1. Introduction
Multiple myeloma (MM) is a malignant hematological disorder that occurs due to uncontrolled proliferation of monoclonal plasma cells (terminally differentiated B-lymphocytes) in the bone marrow, disrupting the process of hematopoiesis and bone tissue remodeling [ 1 , 2 ]. According to the epidemiological data, MM accounts for 10% of all hematological malignancies, while the worldwide incidence of MM is currently 188,000, and mortality is 121,000 patients for the year 2022 [ 3 ].
MM usually develops gradually, through precursor stages: monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM) [ 4 ]. The diagnosis of final MM is established based on the presence of more than 10% of monoclonal plasma cells in the bone marrow, as well as the presence of one or more CRAB (calcium, renal failure, anemia, bone lesions) symptoms: elevated level of calcium (hypercalcemia), kidney failure, anemia, and bone lesions. Thus, dysregulated hematopoiesis and osteolytic bone lesions are key complications in MM [ 5 , 6 ].
At the cellular level, MM is the result of a series of sequential genetic and epigenetic changes that transform plasma cells, leading to the excessive production of monoclonal immunoglobulins or free light chains (M-protein). In addition to becoming the dominant cell population, transformed MM cells in bone marrow actively participate in bone tissue remodeling [ 4 , 7 ]. They stimulate the activity of osteoclasts via increased RANKL expression, while inhibiting osteoblasts that build bone matrix and the production of bone tissue components [ 8 , 9 ]. The result is the weakening of bone tissue and the development of osteolytic lesions, which are clinically manifested by bone pain, pathological fractures and reduced mobility [ 10 , 11 ].
Despite advances in the MM treatment [ 12 , 13 ], this malignancy remains an incurable disease, as almost all patients develop relapses [ 13 ]. Due to MM complexity, therapy requires a comprehensive approach to combine malignancy control but also accompanying symptoms, such as bisphosphonates for bone lesions and erythropoietic agents for anemia, to maintain patient quality of life [ 14 , 15 ]. Significant breakthroughs in the treatment of hematological malignancies have been achieved by the application of epigenetic therapeutics, which modulate reversible changes in gene expression [ 16 ].
Along with the common application in the treatment of myelodysplastic syndrome and myeloid leukemia [ 17 ], current research indicates that N-azacytidine, an epigenetic DNA methyltransferase inhibitor and metabolic reconfigurator, can also be beneficial in the MM treatment [ 18 , 19 ] as dysregulated DNA methylation and histone modification drive disease progression [ 20 ]. In addition to causing direct cytotoxicity of myeloma cells [ 18 , 21 , 22 , 23 ], N-azacitidine reprograms the bone marrow microenvironment in hematologic malignancies [ 24 , 25 , 26 ]. A special component of the bone marrow are mesenchymal stromal cells (BMSCs), a critical cell population in the hematopoiesis process, and at the same time representing the cellular basis for the bone tissue maintenance and remodeling [ 27 , 28 , 29 ]. Thus, since 95% of MM cells remain localized within the bone marrow, the stromal regulation is considered an indispensable therapeutic target in MM [ 30 , 31 , 32 ].
However, data addressing the influence of N-azacitidine on BMSCs of MM patients (MM-MSCs) is limited [ 33 , 34 , 35 ]. Here we demonstrate that in vitro administration of N-azacytidine has an inhibitory effect on the survival of AMO-1 myeloma cells, while preserving the osteogenic and adipogenic differentiation potential of BMSCs, derived from both MM patients and non-malignant controls. In this way, this work provides evidence that the use of N-azacitidine in MM patients at the appropriate dose can represent a promising therapeutic treatment with a protective role on cellular components of the bone marrow microenvironment.
2. Materials and Methods
2.1. Isolation and Cell Culture of MSC and AMO-1 Cells
Primary mesenchymal stromal cells were isolated from bone marrow of non-malignant patients (BMSCs) and multiple myeloma patients (MM-MSCs) after obtaining informed consent from the patients. The study was conducted in accordance with the Declaration of Helsinki. Bone marrow samples of non-malignant patients undergoing hip arthroplasty and bone marrow specimens from newly diagnosed MM patients were obtained from the Banjica Institute of Orthopedics, Belgrade, and the Institute of Oncology and Radiology of Serbia in Belgrade. Upon receipt in the lab, bone marrow samples were washed in PBS (Phosphate-Buffered Saline, Capricorn Scientific, Ebsdorfergrund, Germany) by centrifugation at 1400 rpm for 5 min. The obtained cell pellet was resuspended in PBS and filtered through nylon strainers with a pore size of 70 µm and centrifuged again at 1400 rpm for 5 min. Next, mononuclear cells were isolated based on density gradient centrifugation using Lymphoprep™ (Capricorn, Scientific, Ebsdorfergrund, Germany). Mononuclear cells were seeded in flasks (Thermo Fisher Scientific, Nunc, Waltham, MA, USA), and adherent BMSCs and MM-MSCs were expanded by cultivation in standard growth medium (GM) containing Minimal Essential Medium (MEM; Capricorn Scientific, Ebsdorfergrund, Germany) with 10% fetal bovine serum (FBS; Capricorn Scientific, Ebsdorfergrund, Germany), 1% penicillin/streptomycin (Capricorn Scientific, Ebsdorfergrund, Germany), 10 mM HEPES buffer (PanBiotech, Aidenbach, Germany) and 5 ng/mL fibroblast growth factor (FGF, BioLegend, San Diego, CA, USA). Cells were maintained at 37 °C in a humidified atmosphere containing 5% CO 2 and a hypoxic chamber with 3% O 2 (BioSpherix, Parish, NY, USA) with regular medium exchange. Upon reaching confluency, cells were detached from culture flasks by trypsin/EDTA (Capricorn, Scientific, Ebsdorfergrund, Germany), centrifuged, and washed, while the cell number was determined by Trypan-blue dye (Gibco, Grand Island, New York, NY, USA). All experiments were performed using MSC between passages 2 and 5.
A commercially available human cell line, multiple myeloma AMO-1, was obtained thanks to Prof. Dr. Regina Ebert, University of Würzburg, Germany. AMO-1 cell line was cultured in RPMI medium (Capricorn, Scientific, Ebsdorfergrund, Germany) supplemented with 10% FBS, 2 mM L-glutamine, and 1% penicillin/streptomycin (Capricorn, Scientific, Ebsdorfergrund, Germany) under hypoxic conditions (3% O 2 ). Cells were seeded at an initial density of 1 × 10 5 cells/mL and passaged upon reaching confluency, with medium exchange every 2–3 days.
2.2. Cellular Viability and Metabolic Activity: MTT Assay
Cell viability of both primary stromal cells and AMO-1 cells was assessed using the MTT test. AMO-1 cells were seeded in 96-well plates at a density of 2 × 10 4 cells/well in 200 μL RPMI medium supplemented with 10% FBS and treated with N-azacytidine at concentrations of 50 nM, 100 nM, 200 nM, 500 nM, and 1000 nM for 24, 48, and 72 h under hypoxic conditions (3% O 2 ). Following the treatment, 20 μL of MTT solution (3-(4,5-dimethiltiazol-2yl-)-2,5 diphenyltetrazolium bromide, Sigma Aldrich, Burlington, MA, USA) at a final concentration of 5 mg/mL was added to each well. After 3 h of incubation at 37 °C, the reaction was stopped with 10% SDS in 1 N HCl, dissolving formazan crystals. For adherent cultures, cells were seeded in 96-well plates at 5 × 10 3 cells/well and allowed to attach for 24 h before treatment. After incubation with N-azacytidine for 24, 48, and 72 h, MTT solution (final concentration 5 mg/mL; Sigma-Aldrich, Burlington, MA, USA) was added, incubated for 3 h at 37°, while formazan crystals were dissolved in 100 μL isopropanol (VWR Chemicals, Rosny-sous-Bois, France). The optical density of the obtained solutions was measured at 540 nm using a microplate reader (Labsystems Multiskan PLUS, Helsinki, Finland).
