Bortezomib Reverts Stroma-Mediated Resistant to APO2L/TRAIL in Multiple Myeloma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2806-2806
Author(s):  
Nancy Parquet ◽  
Mark Mead ◽  
Claudio Anasetti ◽  
William S. Dalton ◽  
Lia Perez
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cancer Cells ◽  
Induced Resistance ◽  
Soluble Factor ◽  
Bone Marrow Stroma ◽  
Myeloma Cells ◽  
Bortezomib Treatment ◽  
Trail Receptors ◽  
Marrow Stroma

Abstract Abstract 2806 Poster Board II-782 Hematopoietic stroma provides a sanctuary for multiple myeloma (MM) cells and promotes resistance to immune control mediated by APO2L/TRAIL in part by increasing synthesis of the anti-apoptotic protein c-FLIP (J Immunol, 180: 3; 2008). Bortezomib, a reversible 26S proteosome inhibitor, sensitizes cancer cells to APO2L/TRAIL induced apoptosis in a variety of malignancies including MM. In addition, it has been well established that bortezomib interferes with the protective interaction between cancer cells and the bone marrow stroma. In this study we tested whether bortezomib can reverse the APO2L/TRAIL environmental mediated-immune resistant (EM-IR). Using MM cell lines (RPMI 8226 and U266) or CD138+ positive selected cells from MM patient's bone marrow, we found that exposure to HS5 stroma cells by direct adhesion or in a transwell system induced resistance to bortezomib (10nM for 24 hours) and that pretreatment with bortezomib (10nM for 20 hours) effectively overcomes APO2L/TRAIL resistance (10ng/mL for 4 additional hours). Conditioned medium made from a 14-day culture of MM patient's bone marrow stroma induced resistance to bortezomib and pretreatment with bortezomib sensitized cells to APO2L/TRAIL induced cell death as shown with HS5 cells. Bortezomib showed not to be cytotoxic to HS5 stroma cells and only MCP-2-3 and IL-10 levels were altered in the stroma-MM milieu within cytokines measured by ELISA array. IL-6, a cytokine shown to induce APO2L/TRAIL mediated resistance, remained unchanged with bortezomib treatment. We found that bortezomib increased expression of TRAIL receptors (DR5, DCR1 and DCR2), but such expression did not predict for sensitivity to apoptosis as DR4 demonstrated to be the receptor responsible for activation of APO2L/TRAIL. Soluble factor(s) released by HS5 stroma increased expression of c-FLIP and induced STAT-3 and ERK phosphorylation in myeloma cells. However, only c-FLIP protein expression was effectively reduced by bortezomib. c-FLIP quantitative-PCR found that HS5 increased transcription only after 20 hours bortezomib treatment and significantly reduces soluble factor(s) induced c-FLIP transcription. In a transwell assay, HS5 stroma cells induced NF-κB activation and the addition of bortezomib diminished its activation at 20 hours. These findings provide the rationale to combine bortezomib and APO2L/TRAIL to disrupt the influence of the stroma microenvironment on myeloma cells. Disclosures: Off Label Use: sirolimus for graft-versus-host disease.

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.

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.

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.

Exchange of microRNAs between Breast Cancer Cells and Bone Marrow Stroma: Potential for Avenues To Revisit Hematopoietic Stem Cell Transplantation for Breast and Other Cancers That Show Preference for Bone Marrow.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1199-1199
Author(s):  
Marcelo Taborga ◽  
Pranela Rameshwar
Keyword(s):  
Breast Cancer ◽  
Bone Marrow ◽  
Gap Junctions ◽  
Cancer Cells ◽  
Breast Cancer Cells ◽  
Cancer Metastasis ◽  
Luciferase Activity ◽  
Bone Marrow Stroma ◽  
Cell Type ◽  
Marrow Stroma

