IL-3 expression by myeloma cells increases both osteoclast formation and growth of myeloma cells

Blood ◽  
2004 ◽  
Vol 103 (6) ◽  
pp. 2308-2315 ◽  
Author(s):  
Jun Won Lee ◽  
Ho Yeon Chung ◽  
Lori A. Ehrlich ◽  
Diane F. Jelinek ◽  
Natalie S. Callander ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Destruction ◽  
Osteoclast Formation ◽  
Mrna Levels ◽  
Myeloma Cells ◽  
Protein Levels ◽  
Inflammatory Protein ◽  
Receptor Activator ◽  
Bone Marrow Plasma ◽  
Human Osteoclast

Abstract Macrophage inflammatory protein–1α (MIP-1α) gene expression is abnormally regulated in multiple myeloma (MM) owing to imbalanced expression of the acute myeloid leukemia–1A (AML-1A) and AML-1B transcription factors. We hypothesized that the increased expression ratios of AML-1A to AML-1B also induced abnormal expression of other hematopoietic and bone-specific genes that contribute to the poor prognosis of MM patients with high levels of MIP-1α. We found that interleukin-3 (IL-3) was also induced by the imbalanced AML-1A and AML-1B expression in myeloma. IL-3 mRNA levels were increased in CD138+ purified myeloma cells with increased AML-1A–to–AML-1B expression from MM patients, and IL-3 protein levels were significantly increased in freshly isolated bone marrow plasma from MM patients (66.4 ± 12 versus 22.1 ± 8.2 pg/mL; P = .038). IL-3 in combination with MIP-1α or receptor activator of nuclear factor–kappa B ligand (RANKL) significantly enhanced human osteoclast (OCL) formation and bone resorption compared with MIP-1α or RANKL alone. IL-3 stimulated the growth of interleukin-6 (IL-6)–dependent and IL-6–independent myeloma cells in the absence of IL-6, even though IL-3 did not induce IL-6 expression by myeloma cells. These data suggest that increased IL-3 levels in the bone marrow microenvironment of MM patients with imbalanced AML-1A and AML-1B expression can increase bone destruction and tumor cell growth.

scholarly journals Overexpression of γ-Glutamyltransferase in Transgenic Mice Accelerates Bone Resorption and Causes Osteoporosis

Endocrinology ◽  
2007 ◽  
Vol 148 (6) ◽  
pp. 2708-2715 ◽  
Author(s):  
Kiyoshi Hiramatsu ◽  
Yutaro Asaba ◽  
Sunao Takeshita ◽  
Yuji Nimura ◽  
Sawako Tatsumi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Resorption ◽  
Transgenic Mice ◽  
Bone Destruction ◽  
Nuclear Factor Κb ◽  
Osteoclast Formation ◽  
Expression Cloning ◽  
Nuclear Factor ◽  
Receptor Activator

We previously identified γ-glutamyltransferase (GGT) by expression cloning as a factor inducing osteoclast formation in vitro. To examine its pathogenic role in vivo, we generated transgenic mice that overexpressed GGT in a tissue-specific manner utilizing the Cre-loxP recombination system. Systemic as well as local production of GGT accelerated osteoclast development and bone resorption in vivo by increasing the sensitivity of bone marrow macrophages to receptor activator of nuclear factor-κB ligand, an essential cytokine for osteoclastogenesis. Mutated GGT devoid of enzyme activity was as potent as the wild-type molecule in inducing osteoclast formation, suggesting that GGT acts not as an enzyme but as a cytokine. Recombinant GGT protein increased receptor activator of nuclear factor-κB ligand expression in marrow stromal cells and also stimulated osteoclastogenesis from bone marrow macrophages at lower concentrations. Thus, GGT is implicated as being involved in diseases characterized by accelerated osteoclast development and bone destruction and provides a new target for therapeutic intervention.

