Communication between bone marrow mesenchymal stem cells and multiple myeloma cells: Impact on disease progression

Multiple myeloma (MM) is a hematological malignancy characterized by the accumulation of immunoglobulin-secreting clonal plasma cells at the bone marrow (BM). The interaction between MM cells and the BM microenvironment, and specifically BM mesenchymal stem cells (BM-MSCs), has a key role in the pathophysiology of this disease. Multiple data support the idea that BM-MSCs not only enhance the proliferation and survival of MM cells but are also involved in the resistance of MM cells to certain drugs, aiding the progression of this hematological tumor. The relation of MM cells with the resident BM-MSCs is a two-way interaction. MM modulate the behavior of BM-MSCs altering their expression profile, proliferation rate, osteogenic potential, and expression of senescence markers. In turn, modified BM-MSCs can produce a set of cytokines that would modulate the BM microenvironment to favor disease progression. The interaction between MM cells and BM-MSCs can be mediated by the secretion of a variety of soluble factors and extracellular vesicles carrying microRNAs, long non-coding RNAs or other molecules. However, the communication between these two types of cells could also involve a direct physical interaction through adhesion molecules or tunneling nanotubes. Thus, understanding the way this communication works and developing strategies to interfere in the process, would preclude the expansion of the MM cells and might offer alternative treatments for this incurable disease.     

Pharmacoeconomic impact of up-front use of plerixafor for autologous stem cell mobilization in patients with multiple myeloma

Background aimsStem cell collection can be a major component of overall cost of autologous stem cell transplantation (ASCT). Plerixafor is an effective agent for mobilization; however, it is often reserved for salvage therapy because of its high cost. We present data on the pharmacoeconomic impact of the use of plerixafor as an up-front mobilization in patients with multiple myeloma (MM).MethodsPatients with MM who underwent ASCT between January 2008 and April 2011 at the Mount Sinai Medical Center were reviewed retrospectively. In April 2010, practice changes were instituted for patients with MM to delay initiation of granulocyte-colony-stimulating factor (G-CSF) support from day 0 to day +5 and to add plerixafor to G-CSF as an up-front autologous mobilization. Targets of collection were 5–10 × 10⁶ CD34⁺ cells/kg.ResultsOf 50 adults with MM who underwent ASCT, 25 received plerixafor/filgrastim and 25 received G-CSF alone as an up-front mobilization. Compared with the control, plerixafor mobilization yielded higher CD34⁺ cell content (16.1 versus 8.4 × 10⁶ CD34⁺ cells/kg; P = 0.0007) and required fewer sessions of apheresis (1.9 versus 3.1; P = 0.0001). In the plerixafor group, the mean number of plerixafor doses required per patient was 1.8. Although the overall cost of medications was higher in the plerixafor group, the cost for blood products and overall cost of hospitalization were similar between the two groups.ConclusionsUp-front use of plerixafor is an effective mobilization strategy in patients with MM and does not have a substantial pharmacoeconomic impact in overall cost of hospitalization combined with the apheresis procedure.     

Loss of p53 exacerbates multiple myeloma phenotype by facilitating the reprogramming of hematopoietic stem/progenitor cells to malignant plasma cells by MafB

Multiple myeloma (MM) is a serious, mostly incurable human cancer of malignant plasma cells. Chromosomal translocations affecting MAFB are present in a significant percentage of multiple myeloma patients. Genetically engineered Sca1-MafB mice, in which MafB expression is limited to hematopoietic stem/progenitor cells (HS/P-Cs), display the phenotypic features of MM. Contrary to many other types of cancer, it is not yet known if the p53 gene plays any essential role in the pathogenesis of this disease. Here, we show, taking advantage of the Sca1-MafB MM mouse model, that loss of p53 does not rescue the multiple myeloma disease, but instead accelerates its development and exacerbates the MM phenotype. Therefore, the efficiency of the MafB-induced MM reprogramming of normal HS/P-Cs to terminally differentiated malignant plasma cells is enhanced by p53 deficiency, in analogy to what happens in reprogramming to pluripotency. These results raise caution about interfering with p53 function when treating multiple myeloma.     

