Semaphorin 4D to suppress bone formation in multiple myeloma.

8039 Background: Myeloma bone disease is characterized by osteoclast activation and long-term osteoblast suppression. We investigated if Semaphorin 4D (Sema4D; CD100) plays a role in these processes. Sema4D has been shown to be a potent osteoblast inhibitor (Negishi-Koga T et al, Nat Med. 2011). A study recently identified that the breast cancer cell line MDA-MB-231 utilizes Sema4D to create osteolysis (Yang Y et al, PLOS One 2016). There have been previous data that Sema4D is increased in the serum of myeloma patients (Terpos et al, Blood 2012) and that co-culturing myeloma cell lines with osteocytes increases the expression of Sema4D mRNA in both (Suvannasankha et al, Blood 2016). We sought to investigate if myeloma cells are using Sema4D to suppress bone formation and if they affect the levels of Sema4D produced by osteoclasts. Methods: We used lentivirus carrying shRNA for Sema4D or control (Scr) to knock down the level of the protein in the 5TGM1 murine myeloma cell line. Knockdown was verified by qPCR and Western Blot. We subsequently co-cultured the 5TGM1 cells with the MC3T3-subclone M4 (MC4) murine stromal cell line for 2 days, removed the myeloma cells and then differentiated the MC4 cells using ascorbic acid and β-glycerolphosphate. At day 5, we analyzed the cells for Runx2 (a critical gene for the differentiation of stromal cells into osteoblasts) expression utilizing qPCR. Also, we performed qPCR in primary osteoclast (OCL) mouse cells differentiating into OCL with RANKL with or without pre-treatment with myeloma-conditioned media for 3 days before the addition of RANKL. Results: When 5TGM1-Scr were co-cultured with MC4 cells the expression of Runx2 on day 5 was decreased (p=0.02). Strikingly, the 5TGM1-shSema4D cells when co-cultured with MC4s did not have the same effect and allowed the upregulation of Runx2 expression on day 5 (p=0.01). Myeloma-conditioned media increased Sema4D expression by OCL throughout the 5 days of differentiation 2 to 3-fold (p=0.01 for day 5). Conclusions: The myeloma cells seem to be utilizing Sema4D both directly and indirectly to inhibit bone formation. Targeted therapy against Sema4D may improve outcomes and fracture-free survival for multiple myeloma patients.

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Panobinostat Induces Upregulation of CD38 and Augments the Anti-Myeloma Efficacy of Daratumumab in Pre-Clinical Models

Blood ◽

10.1182/blood.v128.22.4481.4481 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4481-4481 ◽

Cited By ~ 1

Author(s):

Estefania Garcia-Guerrero ◽

Tea Gogishvili ◽

Sophia Danhof ◽

Martin Schreder ◽

Celine Pallaud ◽

...

Keyword(s):

