Diagnosis of MM followed the International Myeloma Working Group (IMWG) criteria (36). The present study was approved by the Ethics Committee of University Hospital and the Faculty of Medicine, Palacky University in Olomouc (Olomouc, Czech Republic; approval no. EK FNOL 112/17) and samples were collected following informed consent from the patients.

Patients were eligible for inclusion if they met the following criteria: i) Signed informed consent; ii) age ≥18 years; iii) diagnosis of plasma cell disorder determined according to IMWG criteria, including newly diagnosed MM, relapsed/progressive MM or MM in remission; and iv) availability of BM aspirate suitable for CD138⁺ plasma cell isolation. A patient with amyloidosis was also included for comparative purposes, as amyloidosis represents an associated plasma cell dyscrasia and was used to complement the spectrum of TET mRNA expression profiles across plasma cell disorders (Table I; Fig. 1). Exclusion criteria comprised insufficient quantity or quality of the BM sample, low plasma cell infiltration preventing reliable CD138⁺ cell sorting or degraded nucleic acids not suitable for downstream analyses.

The CD138⁺ sorted cell population data was prepared from the plasma cells of BM-aspirate, from 24 patients with MM in different stages: i) Active MM, comprising newly diagnosed patients and patients in relapse/progression as per IMWG criteria; and ii) remission phase MM, defined as achieving at least a partial response after finished initial treatment or being in stable remission after stem cell transplant with continued maintenance therapy. The patients were recruited for the present study at the Department of Hemato-Oncology, University Hospital Olomouc (Olomouc, Czech Republic) between August 2018 and November 2019. All experiments involving patient samples were performed at the Department of Immunology and the Department of Clinical and Molecular Pathology, both within the Faculty of Medicine and Dentistry, Palacky University Olomouc (Olomouc, Czech Republic). The clinicopathological characteristics of the entire cohort (n=24) are provided in Table II.

Among the 24 patients (mean age 64±6 years), 3 patients [two with monoclonal gammopathy of undetermined significance (MGUS) and one newly diagnosed MM] were female and the remaining 21 patients were male. For the quantification of DNA methylation (5-mC and 5-hmC) at a specific CCGG site, a subset of four patients (samples W01-W04) was selected from the primary cohort. The selection was based on the availability of sufficient amounts of high-quality genomic DNA and the requirement to include representative samples from distinct disease stages (newly diagnosed MM, remission and relapse). As a result, the final subset consisted exclusively of male patients, as the available samples from female patients did not meet the quality and/or quantity requirements for this restriction enzyme-based assay. The clinicopathological characteristics of these 4 patients are presented in Table SI.

In total, 5 different MM cell lines were used: RPMI8226, U266/B1, OPM-2, KMS12-PE and KMS12-BM. The cell lines RPMI8226 (cat. no. CCL155), U266/B1 (cat. no. TIB-196) and OPM-2 (cat. no. ACC 50) were purchased from the American Type Culture Collection and KMS12-BM (cat. no. JCRB0429) and KMS12-PE (cat. no. JCRB0430) cell lines were obtained from the Japanese Cancer Research Resources Bank (National Institute of Biomedical Innovation). To determine their authenticity, the KMS12-BM and KMS12-PE cell lines were assessed using the AmpF/STR™ IdentiFiler™ (Thermo Fisher Scientific, Inc.) for eight short tandem repeat markers in DNA (Tables SII and SIII). The cells were maintained in RPMI-1640 medium supplemented with 10% or 15% FBS, respectively, 1% penicillin-streptomycin antibiotics, 1% L-glutamine and 100 mM sodium pyruvate, at 37°C with 5% CO₂. Under these conditions, cell lines were treated with 0.2 and 0.5 µmol/l 5-azacytidine (AZA) and/or 5-aza-2′-deoxycytidine (decitabine; DAC) for 48 h with 24 h of re-treatment. Cells treated with an equivalent concentration of DMSO served as vehicle control. All cell line experiments were conducted at the Department of Clinical and Molecular Pathology, Faculty of Medicine and Dentistry, Palacky University Olomouc (Olomouc, Czech Republic).

