The epigenetic treatment remodel genome-wide histone H4 hyper-acetylation patterns and affect signaling pathways in acute promyelocytic leukemia cells

Giedre Valiulien˙ e˙ *, Aida Vitkeviˇciene, uta Navakauskien¯ e ˙

Abstract

Although majority of acute promyelocytic leukemia (APL) patients achieve complete remission after the standard treatment, 5–10% of patients are shown to relapse or develop resistance to treatment. In such cases, medications that target epigenetic processes could become an appealing supplementary approach. In this study, we tested the anti-leukemic activity of histone deacetylase inhibitor Belinostat (PXD101) and histone methyltransferase inhibitor 3-Deazaneplanocin A combined with all-trans retinoic acid in APL cells NB4, promyelocytes resembling HL-60 cells and APL patients’ cells. After HL-60 and NB4 cell treatment, ChIP-sequencing was performed using antibodies against hyper-acetylated histone H4. Hyper-acetylated histone H4 distribution peaks were compared in treated vs untreated HL-60 and NB4 cells. Results demonstrated that in treated HL-60 cells, the majority of peaks were distributed within the regions of proximal promoters, whereas in treated NB4 cells, hyper-acetylated histone H4 peaks were mainly localized in gene body regions. Further ChIP-seq data analysis revealed the changes in histone H4 hyper-acetylation in promoter/gene body regions of genes involved in cancer signaling pathways. In addition, quantitative gene expression analysis proved changes in various cellular pathways important for carcinogenesis. Epigenetic treatment down-regulated the expression of MTOR, LAMTOR1, WNT2B VEGFR3, FGF2, FGFR1, TGFA, TGFB1, TGFBR1, PDGFA, PDGFRA and PDGFRB genes in NB4, HL-60 and APL patients’ cells. In addition, effect of epigenetic treatment on protein expression of aforementioned signaling pathways was confirmed with mass spectrometry analysis. Taken together, these results provide supplementary insights into molecular changes that occur during epigenetic therapy application in in vitro promyelocytic leukemia cell model.

Keywords:
Acute promyelocytic leukemia (APL)
3-Deazaneplanocin A
Belinostat (PXD101)
All-trans retinoic acid
Epigenetic therapy

1. Introduction

Acute promyelocytic leukemia (APL) is a subtype of acute myeloid leukemia, characterized by fusion gene PML-RARA (t(15;17)(q22;q21)). This genetic abnormality causes the blockade of promyelocyte differentiation into granulocytes and clonal promyelocyte expansion (Moosavi and Djavaheri-Mergny, 2019). Historically, APL was considered to be fatal; however, advent of all-trans retinoic acid (RA) revolutionized its treatment. Studies show that following standard treatment with RA or RA with chemotherapy, more than 90% of APL patients achieve complete remission; however, approximately 5–10% of patients are resistant to the treatment or relapse (Sanford et al., 2015).
Usually, (15;17)(q22;q21) chromosomal translocation is not the only aberration in APL cells. Recent study confirmed that various epigenetic aberrations in APL cells are also very common (Rahimi et al., 2019). Moreover, PML-RARα fusion protein recruits various other partners to form a large protein complex and bind retinoic acid-dependent promoters of differentiation genes and suppresses them. Among various PML-RARα protein complex partners, there are epigenetic regulators such as histone deacetylases (HDACs) and histone methyltransferases (HMTs) (Arteaga et al., 2015). Therefore, HDAC and HMT inhibitors might improve conventional APL therapy.
HDAC inhibitors are relatively nontoxic to normal cells. Furthermore, HDAC inhibitors accelerate the degradation of PML-RARα fusion protein (Noack et al., 2017). Belinostat (PXD101, (2E)-N-Hydroxy-3-[3-(phenylsulfamoyl)phenyl]prop-2-enamide)) is a pan-HDAC inhibitor with potency to inhibit class I, II and IV HDAC isoforms. In 2014, it has been approved by FDA for relapsed or refractory peripheral T-cell lymphoma treatment (Campbell and Thomas, 2017). There are ongoing research studies on Belinostat application for the treatment of other cancer types (Zhang et al., 2019). HMT inhibitors have also been actively explored recently. One such inhibitor – 3-Deazaneplanocin A (5R-(4-amino-1H-imidazo[4,5-c]pyridin-1-yl)-3-(hydroxymethyl)-3-cyclopentene-1S,2R-diol) was shown to inhibit cell proliferation and induce apoptosis in several cancer types (Kikuchi et al., 2012; Sharma et al., 2009). 3-Deazaneplanocin A is an S-adenosyl-l-homocysteine hydrolase inhibitor and was shown to inhibit Polycomb repressive complex 2 protein EZH2 and cause concomitant loss of its target H3K27me3 and silenced gene reactivation (Kikuchi et al., 2012).
In previous studies, we demonstrated that 3-Deazaneplanocin A and Belinostat in combination with RA exhibits anti-cancerous effects on APL in vitro, in vivo and ex vivo (Valiuliene et al., 2016, 2017; Vitkeviciene et al., 2019). Our studies revealed that these epigenetic modifiers inhibited NB4 and HL-60 cell proliferation, caused apoptosis, enhanced cell differentiation and remodelled chromatin (Valiuliene et al., 2017). Similar findings were revealed after APL patient cell treatment with 3-Deazaneplanocin A and Belinostat in combination with conventional therapy agents (retinoic acid with Idarubicin) (Vitkeviciene et al., 2019). Moreover, 3-Deazaneplanocin A, Belinostat and RA combination prolonged APL xenograft mice survival and prevented tumor formation (Valiuliene et al., 2016). Since tested epigenetic agents have enhanced conventional therapy effects, the aim of this study was to further examine molecular mechanisms of our proposed epigenetic therapy. Thus, we performed ChIP-sequencing using antibody against hyper-acetylated histone H4 on NB4 and HL-60 cells treated with 3-Deazaneplanocin A and Belinostat in combination with retinoic acid. In addition, using mass spectrometry analysis, we evaluated the effect of these agents on NB4 and HL-60 cells’ protein expression. After result analysis, we revealed changes in various cellular pathways important for carcinogenesis.

