21CIP/WAF (Fig. 6). Association between HOX/TALE activation and epigenetic drivers such as CBP/P300 recruitment, histone acetylation, and alternative recruitment of RNA polymerase II has recently been reported [41, 42]. Together with the data presented herein, this suggests that collaborating oncogenic dysfunction may result in epigenetic disruption that is potentially targetable therapeutically. Congruent with this, a single treatment of Entinostat, to developing A9M-L2 leukemia, resulted in significant extension in survival (∼8 days) compared to control or vehicle-treated mouse recipients. The lack of general cytotoxicity of Entinostat, as demonstrated by it being well-tolerated in NBM cells and failing to impair homing or short-term repopulation, indicates good efficacy which may be of particular value in combination therapies.

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Figure 6. Proposed mechanism of action for Entinostat in A9M leukemia. Hematopoietic development is in balance, with genetic input (HOXA9/MEIS1) and epigenetic maintenance, allowing normal differentiation and apoptosis (upper section). Increased HOXA9/MEIS1 input with potential and associated epigenetic disruption leads to increased proliferation/self-renewal and impaired differentiation. This culminates in leukemia initiation and maintenance. However, maintenance is disrupted by histone deacetylase class I inhibitor (Entinostat) treatment allowing for epigenetic recovery, reduced proliferation/self-renewal, and restored differentiation which results in loss of leukemia maintenance (lower section).

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SUMMARY

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. SUMMARY
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Together these data show that application of sscMap to genetically defined models can identify small molecule inhibitors across species that may be clinically relevant. The ex vivo purging of leukemia maintenance cells and prolonged survival in vivo supports the potential of Entinostat as a therapeutic for HOXA9 and MEIS1 overexpressing leukemias. Whether Entinostat has a direct effect on the proto-oncogenes as recently suggested [43] or indirectly through other mechanisms, in CN-AML, remains to be determined. Entinostat prolonged survival in leukemic mice as a single-agent to a similar level as that reported for continuous infusion of a potent small molecule inhibitor [44]. However, epigenetic priming agents, such as HDACis and hypomethylating molecules will most likely be more effective as part of a combined therapeutic approach in leukemia and cancer [45–47]. HDACis are associated with several modes of antitumor activity including modulation of the immune response [48], cell cycle arrest, promotion of differentiation, senescence, and apoptosis [46]. Harnessing the beneficial properties of such compounds while limiting cytotoxic effects will improve their future use particularly in combination therapy.

Advanced connectivity maps, obtained from in vivo studies, may accelerate drug development and redeployment of approved small molecule inhibitors. Therefore, the generation and treatment of clinically relevant in vivo models becomes more pressing. This study demonstrates that the HDAC class I inhibitor Entinostat inhibits disease maintenance and prolongs survival in a clinically relevant murine model of cytogenetically normal AML.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. SUMMARY
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

We gratefully acknowledge Danielle Gagne, Melanie Fréchette, and Jodie Hay for technical support, as well as the staff within the Biological Resource Unit, Bioinformatics, and Flow Cytometry Cores, Queen's University Belfast. A.T. is a recipient of The American Cancer Society for Beginning Investigator Fellowship from the UICC and supported by Leukemia Lymphoma Research (U.K.) grant numbers 09035 and 07016 and the Northern Ireland Leukemia Research Fund (NILRF). G.D. and L.K. were supported by Leukemia Lymphoma Research (U.K.); G.D. is a recipient of a UICC Fellowship (YY); T.R.L. and K.I.M. were both funded by NILRF, J.M.R. was funded by the Northern Ireland Department of Education and Learning. S.D.Z. was supported by the Biotechnology and Biological Sciences Research Council (BBSRC, U.K.) grant BB/I009051/1. G.S. is a recipient of a Canada Research Chair in molecular genetics of stem cells and is supported by grants from the National Cancer Institute of Canada with funds from the Canadian Cancer Society.