RNA preparation was performed from CD138⁺-enriched BM cells using the Single Cell RNA Purification Kit (Norgen Biotek Corp.) and from treated MM cell lines with TRI Reagent^® BD (Molecular Research Center, Inc.). Reverse transcription of 100 ng of total RNA was performed using the Transcriptor First Strand cDNA Synthesis Kit (Roche Diagnostics), according to the manufacturer's protocol.

RT-qPCR was conducted with Taq-Man™ probes (cat. no. 4331182; Thermo Fisher Scientific) and Xceed qPCR Probe Mix (cat. no. NPCR 10502 S; Xceed; iBioTech), using the Light cycler^® 480 System (Roche Diagnostics). The thermocycling conditions were as follows: Initial denaturation at 95°C for 10 min, followed by 45 cycles of denaturation at 95°C for 15 sec and a combined annealing/extension step at 60°C for 60 sec.

The mRNA expression levels of TET1 (assay ID: Hs04189344 g1), TET2 (assay ID: Hs00766782_s1) and TET3 (assay ID: Hs00896441_m1) were quantified by RT-qPCR from complementary DNA (cDNA) synthesized by reverse transcription of mRNA and normalized to the expression of endogenous housekeeping control β2-microglobulin (assay ID: Hs00984230_m1). All used probes were provided by Thermo Fisher Scientific, Inc. qPCR data were analyzed using the 2^−ΔΔCq quantification method (37), with untreated cells used as the control group.

The human TET1 transcript NM_030625 is located on chromosome 10 (GRCh38/hg38 assembly) at positions 68560337-68694487 on the plus strand. The promoter region was operationally defined as 1 kb upstream and 1 kb downstream of the transcription start site (TSS). The TET2 transcript NM_017628.4 is located on chromosome 4 (GRCh38/hg38 assembly) at positions 105145875-105242771 on the plus strand, with the promoter defined at TSS ± 1 kb. The TET3 transcript NM_001287491.2 is located on chromosome 2 (GRCh38/hg38 assembly) at positions 73984910-74108176 on the plus strand and its promoter was similarly defined relative to TSS.

KMS12-PE and KMS12-BM cell lines were treated with the DNA demethylating agents AZA or DAC at final concentrations of 0.2 and 0.5 µmol/l for 48 h, with retreatment after 24 h. Cells treated with an equivalent concentration of DMSO served as vehicle control.

Total protein lysates were prepared using self-prepared RIPA lysis buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 0.5 EGTA, 140 mM NaCl, 1% Triton X-100, 0.1% sodium deoxycholate and 0.1% SDS) supplemented with protease inhibitors (Roche cOmplete™ Protease Inhibitor Cocktail Tablets; cat. no. 04693124001; Roche Diagnostics). Cells were incubated on ice for 30 min with occasional vortexing and lysates were clarified by centrifugation at 14,000 × g for 15 min at 4°C. Protein concentration was determined using the Bradford assay (Bio-Rad Protein Assay Dye Reagent Concentrate; cat. no. 5000006; Bio-Rad Laboratories, Inc.).

Equal amounts of protein (20 µg per lane) were mixed with Laemmli sample buffer, boiled at 95°C for 5 min and resolved by SDS-PAGE on precast 4–15% gradient polyacrylamide gels (Mini-PROTEAN^® TGX™; cat. no. 4561085; Bio-Rad Laboratories, Inc.). The use of gradient gels allowed efficient separation of proteins across a broad range of molecular weights (TET1 ~235 kDa, TET2 ~130 kDa and GAPDH ~37 kDa). Electrophoresis was performed at 100 V for ~120 min. Proteins were transferred onto nitrocellulose membranes using semi-dry transfer conditions (10 A for 50 min). The membranes were subsequently cut according to molecular weight and processed separately for incubation with the indicated antibodies. Membranes were then blocked in 5% non-fat dry milk in TBS-T (TBS containing 0.1% Tween-20) for 60 min at room temperature.

For detection of TET1, membranes were incubated with rabbit polyclonal anti-TET1 antibody (cat. no. ab191698; Abcam) diluted 1:1,000 in 5% milk/TBS-T overnight at 4°C. For detection of TET2, membranes were incubated with rabbit polyclonal anti-TET2 antibody (cat. no. 21207-1-AP; Proteintech Group, Inc.) diluted 1:1,000 in 5% milk/TBS-T overnight at 4°C.