2. Materials and methods

2.1. NB4 and HL-60 cell cultivation and treatment

NB4 and HL-60 cell lines were purchased from DSMZ (Braunschweig, Germany). Bone marrow samples were obtained from three patients diagnosed with APL (PML-RARA translocation was detected). White mononuclear cells were purified from bone marrow aspirate by Ficoll- Paque PLUS density gradient centrifugation (GE Healthcare Chicago, IL, USA). Ethical permission from Vilnius Regional Biomedical Research Ethics Committee (approval no. 158200-16-824-356) and informed consent of the patients were obtained.
The cells were cultivated in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin (Gibco, Carlsbad, CA, USA) at 37 ◦C in a humidified 5% CO2 atmosphere. For treatment with epigenetic agents NB4 and HL-60 cells were seeded at density 0.5 × 106 cells/ml. Cell samples were treated with 0.5 μM 3-Deazaneplanocin A (Cayman Chemical Company, Ann Arbour, MI, USA) and 0.8 μM Belinostat (PXD101) (Selleckchem, Munich, Germany) combination for 4 h; after the treatment, the cells were washed and treated with 1 μM retinoic acid (Sigma-Aldrich, St. Louis, MO, USA) for 12 and 72 h. As a control, NB4 and HL-60 cells were treated with 1 μM retinoic acid only (Sigma-Aldrich) for 12 and 72 h.

2.2. Cell proliferation, survival and granulocytic differentiation assays

Cell proliferation and survival were evaluated using 0.2% of trypan blue dye (final concentration) by trypan blue exclusion test. Viable and dead (blue coloured) cell numbers were counted in a haemocytometer under the light microscope. Granulocytic differentiation was determined using nitro blue tetrazolium (NBT) (Sigma-Aldrich) assay. Differentiated cells reduce soluble nitro blue tetrazolium to insoluble blue-black formazan after stimulation with phorbolmyristate acetate (PMA) (Sigma-Aldrich). Cells were incubated with 0.1% of NBT and 100 ng/ml PMA (final concentrations) at 37 ◦C for 30 min. Undifferentiated and differentiated (blue coloured) cells were counted in a haemocytometer under the light microscope. Differentiated cell percentage was expressed as the blue coloured cell number relative to viable cell number, which was determined by trypan blue exclusion test.