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  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. SUMMARY
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. SUMMARY
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
sc-12-0925_sm_SupplFigure1.pdf444KFigure S1. Immunophenotype of A9M-L2 leukemic cells. Representative scattergrams (dot plots) demonstrating comparative cell surface immunophenotype of A9M-L2 leukemic and normal bone marrow cells (Control). Bone marrow cells were stained with antibodies that recognize: Sca1, Kit, Lin, Mac1, GR1, IgM, B220, CD43, CD4 and CD8. The target cells were gated based on FSC/SSC and Myeloid, T-Cell, SKL and B-cell populations are marked.
sc-12-0925_sm_SupplFigure2.pdf160KFigure S2. Altered Cellular Dynamics and Differential Gene Expression in Cre-Treated A9M-L2 cells Bone marrow cells were obtained from terminal A9M-L2 leukemic mice and cocultured with naive GP+E cells (Control Non-Infected) or those previously infected with MSCV-Cre-GFP or control vector (MSCV-GFP) virus. (a) Cells were cultured for 48 hours prior to sorting based on eGFP positivity. Transduced cells were capable of forming colonies in methylcellulose within 10 days of culture. (b) A representative bar graph demonstrating reduced colony formation following sorting of Cre-GFP expressing cells compared to control. Mean values ± S.E.M are plotted. Significant differences are denoted p ≤ 0.01 **. (c) Total RNA isolated from Cre- or GFP-Treated A9M-L2 primary cells was converted to cDNA and examined by qRT-PCR. Differential expression of candidate genes, determined by Delta CT values equivalent to ≥4-fold changes, were identified and compared to values obtained from the array analysis of A9M-L2 and normal bone marrow samples. Fourteen genes, previously identified as demonstrating reduced expression in the A9M-L2 model compared to normal bone marrow, showed a measurable increase in expression (dark grey bars) upon deletion of the HOXA9-ires-MEIS1 element. Jarid1b and Irf4 were marked as genes validated by individual assays. (d) Transduced cells were also maintained in liquid culture for up to 14 days in defined media. Images were captured at 20X using an inverted microscope (CKX41) with attached digital camera (E620) both Olympus, Essex, UK. Microscopic analysis showed increased differentiation and adhesion, indicative of a macrophage phenotype in the Cre-Treated A9M-L2 cells compared to GFP-Treated controls.
sc-12-0925_sm_SupplFigure3.pdf26KFigure S3. Comparable levels of HOXA9 and MEIS1 expression in clinical samples and murine model. Comparative bar graph obtained from quantitative RT-PCR analysis of A9M-L2 bone marrow samples, normal mouse bone marrow control (n=5 per group) and a cohort of AML patients classified based on standard prognosis adverse (n=3), favorable (n=6) or intermediate (n=15). Relative expression is displayed as log2 estimated copy number values for HOXA9 and MEIS1 (where Ct 35 = 5 copies). Mean values ± S.E.M are plotted. Comparable gene expression was observed for the Intermediate risk patient group and the A9M-L2 model as determined by the student t-test (ns for non-significant).
sc-12-0925_sm_SupplFigure4.pdf23KFigure S4 Validation of array expression changes by SYBR QPCR. Bar graph of gene expression values denoted by Delta Ct values corrected to 18SrRNA, obtained from both TaqManTM Low Density Array (Array) and individual SYBR Green-based assays (SYBR). Comparable values or similar trends for Array and SYBR assays were demonstrated in 17/21 genes examined for A9M-L2 cells relative to normal bone marrow cells (n=5 per group). Mean values ± S.E.M are plotted.
sc-12-0925_sm_SupplFigure5.pdf55KFigure S5 Functional validation of neutral/weak sscMap connections. (Left panel) a bar chart demonstrating no significant alteration in the expression of a ten gene signature (Figure 3b) in A9M-L2 cells following treatment with three small molecule inhibitors identified as having neutral/weak sscMap connectivity. The combined expression of the gene signature (estimated copy number) was obtained for vehicle control (PBS) or Tamoxifen, Thiamine and Ascorbic acid treated A9M-L2 cells and compared to Entinostat treatment. Mean values ± S.E.M of relative expression (fold of control) was plotted. Significant differences as determined by the student t-test are denoted p ≤ 0.02**, ns= non-significance. (Right Panel) Representative colony images from A9M-L2 cells treated with vehicle (PBS), Tamoxifen (1 μM), Thiamine (100 μM) or Ascorbic acid (100 μM). Leukemic colonies were stained with 1 mg/ml p-iodonitrotetrazolium violet (INT) for 16 hours and images captured by an Olympus CKX41 microscope and camera magnification 40× using an inverted microscope (CKX41) with attached digital camera (E620) both Olympus, Essex, UK.