2.3. Flow Cytometry Analyses of Surface Markers, Proliferation, and Cell Cycle
Expression of CD138 and CD38 surface markers on AMO-1 cells was analyzed by flow cytometry following 72 h of treatment with N-azacytidine (100 nM, 500 nM, and 1000 nM). Cells were collected, washed in cold PBS with 0.5% bovine serum albumin (Capricorn Scientific, Scientific, Ebsdorfergrund, Germany), and incubated for 45 min at 4 °C in the dark with fluorochrome-conjugated antibodies: anti-CD138-APC (BioLegend, San Diego, CA, USA) and anti-CD38-FITC (Beckman Coulter, Villepinte, France). Afterwards, cells were washed in PBS, fixed in 4% paraformaldehyde, and analyzed using a BD FACSLyric flow cytometer (BD Biosciences, San Jose, CA, USA). After 72 h treatment with N-azacytidine (100 nM, 500 nM and 1000 nM), the proliferative capacity of AMO-1 was assessed by Trypan blue exclusion assay, CFSE staining, and cell cycle analysis. Cell number was determined using Trypan blue dye to discriminate between live and dead cells. For cell division distribution, cells were labeled with 5 µM carboxyfluorescein succinimidyl ester (CFSE; Affymetrix, eBioscience, San Diego, CA, USA) in PBS supplemented with 5% fetal bovine serum (FBS; Capricorn Scientific, Ebsdorfergrund, Germany) for 15 min at room temperature in the dark. After washing, cells were seeded in 24-well plates at a density of 5 × 10 4 cells/well and treated with N-azacytidine (Sigma-Aldrich, St. Louis, MO, USA) for 72 h under hypoxic conditions (3% O 2 ). The distribution of the dividing AMO-1 cell population was determined based on the CFSE dilution analyzed by flow cytometry. For cell cycle analyses, following the treatment, cells were collected, washed in cold PBS, fixed in 4% paraformaldehyde, and stained with 1 ug/mL DAPI (4′,6-diamidino-2-phenylindole) (Sigma-Aldrich). DNA content was analyzed by flow cytometry. Flow cytometry was performed using a Cytomics FC 500 cytometer (Cytomics FC 500, Beckman Coulter, Brea, CA, USA). Data was processed using FlowJo v10 software (Tree Star, Ashland, Ashland, OR, USA).
2.4. Immunofluorescence Staining and Microscopy
For Immunofluorescence analysis, BMSCs and MM-MSCs were seeded on glass coverslips in 24-well plates (2 × 10 3 cells/coverslip) and cultivated without or with N-azacytidine (100 nM, 500 nM, and 1000 nM) for 72 h under hypoxic conditions (3% O 2 ). Cells were fixed, permeabilized with 0.1% Triton X-100 (Sigma Aldrich, Burlington, MA, USA), and blocked in PBS containing 1% BSA for 30 min at room temperature. Then, primary antibodies were added: mouse anti-NANOG, rabbit anti-OCT-4, and mouse anti-SOX-2 (all from Cell Signaling Technology, Danvers, MA, USA). Phalloidin–tetramethylrhodamine B isothiocyanate (Sigma-Aldrich) at a concentration of 10 µg/mL was used to stain F-actin. Following the primary antibody incubation, samples were washed 3 times with PBS, and the corresponding FITC-coupled secondary antibodies (Sigma-Aldrich) were added for 2 h. Nuclei were counterstained with 1 ng/mL DAPI (Sigma-Aldrich). Next, cells were washed, and coverslips were mounted with Fluoromount™ Aqueous Mounting Medium (Sigma Aldrich, Burlington, MA, USA). Samples were analyzed using an epifluorescence microscope (Olympus, Tokyo, Japan). Signal quantification was performed by image densitometry in NIH—ImageJ software version 1.54k (LOCI, University of Wisconsin, Madison, WI, USA). For densitometric analysis, images of cells in wells were assessed, with the same image size and magnification used.
2.5. Osteogenic and Adipogenic Differentiation Potential of MSCs
The differentiation potential of BMSCs and MM-MSCs towards osteogenic and adipogenic lineages was examined in vitro under hypoxic conditions. Cells were seeded in 96-well plates at a concentration of 5000 cells/well and, upon reaching subconfluency, GM was replaced with the osteogenic induction medium (OM) or adipogenic induction medium (AM) without or with N-azacytidine (100 nM, 500 nM, and 1000 nM). For OM, GM without FGF was used and contained dexamethasone (10 nM; Applichem GmbH, Darmstadt, Germany), 50 μM ascorbic acid (Sigma-Aldrich), and 10 mM β-glycerophosphate (Sigma-Aldrich, Burlington, MA, USA). As for the N-azacytidine pretreatment effect, MSC were cultivated in GM without or with N-azacytidine (100 nM, 500 nm, and 1000 nM) for 3 days, and subsequently only in OM for an appropriate time. Early osteogenic differentiation was assessed after 7 days by analyzing alkaline phosphatase (ALP) activity by staining with BCIP/NBT solution (Sigma-Aldrich, Burlington, MA, USA), while the late phase of differentiation, i.e., matrix mineralization, was assessed after 14 days by Alizarin red staining (Riedel de Haen, Hanover, Germany). A light microscope (Olympus, Tokyo, Japan) was used to capture cells, while the osteogenic degree was quantified by image densitometry in NIH—ImageJ software version 1.54k (LOCI, University of Wisconsin, Madison, WI, USA). For densitometric analysis, images of cells in wells were assessed, with the same image size and magnification used. Adipogenic differentiation was induced by GM (without FGF) containing 1 μM dexamethasone, 100 μg/mL isobutyl-methylxanthine (IBMX; Sigma-Aldrich), and 10 μg/mL insulin (Sigma-Aldrich). After 14 days of cultivation, lipid droplets were detected by Oil Red O staining (Merck, Darmstadt, Germany), and the level of differentiation was assessed microscopically (Olympus, Tokyo, Japan), by counting Oil Red O-positive cells.
2.6. Colony-Forming Unit-Fibroblast Test (CFU-F)
To assess the clonogenicity of BMSCs and MM-MSCs, a Colony-Forming Unit-Fibroblast (CFU-F) assay was performed. Cells were seeded in 24-well plates (at a concentration of 100 cells/well) in the presence of N-azacytidine (100 nM, 500 nM, or 1000 nM) and cultivated in hypoxic conditions for 10 days in GM or OM. Next, cells were fixed in cold methanol and stained with Crystal violet dye. The percentage of formed colonies relative to the total number of seeded cells in each well was determined as colony efficiency.
2.7. Statistical Analysis
Statistical analysis was performed using GraphPad Prism 10 software (GraphPad Software, San Diego, CA, USA). The number of investigated patients’ specimens (biological replicates) is indicated as n for each experiment in the figure legends. Differences between groups were evaluated using the nonparametric Kruskal–Wallis and Two-Way ANOVA tests, followed by appropriate post hoc analysis. A value of p < 0.05 was considered statistically significant.
3. Results
3.1. N-Azacytidine Inhibits Viability, Proliferative Capacity and Modulates the CD138/CD38 Myeloma Phenotype of the AMO-1 Cell Line
Viability of AMO-1 cells in the presence of increasing concentrations of N-azacytidine (50–1000 nM) at different time points (24 h, 48 h, and 72 h) was assessed by the MTT test. The results showed a statistically significant reduction in metabolic activity that correlates with the concentration and treatment time increase, whereby the greatest decline (the viability of AMO-1 cells about 80%) was observed after 72 h ( Figure 1 A). In line with this result, counting of AMO-1 cells treated with N-azacytidine (100 nM, 500 nM, and 1000 nM) during 72 h, demonstrated a significant reduction in cell proliferation rate of approximately 20%+/−4.49% across all groups (representing the maximum SEM among the groups) ( Figure 1 B). Cell division analyzed by CFSE labeling demonstrated separation of three cell populations in the analyzed AMO-1 cells treated with N-azacytidine (100 nM, 500 nM, and 1000 nM) after 72 h. Results demonstrated the reduction trend of the CFSE low population (actively dividing cells), as well as an increase in the CFSE high population (cells that have undergone few or no divisions) under the influence of N-azacytidine, implying the inhibitory effect of N-azacytidine on the proliferative capacity of AMO-1 cells ( Figure 1 C). Analyses of cell cycle progression of AMO-1 cells following the 72 h treatment with N-azacytidine (100 nM, 500 nM, and 1000 nM) showed a reduction in cell distribution in G2 phase ( Figure 1 D). Along with proliferative capacity, results revealed that N-azacytidine treatment had not changed the percentage of AMO-1 myeloma cells coexpressing CD138 and CD38 (approximately 98.7–97.4% of positive cells) ( Figure 1 E). However, further analysis of the fluorescence intensity of CD138 + and CD38 + positive cells showed that N-azacytidine at concentrations of 500 nM and 1000 nM significantly reduced the intensity of the fluorescence signal ( Figure 1 F), which indicates that N-azacytidine treatment may still be important for the modulation of the phenotype of myeloma cells, as well as the subsequent mechanisms controlled by these markers.