2.3. ChIP-sequencing and analysis

The ChIP assay was performed as follows. Briefly, 2 × 107 HL-60 or NB4 cells were treated with formaldehyde at a final concentration of 1% for 5 min at room temperature to cross-link DNA-protein complexes. After cross-linking, cells were lysed in buffer 10 mM Tris-HCl pH 8.0, 10 mM EDTA, 0.5 mM EGTA, 0.25% Triton X-100, 10 mM sodium butyrate, 20 mM β-glycerophosphate, 1 mM Na3VO4, Protease Inhibitor Cocktail (Roche Diagnostics Risch-Rotkreuz, Switzerland). Crude nuclei were collected by centrifugation, washed with buffer 10 mM Tris-HCl pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 10 mM sodium butyrate, 20 mM β-glycerophosphate, 1 mM Na3V04, Protease Inhibitor Cocktail (Roche Diagnostics) and resuspended in nuclei lysis buffer 50 mM Tris–HCl, pH 8.1, 10 mM EDTA, 1% SDS, 10 mM sodium butyrate, 20 mM β-glycerophosphate, 1 mM Na3VO4 and Protease Inhibitor Cocktail (Roche Diagnostics). Chromatin was fragmented by sonication for 25 cycles, using Bioruptor Pico apparatus (Diagenode, Belgium). The insoluble material was pelleted by centrifugation at 8000×g for 15 min at room temperature. Supernatant with soluble chromatin was diluted 10 times with RIPA buffer and re-concentrated using Centriplus YM 50 concentrators (Millipore, Billerica, MA, USA).
6.5 μg of antibody against hyper-acetylated histone H4 (Upstate Biotechnology, Lake Placid, NY, USA) was used per 500 μl of soluble chromatin in ChIP assay (rotated for 4 h at +4 ◦C). 25 μl of Protein A/G beads (Santa Cruz Biotechnology Dallas, Texas, United States) were added to samples and immunoprecipitated for 2 h with rotation at +4 ◦C. After immunoprecipitation, beads were collected by centrifugation and washed in succession with RIPA, RIPA +500 mM NaCl, LiCl and TE buffers. Protein A/G beads were resuspended in TE buffer and left overnight to revert at +65 ◦C. After elution from Protein A/G, specimens were treated with RNase A (Thermo Fisher Scientific, Waltham, MA, USA) and proteinase K (Thermo Fisher Scientific). After these steps, DNA was finally cleaned using QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany).
Eluted and purified ChIP DNA and input DNA (soluble chromatin before ChIP) were used to prepare the DNA for sequencing. DNA sequencing and analysis was performed at Karolinska Institute, Department of Biosciences and Nutrition, Bioinformatics and Expression Analysis core facility (Sweden). DNA libraries were prepared using the NEBnext ChIPSeq library Prep kit (NEB, Ipswich, MA, USA). Next- Generation Sequencing was carried out using Illumina Genome Analyser IIx system (Illumina, San Diego, CA, United States). Raw ChIP- sequencing data sets are available at the Gene Expression Omnibus (GEO) repository under the accession ID:GSE124725. Raw data were mapped against hg19 reference genome using Bowtie2. Quality control was performed on the BAM files using the FastQC tool. Peak detection and downstream data analysis were performed using HOMER, a software package for ChIP-sequencing analysis.
Subsequent analysis included peak annotation to gene promoters (3 kb upstream to 0.5 kb downstream transcription start site) and gene bodies region, differential promoter and gene body occupancy by peaks using the web server for functional enrichment analysis g:Profiler (version e99_eg46_p14_f929183, database updated on February 07, 2020) and classification system GO PANTHER (15.0).

2.4. Gene expression analysis by RT-qPCR

Total RNA was purified using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), cDNA was synthesized using SensiFAST™ cDNA Synthesis Kit (Bioline, Memphis, TN, USA) and qPCR was performed using SensiFAST™ SYBR® No-ROX Kit (Bioline) on the RotorGene 6000 system (Corbett Life Science, QIAGEN, Hilden, Germany). Primer sequences (Metabion international AG, Planegg/Steinkirchen, Germany) are presented in Table 1 mRNA levels were normalized to GAPDH expression. Relative gene expression was calculated using ΔΔCt method. Data are expressed as mean ± standard deviation (S.D.).

2.5. Liquid chromatography–mass spectrometry analysis (LC-MS)

Treated cells were harvested by centrifugation, washed twice with PBS, resuspended in lysis solution (7 M Urea, 2 M Thiourea, 4% (w/v) CHAPS, 40 mM DTT) and agitated in 37 ◦C for 30 min. After cell lysis, the samples were centrifuged (16,000×g, 10 min) and supernatants were collected. Trypsin digestion was performed according to FASP protocol (Wisniewski et al., 2009). LC-MS data were collected as described previously (Ger et al., 2016). Briefly, liquid chromatographic analysis was performed in a Waters Acquity ultra performance LC system (Waters Corporation, Wilmslow, UK). Peptide separation was performed on an ACQUITY UPLC HSS T3 250 mm analytical column. Data were acquired using Synapt G2 mass spectrometer and Masslynx 4.1 software (Waters Corporation) in positive ion mode using data independent (DIA) acquisition (UDMSE acquisition mode) (Distler et al., 2014). Raw data were lock mass-corrected using the doubly charged ion of [Glu1]-fibrinopeptide B (m/z 785.8426; [M+2H]2+). Raw data files were processed and searched using ProteinLynx Global SERVER (PLGS) version 3.0.1 (Waters Corporation). Data were analysed using trypsin as the cleavage protease. One missed cleavage was allowed and fixed modification was set to carbamidomethylation of cysteines, variable modification was set to oxidation of methionine. Minimum identification criteria included 1 fragment ions per peptide, 3 fragment ions per protein and minimum of 2 peptides per protein. The following parameters were used to generate peak lists: (i) minimum intensity for precursors was set to 135 counts, (ii) minimum intensity for fragment ions was set to 25 counts, (iii) intensity was set to 750 counts. For further analysis and biological pathway identification, Reactome data base (reactome.org) was used.

2.6. Statistical analysis

Unless otherwise specified, all experiments were repeated at least three times. Data were expressed as mean values with S.D. One-way ANOVA with Dunnett post-hoc test in GraphPad Prism software (8.0.1) was used for statistical analysis. Significance was set at P ≤ 0.05