After primary antibody incubation, membranes were washed 3 times in TBS-T and incubated with HRP-conjugated goat anti-rabbit IgG secondary antibody (cat. no. 7074S; Cell Signaling Technology, Inc.; 1:3,000) for detection of TET1 and TET2 or with HRP-conjugated goat anti-mouse IgG secondary antibody (cat. no. 7076S; Cell Signaling Technology, Inc.; 1:5,000) for detection of GAPDH, both for 1 h at room temperature. Following additional washing steps, immunoreactive bands were visualized using the infrared LI-COR Odyssey Imaging System (LI-COR Biosciences) and analyzed using Image Studio software (version 2.0; LI-COR Biosciences). The expected molecular weights were ~235 kDa for TET1 and 130 kDa for TET2. GAPDH was used as a loading control.

Western blotting signals were evaluated qualitatively based on band intensity. Densitometric analysis was not performed, as the experiments were intended to provide supportive determination of expression changes observed at the mRNA level.

Genomic DNA was isolated from the KMS12-PE cell line using the Wizard^® Genomic DNA Purification Kit (Promega Corporation) according to the manufacturer's instructions. DNA concentration and purity were assessed using a NanoDrop™ spectrophotometer (Thermo Fisher Scientific, Inc.) and samples were stored at −20°C. The isolated genomic DNA was used for colorimetric quantification of global 5-hmC using the MethylFlash™ Hydroxymethylated DNA 5-hmC Quantification Kit (Colorimetric; cat. no. P-1036; EpigenTek Group, Inc.). This assay is based on an ELISA-like format that specifically detects 5-hmC without cross-reactivity to 5-mC or unmodified cytosine.

Genomic DNA (200 nm per well) was bound to the light-affinity strip wells provided in the kit. After DNA binding and washing, a primary capture antibody (cat. no. P-1036-96; EpigenTek Group, Inc.; 1,1000) specific for 5-hmC and a corresponding detection antibody (cat. no. P-1036-48; EpigenTek Group, Inc.; 1:1,000), both provided within the MethylFlash™ Hydroxymethylated DNA 5-hmC Quantification Kit (Colorimetric; cat. no. P-1036; EpigenTek Group, Inc.), were applied according to the manufacturer's instructions. A colorimetric signal was generated using the enhancer and developer solutions, and the absorbance was read at 450 nm on microplate spectrophotometer to obtain optical density (OD) values.

Each sample was measured in triplicate, together with kit-provided negative and positive controls. A calibration curve was generated from the positive control dilution series according to the manufacturer's instructions to allow absolute quantification of 5-hmC. The amount of 5-hmC (ng) in each sample was calculated using the standard curve slope generated from linear regression and the global percentage of 5-hmC relative to input DNA was determined as follows: 5-hmC (ng)=(sample OD450-negative control OD450)/slope ×5* and [5-hmC (%)=5-hmC (ng)/S] ×100, whereby ‘S’ refers to the amount of input sample DNA (ng) and ‘5*’ is a factor to normalize 5-hmC in the positive control to 100%, as the positive control contains only 20% of 5-hmC.

Both 5-hmC and 5-mC levels at a particular CCGG site were measured with a restriction enzyme-based assay, the EpiMark 5-hmC and 5-mC analysis kit (EpigenTek Group, Inc.) (26,38), according to the manufacturer's instructions. The assay is based on the differential susceptibility of methylated and hydroxymethylated DNA to cleavage by HpaII and MspI. Genomic DNA (620 ng) was treated with 30 units of T4 β-glucosyltransferase and 80 µl uridine diphosphate-glucose at 37°C for 16 h. Glucosylated DNA was digested with 100 units of MspI, 50 units of HpaII or no enzyme (mock digestion) at 37°C for 4 h, followed by treatment with 1 µl proteinase K (20 mg/ml) at 40°C for 30 min.