Macrophage inflammatory protein-1α is an osteoclastogenic factor in myeloma that is independent of receptor activator of nuclear factor κB ligand

Blood ◽  
2001 ◽  
Vol 97 (11) ◽  
pp. 3349-3353 ◽  
Author(s):  
Je-Ho Han ◽  
Sun Jin Choi ◽  
Noriyoshi Kurihara ◽  
Masanori Koide ◽  
Yasuo Oba ◽  
...  
Keyword(s):  
Stromal Cells ◽  
Nuclear Factor Κb ◽  
Marrow Stromal Cells ◽  
Dependent Manner ◽  
Nuclear Factor ◽  
Myeloma Cells ◽  
Parathyroid Hormone Related Protein ◽  
Inflammatory Protein ◽  
Receptor Activator ◽  
Macrophage Inflammatory Protein

A complementary DNA expression library derived from marrow samples from myeloma patients was recently screened and human macrophage inflammatory protein-1α (hMIP-1α) was identified as an osteoclastogenic factor expressed in these samples. hMIP-1α enhanced osteoclast (OCL) formation in human marrow cultures and by highly purified OCL precursors in a dose-dependent manner (5-200 pg/mL). Furthermore, hMIP-1α enhanced OCL formation induced by human interleukin-6 (IL-6), which is produced by marrow stromal cells when they interact with myeloma cells. hMIP-1α also enhanced OCL formation induced by parathyroid hormone-related protein (PTHrP) and receptor activator of nuclear factor κB ligand (RANKL), factors also implicated in myeloma bone disease. Time-course studies revealed that the hMIP-1α acted during the last 2 weeks of the 3-week culture period. Reverse transcription–polymerase chain reaction analysis showed that the chemokine receptors for hMIP-1α (CCR1 and CCR5) were expressed by human bone marrow and highly purified early OCL precursors. Furthermore, hMIP-1α did not increase expression of RANKL. These data demonstrate that hMIP-1α is an osteoclastogenic factor that appears to act directly on human OCL progenitors and acts at the later stages of OCL differentiation. These data further suggest that in patients with myeloma, MIP-1α produced by myeloma cells, in combination with RANKL and IL-6 that are produced by marrow stromal cells in response to myeloma cells, enhances OCL formation through their combined effects on OCL precursors.

scholarly journals Role for macrophage inflammatory protein (MIP)-1α and MIP-1β in the development of osteolytic lesions in multiple myeloma

Blood ◽  
2002 ◽  
Vol 100 (6) ◽  
pp. 2195-2202 ◽  
Author(s):  
Masahiro Abe ◽  
Kenji Hiura ◽  
Javier Wilde ◽  
Keiji Moriyama ◽  
Toshihiro Hashimoto ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Resorption ◽  
Stromal Cells ◽  
Neutralizing Antibodies ◽  
Bone Cells ◽  
Osteoclast Differentiation ◽  
Bone Destruction ◽  
Myeloma Cells ◽  
Inflammatory Protein ◽  
Macrophage Inflammatory Protein

Abstract Multiple myeloma (MM) cells cause devastating bone destruction by activating osteoclasts in the bone marrow milieu. However, the mechanism of enhanced bone resorption in patients with myeloma is poorly understood. In the present study, we investigated a role of C-C chemokines, macrophage inflammatory protein (MIP)–1α and MIP-1β, in MM cell-induced osteolysis. These chemokines were produced and secreted by a majority of MM cell lines as well as primary MM cells from patients. Secretion of MIP-1α and MIP-1β correlated well with the ability of myeloma cells to enhance osteoclastic bone resorption both in vitro and in vivo as well as in MM patients. In osteoclastogenic cultures of rabbit bone cells, cocultures with myeloma cells as well as addition of myeloma cell-conditioned media enhanced both formation of osteoclastlike cells and resorption pits to an extent comparable to the effect of recombinant MIP-1α and MIP-1β. Importantly, these effects were mostly reversed by neutralizing antibodies against MIP-1α and MIP-1β, or their cognate receptor, CCR5, suggesting critical roles of these chemokines. We also demonstrated that stromal cells express CCR5 and that recombinant MIP-1α and MIP-1β induce expression of receptor activator of nuclear factor-κB (RANK) ligand by stromal cells, thereby stimulating osteoclast differentiation of preosteoclastic cells. These results suggest that MIP-1α and MIP-1β may be major osteoclast-activating factors produced by MM cells.