Loss of p53 exacerbates multiple myeloma phenotype by facilitating the reprogramming of hematopoietic stem/progenitor cells to malignant plasma cells by MafB

Multiple myeloma (MM) is a serious, mostly incurable human cancer of malignant plasma cells. Chromosomal translocations affecting MAFB are present in a significant percentage of multiple myeloma patients. Genetically engineered Sca1-MafB mice, in which MafB expression is limited to hematopoietic stem/progenitor cells (HS/P-Cs), display the phenotypic features of MM. Contrary to many other types of cancer, it is not yet known if the p53 gene plays any essential role in the pathogenesis of this disease. Here, we show, taking advantage of the Sca1-MafB MM mouse model, that loss of p53 does not rescue the multiple myeloma disease, but instead accelerates its development and exacerbates the MM phenotype. Therefore, the efficiency of the MafB-induced MM reprogramming of normal HS/P-Cs to terminally differentiated malignant plasma cells is enhanced by p53 deficiency, in analogy to what happens in reprogramming to pluripotency. These results raise caution about interfering with p53 function when treating multiple myeloma.     

Establishment of a monoclonal antibody to human myeloma cell: relation to chemotherapy and extramedullar infiltration

Resistance of myeloma cells to melphalan (L-PAM) is a serious problem. To investigate mechanisms of drug resistance, we generated a monoclonal antibody, clone O3, to melphalan-resistant myeloma cells, KHM-11R. Western blot analysis showed that molecular weight of O3 antigen was approximately 90 kDa. Expression of O3 antigen was approximately two times higher in KHM-11R than in parental melphalan sensitive cell line, KHM-11. O3 was preferentially expressed in plasma cell, B-cell, and monocytic cell lines, but not in T-cell lines. Analysis of bone marrow samples from myeloma patients revealed that 13 of 23 samples expressed O3 antigen at various levels, and that O3 antigen expression in patients correlate with preceding chemotherapy, advanced clinical stage and extramedullar invasion of myeloma cells. Furthermore, patients expressing O3 antigen at the time of diagnosis tended to have poor prognosis. The investigation of O3 antigen in myeloma cells will be useful to reveal the pathophysiology of extramedullar invasion and the mechanism of cell killing by melphalan.     

Modification of the bone marrow MSC population in a xenograft model of early multiple myeloma

Multiple myeloma (MM) is a hematological malignancy characterized by clonal proliferation of abnormal plasma cells. MM dysregulates the homeostasis of the bone niche cells like osteoclasts and osteoblasts, responsible for the bone maintenance leading to bone loss and hypercalcemia, as well as the normal immune cells leading to immunodeficiency and anemia. Osteoblasts are part of the cell population differentiating from mesenchymal stem cells (MSC). MSC also gives rise to other cell types such as adipocytes and chondrocytes. It has been observed that adipocytes support MM growth by increasing its survival and chemo-resistance. As adipocytes originate from MSC, the understanding of early modifications in the MSC population during the disease progression is of paramount importance and may help for early diagnosis of MM. Herein, we have evaluated the modification of the MSC population in the bone niche in an in vivo model of MM. Our results showed that before an observable engraftment of MM in the bone niche, the proportion of MSC population is significantly decreased, while a significant increase in adipocyte related genes such as PPARγ and CEBPα expression appears, with no difference in osteogenic differentiation. These results suggest that the bone niche is switching to a “fatty” marrow which would create an adequate microenvironment for MM. This led us to screen for and identify modulated adipokines in the sera of this in vivo MM-mice model. Such changes could reflect early signs of MM and potentially be exploited as detection biomarkers of the disease.     

Differentiation of adult rat bone marrow stem cells into epithelial progenitor cells in culture

We have previously obtained monoclonal bone marrow stem cells from adult rats (rMSCs) and induced them into phenotypic neurons. In the present study, we aimed to induce rMSCs into epithelial cells by culturing them onto compartmentalized permeable supports, which have been used for growing a variety of polarized epithelia in culture. Hematoxylin staining showed that after 4 days grown on permeable supports, rMSCs formed an epithelial-like monolayer. Immunofluorescence of the permeably-supported monolayers, but not the rMSCs grown in culture flasks, showed positive signals for epithelial markers, cytokeratin 5 & 8. RT-PCR results also showed the mRNA expression of epithelial sodium channel (ENaC) and cystic fibrosis transmembrane conductance regulator (CFTR) as well as tight junction protein ZO-1 in the rMSC-derived monolayers grown on permeable supports but absent from those grown in culture flasks. However, western blot only detected protein expression of ZO-1 but not ENaC nor CFTR. The short-circuit current measurements showed that the rMSC-derived monolayers grown on permeable supports exhibited a trans-monolayer resistance of 30-50 Omega cm(2); however, the monolayers did not respond to activators or blockers of CFTR or ENaC. The results suggest that compartmentalized or polarized culture conditions provide a suitable environment for rMSCs to differentiate into epithelial progenitor cells with tight junction formation; however, this condition is not sufficient for functional expression of epithelial ion channels associated with well-differentiated epithelia.     