Multiple Myeloma ◽

Flow Cytometry ◽

Cell Line ◽

Myeloma Cell ◽

Target Cells ◽

Myeloma Cell Line ◽

Myeloma Cells ◽

Cd38 Expression ◽

Clinical Models ◽

Pre Treatment

Abstract Background: Immunotherapy with monoclonal antibodies (mAbs) has recently entered the clinical arena in multiple myeloma, including Daratumumab that targets CD38 on malignant plasma cells. The efficacy of mAbs depends on antigen density and expression of accessory ligands on target cells to initiate cell- and complement-dependent effector mechanisms. Here, we investigate the use of the histone deacetylase inhibitor (HDACi) Panobinostat to modulate target antigen expression and ligand profile on myeloma in favor of potent mAb-mediated recognition and destruction. We show that Panobinostat augments CD38 expression specifically on myeloma cells and demonstrate powerful synergy with anti-CD38 mAb Daratumumab in pre-clinical models. Methods: The myeloma cell line MM1.S and primary myeloma cells were treated with titrated doses of Panobinostat (0, 10, 25 nM) and expression of CD38 and a panel of additional target molecules including B-cell maturation antigen (BCMA) and SLAMF7, as well as accessory ligands analyzed by flow cytometry at 24, 48 and 72 hours. Antibody-dependent cellular cytotoxicity (ADCC) against Panobinostat treated and untreated myeloma cells was analyzed at 4 and 20 hours after addition of PBMC at an effector to target ratio of 25:1 in the presence of Daratumumab (1, 10, 50 ug/mL) or an isotype control antibody. Results: We first treated the myeloma cell line MM1.S with Panobinostat and analyzed its direct cytotoxic anti-myeloma effect. Consistent with previous work, the percentage of live MM1.S myeloma cells had decreased to 85% and 50% after 48 hours of exposure to 10 and 25 nM respectively. We analyzed expression of CD38 on residual live, i.e. 7-AAD negative MM1.S cells by flow cytometry and observed a 1.5 (10 nM) and 2-fold (25 nM) increase of CD38 expression by mean fluorescence intensity (MFI) compared to baseline levels and untreated control cells. The increase in CD38 expression was already detectable after 24 hours and plateaued between 48 and 72 hours. We confirmed our observation in primary myeloma cells from multiple donors (n=4) and detected an even stronger increase to 2 (10 nM) and 4-fold (25 nM) higher CD38 expression compared to untreated cells at 48 hours. Interestingly, expression of BCMA and SLAMF7 was not increased after Panobinostat treatment at all tested concentrations and time points in both MM1.S and primary myeloma. We confirmed that Panobinostat-induced upregulation of CD38 specifically occurred in myeloma, and neither observed this phenomenon in a panel of leukemia and lymphoma cell lines including Raji (Burkitt) and JeKo-1 (mantle cell), nor on resting/activated primary CD8+ and CD4+ T cells that we isolated from peripheral blood of several donors (n=3). Next, we were interested in determining whether the increase in CD38 expression enabled superior anti-myeloma activity of the anti-CD38 mAb Daratumumab. Panobinostat pre-treatment was done for 48 hours at 10 nM as this is a clinically achievable serum level with currently approved regimens. Indeed, significantly higher ADCC was mediated by Daratumumab at all tested concentrations (1, 10 and 50 ug/mL) against MM1.S that we had exposed to Panobinostat. At 4 hours, ADCC was 45% and 25% in Panobinostat-treated and untreated MM1.S respectively, and at 20 hours, near-complete, >90% ADCC of Panobinostat-pre-treated MM1.S had occurred, whereas only 65% of MM1.S were eliminated by Daratumumab without Panobinostat pre-treatment. These data were confirmed in multiple experiments with MM1.S and PBMC from different donors, and with primary myeloma cells. Experiments to evaluate synergy of Panobinostat and Daratumumab therapy in a xenograft model (NSG/MM1.S) are ongoing. Conclusions: Our data demonstrate that the HDACi Panobinostat induces upregulation of CD38 on myeloma and a subsequent dramatic increase of Daratumumab-mediated ADCC in pre-clinical models. These data suggest that Panobinostat could be used synergistically with Daratumumab in a clinical setting to increase response rates and extend duration of responses to Daratumumab. Panobinostat has a known ability to modulate the transcriptional profile of myeloma cells and our data demonstrate for the first time that this ability can be utilized to augment the therapeutic index of antibody-based immunotherapy in multiple myeloma. Disclosures Pallaud: Novartis: Employment. Lehmann:Novartis: Employment. Hudecek:Novartis: Research Funding.

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Expression of TRAIL Receptors on the Multiple Myeloma Cells.

Blood ◽

10.1182/blood.v104.11.4868.4868 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4868-4868

Author(s):

Juan Li ◽

Junhe Li ◽

Shaokai Luo ◽

Yin Zhao

Keyword(s):

Multiple Myeloma ◽

Cell Line ◽

Mononuclear Cells ◽

Myeloma Cell ◽

Myeloma Cell Line ◽

Killing Effect ◽

Myeloma Cells ◽

Decoy Receptors ◽

Trail Receptors ◽

Chemotherapy Agents

Abstract Objective To study the different expression of death receptors and decoy receptors on mononuclear cells from patients with multiple myeloma and myeloma cell line KM3 and compare the different expression of TRAIL receptors after chemotherapy or exposure to doxorubicin, to explore the mechanisms by which TRAIL selectively kills tumor cells. Methods Semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and flow cytometry was used to investigate the expression of four receptors on mononuclear cells in 23 multiple myeloma patients and myeloma cell line KM3 and 15 controls, we furthermore compared the changes of expression mode after chemotherapy and incubation of KM3 cell with sub-clinical concentration of Doxorubicin. Results There finds only DR4 and DR5 on KM3 cell line without the expression of DcR1 and DcR2. Expression of DR4 and DR5 on mononuclear cells of MM patients is higher than that of controls (P<0.05), but DcR1 and DcR2 expression was lower than that of controls (P<0.05), after chemotherapy and exposure to Doxorubicin, the expression of DR5 on MM cells was up-regulated (P<0.05) Conclusions The expression of four receptors on myeloma cells and normal controls was significantly different, which might account for the selective killing effect of TRAIL on MM cells. DR5 was up-regulated on KM3 when incubating with Doxorubicin and after chemotherapy which suggests chemotherapy agents might enhance the apopotosis of MM cells through up-regulating of DR5 receptor.