Cell–cell contact between marrow stromal cells and myeloma cells via VCAM-1 and α4β1-integrin enhances production of osteoclast-stimulating activity

Blood ◽  
2000 ◽  
Vol 96 (5) ◽  
pp. 1953-1960 ◽  
Author(s):  
Toshimi Michigami ◽  
Nobuaki Shimizu ◽  
Paul J. Williams ◽  
Maria Niewolna ◽  
Sarah L. Dallas ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Line ◽  
Stromal Cells ◽  
Neutralizing Antibodies ◽  
Bone Destruction ◽  
Marrow Stromal Cells ◽  
Cell Contact ◽  
Tartrate Resistant Acid Phosphatase ◽  
Myeloma Cells ◽  
Bone Resorbing Activity

Abstract Myeloma is a unique hematologic malignancy that exclusively homes in the bone marrow and induces massive osteoclastic bone destruction presumably by producing cytokines that promote the differentiation of the hematopoietic progenitors to osteoclasts (osteoclastogenesis). It is recognized that neighboring bone marrow stromal cells influence the expression of the malignant phenotype in myeloma cells. This study examined the role of the interactions between myeloma cells and neighboring stromal cells in the production of osteoclastogenic factors to elucidate the mechanism underlying extensive osteoclastic bone destruction. A murine myeloma cell line 5TGM1, which causes severe osteolysis, expresses α4β1-integrin and tightly adheres to the mouse marrow stromal cell line ST2, which expresses the vascular cell adhesion molecule-1 (VCAM-1), a ligand for α4β1-integrin. Co-cultures of 5TGM1 with primary bone marrow cells generated tartrate-resistant acid phosphatase-positive multinucleated bone-resorbing osteoclasts. Co-cultures of 5TGM1 with ST2 showed increased production of bone-resorbing activity and neutralizing antibodies against VCAM-1 or α4β1-integrin inhibited this. The 5TGM1 cells contacting recombinant VCAM-1 produced increased osteoclastogenic and bone-resorbing activity. The activity was not blocked by the neutralizing antibody to known osteoclastogenic cytokines including interleukin (IL)-1, IL-6, tumor necrosis factor, or parathyroid hormone-related peptide. These data suggest that myeloma cells are responsible for producing osteoclastogenic activity and that establishment of direct contact with marrow stromal cells via α4β1-integrin/VCAM-1 increases the production of this activity by myeloma cells. They also suggest that the presence of stromal cells may provide a microenvironment that allows exclusive colonization of myeloma cells in the bone marrow.

Requirement of the E2F3 Transcription Factor for BCR/ABL Leukemogenesis.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 33-33
Author(s):  
Anna M. Eiring ◽  
Paolo Neviani ◽  
Ramasamy Santhanam ◽  
Joshua J. Oaks ◽  
Ji Suk Chang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Transcription Factor ◽  
Blast Crisis ◽  
Scid Mice ◽  
Myelogenous Leukemia ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Hnrnp A1 ◽  
Protein Levels ◽  
Abl Kinase

