Aberrant Expression Of miRNA and mRNA Of Cell Cycle and Adhesion-Related Genes In Bone Marrow Stroma Cells Derived From Patients With Multiple Myeloma

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3149-3149 ◽  
Author(s):  
Rimma Berenstein ◽  
Blau Igor Wolfgang ◽  
Axel Nogai ◽  
Marlies Wächter ◽  
Antonio Pezzutto ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Multiple Myeloma ◽  
Bone Marrow ◽  
Genetic Alterations ◽  
Bone Marrow Stroma ◽  
Aberrant Expression ◽  
Healthy Donors ◽  
Marrow Stroma ◽  
Bone Marrow Stroma Cells

Abstract Multiple myeloma (MM) is a B-cell malignancy characterized by accumulation of malignant plasma cells (PC) within the bone marrow. The bone marrow microenvironment such as bone marrow stroma cells (BMSC) supports MM disease progression, resistance to chemotherapy, protects the tumor cells against apoptosis and causes osteolytic bone disease and angiogenesis. The aim of this study was to identify constitutive genetic alterations in BMSC derived from patients with MM (MM-BMSC) in comparison to BMSC from healthy donors. For BMSC selection, mononuclear cells were isolated from fresh bone marrow aspirates and were further expanded in cell culture. We studied 25 MM patients and 5 healthy donors. Senescence status was determined in passage 1 of cell cultures. MM-BSMC displayed a considerably higher percentage of senescence cells (p<0,05). We investigated the expression of cell cycle and adhesion-associated genes (CCNE1, CCND1, CDKN1B, VCAM, ICAM, IKK-alpha) in BMSC (passage 4) using SYBR-Green Real-Time PCR and relative quantification by linear regression. A downregulation of CCNE1 (p=0,05), CDKN1B (p=0,29), and an upregulation of CCND1 (p=0,05), VCAM-1 (p=0,33), ICAM-1 (p=0,33), and IKK-alpha (p=0,05) was demonstrated. Furthermore, the expression profile of miRNAs, targeting the analyzed mRNA genes or correlating with senescence, was studied (miR-16, miR-221, miR-126, miR-223, miR-485-5p and miR-519d). For miRNA detection treatment with Poly(A)-Polymerase and cDNA-Synthesis with a Poly(T)VN-Adaptor primer were carried out following an amplification with an universal reverse primer corresponding to the adaptor sequence and a miRNA-specific primer. miR-16, miR-223, miR-485-5p and miR-519d were significantly upregulated, (p=0,02; p=0,004; p=0,02; and p=0,002, respectively), whereas miR-221 and miR-126 showed no considerable differences to BMSC obtained from healthy donors. Next we investigated incubation of immunmodulatory drug Lenalidomid in BMSC cultures. Cells were treated with 10 µM Lenalidomid over 72 hours and expression was normalized to a 0,01 % DMSO control. Significant difference in gene expression were visible for ICAM-1 (p=0,01). For CDKN1B (p=0,16) and VCAM-1 (p=0,12) we demonstrated changes in gene expression. Our results indicate aberrant expression of cell cycle and adhesion-related genes, such as CCNE1, CCND1 and CDKN1B VCAM-1, ICAM-1 and IKK-alpha in MM-BMSC as compared with healthy donors. Furthermore, we found significant upregulation of miR-16, miR-223, miR-485-5p and miR-519d. Further investigation are needed to determine molecular mechanisms in MM-BMSC and PC interaction that lead to constitutive changes in BMSC characteristics and gene expression patterns. Disclosures: No relevant conflicts of interest to declare.

Establishment and characterization of a novel ALL-L3 cell line (BALM-18): induction of apoptosis by anti-IgM and inhibition of apoptosis by bone marrow stroma cells

1999 ◽  
Vol 23 (6) ◽  
pp. 559-568 ◽  
Author(s):  
Yoshinobu Matsuo ◽  
Akira Sugimoto ◽  
Akira Harashima ◽  
Chiharu Nishizaki ◽  
Fumihiko Ishimaru ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Line ◽  
Bone Marrow Stroma ◽  
Marrow Stroma ◽  
Bone Marrow Stroma Cells ◽  
Induction Of Apoptosis

Discovery and Characterization of Macrocyclic Thiopeptide Proteasome Inhibitors for Hematologic Malignancies

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3669-3669
Author(s):  
Sridevi Ponduru ◽  
Raymond Moellering ◽  
Edward Greenberg ◽  
John Paul Ying-Ching Shen ◽  
Benjamin Z Stanton ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Protein Degradation ◽  
High Throughput ◽  
Active Sites ◽  
Proteasome Inhibitors ◽  
Hematologic Malignancies ◽  
Bone Marrow Stroma ◽  
Shh Signaling ◽  
Marrow Stroma