For qPCR, 1 µl glycosylated/digested DNA was used with promoter-specific primers. Primer sequences were as follows: TET1 gl (−528): Forward (F), 5′-ACTCCCTGAGGTCTGTCCTG-3′ and reverse (R), 5′-CAGGTAGGGCTGCATGACTT-3′; TET2 gl (−421) F, 5′-GAAGGTGGGCCGGGGCGG-3′ and R, 5′-GAGAGGGTGTGCTGCTGAAT-3′; and TET3 gl (−816) F, 5′-AAAGGCCATGGTAGGAAGTG-3′ and R, 5′-TGAAGTAGCGCTGTCCAGAA-3′.

Genomic DNA was isolated from BM aspirates and CD138⁺ sorted cells of patients with MM using a QIAamp DNA Mini Kit (Qiagen GmbH). Bisulfite treatment of extracted genomic DNA was conducted as aforementioned. PCR primers and subsequent pyrosequencing reaction were designed using PyroMark^® Assay Design SW 2.0 (Qiagen GmbH). Pyrosequencing primers were designed to the promoter regions of the TET1 (genomic reference: NC_000010.11, −528 bp upstream of the TSS), TET2 (genomic reference: NC_000004.12, −421 bp upstream) and TET3 (genomic reference: NC_000002.12, −816 bp upstream). The primer sequences used were as aforementioned.

In brief, 1 µl bisulfite-treated DNA was added to a 25 µl PCR reaction mixture containing 12.5 µl 1X PyroMark PCR Master Mix (Qiagen GmbH), 1 µl 25 mM MgCl₂, 2.5 µl 1X CoralLoad Concentrate (Qiagen GmbH), 0.2 µl forward primer and 0.2 µl biotinylated reverse primer. For HotStartTaq Polymerase activation, the PCR reaction mixture was initially denatured at 95°C for 15 min, followed by 45 cycles of denaturation at 94°C for 30 sec, annealing at 56°C for 30 sec, elongation at 72°C for 30 sec and the final extension at 72°C for an additional 10 min after the last cycle. The PCR products were verified by electrophoresis on a 2% agarose gel. The incurred biotinylated PCR product was immobilized on Streptavidin Sepharose^® High Performance (GE Healthcare), precipitated with 70% ethanol, passed through a denaturation step and then a washing step using PyroMark Q96 Vacuum Workstation (Qiagen GmbH). The amplicons were transferred to each well of the PyroMark Q96 plate containing 40 µl of 0.4 µM sequencing primer diluted in annealing buffer (Qiagen GmbH). Control samples (bisulfite unmethylated and methylated DNA; Qiagen GmbH) were part of a set of analyzed samples from patients with MM. The pyrosequencing analysis was performed using the PyroMark CpG software (version 1.0.11; Qiagen GmbH). The methylation value was quantified in terms of the methylation level (MtL) as the mean percentage of cytosines methylated per CpG: MtL (%)=(Σ% methylated cytosines)/no. of CpGs analyzed.

To identify DNA methylation changes associated with MM pathogenesis, the 5-mC/5-hmC percentage abundance was investigated in patients at three different stages of MM: i) Newly diagnosed MM (samples W03 and W04); ii) MM in remission (sample W01); and iii) MM in the relapsed stage (sample W02). Calculation of the methylation status of inner C in CCGG sites was performed using the following formula: C^hmCGG (%)=[M2 × (C1/C2)]-M1]/C1 and C^mCGG (%)=[H1-M2 × (C1/C2)]/C1. Representative values used for these calculations are shown in Table III.

In the calculations (Table SI), the parameters were: M2 (5-hmC), normalized Cq values as target unknown, Tube 1; H2 (5-mC + 5-hmC), normalized Cq values as target unknown, Tube 2; C2, normalized Cq values as target poscalibrator, Tube 3; M1, normalized Cq values as reference unknown, Tube 4; H1, normalized Cq values as reference unknown, Tube 5; C1-normalized Cq values as reference poscalibrator, Tube 6.

The percentages of 5-hmC, 5-mC and cytosine were calculated using the comparative Cq method (Table III) when the HpaII- and MspI-resistant fraction was normalized to the mock digestion control (C2 and C1). The 5-hmC levels were determined directly from the fraction resistant to MspI digestion (M1 and M2), as MspI is inhibited by the presence of 5-hmC but cleaves both unmethylated cytosine and 5-mC. The 5-mC (H2 and H1) levels were obtained by subtracting the 5-hmC contribution from the total HpaII resistance.