Abstract Several RNA binding proteins (RBPs) have been implicated in the progression of chronic myelogenous leukemia (CML) from the indolent chronic phase to the aggressively fatal blast crisis. In the latter phase, expression and function of specific RBPs are altered at transcriptional or post-translational levels by the increased constitutive kinase activity of the BCR/ABL oncoprotein, resulting in enhanced resistance to apoptotic stimuli, growth advantage and differentiation arrest of CD34+ CML blast crisis (CML-BC) progenitors. In the current study, we identified by RIP (RNA immunoprecipitation)-mediated microarray analysis that mRNA encoding the E2F3 transcription factor associates to the BCR/ABL-regulated RBP hnRNP A1. Moreover, RNA electrophoretic mobility shift and UV-crosslinking assays revealed that hnRNP A1 interacts with E2F3 mRNA through a binding site located in the 3’UTR of both human and mouse E2F3 mRNA. Accordingly, E2F3 protein levels were upregulated in BCR/ABL-transformed myeloid precursor cell lines compared to parental cells in a BCR/ABL-kinase- and hnRNP A1 shuttling-dependent manner. In fact, treatment of BCR/ABL-expressing myeloid precursors with the kinase inhibitor Imatinib (2mM, 24 hr) or introduction of a dominant-negative shuttling-deficient hnRNP A1 protein (NLS-A1) markedly reduced E2F3 protein and mRNA levels. Similarly, upregulation of BCR/ABL expression/activity in the doxycycline inducible TonB2.10 cell line resulted in increased E2F3 protein expression. BCR/ABL kinase-dependent induction of E2F3 protein levels was also detected in CML-BCCD34+ compared to CML-CPCD34+ progenitors from paired patient samples and to normal CD34+ bone marrow samples. Importantly, the in vitro clonogenic potential of primary mouse BCR/ABL+ lineage negative (Lin−) progenitors was markedly impaired in BCR/ABL+ E2F3−/− compared to BCR/ABL-transduced E2F3+/+ myeloid progenitors and upon shRNA-mediated downregulation of E2F3 expression (90% inhibition, P<0.001). Furthermore, subcutaneous injection of shE2F3-expressing BCR/ABL+ cells into SCID mice markedly impaired in vivo tumorigenesis (>80% reduction in tumor burden, P<0.01). Accordingly, BCR/ABL leukemogenesis was strongly inhibited in SCID mice intravenously injected with E2F3 shRNA-expressing 32D-BCR/ABL cells and in mice transplanted with BCR/ABL-transduced Lin− bone marrow cells from E2F3−/− mice. Specifically, we demonstrate that reduced or absent levels of E2F3 resulted in dramatically decreased numbers of circulating BCR/ABL+ cells as determined by nested RT-PCR at 4 weeks post-injection (P=0.0001), normal splenic architecture and bone marrow cellularity and the absence of infiltrating myeloid blasts into non-hematopoietic compartments (i.e. liver). By contrast, SCID mice transplanted with vector-transduced 32D-BCR/ABL cells or BCR/ABL+ E2F3+/+ Lin− BM progenitors showed signs of an overt acute leukemia-like process with blast infiltration of hematopoietic and non-hematopoietic organs. Altogether, these data outline the importance of E2F3 expression for BCR/ABL leukemogenesis and characterize a new potential therapeutic target for the treatment of patients with advanced phase CML.

P53 is Activated without RPL11 Upregulation in Diamond-Blackfan Anemia,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3432-3432
Author(s):  
Hong-Yan Du ◽  
M. Tarek Elghetany ◽  
Blanche P Alter ◽  
Akiko Shimamura
Keyword(s):  
Bone Marrow ◽  
Ribosomal Protein ◽  
Ribosomal Proteins ◽  
Mrna Levels ◽  
Protein Levels ◽  
Diamond Blackfan Anemia ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
P53 Activation ◽  
Ribosomal Stress

Abstract Abstract 3432 Diamond-Blackfan anemia (DBA) is an autosomal dominantly inherited bone marrow failure syndrome characterized by red cell aplasia, physical anomalies, and cancer predisposition. DBA is caused by mutations resulting in haploinsufficiency of genes encoding ribosomal proteins. p53 is activated in the erythroid lineage following reduction of ribosomal protein expression; however the mechanism whereby ribosomal stress results in p53 activation in DBA remains unclear. RPL11 has been proposed to play a central role in p53 activation following ribosomal stress. Reduced expression of individual small ribosomal subunit proteins in a tumor cell line resulted in increased translation of RPL11. Excess free RPL11 can bind and inactivate HDM2, an E3 ubiquitin ligase targeting p53 for degradation. The recent demonstration that cellular responses to ribosomal perturbations vary widely between different tissues raised the question of whether RPL11 upregulation contributes to p53 activation following ribosomal stress in hematopoietic progenitors. To address this question, we modeled DBA in human CD34+ cells. Since RPS19 is the most commonly mutated gene in DBA, we used lentiviral vectors expressing short hairpin RNAs to knock down RPS19 expression in primary human CD34+ cells. RPS19 protein levels were reduced to about 50% of control levels in a manner reflecting the haploinsufficient state in DBA. RPS19 depletion resulted in elevated p53 protein levels and increased mRNA levels of p21, a transcriptional target of p53. Total p53 mRNA levels and p53 mRNA translational activity remained unchanged consistent with a post-transcriptional mechanism for p53 activation. Although total RPL11 mRNA levels were not diminished following RPS19 depletion, RPL11 protein levels were significantly decreased consistent with post-transcriptional downregulation. Depletion of RPS19 in human CD34+ cells did not affect polysome loading of RPL11 mRNA. Reduction of additional ribosomal proteins also accompanied RPS19 knockdown consistent with coordinate regulation of multiple ribosomal protein levels. Corticosteroids, which improve anemia in the majority of DBA patients, did not prevent p53 activation, nor did this improve RPS19 or RPL11 protein levels. Expression of p53 was also assessed in bone marrow biopsy slides from 26 DBA patients with the following genotypes: RPS19 (18), RPS24 (2), RPS26 (2), RPS10 (1), RPS17 (1), RPS7 (1), and RPL11 (1). p53 was over-expressed in all but one patient (RPS26), and was clearly over-expressed in the DBA patient harboring the RPL11 mutation. In summary, we find that p53 activation in DBA does not require upregulation of RPL11 translation or elevated RPL11 protein levels. p53 activation persists in DBA caused by RPL11 deficiency. Corticosteroids do not improve ribosomal protein levels nor do they prevent p53 activation. Disclosures: No relevant conflicts of interest to declare.