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Enzastaurin (LY317615), an Oral Protein Kinase C β Inhibitor, Induces Apoptosis in Multiple Myeloma Cell Lines.

Blood ◽

10.1182/blood.v106.11.1577.1577 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1577-1577

Author(s):

Mujahid A. Rizvi ◽

Kulsoom Ghias ◽

Katharine M. Davies ◽

Chunguang Ma ◽

Nancy L. Krett ◽

...

Keyword(s):

Multiple Myeloma ◽

Protein Kinase C ◽

Protein Kinase ◽

Cell Line ◽

Cell Lines ◽

Cancer Cell Line ◽

Colon Cancer Cell Line ◽

Myeloma Cell ◽

Akt Pathway ◽

Kinase C

Abstract The protein kinase C (PKC) family consists of 11 serine/threonine protein kinase isoforms that are involved with cell proliferation and differentiation, gene transcription, and tumor-induced angiogenesis. The PKC pathway has been shown to play a role in the regulation of cell growth in several hematologic malignancies. However, in multiple myeloma (MM), the role of the PKC pathway has not been extensively studied. Enzastaurin (LY317615), an acyclic bisindolylmaleimide, is an oral inhibitor of the PKC β isozyme. Enzastaurin has been reported to induce apoptosis and suppress proliferation in the HCT116 colon cancer cell line by inhibiting the AKT pathway. The objective of this study was to assess the efficacy of Enzastaurin in inducing apoptosis in MM cell lines and to investigate possible mechanisms of apoptosis. A spectrum of MM cell lines, with unique characteristics (dexamethasone-sensitive, dexamethasone-resistant, chemotherapy-sensitive, chemotherapy-resistant) were treated with Enzastaurin. There is evidence of cell death in all cell lines at clinically significant concentrations (1–3μM) after 72 hours of treatment. The dexamethasone-sensitive MM1.S cell line was used to further assess the effect of Enzastaurin in the presence of dexamethasone, insulin-like growth factor-1 (IGF-1), and IL-6. IGF-1 and IL-6 are potent growth factors for MM and have been observed to blunt the cytotoxic activity of dexamethasone. Dexamethasone and Enzastaurin appear to have an additive effect in the induction of apoptosis. In addition, while IGF-1 slightly decreases the effect of Enzastaurin, IL-6 has no effect. Enzastaurin treatment also induces a decrease in GSK3β and AKTSerine473 phosphorylation. GSK3β phosphorylation is thought to be a reliable pharmacodynamic marker for Enzastaurin activity. In conclusion, these data indicate that Enzastaurin induces apoptosis in MM cells and suppression of the AKT pathway may be one of the mechanisms by which Enzastaurin exerts its anti-myeloma activity.

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Multiple Myeloma: Interleukin-15 Antagonizes Bone Morphogenetic Protein-Induced Apoptosis and Growth Inhibition.

Blood ◽

10.1182/blood.v108.11.3444.3444 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3444-3444

Author(s):

Magne Rekvig ◽

Anne-Tove Brenne ◽

Torstein Baade Ro ◽

Anders Waage ◽

Magne Borset ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Growth Inhibition ◽