Genomic DNA was extracted from the KMS12-PE cell line after the AZA and/or DAC (0.2 and 0.5 µmol/l) treatments using the QIAGEN Blood and Cell Culture DNA Kit (Qiagen GmbH) following the manufacturer's protocol. DNA quality and concentration were assessed using a Qubit^® Fluorometer (Invitrogen; Thermo Fisher Scientific, Inc.). Sequencing libraries were prepared using the Oxford Nanopore Technologies Native Barcoding Kit 96 V14 (cat. no. SQK-NBD114.96; Oxford Nanopore Technologies plc) and VAHTS TGS DNA Library Prep Kit for (Vazyme Biotech Co., Ltd.) following the manufacturer's protocol with modifications, including omission of intermediate clean-up steps and adjustment of input DNA and reaction volumes as specified below. The DNA input used was 1,000 ng per sample and end-repair and barcode ligation were performed without intermediate clean-up. After barcode ligation, 2 µl EDTA was added to each sample to stop the reaction prior to pooling. Samples were then pooled and cleaned using AMPure XP beads (cat. no. A63880; Beckman Coulter, Inc.). Adapter ligation was performed using Oxford Nanopore Technologies reagents. DNA concentration was measured with the Qubit^® dsDNA HS Assay Kit (Thermo Fisher Scientific, Inc.) and adjusted as needed. For sequencing, 5 µl of the library was included in a final loading mix with total volume 32 µl, which was loaded onto an R10.4.1 flow cell (FLO-PRO114) and sequenced on the PromethION platform (Oxford Nanopore Technologies plc).

Basecalling and 5-mC methylation calling in a CpG context were performed using the Dorado basecaller within the wf-human-variation pipeline (version 2.6.0; Oxford Nanopore Technologies plc). Reads were aligned to the GRCh38 human reference genome using the minimap2 software (version 2.24; Heng Li, Dana-Farber Cancer Institute) and methylation frequencies were generated using the modkit software (version 0.3.3; Oxford Nanopore Technologies plc) with default probability thresholds. Basic quality control was performed to determine high data integrity with a median mapping accuracy of 99%, a read N50 of 39.3 kb and mean sequencing coverage of 4.05× across targeted regions. Analysis of methylation levels was specifically focused on standardized promoter regions of the TET1, TET2 and TET3 genes (chromosome 10: 68570336-68595336, chromosome 4: 105105874-105319803 and chromosome 2: 73943630-74175498). Amplicons were designed to cover these coordinates. Methylation levels were calculated per CpG site and summarized for each promoter region, following the coordinates presented in Table SIV.

ChIP assays were performed using the Magna ChIP^® A/G Chromatin Immunoprecipitation Kit (cat. no. 17-10085; Merck KGaA) according to the manufacturer's instructions. Sonicated chromatin prepared from KMS12-PE cells (5×10⁶ cell equivalents per immunoprecipitation) was incubated overnight at 4°C with gentle rotation with 2 µg anti-SP1 (cat. no. ab13370; Abcam) or anti-SP3 (cat. no. ab227856; Abcam) antibodies (diluted in a total volume of 515 µl). Normal rabbit IgG (cat. no. 2729S; Cell Signaling Technology, Inc.; 2 µg) was used as a negative control under the same conditions. Immunoprecipitated DNA was purified using the Magna ChIP^® A/G Chromatin Immunoprecipitation Kit (cat. no. 17-10085; Merck KGaA) according to the manufacturer's instructions and subsequently analyzed by qPCR. The ChIP-qPCR conditions were identical to those described above for RT-qPCR. Primers specific for the promoter regions of TET1 (genomic reference: NC_000010.11; −528 bp upstream of TSS), TET2 (genomic reference: NC_000004.12; −421 bp upstream) and TET3 (genomic reference: NC_000002.12; −816 bp upstream) were used for ChIP-qPCR. The primer sequences used for ChIP-qPCR were as aforementioned. The two methods used for ChIP-qPCR data normalization were fold enrichment and percent of input. Fold enrichment is a signal-to-noise ratio comparing the amount of target sequence measured in the immunoprecipitate isolate to the amount measured in a negative control isolate (39). The percent input method compares the amount of target sequence measured in the immunoprecipitate isolate to the total amount of the target sequence in the input isolate (40).