Mir-23b Plays a Critical Role As a Tumor Suppressor miRNA In Multiple Myeloma

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 122-122 ◽  
Author(s):  
Mariateresa Fulciniti ◽  
Nicola Amodio ◽  
Rajya Bandi ◽  
Rao H. Prabhala ◽  
Sophia Adamia ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Proliferation ◽  
Transcription Factor ◽  
Tumor Suppressor ◽  
Critical Role ◽  
Luciferase Reporter ◽  
Mrna Levels ◽  
Myeloma Cells ◽  
Equity Ownership

Abstract Deregulated expression of microRNAs (miR) is a hallmark of cancer. Tumor suppressor miRNAs are generally down-regulated in cancer cells compared to their normal counterpart, and their enforced expression indeed represents a promising strategy for cancer treatment. We have found miR-23b to be downregulated in CD138+ myeloma cells from 38 multiple myeloma (MM) patients and 18 plasma cell leukemia (PCL) patients compared to normal PCs. Decreased expression of miR-23b was further confirmed in an independent dataset of 66 MM patients by TaqMan miRNA assays. The downregulation of miR-23b expression was also observed in several myeloma cells lines when compared with PBMC and BMSC. Interestingly, interaction of BMSC with MM cells resulted in further decrease in miR-23b expression in both cell types. Moreover, Interleukin-6 (IL-6) also suppressed the expression of miR-23b in a time- and dose- dependent pattern, indicating that the human bone marrow microenvironment (huBMM) modulates miR-23b levels. miR-23b is commonly repressed in autoimmune conditions by IL-17, a cytokine shown to promote myeloma cell growth and inhibit its immune function. We have indeed observed further decrease in miR-23b expression in MM cells after IL-17 treatment for 24 hours. We have also observed downregulation of miR-23b in CD19+ Waldenstrom’s Macroglobulinemia (WM) cells compared to CD19+ B cells from healthy donors, which was further decreased in the presence of components of the WM bone marrow milieu. We further assessed the functional significance of miR-23b by both gain- and loss-of-function studies. A significant decrease in cell proliferation and survival, along with induction of caspase 3/7 activity was observed over time in miR-23b mimic–transfected myeloma (H929, KMS11) and WM cell lines (MWCL1) with low miR-23b expression. At the molecular level, we have identified Sp1, a transcription factor endowed with oncogenic activity in MM and WM, as a target of miR-23b. Expression of miR-23b decreased Sp1 mRNA levels via 3’UTR binding, as assessed in luciferase reporter assays. On the other hand, genetic and/or pharmacological inhibition of Sp1 led to miR-23b upregulation, thus highlighting the occurrence of a feedback loop between miR-23b and its target. Of note, miR-23b transfection significantly reduced Sp1-driven NF-kB activity in MM and WM cells. Finally, c-Myc, an important oncogenic transcription factor known to stimulate MM cell proliferation, has been shown to transcriptionally repress miR-23b. Moreover, treatment with the demethylating agent 5-aza-deoxycitidine significantly increase the expression of miR-23b in MM1S and KMS-11 cells suggesting that promoter methylation may be an additional mechanism of miR-23b suppression in myeloma. Thus MYC-dependent miR-23b repression in myeloma cells may allow activation of oncogenic transcription factors Sp1 and NF-κB, representing the first feed forward loop with critical growth and survival role in myeloma. Taken together, these data support a model in which the humoral environment reduces miR-23b expression in tumor cells, suggesting a tumor suppressor role in MM and WM and highlighting the potential of a miR-23b-based replacement therapy to treat these hematologic malignancies. Disclosures: Anderson: gilead: Consultancy; onyx: Consultancy; celgene: Consultancy; sanofi aventis: Consultancy; oncopep: Equity Ownership; acetylon: Equity Ownership.