3.2. Comparison of Morphological Characteristics, Differentiation Potential, and Viability of MM-MSCs and BMSCs
Next, we established mesenchymal stromal cell cultures derived from bone marrow of MM patients (MM-MSCs) and non-malignant control (BMSCs) to precisely determine the effect of N-azacytidine on MM-MSCs and compare them to BMSCs. To observe potential differences, we investigated their basic characteristics. Both cell types are fibroblast-like in shape, whereby this morphology was maintained even during long-term cultivation through passages ( Figure 2 A). In addition, expressions of cytoskeletal proteins, including F-actin ( Figure 2 B), show similarity in its organization in both cell populations.
Bearing in mind the importance of osteogenic and adipogenic differentiation in the bone marrow, a comparative analysis confirmed the ability of osteogenesis and adipogenesis of MM-MSCs and BMSCs, while no differences were observed in their differentiation potential ( Figure 2 C). The obtained results showed that both cell populations exhibited a significant increase in the expression of the enzyme alkaline phosphatase (ALP) when cultured in osteogenic medium compared to cells grown in standard medium (control), as an indicator of the early phase of osteogenic differentiation. In addition, after long-term incubation in the osteogenic medium, Alizarin red staining of deposited calcium into the extracellular matrix did not show a difference between MM-MSCs and BMSCs ( Figure 2 C). Moreover, based on Oil Red O staining, MM-MSCs and BMSCs can be differentiated into adipocytes with formed lipid droplets, with no difference between cells observed ( Figure 2 D). Although additional analysis (e.g., in vivo) is necessary, these basic tests indicated that there was no significant difference in the stem cell properties between MM-MSCs and BMSCs.
Differential gene expression analysis was performed using datasets GSE196297 [ 36 ] from the GEO database to compare MSCs from the control group (non-malignant bone marrow, control MSCs) and active MM disease (MM-MSCs). DNMT1 and POUF51 (OCT4) genes were upregulated, while DNMT3A and DNMT3B were slightly reduced in MM-MSCs, when compared to control MSCs ( Figure 2 E). This suggested hypomethylating agents, such as N-azacytidine, might be efficient in the regulation of proteins of importance for MM-MSCs integrity. The results of the MTT test demonstrate that N-azacytidine does not cause significant changes in the metabolic activity of stromal cells. It does not affect the viability of these cells at any of the investigated time points, as opposed to exhibiting an inhibitory effect on myeloma cells ( Figure 2 F). However, the efficiency of CFU-F formation showed significantly lower potential in MM-MSCs compared to BMSCs. Although not statistically significant, a different trend of the effect of N-azacytidine on MM-MSCs and BMSCs was observed. While the number of BMSCs colonies increased when treated with N-azacytidine, MM-MSCs showed decreased ability to form colonies in the presence of N-azacytidine ( Figure 2 G). Interestingly, in MM-MSCs, N-azacytidine increased, while in BMSCs, it reduced F-actin staining ( Figure 2 H). This suggests that the N-azacytidine may remodel the cytoskeleton in a context-dependent manner, leading to different responses in MM-MSCs and BMSCs to epigenetic modulation, which could influence cell shape, adhesion, migration, and interactions within the bone marrow microenvironment.
3.3. N-Azacytidine Modulates Pluripotency Markers Expression in MM-MSCs and BMSCs
At the molecular level MSC self-renewal capacity, stemness retention and clonogenic proliferation rates are directly determined by pluripotency marker expression, such as OCT4, NANOG, and SOX2. Figure 3 A–C shows expressions of these markers in MM-MSCs and BMSCs determined by immunofluorescence labeling, following the N-azacytidine treatment. Both cell types showed positive expression of OCT4, SOX2, and NANOG. We observed a lack of baseline nucleus localization of pluripotency-related markers in MM-MSCs. Although not statistically significant, N-azacytidine decreased OCT4, SOX2 and NANOG protein expression in BMSCs that was followed by the reduced localization in the nucleus. On the contrary, this was not the case in MM-MSCs. Nevertheless, these results indicate that N-azacytidine has a different action potential on the expression of pluripotency markers depending on the MSC source. At the level of the MM bone marrow niche, it is important that MM-MSCs maintain the expression of these markers in the presence of N-azacytidine, i.e., stemness capacity.
3.4. Effect of N-Azacytidine on the Differentiation Potential of MM-MSCs and BMSCs: Osteogenesis and Adipogenesis
By using alkaline phosphatase (ALP) enzyme detection assay, no significant differences in the presence of N-azacytidine were determined, both in MM-MSCs and BMSCs after 7-day culture in osteogenic medium ( Figure 4 A). This finding indicates that N-azacytidine did not alter the process of early phase osteogenic differentiation. In addition, Alizarin red was used to detect the ability of cells to form a mineralized matrix after 14 days of treatment in an osteogenic medium ( Figure 4 B). The obtained results show that N-azacytidine induces a similar response in MM-MSCs and BMSCs regarding the degree of mineralization. Namely, the concentration of N-azacytidine of 100 nM does not lead to significant changes in the mineralization level of the extracellular matrix compared to the corresponding untreated control in both MM-MSCs and BMSCs. However, in the presence of higher concentrations of this drug (500 nM and 1000 nM), a statistically significant decrease in mineralization compared to the control was shown ( Figure 4 B). Therefore, it can be concluded that N-azacytidine, although it does not affect the initial phase of osteogenic differentiation, has the potential to impair the formation of mineralized extracellular matrix in later stages of the bone-forming process. On the other hand, Oil Red O staining of lipid droplets in adipogenic-differentiated cultures demonstrated that N-azacytidine (100 nM, 500 nM, and 1000 nM) does not affect adipogenesis in both MM-MSCs and BMSCs. Namely, based on the number of cells in which lipid droplets were formed, no significant differences were found in the presence of N-azacytidine compared to untreated cells. Altogether, N-azacytidine selectively preserved early osteogenic commitment and adipogenic differentiation, but when applied at higher concentrations (≥500 nM), it impaired late-stage bone matrix mineralization. This suggests that at lower concentrations (100 nM), N-azacytidine maintained BMSCs’ osteogenic and adipogenic potential, which is important for preserving bone marrow function in MM treatment.
3.5. Effect of N-Azacytidine Pretreatment on the MM-MSC and BMSC Osteogenesis and Clonogenicity Under Osteogenic Conditions
As the biggest changes were determined at the level of osteogenic differentiation, we further conducted additional experiments to fully elucidate the effect of N-azacytidine on the osteogenic potential of MM-MSC and BMSCs. Given that the real conditions of the therapeutic application of chemotherapeutics imply a certain period, after which the therapy is paused, in this study we tried to simulate those conditions in vitro. We investigated the effect of pretreatment of these cells with N-azacytidine, i.e., the capacity of MSC to establish the original capacity of osteogenic differentiation, following the treatment. Namely, cells were grown for 72 h in the presence of N-azacytidine (100 nM, 500 nM, and 1000 nM) in the standard medium, after which the medium was changed, and further induction continued only in the osteogenic medium for 7 or 14 days. The obtained results showed that this treatment regimen did not affect the expression of ALP enzyme, either in MM-MSC or in MM-MSC ( Figure 5 A). Next, results showed that this pretreatment regime also did not modify the ability of the mineralization process of the extracellular matrix, neither in MM-MSCs, nor in BMSCs ( Figure 5 B), which altogether indicates that the effect of N-azacytidine on osteogenesis is reversible, and that N-azacytidine did not lead to loss of osteogenic potential of MM-MSCs and BMSCs. In addition, the graph of colony efficiency in osteogenic conditions ( Figure 5 C) also shows that, as in the growth medium, BMSCs have a higher colony formation capacity compared to MM-MSCs. However, unlike the effects in the growth medium, N-azacytidine statistically significantly reduces the efficiency of BMSCs to form colonies but does not affect the ability of MM-MSCs to form colonies. Therefore, it can be concluded that in the osteogenic setting, N-azacytidine shows different reactivity of clonogenic efficiency depending on the type of cells, but that the colony formation potential is not lost, both in BMSCs and in MM-MSCs.