3. Results

3.1. Hyper-acetylated histone H4 distribution along chromosomes after epigenetic treatment

Acute promyelocytic leukemia (APL) cell lines HL-60 and NB4 were pre-treated with epigenetic agents 0.5 μM 3-Deazaneplanocin A (HMT inhibitor) and 0.8 μM Belinostat (HDAC inhibitor) for 4 h. Pre-treated cells were washed and treated with 1 μM retinoic acid for 12 h. Agent concentrations were determined based on previously published (Valiuliene et al., 2017) and on unpublished data. Also, we demonstrated previously that the combination of 3-Deazaneplanocin A, Belinostat and retinoic acid had stronger anti-cancerous effects than every agent separately. Thus, in this study, we did not test them separately as we aimed to further analyse molecular mechanisms caused by complete combination of 3-Deazaneplanocin A, Belinostat and retinoic acid. 12 h after HL-60 and NB4 cell treatment, ChIP-sequencing was performed using antibodies against hyper-acetylated histone H4. Hyper- acetylated histone H4 distribution peaks were compared in treated and untreated HL-60 and NB4 cells. The peaks were counted in every chromosome and peak numbers detected after the treatment were subtracted from peak numbers detected in untreated cell samples. The highest level of hyper-acetylation enrichment was detected in chromosomes 7, 10 and 19 in all tested samples (Fig. 1A). Peak distribution in these chromosomes is demonstrated in Fig. 1B. The highest number of hyper-acetylated histone H4 distribution peaks was detected in gene- rich regions, while only small amount of these peaks was detected in centromere regions.

3.2. Localization of hyper-acetylated histone H4 peaks in the gene- associated regions of epigenetically treated HL-60 and NB4 cells

To ascertain the effect of epigenetic treatment with HMT inhibitor 3- Deazaneplanocin A and HDAC inhibitor Belinostat in combination with retinoic acid on histone H4 hyper-acetylation patterns in HL-60 and NB4 cells, we studied hyper-acetylated H4 distribution for locations within or near genes (Fig. 2A). After HL-60 cell treatment with epigenetic modifiers, in comparison with untreated (control) cells, we found 1900 gene- associated hyper-acetylated H4 peaks in total (Fig. 2B). 58.5% of those peaks were allocated within the regions of proximal promoters, encompassing 3 kb upstream and 0.5 kb downstream of annotated TSS (transcription start site). Contrary to HL-60 cells, in treated NB4 cells vs untreated NB4 cells, the vast majority of gene-associated hyper-acetylated H4 peaks were found in gene bodies and only 1/5 of all the peaks were distributed across the proximal promoters (Fig. 2B). When we aligned genes that in NB4 and HL-60 cells were enriched with the hyper- acetylated histone H4 peaks in promoter associated regions, we detected 16 matches between the cell lines. Meanwhile, in gene body associated peaks there were 9 matches (Table 2).
Gene ontology and molecular function analysis, which was carried out using the g:Profiler web server for matching genes in promoter and gene body regions, revealed the highest enrichment for genes that code and molecular function of their products. proteins involved in binding, catalytic activity, molecular function regulation, structural molecule activity, etc. (Fig. 2C).

3.3. Functional characteristics of genes enriched with hyper-acetylated histone H4 after the treatment with epigenetic modulators