Statistical analysis was performed using Statistica software (version 14.0.0.15; TIBCO Software, Inc.). Data are expressed as the mean ± SD. Patient samples were analyzed in technical triplicates and MM cell lines experiments were performed in both biological and technical triplicates. Due to the small sample size and limited number of biological replicates, non-parametric statistical methods were applied. Differences between groups were evaluated using the Kruskal-Wallis test followed by Bonferroni post hoc tests for pairwise comparisons. P<0.05 was considered to indicate a statistically significant difference and the adjusted significance level after Bonferroni correction was set at P<0.005.

In the CD138⁺ sorted plasma cells of both newly diagnosed and relapsed patients with MM, TET1 mRNA expression levels were increased when compared with that of TET2 and TET3 (Figs. 1 and 2A). By contrast, quantitative bisulfite pyrosequencing of the CD138⁺ purified plasma cell samples showed a decrease in DNA methylation level at TET1 and TET2 selected promoter regions in both newly diagnosed and relapsed patients with MM (Table SI), while increased levels of DNA methylation at the TET3 gene promoter were determined using the pyrosequencing method (Fig. 2B).

Changes in the mRNA expression levels of TET genes after treatment with both demethylating agents was determined in all myeloma lines used, depending on the demethylation agents (AZA and/or DAC) and concentration (0.2 µmol/l and/or 0.5 µmol/l; Fig. 3). For both the KMS12-BM and KMS12-PE cell lines, demethylation treatment altered the normalized TET2 mRNA expression levels; however, a statistically significant difference between TET1, TET2 and TET3 expression was observed only in the KMS12-PE cell line (P<0.001). The mRNA levels of all three TET genes in the U266/B1, RPMI and OMP2 cell lines were found not to be significantly different (Fig. S1). A further demethylation experiment with KMS12-BM and KMS12-PE cell lines validated the results of the increased normalized TET2 mRNA levels compared with the mRNA expression values of TET1 and TET3 (Fig. 3). Increased normalized TET2 mRNA levels in KMS12-BM did not exhibit a statistical difference. In the KMS12-PE cell line, the normalized value of TET2 mRNA (5.146±0.29) was evaluated as significantly increased after 0.2 µmol/l AZA treatment compared with normalized TET1 (1.321±0.16; P<0.01) and TET3 (1.005±0.19; P<0.05) mRNA expression levels (Fig. 3).

To evaluate the impact of AZA and DAC on the epigenetic regulatory machinery in the KMS12-PE and KMS12-BM cell lines, the protein expression levels of TET1 and TET2 were examined using western blotting analysis. Total protein lysate analysis revealed distinct bands at the expected molecular weights for both TET1 and TET2. Visual inspection of the blots revealed that TET1 protein levels remained stable and did not show an increase following treatment with either AZA or DAC (0.2 and 0.5 µmol/l). By contrast, a marked and consistent increase in TET2 band intensity was observed across all treatment conditions in the KMS12-PE cell line compared with the DMSO control. Representative blots demonstrating these changes are presented in Fig. 3 and repetitive gel experiments demonstrating these findings are provided in Fig. S2. In addition, the original uncropped gels with molecular weight markers are provided in Fig. S3.

Changes in the percentage representation of 5-hmC were evaluated in the KMS12-PE cell line following treatment with demethylating agents AZA (0.2 and 0.5 µmol/l) and DAC (0.2 and 0.5 µmol/l). The percentage of 5-hmC increased from 0.15% in the DMSO control to 0.29 and 0.26% after treatment with AZA at 0.2 and 0.5 µmol/l, respectively and to 0.39 and 0.63% after treatment with DAC at 0.2 and 0.5 µmol/l (Fig. 4). Statistically significant differences in 5-hmC levels were observed across all tested groups (DMSO control, AZA 0.2/0.5 µmol/l and DAC 0.2/0.5 µmol/l; Kruskal-Wallis: P=0.01) and treatment with 0.5 µmol/l DAC differed significantly from the DMSO control (Dunn's test with Bonferroni correction: α=0.005).