sc-12-0925_sm_SupplFigure6.pdf117KFigure S6 Comparable effects of low dose Entinostat and Panobinostat Western blot and densitometry analysis of primary A9M-L2 cells treated with vehicle control (DMSO), Entinostat (Ent; 300 nM) or Panobinostat (Pan-7; 7 nM and Pan-25; 25 nM). Proteins were isolated 24 hours following treatment and probed with specific antibodies for Acetylated Histone-H3/H4. Representative data from three independent experiments demonstrating comparable acetylation levels are shown normalized to β-actin.
sc-12-0925_sm_SupplFigure7.pdf33KFigure S7 Short-term repopulation of NBM cells treated with Entinostat Normal CD45.1+ donor bone marrow cells were obtained by flushing femurs and tibias of C57Bl/6-Ly5.1 mice and treated with vehicle control (DMSO) or Entinostat (300 nM) for 24 hours in maintenance media containing interleukin-6, granulocyte/macrophage colony-stimulating factor, 10 ng/mL; stem cell factor, 100 ng/mL. Recipient CD45.2+ (C57Bl/6J) mice were sublethally irradiated (650 cGy) prior to transplantation with 5 × 105 treated CD45.1+ donor cells (n=4 per group). Recipient mice were housed within a specific pathogen free animal unit (QUB-BRU) for eight weeks after which they were humanely killed by CO2 asphyxiation. Bone marrow and spleen cells were obtained from the recipient mice and treated with APC conjugated anti-mouse CD45.1 and PE conjugated anti-mouse CD45.2 (both eBioscience, Hatfield, UK). Data were acquired and analyzed using an LSRII cytometer and FACSDiva Software 6.1.2 (BD Pharmingen). (a) Representative dot plots obtained from recipient mouse bone marrow or spleen samples demonstrating the percentage retention/repopulation of donor (CD45.1+) cells in the recipient (CD45.2+) background. (b) a bar graph depicting comparable low level of repopulation (∼10%) is observed for the Entinostat or DMSO treated donor cells in the bone marrow of recipient mice compared to unstained control. A higher level of repopulation (∼20%) is observed for repopulation of the spleen for both treatments. No significant difference in repopulation ability was demonstrated in the DMSO or Entinostat treated donor cells. Mean values ± S.E.M are plotted (n=4 per group).
sc-12-0925_sm_SupplFigure8.pdf41KFigure S8 Homing of NBM or A9M-L2 cells following Entinostat treatment Normal and A9M-L2 leukemic CD45.1+ bone marrow cells were obtained by flushing femurs and tibias of donor mice and treated with vehicle control (DMSO) or Entinostat (300 nM) for 24 hours in maintenance media as above. Recipient CD45.2+ (C57Bl/6J) mice were lethally irradiated (1300 cGy) prior to transplantation with 5 × 106 treated CD45.1+ donor cells (n=5 per group). Recipient mice were housed within a specific pathogen free animal unit (QUBBRU) for 24 hours after which time they were humanely killed by CO2 asphyxiation. Bone marrow and spleen cells were obtained from the recipient mice and stained with PE conjugated anti-mouse CD45.1 (Southern Biotech, Alabama, USA). Data were acquired and analyzed using an LSRII cytometer and FACSDiva Software 6.1.2 (BD Pharmingen) or FlowJo software 7.6.5 (TreeStar Inc, Ashland, OR). (a) Bar graph showing reduced bone marrow homing of NBM or A9M-L2 cells following Entinostat treatment compared to DMSO controls. The analysis shows that homing of the donor cells to the spleen is not impaired following Entinostat treatment and that a higher percentage of A9M-L2 cells home to the spleen regardless of treatment. Mean values ± S.E.M are plotted (n=5 per group). Significant differences are denoted p ≤ 0.05 *, p ≤ 0.01 ** as determined by the student t-test. (b) Overlay of representative histogram plots obtained for CD45.1 expression in recipient mouse bone marrow and spleen cells obtained from transplanted DMSO or Entinostat-treated NBM or A9M-L2 donor cells. The treated cells are compared to unstained control.