Cell Line ◽

Caspase 3 ◽

Myeloma Cell ◽

Stimulus Figure ◽

Myeloma Cell Line ◽

Myeloma Cells ◽

Induced Apoptosis

Abstract Multiple myeloma has two distinct features: Expansion of malignant plasma cells within the bone marrow accompanied by skeletal destruction. Bone morphogenetic proteins (BMPs) have been shown to induce apoptosis and inhibit growth in myeloma cells. BMPs are members of the TGF-β superfamily of proteins capable of inducing bone formation, and regulate proliferation, differentiation and apoptosis. We have investigated myeloma cell apoptosis and proliferation with BMP-4 and −6 in concert with the myeloma cell growth factors interleukin (IL)-2, IL-6, IL-10, IL-15, IL-21, tumor necrosis factor (TNF)-α and insulin-like growth factor (IGF)-1. Eight samples of highly purified myeloma cells from patients and a human myeloma cell line, IH-1 (Brenne AT et al. Blood. 2002 May 15;99(10):3756–62.), were used in this study. Cytokine concentrations used in the referred experiments were for BMP-4 20ng/ml, BMP-6 250ng/ml, IL-15 20ng/ml and IL-6 0,1ng/ml, respectively. Growth inhibition was measured in a proliferation assay by methyl-[3H]-thymidine incorporation and apoptosis by annexin V- FITC-binding/PI-uptake on flow cytometry. IL-15 antagonized growth inhibition (Figure A) and prevented apoptosis induced by BMP-4 (Figure B) and BMP-6 in the myeloma cell line IH-1. IL-15 also antagonized the growth inhibition induced by BMP-4 and/or BMP-6 in three out of eight patient samples. Neither IL-6, nor any of the other investigated cytokines were able to rescue the myeloma cells from growth inhibition and apoptosis induced by BMP-4 and -6. Among the investigated cytokines, we found that IL-15 has a unique capability to antagonize BMP- induced apoptosis and growth inhibition in myeloma cells. We examined cleavage of the proapoptotic protein caspase-3 and found that BMP-4 activated caspase-3 in the IH-1 cell line. This activation of caspase-3 was blocked by IL-15 but not by IL-6. We have demonstrated a possible mechanism for myeloma cells to escape apoptosis and growth-inhibition within the bone marrow. Intramedullar levels of IL-15 and BMPs may play a role in the pathogenesis of multiple myeloma. Figure A. Proliferation in response to BMP-4 stimulus Figure A. Proliferation in response to BMP-4 stimulus Figure B. Apoptosis in response to BMP-4 stimulus Figure B. Apoptosis in response to BMP-4 stimulus

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Herbal Extracts from Lycii Radicis Corex and Achyranthes japonica Prevent Multiple Myeloma

Blood ◽

10.1182/blood-2021-153673 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 4918-4918

Author(s):

Carol A. Morris ◽

Syed Mehdi ◽

Jenat Rahman ◽

Pamela Rosales ◽

Sun-Ok Lee ◽

...

Keyword(s):

Multiple Myeloma ◽

Treatment Group ◽

Cell Growth ◽

Cell Line ◽

Myeloma Cell ◽

Herbal Extracts ◽

Osteoblast Cells ◽

Myeloma Cells ◽

Nsg Mice ◽

Achyranthes Japonica

Abstract Introduction Multiple Myeloma (MM) is a plasma cell malignancy that, despite advances in treatment, remains incurable. In over 90% of MM patients, aberrant bone remodeling occurs and results in osteolytic lesions. It severely reduces the patient's quality of life and increases mortality. Korean traditional medicine has a long-standing interest in healthy foods to enhance the immune system, energy-boost, and Yin-and-Yang balance. A healthy diet in Tradition medicine is based on accumulated observations from countless cases over multi-centuries. Traditional medicine has used herbal extracts (HE) from natural plants with fewer side effects and long-term treatment tolerance. In earlier studies, lycii radices cortex (LRC) and achyranthes japonica (AJ) containing herbal extracts (HE) have demonstrated the ability to enhance cell growth and mineralization of osteoblast cells while also inhibiting osteoclast differentiation. Furthermore, a sesquiterpene glucoside, (1'R,3'S,5'R,8'S,2 Z,4 E)-dihydrophaseic acid 3'-O-β-d-glucopyranoside (DPA3G) were isolated from the LRC ethanol extract and shown to be a bioactive compound for enhancement of cell growth and bone anabolic activity (Park et al). The current study investigated the effects of LRC+AJ containing HE on murine 5TGM1 MM-bearing mice. Methods We transplanted 1x10 6 cells of the enforced luciferase-expressing 5TGM1 (5TGM1-Luc) into 8~12-week-old Nod-Scid-IL2Rg null (NSG) mice via tail vein (representing an equal number of each sex). For LRC+AJ containing HE treatment, mice were divided into three groups; i) PBS group; ii) treatment group at 300 mg/kg, gavage, TIW started two weeks before MM transplant (TbT); and iii) treatment group at 300 mg/kg, gavage, TIW two weeks after MM transplant (TaT). We assessed tumor burden by weekly bioluminescence imaging (IVIS 200 Imager, Perkin Elmer). The spine was extracted from carcass and scanned at postmortem by Dual Energy X-ray Absorptiometry using PIXImus Densitometer (G.E. Lunar, Madison, USA) and micro-computed tomography (microCT; Scanco Medical AG, Switzerland). HE effects in cell growth were tested in the myeloma cells (5TGM1 and U266) and preosteoblast cells (MC3T3-E1). The cells were grown in various concentrations (8, 80, 800 ug/ml) of HE up to 96 hours. In addition, Chromatogram and Mass Spectrometry were conducted to identify the DPA3G. Results As shown in Figure 1A, this HE significantly increased the mouse survivals of both TbT and TaT groups with median survivals of undetermined and 52.5 days, respectively, while MM control had a median survival of 42 days. The Mantel-Cox test found that TbT and TaT mice were significantly different from the control group (P=0.0014 and 0.0182, respectively). Furthermore, DEXA scans at postmortem showed a significant increase in bone mineral density (BMD) and bone mineral content (BMC) in TbT and TaT groups than control MM mice. For a histomorphometry analysis, the spines were scanned by micro-CT and revealed that TbT and TaT groups had significantly increased bone volume over total volume (BV/TV) than control. To see if HE affects cell growth of myeloma cells and osteoblast cells, we further investigated the HE on preosteoblast cell line MC3T3-E1, mouse MM cell line 5TGM1-Luc, and human MM cell line U-266. Cells were treated with HE in various concentrations, and viability was assessed at 48 and 96 hours post-treatment. Remarkably, LRC+AJ containing HE increased MC3T3-E1 cell growth while it decreased 5TGM1 and U-266 cell viability. We identified the DAP3G and many other compounds in HE used in this study. Conclusions Our results demonstrated that LRC+AJ HE prevents MM and promotes bone formation in 5TGM1 engrafted NSG mice. We also found that LRC+AJ HE suppresses myeloma cell growth and enhances osteoblast cell survival. Although it is yet to be defined, a correlation of osteoblast activity and myeloma cell inhibition suggests a potential mechanism of the HE action to prevent MM progression. 1. Park E, Kim J, Yeo S, Lim E, Choi CW, Choi S, Li WY, Lee JW, Park JH, Huh D, Jeong SY. Anti-Osteoporotic Effects of Combined Extract of Lycii Radicis Cortexand Achyranthes japonica in Osteoblast and Osteoclast Cells and Ovariectomized Mice. Nutrients. 2019 Nov 9;11(11):2716. doi: 10.3390/nu11112716. PMID: 31717518; PMCID: PMC6893723.' Figure 1 Figure 1. Disclosures Huh: Dongwoodang Pharmacy Company: Current Employment. OffLabel Disclosure: Herbal Extracts from Lycii Radicis Corex and Achyranthes Japonica

