**1. Introduction**

Multiple myeloma (MM) is a B-cell malignancy characterized by an accumulation of malignant plasma cells within the bone marrow (BM) [1]. Contact between MM cells and their microenvironment plays a crucial role in MM survival and proliferation and is able to promote tumor progression and drug resistance. Bone marrow mesenchymal stromal cells (BMMSCs) represent a central component of MM microenvironment supporting its progression and proliferation [2–4]. Alterations in BMSC from MM patients (MM-BMMSC) have become an important research focus. Several studies and our previous data have suggested the genesis of constitutive abnormalities within the BMMSC population through direct and indirect interactions with MM cells [5–8]. The development of a senescence-like state in BMMSC and thereby a modulated secretory profile, worsened osteogenic differentiation potential and inhibition of the T-cell proliferation, was reported [6, 8, 9]. Senescent BMMSCs display an increased senescence-associated β-galactosidase activity (SAβGalA) and irregular cell morphology. Usually, the cell cycle of senescent cells is arrested at the G1 / S-transition point in combination with the overexpression of different cell cycle inhibitors as p21 and p16. In spite of the aberrant growth characteristics, senescent cells remain metabolically active, and therefore, the secretion of pro-inflammatory mediators could promote tumorigenesis in neighboring premalignant cells [10–12]. The secretion of pro-inflammatory mediators by senescent BMMSC could therefore promote tumorigenesis in neighboring premalignant cells [13].

It is known that the increased activation of NF-κB together with the overexpression of adhesion molecules can induce a therapy resistance [4, 26, 28, 29]. It can increase the secretion of tumor supportive soluble factors by BMMSC and may possibly lead to the generation of cell

Molecular Aberrations in Bone Marrow Stromal Cells in Multiple Myeloma

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Furthermore, additional interaction pathway, such as Notch signaling, is a factor between BMMSC and myeloma cells [33]. For example, it has been shown that malignant plasma cells overexpress the Notch ligand Jagged-2. An inhibition of Notch interaction induces myeloma cell apoptosis [34]. Notch signaling is important not only for the interaction of the myeloma cell with its surrounding cells but also for intercellular signaling between the malignant plasma cells. Downstream leads the Notch receptor-ligand interaction to an increased release of angiogenic and tumor-stimulating factors, such as VEGF, IL-6, and IGF-1 [35]. In addition, direct interaction between adhesion molecules (e.g., VCAM-1-VLA-4 interactions) and cell surface receptors such as Notch mediates therapy resistance and disease progression [36]. Furthermore, the formation of exosomes by BMSC, which actively transports modulatory substances, such as chemokines and miRNAs to the malignant MM cell, promotes survival and growth of MM cells. However, further investigations are needed to identify the exact

It is known that cancer cells undergo dramatic alteration of metabolic pathways. Cancer cell survival and proliferation depend on metabolic processes, like glucose-uptake via altered glycolysis, also known as the Warburg effect. Sirtuins (SIRTs) are a family of deacylases and ADP-ribosyltransferases with clear links to regulation of cancer metabolism. Through their unique ability to integrate cellular stress and nutrient status in coordination with metabolic outputs, SIRTs are well poised to play pivotal roles in tumor progression and survival [39]. SIRT3 is the main mitochondrial deacetylase, which controls the activity of many metabolic enzymes in the mitochondria. SIRT3 deacetylates mitochondrial proteins that act in mitochondrial metabolism, including the oxidation of fatty acids, glutamine metabolism, and the production of mitochondrial reactive oxygen species (ROS) [40]. It was found that the increased level of cellular ROS observed with the loss of SIRT3 leads to a change in the cellular metabolism with respect to glycolysis. It is possible that the SIRT3 deficiency leads to a cancer resolution, coordinating the metabolic shift in the Warburg

Despite some knowledge of the constitutive changes in the BMMSC of MM patients, the

BM samples from 116 MM patients were studied: 69 patients with MM at the time of diagnosis and 47 at relapse. All patients had indications for treatment. The main clinical characteristics

molecular mechanisms and pathways that induce abnormalities are largely unknown.

adhesion-mediated drug resistance [30–32].

phenotype [41].

**2. Patients and methods**

**2.1. Patients and donor characteristics**

mechanisms of exosome-mediated tumor promotion [37, 38].