sc-12-0925_sm_SupplFigure9.pdf45KFigure S9 Induction of apoptosis following HDACi treatment To examine the effect of HDACi treatment on apoptosis, A9M-L2 cells were cultured for up to 48 hours with Entinostat (300 nM or 1 μM) or Panobinostat (7 nM or 25 nM) and apoptosis measured using the FITC Annexin V Apoptosis Detection Kit I (BD Biosciences, San Diego, CA) as per the manufacturer's instructions. Data were acquired and analyzed using an LSRII cytometer and FACSDiva Software 6.1.2 (BD Pharmingen). (a) Bar graph demonstrating lack of Annexin V staining over DMSO controls (∼5%) in the low dose 24 hour HDACi treatments (300 nM Entinostat, 7 nM Panobinostat) but an increase in apoptosis that is both dose and time dependent peaking (>35%) with the high dose Panobinostat (25 nM) following 48 hours of treatment in liquid culture. Mean values ± S.E.M are plotted (n=4 per group). Significant differences as determined by the Student's t-test are denoted p ≤ 0.01**. (b) Representative dot plots of Annexin V and Propidium Iodide (PI) staining from the A9M cell line or primary A9M-L2 cells for the lower dose HDACi treatments (300 nM Entinostat, 7 nM Panobinostat) at 24- and 48- hour time points.
sc-12-0925_sm_SupplTable1.pdf37KTable S1. Gene expression array profiling in A9M-L2 model. Summary of the quantitative RT-PCR array profiling results of A9M-L2 model compared to normal mouse bone marrow (NBM). Immune, stem cell pluripotent and transcription factor (Biotrove) array profiles are detailed with average Ct and standard deviation values shown. Tabulated values are from duplicate experiments of five biological replicates.
sc-12-0925_sm_SupplTable2.pdf15KTable S2. Gene ontology and pathway analysis of A9M-L2 leukemia. (a) A list of genes differentially expressed significantly in the A9M-L2 leukaemias compared to normal bone marrow controls (n=5 per group) was submitted for Bioinformatics analysis using the Database for Annotation, Visualization and Integrated Discovery (DAVID) Bioinformatics Resources 6.7 and GO Enrichment Analysis Software Toolkit (GOEAST) platforms. The percentages of genes associated with a particular process or pathway are ranked and tabulated and total numbers of genes defined within a process or pathway are tabulated in parenthesis. The upper panel (light grey) indicates statistically significant association with biological processes (DAVID) the lower panel (dark grey) with pathway associations (Kyoto Encyclopedia of Genes and Genomes, KEGG). (b) A table of process-associated genes identified in both the GOEAST and DAVID platforms. The subset of processes examined includes cell proliferation, cell activation, apoptosis, and transcription. Genes in bold font are common to both platforms demonstrating a level of robustness in the analysis.
sc-12-0925_sm_SupplTable3.pdf14KTable S3. Gene ontology of differentially expressed genes in A9M-L2 cells. Gene Ontology Enrichment Analysis Software Toolkit (GOEAST) was used to cluster molecular function (white), biological processes (light grey) and chemical component characteristics (dark grey) from the A9M-L2 gene lists obtained from array profiling experiments. The number of genes associated with a particular process and statistical measures of the possibility that the associations were made by chance (log of odds ratio of enrichment and p values) are tabulated. High log values (≥ 1.5) and low p values ≤ 0.05) indicate the GOterm associations are unlikely to be due to chance.
sc-12-0925_sm_SupplTable4.pdf13KTable S4. Genes and Probeset IDs used for sscMap Analysis Differentially expressed genes were identified from the comparative analysis of the mouse model (A9M-L2 v NBM) and patient cohort MILE data (MILE 13 v MILE 18) using two-sample t test analysis. A gene-signature with 47 Affymetrix probeset IDs, representing 30 individual genes, provided the minimum set of genes which returned significant connections to drugs at 1% false discovery rate (FDR=0.01). For the MILE study, the Affymetrix MAS5 value on the log scale was used as a measure of gene expression level. A gene signature with 24 Affymetrix probeset IDs, representing 21 individual genes, was constructed which returned significant connections to drugs at 1% false discovery rate (FDR=0.01).

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