DNA repair pathways in human multiple myeloma

Every day, cells are faced with thousands of DNA lesions, which have to be repaired to preserve cell survival and function. DNA repair is more or less accurate and could result in genomic instability and cancer. We review here the current knowledge of the links between molecular features, treatment, and DNA repair in multiple myeloma (MM), a disease characterized by the accumulation of malignant plasma cells producing a monoclonal immunoglobulin. Genetic instability and abnormalities are two hallmarks of MM cells and aberrant DNA repair pathways are involved in disease onset, primary translocations in MM cells, and MM progression. Two major drugs currently used to treat MM, the alkylating agent Melphalan and the proteasome inhibitor Bortezomib act directly on DNA repair pathways, which are involved in response to treatment and resistance. A better knowledge of DNA repair pathways in MM could help to target them, thus improving disease treatment.     

Local signals in stem cell-based bone marrow regeneration

The cellular basis of bone marrow （BM） tissue development and regeneration is mediated through hematopoietic stem cells （HSCs） and mesenchymal stem cells （MSCs）. Local interplays between hematopoietic cells and BM stromal cells （BMSCs） determine the reconstitution of hematopoiesis after myelosuppression. Here we review the BM local signals in control of BM regeneration after insults. Hematopoietic growth factors （HGFs） and cytokines produced by BMSCs are primary factors in regulation ofBM hematopoiesis. Morphogens which are critical to early embryo development in multiple species have been added to the family of HSCs regulators, including families of Wnt proteins, Notch ligands, BMPs, and Hedgehogs. Global gene expression analysis of HSCs and BMSCs has begun to reveal signature groups of genes for both cell types. More importantly, analysis of global gene expression coupled with biochemical and biological studies of local signals during BM regeneration have strongly suggested that HGFs and cytokines may not be the primary local regulators for BM recovery, rather chemokines （SDF- 1, FGF-4） and angiogenic growth factors （VEGF-A, Ang- 1） play instructive roles in BM reconstitution after myelosuppression. A new direction of management of BM toxicity is emerging from the identification of BM regenerative regulators.     

A novel molecular mechanism involved in multiple myeloma development revealed by targeting MafB to haematopoietic progenitors

Understanding the cellular origin of cancer can help to improve disease prevention and therapeutics. Human plasma cell neoplasias are thought to develop from either differentiated B cells or plasma cells. However, when the expression of Maf oncogenes (associated to human plasma cell neoplasias) is targeted to mouse B cells, the resulting animals fail to reproduce the human disease. Here, to explore early cellular changes that might take place in the development of plasma cell neoplasias, we engineered transgenic mice to express MafB in haematopoietic stem/progenitor cells (HS/PCs). Unexpectedly, we show that plasma cell neoplasias arise in the MafB-transgenic mice. Beyond their clinical resemblance to human disease, these neoplasias highly express genes that are known to be upregulated in human multiple myeloma. Moreover, gene expression profiling revealed that MafB-expressing HS/PCs were more similar to B cells and tumour plasma cells than to any other subset, including wild-type HS/PCs. Consistent with this, genome-scale DNA methylation profiling revealed that MafB imposes an epigenetic program in HS/PCs, and that this program is preserved in mature B cells of MafB-transgenic mice, demonstrating a novel molecular mechanism involved in tumour initiation. Our findings suggest that, mechanistically, the haematopoietic progenitor population can be the target for transformation in MafB-associated plasma cell neoplasias.     