4. Discussion
In this study we aimed to investigate the potential of an epigenetic and metabolic modulator, N-azacytidine, to inhibit the growth of the myeloma cells, along with emphasis on bone marrow mesenchymal stromal cells (BMSCs) response. Our results show that this drug, in addition to stopping the proliferative capacity of malignant cells, simultaneously maintains the osteogenic potential of both control BMSCs and BMSCs derived from MM patients (MM-MSCs).
N-azacytidine, an epigenetic DNA methyltransferase inhibitor (DNMTi), has been shown to have multiple mechanisms of action in hematologic malignancies, both on cancer cells, as well as on the tumor microenvironment [ 37 , 38 , 39 , 40 , 41 ]. As recent encouraging findings indicate [ 18 , 19 , 42 ], N-azacytidine may be a significant candidate for further clinical trials and a potential addition to modern, clinical therapeutic protocols for MM treatment. However, the effect of this drug in MM patients is still unclear, requiring more detailed investigations related to the dosage, interaction response with other MM drugs, and long-term impact on the myeloma bone marrow niche.
In this study we used AMO-1 myeloma cell line as a well-established line from a relapsed MM patient with a representative phenotype, being an excellent model for exploring anti-myeloma drugs [ 43 ]. Our results clearly confirm that N-azacytidine (50–1000 nM) effectively inhibits the growth of AMO-1 myeloma cells. After 72 h of treatment, AMO-1 cells showed a significant reduction in metabolic activity, proliferation rate, and cell cycle progression of myeloma cells. These findings are supported by several studies showing that this drug also works across different myeloma cell lines, while the effect is dependent on the dose and time of treatment [ 21 , 44 , 45 , 46 ]. Importantly, even a ~20% reduction in MM cell viability is biologically significant in the context of epigenetic therapy, which often works via differentiation or sensitization rather than direct cytotoxicity. Analysis of CD138 and CD38 expression, two most important phenotypic markers for identification and monitoring plasma cells in MM [ 46 , 47 ], showed that N-azacytidine does not affect the total percent of CD138+CD38+ AMO-1. However, deeper analysis revealed reduced expression of these molecules on individual cells in the presence of N-azacytidine, suggesting its potential to alter phenotypic characteristics linked to malignant cell survival and MM progression [ 48 , 49 ]. Although qualitative changes were not observed, small to moderate changes in the surface density of these proteins directly alter cell signaling, metabolic rates, adhesion dynamics, and microenvironmental interactions. Fluctuations in marker density directly modulate how strongly a cell senses microenvironmental signals, adheres to matrix structures, and processes metabolic substrates [ 50 , 51 , 52 , 53 ]. By continuously degrading NAD + , CD38 directly controls intracellular and extracellular NAD + /NADH ratios, altering glycolysis and mitochondrial function promoting tumor survival [ 54 , 55 , 56 ]. Thus, inhibited CD38 expression by N-azacytidine could be the underlying mechanism that directly reverses the metabolic state of MM cells, triggering cell cycle arrest and tumor cell death, as detected in our study. Along with CD38, CD138 binds and concentrates essential growth factors (like IL-6, HGF, and VEGF) at the cell surface that support tumor growth [ 57 ]. Therefore, inhibition of CD138 caused by the action of N-azacytidine may also be an additional mechanism involved in the reduction in metabolic activity, cell cycle progression and proliferation of myeloma cells. Namely, CD38 is the direct target for highly successful monoclonal antibodies like Daratumumab and Isatuximab. The quantitative density of CD38 on the cell surface directly dictates the efficacy of these therapies. Lower expression or shedding of CD138 correlates with advanced disease stages, high-risk features, and poor overall survival. Nevertheless, due to the complex role of CD38 and CD138 in MM and their significance in therapy [ 50 , 51 , 52 , 53 , 58 , 59 , 60 , 61 ] – further analysis is needed to elucidate the contribution of N-azacytidine in regulating the expression of these key markers for tracking myeloma cells.
Since BMSCs are an important contributor in bone marrow niche regulation, their status is crucial, but in MM it may be altered [ 62 , 63 , 64 , 65 ]. Research by [ 31 ] revealed that DNMT inhibitors in MM revert pathological MSC state to a healthy state, restoring their osteogenic potential and suppressing pro-tumor inflammatory cytokines like IL-6 and TNF-α. Comparison of basic morphology and differentiation potential between BMSCs and MM-MSCs in our study showed no differences, whereby the overall sensitivity to N-azacytidine was largely similar between BMSC and MM-MSCs, as well as their reactivity to N-azacytidine (50–1000 nM). Unchanged stromal cell viability in the presence of higher doses of N-azacytidine (50–1000 nM) is consistent with the published data [ 44 , 66 , 67 ]. Previous studies reported high resistance of BMSCs to N-azacytidine depending on the dose and treatment duration [ 66 , 67 , 68 , 69 , 70 ] whereby it is suggested that the effects on BMSCs proliferation and cell cycle can be reversible [ 68 ]. In our study, the most significant differences between BMSCs and MM-MSCs were determined at the level of colony formation capacity based on the CFU-F test. BMSCs showed a significantly higher efficiency. Although additional analyzes are needed, this result may be explained as the consequence of their functional abnormalities developed due to residence in the tumor microenvironment [ 71 ]. In addition, differences in colony formation potential and F-actin organization in the presence of N-azacytidine were also detected. Although the result is not statistically significant, N-azacytidine stimulated colony formation in BMSCs and inhibited the intensity of F-actin staining. In contrast, in MM-MSCs, N-azacytidine led to a decrease in the number of colonies formed, but more intense F-actin staining. Bearing in mind the importance of F-actin organization for numerous cellular functions, including the maintenance of stem cell pull [ 72 ], it can be speculated that there is a correlation between colony formation and cytoskeleton organization in the presence of F-actin in MSCs from the bone marrow. In brief, increased colony formation of BMSCs in the presence of N-azacytidine, along with decreased F-actin level may imply improved stemness capacity, in contrast to the trend achieved by MM-MSC. However, immunofluorescence analysis also showed that N-azacytidine may decrease pluripotency marker expression (OCT4, NANOG, and SOX2) in BMSCs, with no changes induced in MM-MSC, indicating different responsiveness of these cells on this drug. Moreover, we considered F-actin and pluripotency markers might be involved in the N-azacitidine effects shown on MSC clonogenicity and differentiation (main stem cell properties). Namely, the F-actin cytoskeleton acts as a dynamic biophysical processor that links stemness, clonogenicity, and cell fate/differentiation [ 68 ]. Since different responses in clonogenicity, actin organization, and pluripotency marker expression were detected after treatment in MM-MSCs and BMSCs, it may be speculated that cellular source, i.e., the specific microenvironment that MM-MSC and BMSC come from, underlie these differences. Overall, it can be concluded that additional analyses are necessary to elucidate the molecular mechanisms underlying the effect of N-azacytidine on the stemness properties and fate of bone marrow MSCs.