Abstract The ubiquitin proteasome pathway comprises a coordinated, dynamic cellular system critical to cellular metabolism, signaling and proliferation. The expanding clinical utility of the peptide boronate, bortezomib, in the treatment of patients with multiple myeloma and other hematologic malignancies has established the human 26S proteasome as a validated target in cancer. Still, only one FDA-approved proteasome inhibitor presently exists. Restricted activity against one enzymatic function of the proteasome and dose-limiting toxicities associated with bortezomib warrant further discovery efforts aimed at the identification of structurally and functionally distinct protein degradation inhibitors (PDIs). Here, we report a novel family of natural product proteasome inhibitors discovered by high-throughput, high-content screening at the National Cancer Institute Initiative for Chemical Genetics. A primary screen of 14,000 small molecules was performed in 384-well plate format using a cell line stably transfected with a destabilized fluorescent protein chimera. Assay positives were retested in the primary screen in dose-response format. Thiostrepton was selected for further characterization due to its unique macrocyclic chemical structure, the recent publication of its total synthesis, reports of anticancer properties and the lack of prior annotation as a PDI. First, thiostrepton was linked to previously characterized molecules acting on the protein degradation pathways by transcriptional small molecule connectivity mapping (CMAP). Subsequent cell-state analyses confirmed strong induction of functional and annotated gene sets associated with misfolded protein stress and proteasome inhibition. Mechanism of action was confirmed by biochemical profiling of human 20S proteasome active site inhibition and specificity using homogeneous assays and selective substrates for each of three catalytic active sites. Importantly, inhibitory activity of thiostrepton differs from bortezomib by blocking both the chymotryptic-like and PGPH active sites with sub-micromolar potencies. Dose-dependent inhibition of multiple myeloma cell growth was observed, with a concomitant increase in polyubiquitinated protein stress and induction of apoptosis. Inhibition of conferred proliferation by bone marrow stroma was confirmed using a novel miniaturized high-content assay modeling the bone marrow stroma-multiple myeloma microenvironment. Structurally related compounds to thiostrepton, nosiheptide and siomycin, were confirmed also as proteasome inhibitors as above. Our discovery of this class of natural products as proteasome inhibitors and a recent report of siomycin inhibition of Sonic Hedgehog (Shh) signaling begged the question whether established proteasome inhibitors would inhibit Shh signaling in human cancer. This hypothesis was confirmed in a set of reporter-gene assays. In sum, these studies identify thiopeptide macrocycles as a class of naturally-occurring proteasome inhibitors poised for clinical development in hematologic malignancies, establish novel high-throughput assays for modeling MM-stroma microenvironment interactions and pave the way for the development of proteasome inhibitors in disease states where Shh signaling is central to pathogenesis.

Axl blockade in vitro and in patients with high-risk MDS by the small molecule inhibitor BGB324.

2017 ◽  
Vol 35 (15_suppl) ◽  
pp. 7059-7059 ◽  
Author(s):  
Sonja Loges ◽  
Isabel Ben Batalla ◽  
Michael Heuser ◽  
Nikolas Berenbrok ◽  
Thomas Schroeder ◽  
...  
Keyword(s):  
Bone Marrow ◽  
High Risk ◽  
Adverse Events ◽  
Small Molecule ◽  
Small Molecule Inhibitor ◽  
Loading Dose ◽  
Bone Marrow Stroma ◽  
Healthy Donors ◽  
Molecule Inhibitor ◽  
Marrow Stroma

7059 Background: The interplay with bone marrow stroma plays an important role in the pathobiology of MDS. Gas6 is secreted by mesenchymal bone marrow stroma cells and promotes survival and therapy resistance of AML cells expressing the Axl receptor. We hypothesized that inhibiting Axl by the small molecule inhibitor BGB324 might hold therapeutic potential in MDS. Methods: We investigated the inhibitory effect of BGB324 on primary bone marrow mononucleated cells (BMMNC) and mesenchymal stroma cells (MSC) from MDS patients in comparison to healthy donors. In the ongoing first-in-patient Phase 1a/b trial BGBC003 A standard 3 + 3 dose escalation study was performed to identify the maximum tolerated dose of BGB324 in patients with previously treated high risk MDS or AML. BGB324 was administered as an oral loading dose on days one and two followed by a reduced daily maintenance. Three dose levels were explored 400/100mg, 600/200mg and 900/300mg. Results: We found that BGB324 inhibited BMMNC from low- and high-risk MDS patients with an IC50 of 2.1 µM and 3.8 µM, respectively (n = 5). In comparison, BMNNC from healthy donors were resistant to BGB324 (IC50 9.4 µM, p < 0.05, n = 10). Axl expression was present in MSC isolated from the BM of MDS patients and BGB324 inhibited the proliferation of MSC from low- and high-risk MDS patients (IC50 2.5 µM and 2.7 µM, respectively; n = 7/5).To date, 3 patients with MDS were treated with 400 mg loading dose and 100 mg maintenance dose of BGB324. Therapy has been well-tolerated and the MTD has not yet been reached. The majority of adverse events reported have been Grade 1 and 2. The most common related adverse events are diarrhea and fatigue. One patient with MDS was treated for 80 weeks and experienced a PR. Evidence of target inhibition was demonstrated by almost complete inhibition of Axl phosphorylation accompanied by reduction in phosphoErk and phosphoAkt signalling at day 21 of treatment. Conclusions: BGB324 is well-tolerated and might represent a promising novel treatment approach in MDS. Safety and efficacy of BGB324 will be explored further in clinical trials. Clinical trial information: NCT02488408.