Frequent harvesting from perfused bone marrow cultures results in increased overall cell and progenitor expansion

The establishment of prolific long-term human bone marrow cultures has led to the development of hematopoietic bioreactor systems. A single batch expansion of bone marrow mononuclear cell populations leads to a 10- to 30-fold increase in total cell number and in the number of colony forming units-granulocyte/macrophage (CFU-GMs), and a four- to tenfold increase in the number of long-term culture initiating cells (LTC-ICs). In principle, unlimited expansion of cells should be attainable from a pool of stem cells if all the necessary requirements leading to stem cell maintenance and division are met. In this article, we take the first step toward the identification of factors that limit single batch expansion of ex vivo bone marrow cells in perfusion-based bioreactor systems. One possible constraint is the size of the growth surface area required. This constraint can be overcome by harvesting half the cell population periodically. We found that harvesting cells every 3 to 4 days, beginning on day 11 of culture, led to an extended growth period. Overall calculated cell expansion exceeded 100-fold and the CFU-GM expansion exceeded 30-fold over a 27-day period. These calculated values are based on growth that could be obtained from the harvested cell population. Growth of the adherent cell layer was stable, whereas the nonadherent cell population diminished with increasing number of passages. These results show that the bioreactor protocols published to date are suboptimal for long-term cultivation, and that further definition and refinement is likely to lead to even greater expansion of hematopoietic cell populations obtained from bone marrow. More importantly, these results show that the LTC-IC measured during the single pass expansion do have further expansion potential that can be realized by frequent harvesting. Finally, the present culture conditions provide a basis for an assay system for the identifications provide a basis for an assay system for the identification of the factors that determine the long-term maintenance and replication of human stem cells ex vivo. (c) 1994 John Wiley & Sons, Inc.     

Effects of epigenetic-based anti-cancer drugs in leukaemia and multiple myeloma cells

Here, we focus on epigenetic changes in leukaemia and MM (multiple myeloma) cells. We show how the histone signature, DNA methylation and levels of select tumour-suppressor proteins can be affected by inhibitors of HDACs (histone deacetylases) and Dnmts (DNA methyltransferases). Both inhibitors, TSA (trichostatin A) and 5-AZA (5-azacytidine), have the ability to change the histone signature in a tumour-specific manner. In MM cells, we observed changes in H3K4 methylation, while in leukaemia cells, H3K9 methylation was especially affected by select inhibitors. Compared with normal peripheral blood lymphocytes, tumour cell samples were characterized by increased H3K9 acetylation, increased H3K4me2, H3K9me2 and HP1α (heterochromatin protein 1α) levels and specific changes were also observed for DNA methylation. Additionally, we showed that the tumour suppressor pRb1 (retinoblastoma protein) is more sensitive to epigenetic-based anti-cancer stimuli than p53. We have found significant decrease in the levels of pRb1 and p53 in both myeloma and leukaemia cells after HDAC inhibition.     

Ogt controls neural stem/progenitor cell pool and adult neurogenesis through modulating Notch signaling

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Wnt and BMP signaling pathways co-operatively induce the differentiation of multiple myeloma mesenchymal stem cells into osteoblasts by upregulating EMX2

Osteoblast differentiation, defined as the process whereby a relatively unspecialized cell acquires the specialized features of an osteoblast, is directly linked to multiple myeloma (MM) bone disease. Wnt and bone morphogenetic protein (BMP) are proved to be implicated in the pathological or defective osteoblast differentiation process. This study aims to test the involvement of Wnt, bone morphogenetic proteins (BMP) pathways, and empty spiracles homeobox 2 (EMX2) in osteoblast differentiation and MM development. Initially, differentially expressed genes in bone marrow mesenchymal stem cells (MSCs) from MM patients and healthy donors were identified using microarray-based gene expression profiling. The functional role of Wnt and BMP in MM was determined. Next, we focused on the co-operative effects of Wnt and BMP on calcium deposition, alkaline phosphatase (ALP) activity, the number of mineralized nodules, and osteocalcin (OCN) content in MSCs. The expression patterns of Wnt and BMP pathway–related genes, EMX2 and osteoblast differentiation-related factors were determined to assess their effects on osteoblast differentiation. Furthermore, regulation of Wnt and BMP in ectopic osteogenesis was also investigated in vivo. An integrated genomic screen suggested that Wnt and BMP regularly co-operate to regulate EMX2 and affect MM. EMX2 was downregulated in MSCs. The activated Wnt and BMP resulted in more calcium salt deposits, mineralized nodules, and a noted increased in ALP activity and OCN content by upregulating EMX2, leading to induced differentiation of MSCs into osteoblasts. Collectively, this study demonstrated that Wnt and BMP pathways could co-operatively stimulate differentiation of MSCs into osteoblasts and inhibit MM progression, representing potential targets for MM treatment.     

Negative elongation factor regulates muscle progenitor expansion for efficient myofiber repair and stem cell pool repopulation

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