N-azacytidine was also reported to induce various changes in differentiation potential of stromal cells [ 73 , 74 , 75 , 76 ]. In the context of bone marrow niche and MM, we were focused on investigating MSCs osteogenesis and adipogenesis. Our study shows that N-azacytidine (100–1000 nM) does not significantly affect the early phase of osteogenic differentiation, but in higher concentrations (500 and 1000 nM) leads to reduced extracellular mineralization matrix, while no differences were observed in the response between MM-MSCs and BMSCs. On the contrary, data from [ 68 , 76 , 77 ] imply pro-osteogenic potential of N-azacytidine. As for adipogenesis, it has been reported that BMSCs from MDS and AML patients show increased adipogenic potential [ 78 , 79 ]; however, we did not detect differences in terms of BMSCs and MM-MSCs potential, i.e., MM. Also, no changes in presence of N-azacytidine were noticed, although its stimulatory influence on lipid droplets formation at 1 uM and 10 uM was published [ 77 , 80 ]. These discrepancies with previous data may be due to different cell sources, inter-individual donor variations, and cultivation conditions. Still, considering that N-azacytidine had the most pronounced effect on the ability to mineralize the extracellular matrix of MSCs, we further extended analysis of osteogenic potential. We analyzed the effect of pretreatment to determine the degree of reversibility of osteogenic potential after treatment, capacity of colony formation in osteogenic medium and actin organization. Following the 72 h of treatment, N-azacytidine was removed, cells were induced to osteogenesis and no changes in osteogenic potential in both BMSCs and MM-MSCs were detected. The sustained expression of ALP, as well as the reversible capacity of mineralization, indicate that under these conditions the differentiation potential of MSC may remain preserved. Moreover, in BMSCs, N-azacytidine reduced colony efficiency under osteogenic settings, while this was not the case in MM-MSCs, implying again different reactivity of these two cell types in terms of colony formation in presence of N-azacytidine. In the osteogenic medium, there are also differences in the organization of F-actin, both between BMSCs and MM-MSCs and in their response to N-azacytidine. As the increased polymerization of actin is related with promoted osteogenesis [ 81 ], we may assume that in MM-MSCs N-azacytidine in higher doses (500 nM and 1000 nM) could support osteogenic commitment, unlike in BMSCs. Overall, these results indicate that the effect of N-azacytidine on osteogenesis is reversible, i.e., N-azacytidine does not impair the osteogenic potential of bone marrow MSCs, while additional molecular analyses are needed to reveal mechanisms of its action.
Nevertheless, this study has several limitations that should be acknowledged. First, although we used a single myeloma cell line (AMO-1), which limits generalizability across MM, the selected AMO-1 cell line is a well-established line from a relapsed MM patient with a representative phenotype, being an excellent model for exploring anti-myeloma drugs [ 43 ]. Next, the small biological sample size (limited patient numbers) for both BMSCs and MM-MSCs may not fully capture inter-patient variability. Further studies, with additional patients need to be performed, including samples from treatment-naïve and relapsed patients, to better characterize donor-to-donor variability and to assess niche interactions in more physiological contexts (xenografts). However, directly comparing non-malignant versus MM-derived MSCs addresses a critical question on how the N-azacytidine affects the bone marrow niche in disease versus non-cancer conditions. Although our in vitro study lacks in vivo or ex vivo validation to confirm whether observed effects translate to the disease context, obtained results justify progression to in vivo models and combination therapy studies in future work.
5. Conclusions
Here, we showed the in vitro effects of the DNMT inhibitor N-azacytidine as a potential therapeutic agent for myeloma. Since N-azacytidine is already approved for myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), demonstrating its efficacy in myeloma while preserving bone marrow mesenchymal stromal cell function has promising translational potential. This study addresses a critical gap by showing anti-myeloma activity without compromising the osteogenic niche—an important advance given the myeloma bone. Along with decreased metabolic activity, proliferation, cell cycle progression, and CD138 and CD38 expression in myeloma cells, here we demonstrated that N-azacytidine does not interfere with osteogenesis and adipogenesis of stromal cells of both multiple myeloma (MM-MSCs) and non-malignant patients (BMSCs). Although additional analysis is needed, these results indicate that N-azacytidine may be considered as a significant additional or adjuvant drug used in the therapy of MM.
Acknowledgments
We would like to thank to Snežana Marković and Vesna Masarik for their technical assistance. Image(s) provided by Servier Medical Art ( https://smart.servier.com , accessed on 27 July 2026), licensed under CC BY 4.0 ( https://creativecommons.org/licenses/by/4.0/ , accessed on 27 July 2026).
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.
Author Contributions
Conceptualization, T.K.; methodology, A.S., D.A. and D.T.; software, D.T. and T.K.; validation, D.T., T.K. and M.P.; formal analysis, D.T. and T.K.; investigation, T.K. and D.T.; resources, M.V., N.B. and M.P.; data curation, D.T. and A.J.; writing—original draft preparation T.K.; writing—review and editing, all authors; visualization, T.K.; supervision, D.T. and A.J.; project administration, D.T.; funding acquisition, D.T. and A.J. All authors have read and agreed to the published version of the manuscript.
Institutional Review Board Statement
The study was conducted in accordance with the Declaration of Helsinki, and approved by Ethics Committees obtained from the Institute for Orthopedy Banjica, Belgrade (Decisions of the Ethics Committee I-113/25.3 and I-97/14.3; approval date: 28 December 2023 and 31 May 2022) and the Institute of Oncology and Radiology of Serbia, Belgrade (Decision of the Ethics Committee 01-1/2024/1366; approval date: 26 April 2024), and the Institute for Medical Research, National Institute of the Republic of Serbia, University of Belgrade (Decision EO174/2026; 1 April 2026).
Informed Consent Statement
Informed consent was obtained from all subjects involved in the study.
Data Availability Statement
All data generated during this study are included in this published article.
Conflicts of Interest
The authors declare no conflicts of interest.
References
1. Padala S.A. Barsouk A. Barsouk A. Rawla P. Vakiti A. Kolhe R. Kota V. Ajebo G.H. Epidemiology, Staging, and Management of Multiple Myeloma Med. Sci. 2021 9 3 10.3390/medsci9010003 33498356 PMC7838784
