Bone Marrow Stroma Confers Resistance to Apo2 Ligand/TRAIL in Multiple Myeloma in Part by Regulating c-FLIP

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.

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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 ◽

10.1182/blood.v122.21.3149.3149 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3149-3149 ◽

Cited By ~ 3

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.

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

Cancer Cell ◽

10.1016/s1535-6108(04)00019-4 ◽

2004 ◽

Vol 5 (2) ◽

pp. 191-199 ◽

Cited By ~ 230

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

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

Blood ◽

10.1182/blood-2009-03-211045 ◽

2010 ◽

Vol 115 (14) ◽

pp. 2827-2834 ◽

Cited By ~ 64

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.

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

Blood ◽

10.1182/blood.v114.22.2806.2806 ◽

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.

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Abstract 1518: Multiple myeloma cells cross talk with bone marrow stroma lead to induction of DKK1 expression and produce bortezomib resistance

10.1158/1538-7445.am2012-1518 ◽

2012 ◽

Author(s):

Jiyeon Ryu ◽

Woo-june Jung ◽

Chansu Lee ◽

Soyoung Park ◽

Juwon Park ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Cross Talk ◽

Bone Marrow Stroma ◽

Myeloma Cells ◽

Dkk1 Expression ◽

Marrow Stroma

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Human bone marrow stroma-dependent cell line MOLP-5 derived from a patient in leukaemic phase of multiple myeloma

British Journal of Haematology ◽

10.1046/j.1365-2141.2000.01985.x ◽

2000 ◽

Vol 109 (1) ◽

pp. 54-63 ◽

Cited By ~ 14

Author(s):

Yoshinobu Matsuo ◽

Hans G. Drexler ◽

Chiharu Nishizaki ◽

Akira Harashima ◽

Shunichi Fukuda ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Cell Line ◽

Human Bone ◽

Human Bone Marrow ◽

Bone Marrow Stroma ◽

Dependent Cell ◽

Marrow Stroma

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Preclinical studies of the pan-Bcl inhibitor obatoclax (GX015-070) in multiple myeloma

Blood ◽

10.1182/blood-2006-10-047951 ◽

2007 ◽

Vol 109 (12) ◽

pp. 5430-5438 ◽

Cited By ~ 152

Author(s):

Suzanne Trudel ◽

Zhi Hua Li ◽

Jennifer Rauw ◽

Rodger E. Tiedemann ◽

Xiao Yan Wen ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Single Agent ◽

Myeloma Cell ◽

Bone Marrow Stroma ◽

Cytochrome C Release ◽

Marrow Stroma ◽

Combination Studies ◽

Binding Groove ◽

Cytotoxic Responses

Abstract Bcl family members Bcl-2, Bcl-xL, and Mcl-1, are frequently expressed and implicated in the survival of myeloma cells. Obatoclax (GX015-070) is a novel, small-molecule antagonist of the BH3-binding groove of the Bcl family of proteins. We show that GX015-070 inhibits the binding of Bak to Mcl-1, up-regulates Bim, induces cytochrome c release, and activates capase-3 in human myeloma cell lines (HMCLs), confirming the predicted mechanism of action. Consequently, GX015-070 potently inhibited the viability of 15 of 16 HMCLs (mean IC50 of 246 nM), including those resistant to melphalan and dexamethasone. In combination studies, GX015-070 enhanced the antimyeloma activity induced by melphalan, dexamethasone, or bortezomib. Sensitivity to GX015-070 correlated with the absence or near absence of Bcl-xL. Coculture with interleukin-6 or adherence to bone marrow stroma conferred modest resistance; however, it did not overcome GX015-070–induced cytotoxicity. Of importance, GX015-070 as a single agent induced potent cytotoxic responses against patient-derived tumor cells. GX015-070 inhibited normal bone marrow–derived colony formation; however, cytotoxicity to human blood lymphocytes was not observed. Taken together, these studies describe a novel BH3 mimic with selectivity for Mcl-1, and support the therapeutic application of GX015-070 for diverse neoplasias including multiple myeloma.

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Ectopic expression of BIRC5-targeting miR-101-3p overcomes bone marrow stroma-mediated drug resistance in multiple myeloma cells

BMC Cancer ◽

10.1186/s12885-019-6151-x ◽

2019 ◽

Vol 19 (1) ◽

Cited By ~ 5

Author(s):

Jahangir Abdi ◽

Nasrin Rastgoo ◽

Yan Chen ◽

Guo An Chen ◽

Hong Chang

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Drug Resistance ◽

Ectopic Expression ◽

Luciferase Assay ◽

Bone Marrow Stroma ◽

Plate Reader ◽

Marrow Stroma ◽

Direct Target ◽

Fluorescence Plate Reader

Abstract Background Multiple myeloma (MM) cells gain protection against drugs through interaction with bone marrow stromal cells (BMSCs). This form of resistance largely accounts for resistance to therapy in MM patients which warrants further exploration to identify more potential therapeutic targets. Methods We performed miRNA/mRNA qPCR arrays and western blotting to analyze transcriptional and translational changes in MM cells co-cultured with BMSCs. Drug cytotoxicity and apoptosis in MMGFP-BMSC co-cultures were measured using fluorescence plate reader and flowcytometry, respectively. miRNA was overexpressed in MM cell lines using Lentiviral transduction, miRNA-3’UTR binding was examined using luciferase assay. Results We found that BMSCs downregulated miR-101-3p and upregulated survivin (BIRC5) in MM cells. Survivin was downregulated by miR-101-3p overexpression and found to be a direct target of miR-101-3p using 3’UTR luciferase assay. Overexpression of survivin increased viability of MM cells in the presence of anti-myeloma drugs, and miR-101-3p inhibition by anti-miR against miR-101-3p upregulated survivin. Furthermore, overexpression of miR-101-3p or silencing of survivin triggered apoptosis in MM cells and sensitized them to anti-myeloma drugs in the presence of BMSCs overcoming the stroma-induced drug resistance. Conclusions Our study demonstrates that BMSC-induced resistance to drugs is associated with survivin upregulation which is a direct target of miR-101-3p. This study also identifies miR-101-3p-survivin interaction as a druggable target involved in stroma-mediated drug resistance in MM and suggests it for developing more efficient therapeutic strategies.

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