scholarly journals OP9 Bone Marrow Stroma Cells Differentiate into Megakaryocytes and Platelets

PLoS ONE ◽  
2013 ◽  
Vol 8 (3) ◽  
pp. e58123 ◽  
Author(s):  
Yumiko Matsubara ◽  
Yukako Ono ◽  
Hidenori Suzuki ◽  
Fumio Arai ◽  
Toshio Suda ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Stroma ◽  
Marrow Stroma ◽  
Bone Marrow Stroma Cells

scholarly journals Overexpression of c-maf is a frequent oncogenic event in multiple myeloma that promotes proliferation and pathological interactions with bone marrow stroma

Cancer Cell ◽  
2004 ◽  
Vol 5 (2) ◽  
pp. 191-199 ◽  
Author(s):  
Elaine M Hurt ◽  
Adrian Wiestner ◽  
Andreas Rosenwald ◽  
A.L Shaffer ◽  
Elias Campo ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Bone Marrow Stroma ◽  
Marrow Stroma

scholarly journals The sumoylation pathway is dysregulated in multiple myeloma and is associated with adverse patient outcome

Blood ◽  
2010 ◽  
Vol 115 (14) ◽  
pp. 2827-2834 ◽  
Author(s):  
James J. Driscoll ◽  
Dheeraj Pelluru ◽  
Konstantinos Lefkimmiatis ◽  
Mariateresa Fulciniti ◽  
Rao H. Prabhala ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Mammalian Cells ◽  
Treatment Strategies ◽  
Dominant Negative ◽  
Bone Marrow Stroma ◽  
Stroma Cell ◽  
Bone Marrow Stroma Cell ◽  
Marrow Stroma ◽  
Conjugating Enzyme

Abstract Multiple myeloma (MM) is a plasma cell neoplasm that proceeds through a premalignant state of monoclonal gammopathy of unknown significance; however, the molecular events responsible for myelomagenesis remain uncharacterized. To identify cellular pathways deregulated in MM, we addressed that sumoylation is homologous to ubiquitination and results in the attachment of the ubiquitin-like protein Sumo onto target proteins. Sumoylation was markedly enhanced in MM patient lysates compared with normal plasma cells and expression profiling indicated a relative induction of sumoylation pathway genes. The Sumo-conjugating enzyme Ube2I, the Sumo-ligase PIAS1, and the Sumo-inducer ARF were elevated in MM patient samples and cell lines. Survival correlated with expression because 80% of patients with low UBE2I and PIAS1 were living 6 years after transplantation, whereas only 45% of patients with high expression survived 6 years. UBE2I encodes the sole Sumo-conjugating enzyme in mammalian cells and cells transfected with a dominant-negative sumoylation-deficient UBE2I mutant exhibited decreased survival after radiation exposure, impaired adhesion to bone marrow stroma cell and decreased bone marrow stroma cell–induced proliferation. UBE2I confers cells with multiple advantages to promote tumorigenesis and predicts decreased survival when combined with PIAS1. The sumoylation pathway is a novel therapeutic target with implications for existing proteasomal-based treatment strategies.

Ruxolitinib Does Not Affect Bone Marrow Stromal Cell (BMSC) Clonogenicity and Growth but Interferes with JAK Signaling Under Inflammatory Conditions - Implications for Fibrosis in Myeloproliferative Diseases?