2. Lungu O. Toscani D. Giuliani N. Mechanistic insights into bone destruction in multiple myeloma: Cellular and molecular perspectives J. Bone Oncol. 2025 51 100668 10.1016/j.jbo.2025.100668 40124903 PMC11928850
3. Mafra A. Laversanne M. Marcos-Gragera R. Chaves H.V.S. Mcshane C. Bray F. Znaor A. The global multiple myeloma incidence and mortality burden in 2022 and predictions for 2045 J. Natl. Cancer Inst. 2025 117 907 914 10.1093/jnci/djae321 39658225
4. Kuehl W.M. Bergsagel P.L. Molecular pathogenesis of multiple myeloma and its premalignant precursor J. Clin. Investig. 2012 122 3456 3463 10.1172/jci61188 23023717 PMC3461901
5. Rajkumar S.V. Multiple myeloma: 2022 update on diagnosis, risk stratification, and management Am. J. Hematol. 2022 97 1086 1107 10.1002/ajh.26590 35560063 PMC9387011
6. Abduh M.S. An overview of multiple myeloma: A monoclonal plasma cell malignancy’s diagnosis, management, and treatment modalities Saudi J. Biol. Sci. 2024 31 103920 10.1016/j.sjbs.2023.103920 38283805 PMC10818257
7. Ram M. Fraser M.R. Vieira dos Santos J. Tasakis R. Islam A. Abo-Donia J.U. Parekh S. Lagana A. The Genetic and Molecular Drivers of Multiple Myeloma: Current Insights, Clinical Implications, and the Path Forward Pharmacogenomics Pers. Med. 2024 17 573 609 10.2147/PGPM.S350238 39723112 PMC11669356
8. Reagan M.R. Liaw L. Rosen C.J. Ghobrial I.M. Dynamic interplay between bone and multiple myeloma: Emerging roles of the osteoblast Bone 2015 75 161 169 10.1016/j.bone.2015.02.021 25725265 PMC4580250
9. Terpos E. Ntanasis-Stathopoulos I. Gavriatopoulou M. Dimopoulos M.A. Pathogenesis of bone disease in multiple myeloma: From bench to bedside Blood Cancer J. 2018 8 7 10.1038/s41408-017-0037-4 29330358 PMC5802524
10. Stavropoulos N. Papadoyiannis A. Maltezas D. Stavrou P. Babis G. Papagelopoulos P. Pangalis G. Kyrtsonis M. Biology and Treatment of Skeletal Manifestations in Multiple Myeloma J. Cancer Ther. 2014 5 387 402 10.4236/jct.2014.54045
11. Shin D. Kim M.J. Chun S. Kim D. Lee C. Ahn K.S. Jung E. Kim D. Lee B.C. Hwang D. Elucidation of molecular basis of osteolytic bone lesions in advanced multiple myeloma Haematologica 2024 109 2207 2218 10.3324/haematol.2023.283784 38205555 PMC11215386
12. Zavaleta-Monestel E. Quesada-Villaseñor R. Barrantes-López M. Arguedas-Chacón S. Campos-Hernández J. Rojas-Chinchilla C. García-Montero J. Castro-Ulloa J. Anchía-Alfaro A. Montenegro-Chaves J.R. Advancements in the Treatment of Multiple Myeloma Cureus. 2024 16 e74970 10.7759/cureus.74970 39744254 PMC11691229
13. Ramasamy K. Gay F. Weisel K. Zweegman S. Mateos M.V. Richardson P. Improving outcomes for patients with relapsed multiple myeloma: Challenges and considerations of current and emerging treatment options Blood Rev. 2021 49 100808 10.1016/j.blre.2021.100808 33863601
14. Bird S.A. Boyd K. Multiple myeloma: An overview of management Palliat. Care Soc. Pract. 2019 13 1178224219868235 10.1177/1178224219868235 32215370 PMC7065505
15. Geraldes C. Roque A. Sarmento-Ribeiro A.B. Neves M. Ionita A. Gerivaz R. Tomé A. Afonso S. Silveira M.P. Sousa P. Practical management of disease-related manifestations and drug toxicities in patients with multiple myeloma Front. Oncol. 2024 14 1282300 10.3389/fonc.2024.1282300 38585008 PMC10995327
16. Esfahani M.A. Servatian N. Al-Athari A.J.H. Khafaja E.S.M. Seraji H.R. Samarkhazan H.S. The epigenetic revolution in hematology: From benchside breakthroughs to clinical transformations Clin. Exp. Med. 2025 25 230 10.1007/s10238-025-01783-z 40593271 PMC12213975
17. Jin N. George T.L. Otterson G.A. Verschraegen C. Wen H. Carbone D. Herman J. Bertino E.M. He K. Advances in epigenetic therapeutics with focus on solid tumors Clin. Epigenet. 2021 13 83 10.1186/s13148-021-01069-7 33879235 PMC8056722
18. Khouri J. Faiman B.M. Grabowski D. Mahfouz R.Z. Khan S.N. Wei W. Valent J. Dean R. Samaras C. Jha B.K. DNA methylation inhibition in myeloma: Experience from a phase 1b study of low-dose continuous azacitidine in combination with lenalidomide and low-dose dexamethasone in relapsed or refractory multiple myeloma Semin. Hematol. 2021 58 45 55 10.1053/j.seminhematol.2020.12.004 33509443
19. Wang Y. Rao B. Sun S. Zhu H. Combination therapy of BCL-2 antagonist venetoclax and demethylase inhibitor azacitidine for the treatment of multiple myeloma: A clinical study Leuk. Lymphoma 2026 7 608 618 10.1080/10428194.2025.2606209 41502410
20. Kumar S. Kats L.M. Gruber E. Epigenetic reprogramming in multiple myeloma-Challenges and opportunities Int. J. Cancer 2026 158 423 432 10.1002/ijc.35426 40171810 PMC12628036
21. Khong T. Sharkey J. Spencer A. The effect of azacitidine on interleukin-6 signaling and nuclear factor-kappaB activation and its in vitro and in vivo activity against multiple myeloma Haematologica 2008 93 860 869 10.3324/haematol.12261 18443271
22. Tian E. Tang H. Xu R. Liu C. Deng H. Wang Q. Azacytidine induces necrosis of multiple myeloma cells through oxidative stress Proteome Sci. 2013 11 24 10.1186/1477-5956-11-24 23764212 PMC3718702
23. Wang W.M. Wang J. Zhu M.X. Wang Y.F. Liu Y.Y. Jing H.M. Inductive Effect of 5-Azacitidine on Apoptosis of Multiple Myeloma Cell lines and Its Mechanism Zhongguo Shi Yan Xue Ye Xue Za Zhi 2016 24 110 116 10.7534/j.issn.1009-2137.2016.01.022 26913405
24. Wenk C. Garz A.K. Grath S. Huberle C. Witham D. Weickert M. Malinverni R. Niggemeyer J. Kyncl M. Hecker J. Direct modulation of the bone marrow mesenchymal stromal cell compartment by azacitidine enhances healthy hematopoiesis Blood Adv. 2018 2 3447 3461 10.1182/bloodadvances.2018022053 30518537 PMC6290099
25. Menezes D.L. See W.L. Risueño A. Tsai K.T. Lee J.K. Ma J. Khan R. Prebet T. Skikne B. Beach C.L. Oral azacitidine modulates the bone marrow microenvironment in patients with acute myeloid leukaemia in remission: A subanalysis from the QUAZAR AML-001 trial Br. J. Haematol. 2023 201 1129 1143 10.1111/bjh.18783 36990798
26. Lehner K.M. Gopalakrishnapillai A. Kolb E.A. Barwe S.P. Bone Marrow Microenvironment-Induced Chemoprotection in KMT2A Rearranged Pediatric AML Is Overcome by Azacitidine–Panobinostat Combination Cancers 2023 15 3112 10.3390/cancers15123112 37370721 PMC10296268
27. Anthony B.A. Link D.C. Regulation of hematopoietic stem cells by bone marrow stromal cells Trends Immunol. 2014 35 32 37 10.1016/j.it.2013.10.002 24210164 PMC3947365
28. García-García A. de Castillejo C.L. Méndez-Ferrer S. BMSCs and hematopoiesis Immunol. Lett. 2015 168 129 135 10.1016/j.imlet.2015.06.020 26192443
29. Wu J. Zhang W. Ran Q. Xiang Y. Zhong J.F. Li S.C. Li Z. The Differentiation Balance of Bone Marrow Mesenchymal Stem Cells Is Crucial to Hematopoiesis Stem Cells Int. 2018 2018 1540148 10.1155/2018/1540148 29765406 PMC5903338
30. Ria R. Vacca A. Bone Marrow Stromal Cells-Induced Drug Resistance in Multiple Myeloma Int. J. Mol. Sci. 2020 21 613 10.3390/ijms21020613 31963513 PMC7013615