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Sphingolipids as a novel target for the treatment of multiple myeloma.

Journal of Clinical Oncology ◽

10.1200/jco.2013.31.15_suppl.e19534 ◽

2013 ◽

Vol 31 (15_suppl) ◽

pp. e19534-e19534

Author(s):

Yubin Kang ◽

Jagadish Kummetha Venketa

Keyword(s):

Multiple Myeloma ◽

Cell Death ◽

Cell Line ◽

Cell Lines ◽

Apoptotic Cell ◽

Myeloma Cell ◽

Sphingolipid Metabolism ◽

Myeloma Cell Line ◽

Myeloma Cells ◽

Novel Target

e19534 Background: Multiple myeloma (MM) is the second most common hematological malignancy in the United States and accounts for ~10,600 deaths annually. MM remains an incurable disease and almost all patients will eventually relapse and become refractory to currently available therapeutic agents. There is an unmet need for better understanding the disease’s molecular pathways and for identifying novel therapeutic targets. Sphingolipid metabolism is being increasingly recognized as a key pathway in tumor cell proliferation and in tumor sensitivity to anticancer drugs. We hypothesize that altered sphingolipid metabolism plays an important role in the pathogenesis of MM, thus providing a novel target in the treatment of MM. Methods: We first assayed sphingolipid metabolism including sphingolipid metabolites and sphingolipid metabolizing genes in myeloma cell lines, in freshly isolated human primary CD138+myeloma cells, and in publically available dataset. We then tested the efficacy of the selective SK2 inhibitor (ABC294640) and the SK2 shRNA in killing myeloma cells in vitro. Results: 1) Compared to immortalized B cells, the levels of pro-apoptotic ceramides were decreased whereas the proliferative sphingosine 1-phosphate (S1P) was increased in myeloma cell lines. 2) The expression of several key sphingolipid-metabolizing genes including sphingosine kinase (SK) 1 and 2 was altered in freshly isolated human primary bone marrow myeloma cells and in publically available microarray dataset. 3) The selective SK2 inhibitor (ABC294640) induces apoptotic cell death and inhibits myeloma cell growth with an IC50of ~20 μM in 9 myeloma cell lines. 4) Interestingly, OPM-1 myeloma cell line was extremely sensitive to ABC294640 with an IC50of <5 µM whereas U266 myeloma cell line was resistant to ABC294640. SK2 shRNA induced apoptotic cell death in OPM-1, but not in U266 cells. We are currently investigating the molecular mechanisms underlying the resistance of U266 myeloma cells to ABC294640. Conclusions: Our data demonstrated that sphingolipid metabolism provides an attractive target in the treatment of refractory/relapased multiple myeloma.