Functional analysis with PANTHER Classification System was performed on genes enriched with hyper-acetylated histone H4 peaks annotated to NB4 and HL-60 cells’ gene promoters and gene bodies (epigenetically active compound treated cells vs untreated cells). We examined the peak-enriched genes according to their coded protein classes and regulated pathways (Fig. 3; exact numerical values presented in Supplement 1). PANTHER Protein Class analysis demonstrated that after the treatment with 3-Deazaneplanocin A and Belinostat in combination with retinoic acid, hyper-acetylated H4 enriched promoters belonged to the genes which coded transcription factors (15% in HL-60 cells, 7% in NB4 cells and 10% of all matches between HL-60 and NB4 cells), nucleic acid binders (22%, 7% and 10%, respectively), enzyme modulators (8%, 19% and 10%, respectively), as well as hydrolases (13% in HL-60 cells and 19% in NB4 cells), etc. (Fig. 3A). Gene body associated hyper-acetylated H4 peaks were demonstrated to belong to the genes that are mainly responsible for coding transcription factors (14% in HL-60 cells and 9% in NB4 cells), enzyme modulators (11% in HL-60 cells and 16% in NB4 cells), hydrolases (11% in HL-60 cells and 9% in NB4 cells) and nucleic acid binding proteins (13% in HL-60 cells and 16% in NB4 cells) (Fig. 3B).
PANTHER Pathways analysis revealed that genes that were enriched in hyper-acetylated histone H4 in promoter regions were implicated in Wnt signaling (counted for 7% in HL-60 cells and 3% in NB4 cells), inflammation mediated by chemokine and cytokine signaling pathways (counted for 4% in HL-60 cells and 7% in NB4 cells), VEGF signaling receptor pathway (counted for 3% in HL-60 and NB4 cells), EGF signaling pathway (counted for 3% in HL-60, as well as in NB4 cells), etc. (Fig. 3C). Similar results were found in gene bodies associated peaks (Fig. 3D).
In general from the data presented in Fig. 3, we could suggest that in HL-60 and NB4 cells, pre-treatment with epigenetic agents, HMT inhibitor 3-Deazaneplanocin A together with HDAC inhibitor Belinostat and further treatment with retinoic acid, resulted in changes of histone H4 hyper-acetylation at various genes, which are involved in many processes crucial for cell proliferation, survival, differentiation, as well as maintenance of cancerous state. The most relevant molecular pathways and genes involved in these pathways, which were identified in our study, are depicted in Table 3. Several pathways from these groups, related to Fibroblast growth factor (FGF), Platelet-derived growth factor (PDGF), Transforming growth factor beta (TGF-β), Mammalian target of rapamycin (mTOR), Vascular endothelial growth factor (VEGF) and Wnt signaling cascades, were chosen for further gene expression analysis. 3.4. Impact of HDAC and HMT inhibitors on APL cells’ gene expression
We aimed to elucidate, if the treatment with epigenetic agents affect NB4 and HL-60 cells‘, as well as APL patients cells‘, gene expression of main genes involved in the signaling pathways of FGF, PDGF, TGF, mTOR, VEGF and Wnt, as it is widely accepted that these pathways are over-activated in cancer (Harvey, 2019). Instead of studying those specific genes, which in our study were found to be enriched in hyper-acetylated histone H4 mark, we decided to study the expression of genes that are generally the main players in aforementioned signaling pathways. For Wnt pathway, gene expression of WNT1 and WNT2B was evaluated; for mTOR pathway, we examined MTOR and LAMTOR1; for VEGF pathway, VEGFA and VEGFR3 were examined; for FGF pathway − FGF2 and FGFR1; for TGF pathways we examined TGFA, TGFB1 and TGFR1; whereas, for PDGF signaling pathway, we have focused on PDGFA, PDGFRA and PDGFRB. For gene expression evaluation NB4, HL-60 and APL patients‘ (n = 3) cells were pretreated with 0.5 μM 3-Deazaneplanocin A and 0.8 μM Belinostat for 4 h. Pre-treated cells were then washed and treated with 1 μM retinoic acid for 12 and 72 h (in figures denoted as “DBR (12 h)” and “DBR (72 h)”). Cells were also treated with 1 μM retinoic acid alone for 12 and 72 h (in figures denoted as “RA (12 h)” and “RA (72 h)”).
The results revealed similar gene expression patterns of WNT1 and WNT2B in APL cell lines’ and APL patients’ cells after 12 and 72 h treatment with 3-Deazaneplanocin A and Belinostat in combination with retinoic acid and after the treatment with retinoic acid alone (Fig. 4A). The increase in WNT1 gene expression was found in HL-60 cells after 72 h treatments with 3-Deazaneplanocin A, Belinostat and retinoic acid and retinoic acid alone. In APL patients’ cells increase in WNT1 gene expression was the most evident after 12 h treatments. Contrary, in NB4 cells, gene expression levels of WNT1 were not affected by the treatments. However, in all cells that were tested treatment with retinoic acid down-regulated gene expression of WNT2B (after 72 h treatment in HL- 60 cells WNT2B gene expression was diminished approx. 20-fold, in NB4 cells 7-fold and in APL patients’ cells approx. 1.5-fold). In HL-60 cells combined treatment for 12 h had more pronounced effect on WNT2B gene expression reduction compared to action of retinoic acid alone.
Though, in both, HL-60 and NB4 cells, combined treatment with HDAC, HMT inhibitors and retinoic acid, as well as with retinoic acid alone, significantly down-regulated MTOR and LAMTOR1 gene expression (Fig. 4B), in APL patients’ cells, the effect was negligible. However, similar patterns in all tested cells were registered for VEGFR3 gene expression regulation (Fig. 5A): treatment with 3-Deazaneplanocin A, Belinostat and retinoic acid for 72 h significantly decreased the quantity of VEGFR3 mRNA. For FGF2 and FGFR1 gene expression, the same patterns were registered in NB4 and APL patients’ cells (Fig. 5B): the most significant down-regulation of FGF2 gene expression was evident upon combined treatment with 3-Deazaneplanocin A, Belinostat and retinoic acid for 72 h.
Our analysis also demonstrated that treatment with 3-Deazaneplanocin A and Belinostat in combination with retinoic acid in HL-60 and NB4 cells significantly down-regulated gene expression of TGFA, TGFB1 and TGFBR1 (Fig. 6A), as well as expression of PDGFA, PDGFRA and PDGFRB (Fig. 6B). For HL-60 cells the most significant effect was registered on TGFA (after 12 h treatment gene expression was down-regulated more than 33-fold, P ≤ 0.0001), TGFB1 (after 72 h treatment gene expression was down-regulated 25-fold, P ≤ 0.0001), TGFBR1 (after 12 h treatment gene expression was down-regulated more than 9-fold; P ≤ 0.0005), as well as on PDGFA (after 72 h treatment gene expression was inhibited 250-fold; P ≤ 0.0001) and PDGFRA (after 12 h treatment gene expression was inhibited 20-fold; P ≤ 0.0005). Results for TGFA, TGFB1 and
TGFBR1 gene expression in NB4 cells were also similar (though, gene expression inhibition was less pronounced). In APL patients’ cells combined treatment with 3-Deazaneplanocin A, Belinostat and retinoic acid for 72 h reduced TGFA gene expression approx. twice, however, results were not statistically significant (Fig. 6A). Combined epigenetic treatment had more notable effect on APL patients’ cells expression of PDGF pathway genes (Fig. 6B), as treatment with 3-Deazaneplanocin A, Belinostat and retinoic acid diminished PDGFA, PDGFRA and PDGFRB mRNA levels.
Overall, this data shows that combination of HMT inhibitor 3-Deazaneplanocin A, HDAC inhibitor Belinostat and granulocytic differentiation inducer retinoic acid down-regulated expression of WNT2B, MTOR, LAMTOR1, VEGFR3, FGF2, FGFR1, TGFA, TGFB1, TGFBR1, PDGFA, PDGFRA and PDGFRB genes in promyelocytic leukemia cells. In general, NB4 cells and APL patients’ cells, compared to HL-60 cells, were found to be less affected by the combined treatments. Regarding the effect of combined treatments on APL patients’ cells, the strongest down- regulation of gene expression was registered in FGF and PDGF pathways.