31. Garcia-Gomez A. Li T. de la Calle-Fabregat C. Rodríguez-Ubreva J. Ciudad L. Català-Moll F. Godoy-Tena G. Martín-Sánchez M. San-Segundo L. Muntión S. Targeting aberrant DNA methylation in mesenchymal stromal cells as a treatment for myeloma bone disease Nat. Commun. 2021 12 421 10.1038/s41467-020-20715-x 33462210 PMC7813865
32. Binder M. Szalat R.E. Talluri S. Fulciniti M. Avet-Loiseau H. Parmigiani G. Samur M.K. Munshi N.C. Bone marrow stromal cells induce chromatin remodeling in multiple myeloma cells leading to transcriptional changes Nat. Commun. 2024 15 4139 10.1038/s41467-024-47793-5 38755155 PMC11098817
33. Gunn W.G. Conley A. Deininger L. Olson S.D. Prockop D.J. Gregory C.A. A crosstalk between myeloma cells and marrow stromal cells stimulates production of DKK1 and interleukin-6: A potential role in the development of lytic bone disease and tumor progression in multiple myeloma Stem Cells 2006 24 986 991 10.1634/stemcells.2005-0220 16293576
34. Fowler J.A. Mundy G.R. Lwin S.T. Edwards C.M. Bone marrow stromal cells create a permissive microenvironment for myeloma development: A new stromal role for Wnt inhibitor Dkk1 Cancer Res. 2012 72 2183 2189 10.1158/0008-5472.CAN-11-2067 22374979 PMC3775476
35. Garcia-Gomez A. Sanchez-Guijo F. Del Canizo M.C. San Miguel J.F. Garayoa M. Multiple myeloma mesenchymal stromal cells: Contribution to myeloma bone disease and therapeutics World J. Stem Cells. 2014 6 322 343 10.4252/wjsc.v6.i3.322 25126382 PMC4131274
36. Heinemann L. Möllers K.M. Ahmed H.M.M. Wei L. Sun K. Nimmagadda S.C. Frank D. Baumann A. Poos A.M. Dugas M. Inhibiting PI3K-AKT-mTOR Signaling in Multiple Myeloma-Associated Mesenchymal Stem Cells Impedes the Proliferation of Multiple Myeloma Cells Front. Oncol. 2022 12 874325 10.3389/fonc.2022.874325 35795041 PMC9251191
37. Khan R. Schmidt-Mende J. Licht J.D. Azacitidine: Mechanisms of action in the treatment of hematologic malignancies Leuk. Res. 2012 36 431 439 10.1016/j.leukres.2011.11.021
38. Gang A. Frøsig T. Brimnes M. Lyngaa R. Treppendahl M.B. Grønbæk K. Dufva I.H. Straten P.T. Hadrup S.R. 5-Azacytidine treatment sensitizes tumor cells to T-cell mediated cytotoxicity and modulates NK cells in patients with myeloid malignancies Blood Cancer J. 2014 4 e197 10.1038/bcj.2014.14 24681961 PMC3972700
39. Grimm J. Simnica D. Jäkel N. Paschold L. Willscher E. Schulze S. Dierks C. Al-Ali H.K. Binder M. Azacitidine-induced reconstitution of the bone marrow T cell repertoire is associated with superior survival in AML patients Blood Cancer J. 2022 12 19 10.1038/s41408-022-00615-7 35091554 PMC8799690
40. Ebelt N.D. Manuel E.R. 5-Azacytidine-Mediated Modulation of the Immune Microenvironment in Murine Acute Myeloid Leukemia Cancers 2023 15 118 10.3390/cancers15010118 36612115 PMC9817798
41. Šimoničová K. Janotka L. Kavcová H. Sulová Z. Breier A. Messingerova L. Different mechanisms of drug resistance to hypomethylating agents in the treatment of myelodysplastic syndromes and acute myeloid leukemia Drug Resist Updat. 2022 61 100805 10.1016/j.drup.2022.100805 35227933
42. Flanagan L. Coughlan A. Cosgrove N. Roe A. Wang Y. Gilmore S. Drozdz I. Comerford C. Ryan J. Minihane E. Steroid-free combination of 5-azacytidine and venetoclax for the treatment of multiple myeloma Haematologica 2024 109 2930 2943 10.3324/haematol.2023.283771 38511268 PMC11367189
43. Sarin V. Yu K. Ferguson I.D. Gugliemini O. Nix M.A. Hann B. Sirota M. Wiita A.P. Evaluating the efficacy of multiple myeloma cell lines as models for patient tumors via transcriptomic correlation analysis Leukemia 2020 34 2754 2765 10.1038/s41375-020-0785-1 32123307 PMC7483300
44. Kiziltepe T. Hideshima T. Catley L. Raje N. Yasui H. Shiraishi N. Okawa Y. Ikeda H. Vallet S. Pozzi S. 5-Azacytidine, a DNA methyltransferase inhibitor, induces ATR-mediated DNA double-strand break responses, apoptosis, and synergistic cytotoxicity with doxorubicin and bortezomib against multiple myeloma cells Mol. Cancer Ther. 2007 6 1718 1727 10.1158/1535-7163.MCT-07-0010 17575103
45. Murakami Y. Kimura Y. Kawahara A. Mitsuyasu S. Miyake H. Tohyama K. Endo Y. Yoshida N. Imamura Y. Watari K. The augmented expression of the cytidine deaminase gene by 5-azacytidine predicts therapeutic efficacy in myelodysplastic syndromes Oncotarget 2019 10 2270 2281 10.18632/oncotarget.26784 31040918 PMC6481348
46. Atienza Párraga A. Nylund P. Diamanti K. Garrido-Zabala B. Tziola S.I. Vasquez L. Pyl P.T. Raykova D. Skaftason A. Ma A. Combinatorial DNMTs and EZH2 inhibition reprograms the H3K27me3 and DNAme-mediated onco-epigenome to suppress multiple myeloma proliferation Sci. Rep. 2025 15 31568 10.1038/s41598-025-17093-z 40866476 PMC12391466
47. Rawstron A.C. Orfao A. Beksac M. Bezdickova L. Brooimans R.A. Bumbea H. Dalva K. Fuhler G. Gratama J. Hose D. Report of the European Myeloma Network on multiparametric flow cytometry in multiple myeloma and related disorders Haematologica 2008 93 431 438 10.3324/haematol.11080 18268286
48. Hur M. Phenotypic consensus markers for plasma cell myeloma Korean J. Hematol. 2012 47 239 240 10.5045/kjh.2012.47.4.239 23319998 PMC3538791
49. Costa F. Dalla Palma B. Giuliani N. CD38 Expression by Myeloma Cells and Its Role in the Context of Bone Marrow Microenvironment: Modulation by Therapeutic Agents Cells. 2019 8 1632 10.3390/cells8121632 31847204 PMC6952797
50. Kawano Y. Fujiwara S. Wada N. Izaki M. Yuki H. Okuno Y. Iyama K. Yamasaki H. Sakai A. Mitsuya H. Multiple myeloma cells expressing low levels of CD138 have an immature phenotype and reduced sensitivity to lenalidomide Int. J. Oncol. 2012 41 876 884 10.3892/ijo.2012.1545 22766978 PMC3582943
51. Kawano Y. Kikukawa Y. Fujiwara S. Wada N. Okuno Y. Mitsuya H. Hata H. Hypoxia reduces CD138 expression and induces an immature and stem cell-like transcriptional program in myeloma cells Int. J. Oncol. 2013 43 1809 1816 10.3892/ijo.2013.2134 24126540 PMC3834117
52. Nijhof I.S. Casneuf T. van Velzen J. van Kessel B. Axel A.E. Syed K. Groen R.W.J. van Duin M. Sonneveld P. Minnema M.C. CD38 expression and complement inhibitors affect response and resistance to daratumumab therapy in myeloma Blood 2016 128 959 970 10.1182/blood-2016-03-703439 27307294
53. van de Donk N.W.C.J. Usmani S.Z. CD38 antibodies in multiple myeloma: Mechanisms of action and modes of resistance Front. Immunol. 2018 9 2134 10.3389/fimmu.2018.02134 30294326 PMC6158369
54. Riccardi F. Tangredi C. Dal Bo M. Toffoli G. Targeted therapy for multiple myeloma: An overview on CD138-based strategies Front. Oncol. 2024 14 1370854 10.3389/fonc.2024.1370854 38655136 PMC11035824
55. Horenstein A.L. Bracci C. Morandi F. Malavasi F. CD38 in Adenosinergic Pathways and Metabolic Re-programming in Human Multiple Myeloma Cells: In-tandem Insights From Basic Science to Therapy Front. Immunol. 2019 10 760 10.3389/fimmu.2019.00760 31068926 PMC6491463