Myeloma Cells Switch Osteoblastogenesis to Adipogenesis and Suppress Bone Formation

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3386-3386 ◽  
Author(s):  
Jing Yang ◽  
Zhiqiang Liu ◽  
Huan Liu ◽  
Jin He ◽  
Pei Lin ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Formation ◽  
Osteoblast Differentiation ◽  
Conflicts Of Interest ◽  
Bone Destruction ◽  
Current Treatment ◽  
New Bone Formation ◽  
Myeloma Cells ◽  
Dickkopf 1

Abstract Bone destruction is a hallmark of myeloma, and has a severe impact on patients’ quality of life and survival. Unfortunately, current treatment only offers moderate palliative effects, and this disease remains incurable. The bone changes in myeloma patients results from increased osteoclast-mediated bone resorption and decreased osteoblast-mediated bone formation. In particular, new bone formation that usually occurs at sites of previously resorbed bones is deeply suppressed; as a result, areas of bone destruction rarely heal. Previous studies have shown that myeloma cells inhibit osteoblast differentiation from mesenchymal stem cells (MSCs), and the Wnt/b-catenin signaling pathway is suppressed via myeloma-produced Wnt antagonists such as dickkopf-1. However, the role of dickkopf-1 in myeloma-induced inhibition of bone formation remains controversial since myeloma cells alone do not produce sufficient dickkopf-1 to suppress osteoblast differentiation. In addition, the administration of an antibody against dickkopf-1 in myeloma patients failed to restore new bone formation, indicating there must be an additional mechanism for inhibition of osteoblast differentiation seen in myeloma. While MSCs can differentiate into mature osteoblasts, they are also capable of differentiating into adipocytes, which is a major cell type in marrow stroma. We observed that myeloma cells (cell lines and primary cells isolated from myeloma patients’ bone marrow) injected into human or mouse bone not only reduced osteoblast number, but also increased adipocyte number and activity in bone marrow. Similar observations were seen in the clinical setting where collections of adipocytes were found in the bone marrow of newly diagnosed, untreated myeloma patients. Patients with greater bone destruction had higher adipocyte numbers than those in patients with less bone destruction, indicating a relationship among myeloma cells, adipogenesis, and osteoblastogenesis. We hypothesized that inhibition of osteoblast differentiation is a consequence of myeloma-dependent alterations in the control of the MSCs’ fate into osteoblasts or into adipocytes. In our studies, we co-cultured MSCs with myeloma cells in a mixed medium (that contained both adipocyte and osteoblast media), and we observed co-culture with myeloma cells induced more adipocyte than osteoblast formation. Moreover, co-culture with myeloma cells enhanced adipocyte differentiation in vitro. Interestingly, separation of the cells by transwell inserts significantly reduced such effect. By analysis of the adhesion molecules in myeloma cells, we identified integrin α4β1 as a novel contributor in regulation of adipogenesis and osteoblastogenesis. Thus, our studies indicate that in the presence of myeloma cells, MSCs may be more prone to differentiate into adipocytes than into osteoblasts via α4β1. Our studies also suggest the development of new strategies to improve the care of myeloma patients with bone destruction by targeting α4β1 and its signaling pathways. Disclosures No relevant conflicts of interest to declare.