3.5. Effect of HDAC and HMT inhibitors on NB4 cells’ protein expression

We further tested the effect of HMT inhibitor 3-Deazaneplanocin A and HDAC inhibitor Belinostat on NB4 cells protein expression regulation of proteins involved in Wnt, mTOR, VEGF, FGFR, TGFB and PDGF signaling pathways (Figs. 7–9). For this purpose, NB4 cells were treated with 0.5 μM 3-Deazaneplanocin A and 0.8 μM Belinostat combination for 4 h; after the treatment, the cells were washed and treated with 1 μM retinoic acid for 24 h (denoted as “DBR (24 h)”). NB4 cells were also treated with 1 μM retinoic acid alone for 24 h (denoted as “RA (24 h)”), with 0.5 μM 3-Deazaneplanocin A for 4 and 24 h (denoted as “D (4 h)” and “D (24 h)”), and with 0.8 μM Belinostat for 4 and 24 h (denoted as “B (4 h)” and “B (24 h)”). In addition, NB4 cells were treated with combination of 0.5 μM 3-Deazaneplanocin A and 0.8 μM Belinostat for 4 and 24 h (denoted as “DB (4 h)” and “DB (24 h)”). Untreated NB4 cells were considered as control (noted as “Ctrl”). After cell incubation protein expression was evaluated using liquid chromatography–mass spectrometry analysis (LC-MS).
In our specimens Reactome pathway analysis identified in total 98 proteins associated with WNT signaling pathway, while mTOR signaling pathway was represented by 15 different proteins (Fig. 7A–B). For relative protein expression evaluation, protein quantities were represented as a fold change to control (detailed values presented in Supplement 2). Interestingly, expression of Wnt-5a (UniProt accession code: P41221) and Wnt-7a (O00755) were completely diminished after treatment with RA, 3-Deazaneplanocin A and Belinostat, when used separately, as well after 4 h long treatment with combination with 3- Deazaneplanocin A and Belinostat (DB (4 h)) (Fig. 7A). However, after treatment with this combination for 24 h (DB (24 h)), as well as after treatment with the triple combination together with RA (DBR (24 h)), the level of Wnt-5a protein expression was restored. Down-regulation of Wnt-7a protein expression was more constant and remained ceased after 24 h long treatment with all epigenetic agents and all their combinations. Worth to notice, that triple combination (DBR (24 h)) was more effective than treatment with retinoic acid alone (RA (24 h)) in down- regulation of Calcineurin subunit B type 1 (P63098), 26S proteasome non-ATPase regulatory subunit 1 (Q99460), AP-2 complex subunit beta (P63010), Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2 (P59768), as well as Ubiquitin carboxyl-terminal hydrolase 34 (Q70CQ2).
Regulator complex proteins’ LAMTOR1 (Q6IAA8) and LAMTOR2 (Q9Y2Q5) expression remained constant or were up-regulated upon all treatments (to 1.7 fold), except the treatment with 3-Deazaneplanocin A and Belinostat, for 24 h, which decreased LAMTOR1 protein expression > 20% (Fig. 7B). In addition, expression of Serine/threonine-protein kinase mTOR (P42345) was diminished the most significantly after 24 h long treatment with 3-Deazaneplanocin A and Belinostat, as well.
Expression of Vascular endothelial growth factor receptors was affected differently by used epigenetic treatments (Fig. 8A). For example, VEGFR1 (P17948) was up-regulated upon all used treatments (e.g. up to 3.6 fold after 4 h treatment with Belinostat); up-regulation of VEGFR2 (P35968) expression was more modest (up to 1.9 fold after 24 h treatment with “DBR”), while VEGFR3 (P35916) protein expression was reduced approx. 4 times (the strongest inhibition of VEGFR3 expression was detected upon 24 h treatment with “DBR”; precise numerical values presented in Supplement 2).
For FGFR signaling pathway (Fig. 8B) the strongest effect of used epigenetic treatment with 3-Deazaneplanocin A and Belinostat in combination with retinoic acid was registered on Epithelial splicing regulatory protein 2 (Q9H6T0) reduction. In addition, 3-Deazaneplanocin A and Belinostat (DB (24 h)) significantly inhibited protein expression of some proteins involved in TGFB signaling pathway (Fig. 9A), such as Transforming protein RhoA (P61586), Serine-threonine kinase receptor- associated protein (Q9Y3F4), Exportin-1 (O14980), Cyclin-dependent kinase 9 (P50750), as well as E3 ubiquitin-protein ligase CBL (P22681). However, for Platelet-derived growth factor receptor beta (P09619) expression reduction (Fig. 9B), the strongest effect was registered when NB4 cells were treated separately with 3-Deazaneplanocin A or Belinostat for 4 h (“D (4 h)” and “B (4 h)”).
In general, results obtained from mass spectrometry analysis confirmed that used epigenetic treatments do effect protein expression of cancer associated signaling pathways, such as Wnt, mTOR, VEGF, FGFR, TGFB and PDGF.