56. Becherini P. Soncini D. Ravera S. Gelli E. Martinuzzi C. Giorgetti G. Cagnetta A. Guolo F. Ivaldi F. Miglino M. CD38-Induced Metabolic Dysfunction Primes Multiple Myeloma Cells for NAD+-Lowering Agents Antioxidants. 2023 12 494 10.3390/antiox12020494 36830052 PMC9952390
57. McCarron M.J. Park P.W. Fooksman D.R. CD138 mediates selection of mature plasma cells by regulating their survival Blood 2017 129 2749 2759 10.1182/blood-2017-01-761643 28381397 PMC5437827
58. Muz B. de la Puente P. Azab F. Luderer M.J. King J. Vij R. Azab A.K. A CD138-independent strategy to detect minimal residual disease and circulating tumour cells in multiple myeloma Br. J. Haematol. 2016 173 70 81 10.1111/bjh.13927 26729247 PMC4918510
59. Morandi F. Horenstein A.L. Costa F. Giuliani N. Pistoia V. Malavasi F. CD38: A Target for Immunotherapeutic Approaches in Multiple Myeloma Front. Immunol. 2018 9 2722 10.3389/fimmu.2018.02722 30546360 PMC6279879
60. García-Guerrero E. Götz R. Doose S. Sauer M. Rodríguez-Gil A. Nerreter T. Kortüm K.M. Pérez-Simón J.A. Einsele H. Hudecek M. Publisher Correction: Upregulation of CD38 expression on multiple myeloma cells by novel HDAC6 inhibitors is a class effect and augments the efficacy of daratumumab Leukemia 2022 36 297 Erratum in Leukemia 2021 , 35 , 201–214 10.1038/s41375-020-0840-y 34845317 PMC8727283
61. Yu J. Tan L. Wu Q. Rao Y. Ao J. Yang W. Zou B. Chen J. Multiple myeloma with CD138 changed from positive to negative: A case report Cytom. Part B Clin. Cytom. 2021 100 249 253 10.1002/cyto.b.21869 31967404
62. Reagan M.R. Ghobrial I.M. Multiple myeloma mesenchymal stem cells: Characterization, origin, and tumor-promoting effects Clin. Cancer Res. 2012 18 342 349 10.1158/1078-0432.CCR-11-2212 22065077 PMC3261316
63. Xu S. De Veirman K. De Becker A. Vanderkerken K. Van Riet I. Mesenchymal stem cells in multiple myeloma: A therapeutical tool or target? Leukemia 2018 32 1500 1514 10.1038/s41375-018-0061-9 29535427 PMC6035148
64. Kamrani S. Naseramini R. Khani P. Razavi Z.S. Afkhami H. Atashzar M.R. Nasri F. Alavimanesh S. Saeidi F. Ronaghi H. Mesenchymal stromal cells in bone marrow niche of patients with multiple myeloma: A double-edged sword Cancer Cell Int. 2025 25 117 10.1186/s12935-025-03741-x 40140850 PMC11948648
65. Wang L. Yi W. Ma L. Lecea E. Hazlehurst L.A. Adjeroh D.A. Hu G. Inflammatory Bone Marrow Mesenchymal Stem Cells in Multiple Myeloma: Transcriptional Signature and In Vitro Modeling Cancers 2023 15 5148 10.3390/cancers15215148 37958322 PMC10650304
66. Zhang F. Wu J. Lu M. Wang H. Feng H. 5-Azacytidine inhibits proliferation and induces apoptosis of mouse bone marrow-derived mesenchymal stem cells Toxin Rev. 2013 32 55 59 10.3109/15569543.2013.846382
67. Ong A.L.C. Lee S.H. Aung S.W. Khaing S.L. Ramasamy T.S. 5-Azacytidine pretreatment confers transient upregulation of proliferation and stemness in human mesenchymal stem cells Cells Dev. 2021 165 203659 10.1016/j.cdev.2021.203659 34024336
68. Zhou G.S. Zhang X.L. Wu J.P. Zhang R.P. Xiang L.X. Dai L.C. Shao J.Z. 5-Azacytidine facilitates osteogenic gene expression and differentiation of mesenchymal stem cells by alteration in DNA methylation Cytotechnology 2009 60 11 22 10.1007/s10616-009-9203-2 19557538 PMC2780539
69. Kadekar S. Barbe L. Stoddart M. Varghese O.P. Tenje M. Mestres G. Effect of the Addition Frequency of 5-Azacytidine in Both Micro- and Macroscale Cultures Cell. Mol. Bioeng. 2020 14 121 130 10.1007/s12195-020-00654-9 33633814 PMC7878657
70. Kornicka K. Marycz K. Marędziak M. Tomaszewski K.A. Nicpoń J. The effects of the DNA methyltranfserases inhibitor 5-Azacitidine on ageing, oxidative stress and DNA methylation of adipose derived stem cells J. Cell. Mol. Med. 2017 21 387 401 10.1111/jcmm.12972 27998022 PMC5264131
71. Maiso P. Mogollón P. Ocio E.M. Garayoa M. Bone Marrow Mesenchymal Stromal Cells in Multiple Myeloma: Their Role as Active Contributors to Myeloma Progression Cancers 2021 13 2542 10.3390/cancers13112542 34067236 PMC8196907
72. Ambriz X. de Lanerolle P. Ambrosio J.R. The Mechanobiology of the Actin Cytoskeleton in Stem Cells during Differentiation and Interaction with Biomaterials Stem Cells Int. 2018 2018 2891957 10.1155/2018/2891957 30402108 PMC6196919
73. Sotthibundhu A. Promjantuek W. Kunhorm P. Natphopsuk S. Chaicharoenaudomrung N. Noisa P. 5-Azacytidine Enhances Differentiation of Human Placenta-derived Mesenchymal Stem Cells Towards Schwann-like Cells In Vivo 2026 40 235 248 10.21873/invivo.14187 41482420 PMC12764216
74. Saheli M. Pirhajati Mahabadi V. Mesbah-Namin S.A. Seifalian A. Bagheri-Hosseinabadi Z. DNA methyltransferase inhibitor 5-azacytidine in high dose promotes ultrastructural maturation of cardiomyocyte Stem Cell Investig. 2020 7 22 10.21037/sci-2020-007 33437842 PMC7791156
75. Qian Q. Qian H. Zhang X. Zhu W. Yan Y. Ye S. Peng X. Li W. Xu Z. Sun L. 5-Azacytidine induces cardiac differentiation of human umbilical cord-derived mesenchymal stem cells by activating extracellular regulated kinase Stem Cells Dev. 2012 21 67 75 10.1089/scd.2010.0519 21476855 PMC3245671
76. Yan X. Ehnert S. Culmes M. Bachmann A. Seeliger C. Schyschka L. Wang Z. Rahmanian-Schwarz A. Stöckle U. De Sousa P.A. 5-azacytidine improves the osteogenic differentiation potential of aged human adipose-derived mesenchymal stem cells by DNA demethylation PLoS ONE 2014 9 e90846 10.1371/journal.pone.0090846 24603866 PMC3946260
77. Bae Y.J. Kwon Y.R. Kim H.J. Lee S. Kim Y.J. Enhanced differentiation of mesenchymal stromal cells by three-dimensional culture and azacitidine Blood Res. 2017 52 18 24 10.5045/br.2017.52.1.18 28401097 PMC5383582
78. Weickert M.T. Hecker J.S. Buck M.C. Schreck C. Rivière J. Schiemann M. Schallmoser K. Bassermann F. Strunk D. Oostendorp R.A.J. Bone marrow stromal cells from MDS and AML patients show increased adipogenic potential with reduced Delta-like-1 expression Sci. Rep. 2021 11 5944 10.1038/s41598-021-85122-8 33723276 PMC7961144
79. Wu Y. Aanei C.M. Kesr S. Picot T. Guyotat D. Campos Catafal L. Impaired Expression of Focal Adhesion Kinase in Mesenchymal Stromal Cells from Low-Risk Myelodysplastic Syndrome Patients Front. Oncol. 2017 7 164 10.3389/fonc.2017.00164 28848706 PMC5551509
80. Ceccarelli S. Gerini G. Megiorni F. Pontecorvi P. Messina E. Camero S. Anastasiadou E. Romano E. Onesti M.G. Napoli C. Inhibiting DNA methylation as a strategy to enhance adipose-derived stem cells differentiation: Focus on the role of Akt/mTOR and Wnt/β-catenin pathways on adipogenesis Front. Cell Dev. Biol. 2022 10 926180 10.3389/fcell.2022.926180 36120582 PMC9478209
81. Sun B. Qu R. Fan T. Yang Y. Jiang X. Khan A.U. Zhou Z. Zhang J. Wei K. Ouyang J. Actin polymerization state regulates osteogenic differentiation in human adipose-derived stem cells Cell. Mol. Biol. Lett. 2021 26 15 10.1186/s11658-021-00259-8 33858321 PMC8048231