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Expression of Heat Shock Protein 90 and the Effects of HSP90 Inhibitor (17-AAG) in Multiple Myeloma.

Blood ◽

10.1182/blood.v104.11.4916.4916 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4916-4916

Author(s):

Serhan Alkan ◽

Jan Duus ◽

Ismail H. Bahar ◽

Keith F. Izban ◽

Hytham Al-Masri ◽

...

Keyword(s):

Multiple Myeloma ◽

Heat Shock ◽

Heat Shock Protein ◽

Cell Line ◽

Flow Cytometric Analysis ◽

Heat Shock Protein 90 ◽

Myeloma Cell ◽

Immunoperoxidase Staining ◽

Myeloma Cells ◽

Innovative Strategy

Abstract Heat Shock Protein 90 (HSP90) is required for structural folding and maintenance of conformational integrity of various proteins, including several associated with cellular signaling. Recent studies utilizing 17-allylamino-17-demethoxygeldanamycin (17-AAG), an inhibitor of HSP90, demonstrated antitumor effect in solid tumors. In order to test whether HSP90 could be targeted in multiple myeloma (MM) patients, we first investigated expression of HSP90 by immunofluorescence and flow cytometric analysis in a myeloma cell line (U266) and primary myeloma cells. Following demonstration of HSP90 expression in myeloma cells, archival samples of 32 MM patients were analyzed by immunoperoxidase staining. Myeloma cells in all patients showed strong cytoplasmic expression of HSP90 in all samples and 55 % also demonstrated concurrent nuclear immunopositivity. Treatment of U266 and primary MM cells with 17AAG resulted in significantly increased apoptosis compared to untreated control cells. Analysis of anti-apoptotic BCL2 family proteins in MM cells incubated with 17-AAG revealed down-regulation of BCL-2, BCL-XL and MCL-1. Furthermore, while low concentration of bortezomib had no cell death, combination of 17AAG and bortezomib treatment revealed a synergistic apoptotic effect on the U266 cell line. These data suggest that targeted inhibition of HSP90 may prove to be a valid and innovative strategy for the development of future therapeutic options for MM patients.

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Myeloma Cells Induce Osteoblast Suppression through Sclerostin Secretion

Blood ◽

10.1182/blood.v116.21.2961.2961 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2961-2961 ◽

Cited By ~ 3

Author(s):

Silvia Colucci ◽

Giacomina Brunetti ◽

Angela Oranger ◽

Giorgio Mori ◽

Francesca Sardone ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Formation ◽

Bone Disease ◽

Culture System ◽

Conflicts Of Interest ◽

Negative Regulator ◽

Type I ◽

Myeloma Cells ◽

Myeloma Bone Disease ◽

Osteoblast Activity

Abstract Abstract 2961 Reduced osteoblast activity contributes to the development of multiple myeloma-bone disease. Wingless-type (Wnt) signalling pathway is critical in osteoblastogenesis, and it is negatively regulated by molecules such as frizzled-related proteins (sFRPs), Dickkopf proteins (DKKs) and sclerostin. Myeloma cells are known to induce inhibition of osteoblastogenesis through Wnt antagonists such as DKK-1 and sFRP-2 and -3 whereas the role of sclerostin, an osteocyte-expressed negative regulator of bone formation, has not been yet investigated. We provide novel evidence showing sclerostin expression by myeloma cells from patients with multiple myeloma-bone disease and human myeloma cell lines (HMCLs). By means of a co-culture system of bone marrow stromal cells (BMSCs) and HMCLs, we demonstrated that sclerostin expression by myeloma cells and HMCLs is responsible for reduced expression of major osteoblastic specific proteins namely bone-specific alkaline phosphatase, collagen-type I, bone sialoprotein II and osteocalcin as well as decreased mineralized nodule formation and attenuated expression of member of the AP-1 transcription factor family (i.e. Fra-1, Fra-2 and Jun-D). The addition of a neutralizing anti-sclerostin antibody to our co-culture system can restore the above parameters, through the intranuclear accumulation of β-catenin in BMSCs. On the other hand, we demonstrated that sclerostin is also involved in inducing increased receptor activator of nuclear factor-k B ligand (RANKL) and decreased osteoprotegerin (OPG) expression in osteoblasts, contributing to the enhanced osteoclast activity occurring in patients with multiple myeloma-bone disease. Our data suggest that myeloma cells contribute to the suppression of bone formation through sclerostin secretion. Disclosures: No relevant conflicts of interest to declare.

