scholarly journals CD38 Deregulation As Strategy to Make Multiple Myeloma Cells More Sensitive to NAD+ Depleting Agents

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5671-5671
Author(s):  
Michele Cea ◽  
Antonia Cagnetta ◽  
Debora Soncini ◽  
Paola Minetto ◽  
Micaela Bergamaschi ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Therapeutic Target ◽  
Research Funding ◽  
Annexin V ◽  
Metabolic Reprogramming ◽  
Mechanistic Studies ◽  
Nicotinamide Phosphoribosyltransferase ◽  
Myeloma Cells ◽  
Innovative Strategy ◽  
Promising Therapeutic Target

Abstract Background: The upregulated NAD+ biosynthesis, which is needed to face increased proliferation and metabolic processes, represents an important feature distinguishing cancer cells from their normal counterparts. As a result, the NAD+ biosynthetic apparatus emerges as highly promising therapeutic target for tumors, as suggested by the use of nicotinamide phosphoribosyltransferase (NAMPT) inhibitors in a number of malignancies, including Multiple Myeloma (MM) and leukemia (Cea et al. Blood 2012; Cagnetta et al. Blood 2013; Cagnetta et al. CCR 2015). The ecto-enzyme CD38 is emerging as a novel therapeutic target for patients with hematological malignancies, including MM, with several monoclonal antibodies already tested in clinical trials with promising results. Importantly, CD38 by regulating intracellular NAD+ stores acts as member of the intricate network supporting metabolic reprogramming associated with cancer. Based on these assumptions, here we explored CD38 activity as innovative strategy to enhance the anti-tumor activity of NAMPT inhibition in Multiple Myeloma cells. Methods: A panel of different MM cell lines and primary cells, both sensitive and resistant to conventional and novel anti-MM therapies, was used in the study. The effects of Nampt inhibition was evaluated in presence of CD38-gene editing (loss/gain of approaches) by using CTG assay and Annexin-V/propidium iodide staining. Next, the anti-MM effects of chemicals affecting CD38 activity were also evaluated in combination with low doses of Nampt inhibitors. Mechanistic studies were performed with Western-blotting, lentivirus-mediated shRNAs and enzymatic assays. Results and Conclusions: By using different approaches, we found that CD38 deregulation makes MM cells more vulnerable to NAD+ depleting agents. Ongoing mechanistic studies suggest the central role played by energetic metabolism in the observed synergism. Overall our data provide the mechanistic preclinical rationale to enhance anti-MM activity of Nampt targeting agents, in order to both overcome drug resistance and improve patients outcome. Disclosures Gobbi: Mundipharma: Consultancy, Research Funding; Takeda: Consultancy; Roche: Honoraria; Celgene: Consultancy; Gilead: Honoraria; Novartis: Consultancy, Research Funding; Janssen: Consultancy, Honoraria.

Skeletal-Muscle Metabolic Reprogramming in ALS-SOD1 G93G Mice Predates Disease Onset and is a Promising Therapeutic Target

2019 ◽  
Author(s):  
Silvia Scaricamazza ◽  
Illari Salvatori ◽  
Giacomo Giacovazzo ◽  
Jean Philippe Loeffler ◽  
Frederique Renè ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Therapeutic Target ◽  
Disease Onset ◽  
Metabolic Reprogramming ◽  
Promising Therapeutic Target

scholarly journals Multiple Myeloma Cells Induce Lipolysis in Adipocytes and Uptake Fatty Acids through Fatty Acid Transporter Proteins

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 41-42
Author(s):  
Cristina Panaroni ◽  
Keertik Fulzele ◽  
Tomoaki Mori ◽  
Chukwuamaka Onyewadume ◽  
Noopur S. Raje
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Fatty Acid ◽  
Board Of Directors ◽  
Metabolic Reprogramming ◽  
Glycerol Production ◽  
Advisory Committees ◽  
Myeloma Cells ◽  
Long Chain