Abstract Previous failure of autologous bone marrow transplantation as a viable treatment of breast cancer opens the field for investigational studies since the bone marrow, as a source of cancer metastasis, remains a clinical dilemma. This study hypothesizes that a subset of breast cancer cells enter the bone marrow at an early period of disease development, perhaps prior to clinical detection, where they evade chemotherapeutic damage even at high doses. An understanding of this cell population might be crucial for the future success of bone marrow transplants in patients suffering from breast cancer metastasis to the bone marrow. In vivo and in vitro studies have demonstrated gap junctions between bone marrow stroma and breast cancer cells. The gap junctions can passage CFDA cell tracer between stroma and cancer cells. This exchange can be blocked by the gap junctional intercellular communication inhibitor, 1-Octanol, suggesting functional gap junctions. We have now implemented quantitative studies to determine the role of specific microRNAs (miRNAs) in blunting of mRNA translation of the Tac1 gene in cancer cells. The first set of studies optimized the effects of miRNAs, 130a and 206 in the suppression of Tac1 mRNA in the low invasive T47D and the aggressive MDA-MB-231 breast cancer cell lines. The miRNAs were transfected into cells containing the pMIR-R/Tac1/SG vector, which includes the 3′ UTR of Tac1 mRNA. Luciferase activity in cell lysates showed synergism between the two miRNAs, indicating that the predicted miRNAs can inhibit Tac1 translation. The next set of studies determined whether miRNAs 130a and 206 can cross gap junctions between stroma and cancer cells to suppress Tac1 mRNA. The miRNAs were transfected in one cell type and pMIR-R/Tac1/SG in the other cell type. Luciferase activity and parallel studies with 1-Octanol verified movement of miRNAs via the gap junction. These studies recapitulate the downregulation of endogenous Tac1 and other cytokines in the cancer cells. In summary, this study shows a role for miRNA exchange, between bone marrow stroma and breast cancer cells, as a mechanism by which the cancer cells might evade detection and current methods of treatment. An understanding of these mechanisms might lead to new methods to remove cancer from bone marrow in future therapies.

Soluble IL-6 Receptor Synergistically Stimulates the Growth of Multiple Myeloma with IL-6 and Confers Resistance to Bortezomib.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4741-4741
Author(s):  
Young Y. Lee ◽  
Joowon Park ◽  
Sung-Soo Yoon ◽  
Jung H. Choi ◽  
Byoung B. Park ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Proinflammatory Cytokines ◽  
Combined Treatment ◽  
Control Group ◽  
Myeloma Cells ◽  
Bortezomib Treatment ◽  
Anti Cancer ◽  
Apoptotic Effect ◽  
Bone Marrow Plasma

Abstract Proinflammatory cytokines including Interleukin-6 (IL-6), TNFa, and IL-8, involved in a variety of cellular responses including proliferation, differentiation, and inflammatory processes. They play a pivotal role in the pathogenesis of multiple myeloma (MM). Elevated IL-6 and souble IL-6 receptor (sIL-6R) levels have been found in MM patients indicating that its production is not only coordinated as part of a disease progression but also strongly associated with outcome of anti-cancer therapy. Even though Bortezomib, one of proteosome inhibitors, is known to effectively induce the apoptosis of U266 myeloma cells via caspase-dependent pathway and inactivation of NF-kB, these apoptotic effects by Bortezomib could be decreased in U266 myeloma cells exposed to various proinflammatory cytokines. However, roles of IL-6 and sIL-6R in the progression of MM and response to anti-cancer treatment still remain to be explored. In this study, ELISA assay showed higher levels of IL-6, sIL-6R, and IL-8 in bone marrow plasma of MM patients compared to control group, which is the similar results of other investigators. Bortezomib-induced apoptotic effect in U266 myeloma cells was reduced by treatment of IL-6. This Bortezomib-induced apoptotic effect was more reduced in U266 myeloma cells following combined treatment with IL-6 and sIL-6R, although sIL-6R only did not exert on apoptotic effect. Caspase-3 activity by Bortezomib treatment decreased when U266 cells exposed to combined IL-6 and sIL-6R. In addition, U266 cells treated with combined IL-6 and sIL-6R synergically activated the phosphorylation of STAT-3 and ERK resulting in prolong survival of U266 cells. Phosphorylation of STAT-3 and ERK in U266 myeloma cells following combined treatment of IL-6 and sIL-6R did not affect by Bortexomib treatment. These results suggest that the levels of IL-6 and sIL-6R in bone marrow plasma may strongly be associated with clinical outcome of Bortezomib treatment in MM patients.

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