To further investigate the increased TET1 mRNA expression levels and the levels of CCGG site methylation of TET3 promoter in both the newly diagnosed patients with MM and in relapsed stage (Fig. 1), CCGG site methylation patterns of selected promoter TET regions in CD138⁺ purified plasma cells of newly diagnosed patients with MM (samples W03 and W04), MM patient in remission (sample W01) and MM patient in relapsed stage (sample W02) were investigated.

The adequacy of the dissociation curves shown in Fig. 5 was determined by highly specific and reproducible melting profiles. Each panel displays overlapping curves from replicates, demonstrating a specific PCR product. Since 5-mC and 5-hmC share the same DNA sequence, they possess identical melting temperatures and thus produce a single dissociation profile per locus. This specificity ensured reliable Cq determination for the enzymatic digestion-based qPCR used to calculate the relative percentages of 5-mC and 5-hmC.

In the newly diagnosed patients with MM, the TET1 promoter region contained 4.98% (W03) and 7.33% (W04) values of 5-mC without the presence of 5-hmC; whereas 5-hmC was detected in both patients in remission, W01 (6.64%) and patient in relapsed stage W02 (1.92%). Similarly, in the newly diagnosed patients with MM, the TET2 promoter showed 3.80% (W03) and 11.31% (W04) 5-mC percentage values and in the TET3, increased percentage values 34.93% (W03) and 16.59% (W04) as compared with that of TET1 were determined. The 5-hmC abundance was not found in samples of the newly diagnosed patients (W03 and W04). For all three TET1, TET2 and TET3 genes investigated, it was determined that the highest 5-hmC levels were in the patient MM in remission (W01) group: 6.64, 8.29 and 1.49% respectively; while patients in the relapsed stage (W02) showed low levels of 5-hmC: 1.92, 1.63 and 1.70% for TET1, TET2 and TET3 respectively. In comparison with both W01 and W02 patients, the highest incidence of DNA methylation changes in the promoter of all tested TET genes was found in both newly diagnosed patients with MM (W03 and W04; Fig. 5A).

Analysis of DNA methylation patterns in the TET genes of the KMS12-PE cell line demonstrated a significant reduction in methylation levels in samples treated with demethylating agents compared with that of the untreated control. The control group exhibited a mean methylation frequency ~37% in the TET1 gene, 41% in TET2 and 41% in TET3, with minimal variability as determined by Nanopore sequencing and methylation calling (Fig. 6B). By contrast, samples treated with AZA and DAC showed markedly decreased methylation levels across all three genes. The methylation frequencies for 0.2 µmol/l AZA were ~20% (TET1), 12% (TET2) and 20% (TET3), while for cells treated with 0.5 µmol/l AZA, the frequencies were 16% (TET1), 18% (TET2) and 14% (TET3), all showing high variability. After treatment with 0.2 µmol/l DAC, the frequency of methylation was ~22% (TET1), 14% (TET2) and 19% (TET3). The percentage of methylation after 0.5 µmol/l DAC treatment was 27% (TET1), 8% (TET2) and 16% (TET3), also with high variability.

ChIP analysis was performed with KMS12-PE cells to determine the specific binding of Sp1/Sp3 to the individual promoter sequence of TET genes under previously applied demethylation conditions (AZA and/or DAC at concentrations of 0.2 µmol/l and/or 0.5 µmol/l). Fig. 7 shows that Sp1 recruitment to the TET1 and TET3 promoters significantly increased following DAC treatment, peaking at 0.2 µmol/l DAC for both. While Sp1 binding to the TET2 promoter remained low at 0.2 µmol/l DAC, a notable increase was observed at 0.5 µmol/l DAC. In addition, 0.5 µmol/l AZA treatment moderately enhanced Sp1 binding across all three TET promoters, whereas the 0.2 µmol/l AZA dose resulted in negligible enrichment.

By contrast to Sp1, Sp3 exhibited a more pronounced response to AZA treatment at the higher concentration (0.5 µmol/l), which resulted in the highest enrichment for both genes TET2 and TET3. DAC treatment led to a slight, dose-dependent increase in Sp3 binding at the TET3 promoter but had minimal impact on TET2 (Fig. 8).