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Quantitative Proteomic Analysis of Relapsed Multiple Myeloma Identifies CDK6 Upregulation As a Potential Targetable Resistance Mechanism for Lenalidomide

Blood ◽

10.1182/blood-2019-130682 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2567-2567

Author(s):

Yuen Lam Dora Ng ◽

Stephan Bohl ◽

Evelyn Ramberger ◽

Oliver Popp ◽

Imke Bauhuf ◽

...

Keyword(s):

Multiple Myeloma ◽

Protein Expression ◽

Cell Lines ◽

Myeloma Cell ◽

P Value ◽

Rna Expression ◽

Myeloma Cells ◽

Multiple Myeloma Cell ◽

Protein Levels ◽

Pre Treatment

Lenalidomide, an immunomodulatory drug (IMiD), is highly active and broadly used for the treatment of multiple myeloma. Despite high initial remission rates, patients frequently relapse and become resistant to the drug. Comprehensive analyses of gene mutations and RNA expression have identified inactivating mutations and RNA downregulation in cereblon (CRBN), the primary target of lenalidomide, in some of the resistant patients. However, the underlying resistance mechanism for the majority of cases remains unknown. Here, we performed quantitative tandem mass tag (TMT)-based proteomic analyses and RNA sequencing in five paired pre-treatment and relapse samples from multiple myeloma patients treated with drug combinations comprising lenalidomide to identify changes in protein expression associated with resistance. Using a stringent cut-off with an adjusted P value < 0.1 and log2 fold change (FC) > 2, we found 7 proteins to be significantly upregulated and 10 proteins to be downregulated in the relapsed versus pre-treatment multiple myeloma samples. Of these 17 deregulated proteins at relapse, only two were also found to be deregulated on the RNA expression level (adjusted P value < 0.1) as assessed by RNA sequencing. In general, correlation between protein expression levels and RNA expression levels were weak (median Pearson correlation coefficient r=0.35). Among the top upregulated proteins in relapse samples was cyclin-dependent kinase 6 (CDK6) with an average log2 FC of 2.1. Protein and RNA levels of CDK6 showed only weak correlation (r=0.4) and CDK6 RNA was not differentially expressed between the relapse and pretreatment samples. To validate the findings of the proteomic analysis, we assessed CDK6 protein levels by western blot in additional patient samples obtained at diagnosis (N=4) and at relapse (N=9). This confirmed a high CDK6 protein expression in 6 of 9 relapse samples while CDK6 could not be detected in the 4 pre-treatment samples. In order to determine the impact of CDK6 on drug sensitivity, we overexpressed CDK6 using either a retro- or lentiviral vector system in multiple myeloma cell lines. In two multiple myeloma cell lines tested, MM.1S and OPM2, CDK6 overexpression reduced sensitivity to lenalidomide and pomalidomide, but not to melphalan, bortezomib, or dexamethasone. To examine whether lowered IMiD-sensitivity can be overcome by CDK6 inhibition, we treated multiple myeloma cell lines either with the CDK6 inhibitor palbociclib, an IMiD-based CDK6-selective proteolysis targeting chimera (PROTAC) or a non-selective CDK6-PROTAC which is also capable of pomalidomide-mediated degradation of IKZF1 and IKZF3 (Brand et al., Cell Chem Biol 2019). Both palbociclib and CDK6-selective PROTAC as single treatments had only mild effects on the majority of multiple myeloma cells, implying that multiple myeloma cells are generally not dependent on CDK6. In contrast, the combination treatment of palbociclib with lenalidomide, or the non-specific CDK6/IKZF1/IKZF3-targeting PROTAC significantly inhibited proliferation, producing synergistic effects on the decrease of myeloma cell viability in 6 multiple myeloma cell lines, including those with a low IMiD sensitivity like RPMI-8226 and L363. This demonstrates that CDK6 inhibition or degradation enhances the cytotoxic effects of IMiDs. In order to investigate a potential mechanism for the synergistic effects of CDK6 inhibition and IMiDs, we analyzed protein levels in treated cells. CDK6 inhibition or degradation had no effect on CRBN protein levels nor on lenalidomide-induced degradation of IKZF1 and IKZF3. In contrast, combined degradation of CDK6, IKZF1, and IKZF3 revealed decreased protein levels of c-MYC, which was not observable in cells treated with palbocicilib, CDK6-selective PROTAC or pomalidomide alone. In conclusion, quantitative proteomics in primary multiple myeloma samples identified new druggable candidates including CDK6 in relapse that were overlooked by RNA expression analyses. Inhibition of CDK6 by palbociclib or a PROTAC sensitizes multiple myeloma cells to IMiDs and results in synergism when used in combination. Disclosures Bohl: Pfizer: Honoraria. Bullinger:Menarini: Honoraria; Novartis: Honoraria; Pfizer: Honoraria; Sanofi: Honoraria; Seattle Genetics: Honoraria; Janssen: Honoraria; Jazz Pharmaceuticals: Honoraria; Amgen: Honoraria; Astellas: Honoraria; Bristol-Myers Squibb: Honoraria; Celgene: Honoraria; Daiichi Sankyo: Honoraria; Gilead: Honoraria; Hexal: Honoraria; Bayer: Other: Financing of scientific research; Abbvie: Honoraria. Kroenke:Celgene: Consultancy, Honoraria; Takeda: Consultancy.