4. Discussion

In this study, we investigated the effect of HMT inhibitor 3-Deazaneplanocin A and HDAC inhibitor Belinostat in combination with granulocytic differentiation inducer retinoic acid on acute promyelocytic leukemia cells NB4, APL patients’ cells and HL-60 cells, which resemble promyelocytes, though, do not bear typical translocation t(15;17)(q22; q21). We tested the role of epigenetic therapy on histone H4 hyper- acetylation patterns genome-wide using ChIP-sequencing technique. In addition, we tested an effect on gene and protein expression of various signaling pathways that are involved in development or maintenance of the cancerous state.
Our previous studies have demonstrated that Belinostat, which is a potent hydroxamate-type HDAC inhibitor, up-regulates basal histone H4 hyper-acetylation levels in promyelocytic leukemia cells (Valiuliene et al., 2015). More significant effect was demonstrated for Belinostat in combination with retinoic acid, compared to Belinostat or retinoic acid acting alone. Mass spectrometry analysis also revealed therapeutically relevant results: in untreated NB4 cells, hyper-acetylated histone H4 was found in association with proteins crucial for DNA replication and transcription processes, whereas after treatment with Belinostat it was found in close proximity with proteins involved in pro-apoptotic processes, tumor suppression and oxidative stress defense (Valiuliene et al., 2015). In addition, we have previously demonstrated that stronger anti-leukemic effect may be achieved combining Belinostat with HMT inhibitor 3-Deazaneplanocin A and retinoic acid (Valiuliene et al., ˙ 2017). Based on these results, in the current study, we performed distribution analysis of histone H4 hyper-acetylation sites in HL-60 and NB4 cells, which were treated with combination of Belinostat, 3-Deazaneplanocin A and retinoic acid. ChIP-sequencing data analysis revealed that epigenetic treatment acted globally and up-regulated the number of hyper-acetylated histone H4 peaks in the chromosomes 7, 10 and 19. Deeper analysis showed that the great majority of peaks in HL-60 cells were allocated within the regions of proximal promoter, encompassing 3 kb upstream and 0.5 kb downstream of annotated TSS. In contrast, in treated NB4 cells vs untreated NB4 cells, hyper-acetylated histone H4 peaks were mainly localized in gene body regions. Thus, combined treatment with Belinostat, 3-Deazaneplanocin A and retinoic acid is likely to have HL-60 and NB4 cell specific effect on histone H4 hyper-acetylation. Furthermore, this could also partially explain our observation that treatment with HDAC and HMT inhibitors in combination with retinoic acid had less pronounced effect on NB4 cells gene expression regulation, compared to HL-60 cells.
Generally, up-regulation of histone H4 acetylation levels may be regarded as a beneficial and therapeutically promising drug effect, as lowered histone H3 and H4 acetylation levels are characteristic for acute myeloid leukemia blasts (Sauer et al., 2015). Increased acetylation was demonstrated to unlock chromatin for transcription of tumor-suppressor genes and other anti-cancerous acting genes (Di Cerbo and Schneider, enzymes and affecting signaling pathways, which are crucial for control of cell metabolism (Wong et al., 2017). In this study, we have demonstrated that promyelocytic leukemia cell treatment with epigenetically active agents Belinostat and 3-Deazaneplanocin in combination with retinoic acid increased histone H4 hyper-acetylation in gene-associated regions of genes implicated in mTOR signaling cascades. It is well-known that mTOR pathway is employed in various cancers in order to sustain growth under limited nutrition conditions (Magaway et al., 2019). In HL-60 cells, epigenetic treatment enriched LAMTOR1 gene with histone H4 hyper-acetylation mark. Therefore we performed gene expression analysis in order to evaluate the effect of epigenetic treatment to gene expression of LAMTOR1, as well as MTOR. In both HL-60 and NB4 cells, combined treatment with HDAC and HMT inhibitors profoundly diminished expression of MTOR and LAMTOR1 genes. In addition, mass spectrometry analysis results confirmed that in NB4 cells combined epigenetic treatments also inhibit expression of LAMTOR1 at the protein level. However, as evaluated with qPCR, combination of 3-Deazaneplanocin A, Belinostat and retinoic acid had no effect on APL patients‘ MTOR and LAMTOR gene expression.