Targeting EZH2 in Multiple Myeloma Could be Promising for a Subgroup of MM Patients in Combination with IMiDs

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 311-311 ◽  
Author(s):  
Laurie Herviou ◽  
Alboukadel Kassambara ◽  
Stephanie Boireau ◽  
Nicolas Robert ◽  
Guilhem Requirand ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Board Of Directors ◽  
Research Funding ◽  
Plasma Cells ◽  
Newly Diagnosed ◽  
Advisory Committees ◽  
Myeloma Cells ◽  
Ezh2 Expression ◽  
Bone Marrow Plasma

Abstract Multiple Myeloma is a B cell neoplasia characterized by the accumulation of clonal plasma cells within the bone marrow.Epigenetics is characterized by a wide range of changes that are reversible and orchestrate gene expression. Recent studies have shown that epigenetic modifications play a role in multiple myeloma (MM) by silencing various cancer-related genes. We investigated the epigenetic genes differentially expressed between normal bone marrow plasma cells (BMPC ; N=5) and MM plasma cells from patients (N=206). Using SAM (Significance Analysis of Microarrays) analysis, only 12 genes significantly differentially expressed between BMPC and MM cells (ratio > 2 and FDR (false discovery rate) < 5%) were identified, including the EZH2 histone methyltransferase. EZH2, the enzymatic subunit of Polycomb Repressive Complex 2, is a histone methyltransferases able to repress gene expression by catalyzing H3K27me3 histone mark. EZH2 overexpression has been associated with numerous hematological malignancies, including MM. We thus studied EZH2 role in MM physiopathology and drug resistance. EZH2 expression was analyzed in normal bone marrow plasma cells (BMPCs; N=5), primary myeloma cells from newly diagnosed patients (MMCs; N=206) and human myeloma cell lines (HMCLs; N=40) using Affymetrix microarrays. EZH2 gene is significantly overexpressed in MMCs of patients (median 574, range 105 - 4562) compared to normal BMPCs (median = 432; range: 314 - 563) (P < 0.01). The expression is even higher in HMCLs (median 4481, range 581 - 8455) compared to primary MMCs or BMPCs (P < 0.001). High EZH2 expression is associated with a poor prognosis in 3 independent cohorts of newly diagnosed patients (Heidelberg-Montpellier cohort - N=206, UAMS-TT2 cohort - N=345 and UAMS-TT3 cohort - N =158). Furthermore, GSEA analysis of patients with high EZH2 expression highlighted a significant enrichment of genes involved in cell cycle, downregulated in mature plasma cells vs plasmablasts, and EZH2 targets. Specific EZH2 inhibition by EPZ-6438 EZH2 inhibitor induced a significant decrease of global H3K27me3 in all the HMCLs tested (P < 0.01) and inhibited MM cell growth in 5 out of the 6 HMCLs tested. The inhibitory effect of EZH2 inhibitor on MM cell growth appeared at day 6 suggesting that it is mediated by epigenetic reprogramming. To confirm that EZH2 is also required for the survival of primary MMCs from patients, primary MM cells (n = 17 patients) co-cultured with their bone marrow microenvironment and recombinant IL-6 were treated with EPZ-6438. As identified in HMCLs, EZH2 inhibition significantly reduced the median number of viable myeloma cells by 35% (P = 0.004) from a subset of patients (n=9) while the other group (n=8) was resistant. Of interest, EPZ-6438 induced a significant global H3K27me3 decrease in both groups of patient. RNA sequencing of 6 HMCLs treated with EPZ-6438 combined with H3K27me3 ChIP analyses allowed us to create an EZ GEP-based score able to predict HMCLs and primary MM cells sensitivity to EZH2 inhibitors. We also observed a synergy between EPZ-6438 and Lenalidomide, a conventional drug used for MM treatment. More interestingly, pretreatment of myeloma cells with EPZ-6438 significantly re-sensitize drug-resistant MM cells to Lenalidomide. Investigating the effect of EPZ-6438/Lenalidomide combination in MMC, we identified that IKZF1, IRF4 and MYC protein levels were significantly more inhibited by the combination treatment (65.5%, 63.9% and 14.8% respectively) compared with Lenalidomide (51.5%, 43% and 2.2%) or EPZ-6438 (45.2%, 38.7% and 6.2%) alone. Clinical trials are ongoing with EZH2 inhibitors in lymphoma and could be promising for a subgroup of MM patients in combination with IMiDs. Furthermore, the EZ score enables identification of MM patients with an adverse prognosis and who could benefit from treatment with EZH2 inhibitors. Disclosures Goldschmidt: Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Onyx: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Millennium: Membership on an entity's Board of Directors or advisory committees, Research Funding; Chugai: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees. Hose:EngMab: Research Funding; Takeda: Other: Travel grant; Sanofi: Research Funding.

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