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4096-4096
Author(s):  
Dimitra Zacharaki ◽  
Hongzhe Li ◽  
Roshanak Ghazanfari ◽  
Stefan Scheding
Keyword(s):  
Bone Marrow ◽  
Colony Size ◽  
Bone Marrow Stromal Cell ◽  
Bone Marrow Stroma ◽  
Short Term ◽  
Cytokine Expression Profile ◽  
Marrow Stroma ◽  
Bone Marrow Stroma Cells ◽  
One Way Anova

Abstract The JAK2 V617F gain-of-function mutation is found in hematopoietic cells of nearly all patients with myeloproliferative diseases (MPDs). Ruxolitinib (Ruxo), a potent oral JAK1/JAK2 inhibitor, attenuates cytokine signaling by inhibition of JAK signaling, leading to anti-proliferative and pro-apoptotic effects. In patients with myelofibrosis (MF), effects of Ruxolitinib on splenomegaly, symptom improvement and possibly survival were successfully demonstrated in the two COMFORT studies. Furthermore, resolution of bone marrow fibrosis after long-term treatment with Ruxo was recently reported (Wilkins et al., Haematologica 2013). However, bone marrow stromal cells (BMSC), which contribute to fibrosis, do not harbor the JAK2 mutation or other chromosomal abnormalities that can be found in hematopoietic stem/progenitor cells. Therefore, the current study aimed to investigate possible effects of Ruxo on these non-hematopoietic cells. Bone marrow mononuclear cells were harvested from consenting donors and Ruxo effects on stroma progenitor cells were investigated using the standard CFU-F (colony-forming unit, fibroblast) assay. Our data showed that CFU-F numbers were not decreased by Ruxo at doses ranging from 0.2 to 10 μM (n = 9, p = 0.41, one-way ANOVA), but were even found to be increased at the 5 μΜ dose as indicated by a Ruxo/DMSO ratio of over one (p = 0.04, multiple comparisons test, control-vs-5 μΜ) (Figure 1). In contrast, statistically significant differences were observed in CFU-F colony size with a tendency to decreased sizes with higher doses of Ruxo (p < 0.0001, one-way ANOVA) (Figure 1). Next, we tested if Ruxo affected standard culture-derived bone marrow stromal cell (BMSC) growth, both, in short-term (6 h, 12 h, 24 h and 48 h, n = 3) and in long-term exposure experiments (up to a total of 21 days, n = 3). Neither short-term nor long-term exposure with Ruxo at 0.2 μΜ, 0.5 μΜ, 1 μΜ, 5 μΜ and 10 μΜ caused significant changes of BMSC numbers when compared to their corresponding DMSO controls (p > 0.05, two-way ANOVA), however, a trend to lower BMSC counts with higher Ruxo doses was observed. BMSC were reduced by maximum 25.3±5.7 % (mean ± SD) after 24-hour exposure with 10 μΜ Ruxo. After 21 days drug exposure, ratios of Ruxo-treated BMSC relative to their corresponding DMSO controls were 1.1±0.2, 1.1±0.3, 0.9±0.2, 0.7±0.4%, and 0.6±0.2 for 0.2 μΜ, 0.5 μΜ, 1 μΜ, 5 μΜ and 10 μΜ Ruxo, respectively. These data indicated that Ruxo might have a cytotoxic effect on BMSC, however, only at very high concentrations. We therefore went on to study Ruxo effects on JAK signaling in BMSC after stimulation with IL-6, in order to mimic the inflammatory environment in MPDs. Western blot assays revealed that IL-6 treatment (100 ng/ml) induced pJAK2 in BMSC, which was not detectable in unstimulated BMSC. Furthermore, exposure of IL-6 stimulated BMSC to Ruxo (1 μΜ) diminished pSTAT3 and reduced downstream STAT targets such as pAKT and pMAPK. Furthermore, preliminary results on the cytokine expression profile of supernatants from IL-6-activated BMSC treated with Ruxo showed significant differences compared to controls. Specifically, IL-23 and CCL4 were elevated in Ruxo-treated/IL-6 activated BMSC whereas MCP-1 was strongly reduced compared to the non-Ruxo treated controls. Taken together, these data show that clinically-relevant doses of Ruxo did not affect the clonogenic potential and proliferation of primary marrow stroma cells, indicating that Ruxo most likely has no or little direct effect on the fibrosis-causing cells in MPD. However, Ruxo considerably affects JAK-STAT signaling in activated BMSCs, leading to an altered cytokine expression profile which potentially contributes to the amelioration of fibrosis, and, accordingly, ongoing experiments address this question. Figure 1. Effects of increasing doses of Ruxo on a) clonogenic bone marrow stroma cells expressed as ratio of the number of Ruxo-treated CFU-F and corresponding DMSO controls, and b) colony size (*p < 0.05, **p < 0.01, ***p < 0.001) Figure 1. Effects of increasing doses of Ruxo on a) clonogenic bone marrow stroma cells expressed as ratio of the number of Ruxo-treated CFU-F and corresponding DMSO controls, and b) colony size (*p < 0.05, **p < 0.01, ***p < 0.001) Disclosures No relevant conflicts of interest to declare.

Sign in / Sign up

Export Citation Format

Share Document