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Bivalent Histone Modifications By Reiibp Leads to Transcriptional Reprogramming and TLR7-BTK Driven IL-6 Pro-Inflammatory Response in t(4;14) Myelomagenesis

Blood ◽

10.1182/blood-2021-145839 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2202-2202

Author(s):

Phyllis SY Chong ◽

Julia Lim ◽

Jing Yuan Chooi ◽

Wee-Joo Chng

Keyword(s):

Cell Line ◽

Research Funding ◽

Plasma Cells ◽

Cancer Cell Line ◽

Transcriptome Profiling ◽

Myeloma Cell ◽

Stable Cell Line ◽

Dependent Manner ◽

Myeloma Cells ◽

Chip Sequencing

Abstract Multiple Myeloma (MM) is characterized by the uncontrolled proliferation of malignant plasma cells that are incapable of producing functional antibodies. Current treatment regime involves novel drug classes such as proteasome inhibitors, immunomodulatory drugs and monoclonal antibodies, which have significantly improved survival outcomes in patients. Patients with t(4;14) translocation represents ~15% of MM cases, and displays a dysregulation of the MMSET locus. It has one of the worst prognosis when compared to other subgroups, but represents an intermediate-risk group given its response towards bortezomib. REIIBP is a t(4;14)-deregulated isoform arising from alternative promoter usage within the MMSET locus. Despite sharing identical sequence with the C-terminus of MMSET II, we found that REIIBP displayed mutually exclusive expression with the full-length MMSET II arising from MB4-1 breakpoint. Additionally, the expression of REIIBP can be regulated through microRNAs by another histone methyltransferase, EZH2 in a Dicer-dependent manner. We generated a stable cell line that overexpresses REIIBP in the t(4;14)-negative human myeloma cells, RPMI8226, and revealed REIIBP as an epigenetic regulator of repressive H3K27me3 and active H3K4me3 modifications. Transcriptome profiling and ChIP-sequencing identified an upregulation of Toll-like receptor 7 (TLR7) by REIIBP through the enrichment of H3K4me3 on its promoter coupled with decrease in intragenic H3K27me3. This led to a BCR-independent activation of Bruton's tyrosine kinase (BTK) and NF-ĸB signaling in t(4;14) myeloma cells. Using Cancer Cell Line Encyclopedia and DepMap portal (Broad Institute) to compare the expression of BTK and BTK dependency scores across lineages revealed that BTK is an important target in MM, and REIIBP expression correlated with BTK in the CoMMpass dataset. Activation of TLR7-BTK by REIIBP conferred bortezomib resistance through the dysregulation of pro-inflammatory cytokine expression such as IL-6. Importantly, cells with REIIBP overexpression displayed enhanced lethality towards BTK inhibitor Ibrutinib, and combination with Bortezomib potentiated inhibition in myeloma cell lines and mice engrafted with RPMI8226-REIIBP tumors. Altogether, our results indicated that blockade of REIIBP in t(4;14) cells through combining proteasome and BTK inhibitors is a therapeutic strategy in the clinic for further evaluation. Disclosures Chng: Novartis: Honoraria, Research Funding; Antengene: Honoraria; Pfizer: Honoraria; Sanofi: Honoraria; AbbVie: Honoraria; Amgen: Honoraria; BMS/Celgene: Honoraria, Research Funding; Johnson & Johnson: Honoraria, Research Funding; Takeda: Honoraria; Aslan: Research Funding.

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