Although histone acetylation is generally regarded as transcription- activating modification and is associated with chromatin decondensation (Stillman, 2018), scientific data shows that increased acetylation does not necessarily correlate with transcriptional activation (Deckert and Struhl, 2001). In addition, histone acetylation can not be regarded as errorless predictor of gene expression, following treatment with HDAC inhibitors, as stated in Ellis et al. (2008). Even though our results of gene expression analysis do not precisely fit the dogma of histone H4 hyper-acetylation as gene expression-activating mark, the patterns of gene expression of FGF, PDGF, TGF, mTOR, VEGF and Wnt signaling pathway genes confirm the anti-leukemic activity of HDAC and HMT inhibitors. For instance, treatment with Belinostat and 3-Deazaneplanocin in combination with retinoic acid significantly down-regulated gene expression of WNT2B, the component of Wnt pathway, which is known to be over-activated in acute myeloid leukemia cells (Gruszka et al., 2019). In addition, LC-MS analysis revealed that epigenetic treatments significantly inhibit NB4 cells’ Wnt-5a and Wnt-7a protein expression. Generally, higher activity of Wnt pathway signaling in APL depends on PML-RARα, and PLZF-RARα fusion genes, as they activate γ-catenin, the homologue of β-catenin, and consequently up-regulates Wnt signaling (Staal et al., 2016). Therefore, medications targeting Wnt pathway in APL could be of potential clinical value. In addition, numerous studies confirmed that β-catenin is regulated by HDAC-dependent deacetylation (Kim et al., 2012; Li et al., 2008; Lu et al., 2019). Consequently, this explains an additional mechanism by which HDAC inhibitors restrict cancer growth.
As it was previously demonstrated by Gars et al. (2010), acute myeloid leukemia cells depend on the combined signaling of VEGF and PDGF. In accordance to that, our study revealed that epigenetic treatment inhibited aforementioned signaling pathways by reducing gene expression of VEGFR3, PDGFA, PDGFRA and PDGFRB. Furthermore, treatment with 3-Deazanplanocin A and Belinostat, in combination with retinoic acid, significantly diminished VEGFR3 protein expression in NB4 cells, as demonstrated with LC-MS. In addition, inhibition of gene expression of TGF-α pathway gene TGFA was detected in APL cells. It is well known that TGF-α, protein involved in cell proliferation, differentiation and development processes, is highly expressed by promyelocytes, myelocytes, eosinophil precursors and megakaryocytes. However, in mature neutrophils protein levels of TGF-α are diminished (Kavanagh et al., 2016). Our results are in accordance with this data, demonstrating that in acute promyelocytic leukemia cells NB4, APL patients’ cells and in promyelocytes resembling HL-60 cells after treatment with HMT inhibitor 3-Deazaneplanocin A and HDAC inhibitor Belinostat in combination with retinoic acid, expression of TGFA was significantly down-regulated. The effect was more pronounced using combined treatment compared to treatment with retinoic acid alone. This effect also coincide with our previous findings that combined treatment exaggerated retinoic acid induced granulocytic differentiation of HL-60 and NB4 cells, as well APL patients’ blasts (Vitkeviciene et al., 2019; Valiuliene et al., 2017).
In this study, HL-60 and NB4 cell treatment with 3-Deazaneplanocin A and Belinostat in combination with retinoic acid also down-regulated expression of TGF-β pathway genes (TGFB1 and TGFBR1), as well as FGF pathway genes (FGF2, FGFR1). In addition, combined epigenetic treatment also diminished APL patients’ blast expression of FGF2 gene. It is known that TGF-β signaling in cancer is dual: it may act as tumor- suppressor in the early stages of the disease, while in the later stages, TGF-β mainly promotes cancer progression (Gu and Feng, 2018). Meanwhile, FGF signaling pathway plays a crucial role in hematopoiesis (Allouche and Bikfalvi, 1995). Interestingly, it was demonstrated by Traer et al. (2016) that in acute myeloid leukemia cases FGF2 signaling pathway is “hijacked” and exploited for cell survival strategies, as FGF2 signaling activation promotes resistance to FLT3 inhibitors.
Taken all together, we have demonstrated that epigenetically active compounds studied in this research are capable of remodeling genome- wide distribution of histone H4 hyper-acetylation sites in promyelocytic leukemia cells, as well as down-regulating gene and protein expression of certain genes and proteins, involved in development and maintenance of the cancerous state.

5. Conclusions

The role of epigenetic modifications in the pathogenesis of APL has been linked to the PML-RARα fusion protein and its protein complex partners, histone deacetylases and histone methyltransferases. In this study, we demonstrated that HDAC inhibitor Belinostat and HMT inhibitor 3-Deazaneplanocin A in combination with granulocytic differentiation inducer retinoic acid, remodel genome-wide distribution of histone H4 hyper-acetylation sites in promyelocytic leukemia HL-60 and NB4 cells. In addition, cells, treated with these agents, demonstrated down-regulation of genes and proteins involved in cancer-promoting signaling pathways, such as FGF, PDGF, TGF, mTOR, VEGF and Wnt signaling cascades. Taken together, our results provide additional molecular insights into the epigenetic, gene and protein expression changes that occur during promyelocytic leukemia cell treatment with epigenetic modifiers.

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