Two imprinted clusters in the human genome might contribute to the generation of senescence and the induction of cellular changes in MM-BMMSC [14–17]. The DLK1-DIO3 imprinted domain is located on chromosome 14q32.2, and cluster C19MC is located on chromosome 19q13. The DLK1-DIO3 expresses the non-coding transcripts MEG3, anti-RTL1, 53 microR-NAs (miRNA), and 2 snoRNA clusters on the maternal chromosome. The paternal chromosome is responsible for the transcription of the protein-coding genes DLK1, RTL1, and DIO3 [18, 19]. Allelic expression of these genes is controlled through methylation of a regulatory region (IG-DMR) located upstream of the cluster [20]. The C19MC codes for 59 miRNAs are processed into one primary transcript from the paternal chromosome. Its expression strongly correlates with the epigenetic modulation of a CpG site located upstream [21].

In addition, there are evidence that the presence of cancer-associated fibroblasts (CAFs), characterized by high α-SMA, FAP, and FSP-1 expression, in the BM samples of MM patients, contributes to altered, tumor favorable, cell-cell interactions and cytokine secretions [22–24].

BMMSC represents an essential part for assistance of MM partly by the secretion of tumor supportive cytokines as interleukin 6 (IL-6) and vascular endothelial growth factor (VEGF) [2]. Both of them play a major role in the aberration of multiple signaling pathways such as PI3K, JAK/STAT3, Raf, and NF-κB [25]. In addition, MM cells produce inflammatory molecules, such as TGFβ and TNFα, which lead to NF-κB activation tumor-promoting effects. The activation of the NF-κB pathway in both MM cells and BMMSC results in the downstream upregulation of adhesion molecules and a consequent increase in cell-cell interactions [26, 27]. It is known that the increased activation of NF-κB together with the overexpression of adhesion molecules can induce a therapy resistance [4, 26, 28, 29]. It can increase the secretion of tumor supportive soluble factors by BMMSC and may possibly lead to the generation of cell adhesion-mediated drug resistance [30–32].

Furthermore, additional interaction pathway, such as Notch signaling, is a factor between BMMSC and myeloma cells [33]. For example, it has been shown that malignant plasma cells overexpress the Notch ligand Jagged-2. An inhibition of Notch interaction induces myeloma cell apoptosis [34]. Notch signaling is important not only for the interaction of the myeloma cell with its surrounding cells but also for intercellular signaling between the malignant plasma cells. Downstream leads the Notch receptor-ligand interaction to an increased release of angiogenic and tumor-stimulating factors, such as VEGF, IL-6, and IGF-1 [35]. In addition, direct interaction between adhesion molecules (e.g., VCAM-1-VLA-4 interactions) and cell surface receptors such as Notch mediates therapy resistance and disease progression [36]. Furthermore, the formation of exosomes by BMSC, which actively transports modulatory substances, such as chemokines and miRNAs to the malignant MM cell, promotes survival and growth of MM cells. However, further investigations are needed to identify the exact mechanisms of exosome-mediated tumor promotion [37, 38].

It is known that cancer cells undergo dramatic alteration of metabolic pathways. Cancer cell survival and proliferation depend on metabolic processes, like glucose-uptake via altered glycolysis, also known as the Warburg effect. Sirtuins (SIRTs) are a family of deacylases and ADP-ribosyltransferases with clear links to regulation of cancer metabolism. Through their unique ability to integrate cellular stress and nutrient status in coordination with metabolic outputs, SIRTs are well poised to play pivotal roles in tumor progression and survival [39]. SIRT3 is the main mitochondrial deacetylase, which controls the activity of many metabolic enzymes in the mitochondria. SIRT3 deacetylates mitochondrial proteins that act in mitochondrial metabolism, including the oxidation of fatty acids, glutamine metabolism, and the production of mitochondrial reactive oxygen species (ROS) [40]. It was found that the increased level of cellular ROS observed with the loss of SIRT3 leads to a change in the cellular metabolism with respect to glycolysis. It is possible that the SIRT3 deficiency leads to a cancer resolution, coordinating the metabolic shift in the Warburg phenotype [41].

Despite some knowledge of the constitutive changes in the BMMSC of MM patients, the molecular mechanisms and pathways that induce abnormalities are largely unknown.