Multiple myeloma (MM) originates in the bone marrow where adipocytes occupy 65% of the cellular volume in a typical myeloma patient. Cancer associated adipocytes support the initiation, progression, and survival of solid tumors via mechanisms including adipokine secretion, modulation of the tumor microenvironment, and metabolic reprogramming of cancer cells. Although MM cells are surrounded by abundant bone marrow adipocytes (BMAd), the nature of their interaction remains unclear. Recent studies have elucidated the role of BMAds in supporting the survival of MM cells, in part, through secreted adiponectin. Increased fatty acid (FA) metabolism may result in metabolic reprogramming of cancer cells impacting their growth and survival. Here, we hypothesize that MM cells extract FA from adipocytes for their growth. We first characterized mesenchymal stem cells (MSCs) from MGUS, smoldering MM (SMM), and newly diagnosed MM (NDMM) patients by flow cytometry analysis. MSCs showed significant increase in Pref1, leptin receptor and perilipin A, suggesting increased adipogenic commitment. MSCs from healthy donors (HD), MGUS, SMM, and NDMM patients were induced to differentiate into adipocytes and then co-cultured with human MM MM.1S cells. After 72 hr of co-culture, CyQUANT assay demonstrated significant increase in proliferation of MM.1S cells in the presence of BMAd from HD; this was further increased in the presence of BMAd from MGUS/SMM and NDMM. These data suggest that the BMAd support the growth of MM cells and this effect is more pronounced in patient derived BMAd. A PCR-array targeting lipid metabolism on BM fat aspirates showed significant deregulation of genes involved in FA synthesis and lipolysis. Taken together, our data suggest that BMAd in MM patients are altered to further support the aggressive expansion of MM cells. The proliferative-supportive role of adipocytes was further validated in co-culture of OP9 murine BM stromal preadipocytes with 5TGM1 murine MM cells. To study the bidirectional interaction of MM/ BMAd, mature OP9 adipocytes were co-cultured with 5TGM1 or human OPM2 MM cells for 24 hr. Intracellular lipid droplets were labelled with Deep Red LipidTox stain. The lipid droplet sizes were significantly decreased in the presence of both 5TGM1 and OPM2 cells compared to OP9 alone. The decrease in lipid size suggested that MM cells may induce lipolysis in adipocytes. Indeed, 24hr co-culture of 5TGM1 cells with OP9 mature adipocytes significantly increased lipolysis 3-fold as measured by glycerol secretion in conditioned media. Co-culture of OP9 adipocytes with other MM cell lines of human origin, MM.1S, INA6, KMS-12 PE, and OPM2 also significantly increased the glycerol production as much as 4-fold. Taken together these data indicate that MM cells induce lipolysis in adipocytes. In contrast, treatment of 5TGM1 cells with synthetic catecholamine isoproterenol did not induce lipolysis, or glycerol production, indicating lack of triglyceride storage. Next, we hypothesized that the free FAs released from adipocytes are taken up by MM cells for various biological processes. To test this, 5TGM1, MM.1S and OPM2 cells were incubated with BODIPY-C12 and BODIPY-C16, the BODIPY-fluorophore labelled 12-carbon and 16-carbon long chain FA. All MM cells showed saturated uptake of the FA within 10 minutes suggesting that MM cells have efficient FA transporters. To confirm this uptake, unstained 5TGM1, OPM2 and KMS12 PE cells were co-cultured with the LipidTox-labelled OP9 mature adipocytes. After 24 hours, flow cytometric analysis showed LipidTox signal in MM cells. These data demonstrate that FAs released by MM induced adipocyte lipolysis are taken up by MM cells. Long-chain FAs such as BODIPY-C12 and BODIPY-C16 are transported into cells through FA transporter protein (FATP) family of lipid transporters. We therefore analyzed patient samples which showed that CD138+ plasmacells and myeloma cells expressed high levels of FATP1 and FATP4 whereas, their expression was absent in lineage-sibling T-cells. Moreover, pretreatment with Lipofermata, a FATP inhibitor, was able to decrease the uptake of BODIPY-C12 and -C16 in 5TGM1 cells. Taken together, our data show that myeloma cells induce lipolysis in adipocytes and the released free FAs are then uptaken by myeloma cells through FATPs. Inhibiting myeloma cell induced lipolysis or uptake of FA through FATPs may be a potential anti-tumor strategy. Disclosures Fulzele: FORMA Therapeutics, Inc: Current Employment, Other: Shareholder of Forma Therapeutics. Raje:Amgen: Consultancy; bluebird bio: Consultancy, Research Funding; Caribou: Consultancy, Membership on an entity's Board of Directors or advisory committees; Immuneel: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Consultancy; Celgene: Consultancy; Immuneel: Consultancy; Janssen: Consultancy; Karyopharm: Consultancy; Takeda: Consultancy.

ITF2357, a Novel Histone Deacetylase Inhibitor, Demonstrates Significant Apoptotic Activity Against Human Multiple Myeloma Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4791-4791
Author(s):  
Michael Kline ◽  
Kathleen A. Donovan ◽  
John A. Lust
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Histone Deacetylase ◽  
Cell Lines ◽  
Hydroxamic Acid ◽  
Stromal Cell ◽  
Hdac Inhibitors ◽  
Annexin V ◽  
Myeloma Cells

Abstract We have evaluated the efficacy of a novel hydroxamic acid-derived histone deacetylase (HDAC) inhibitor, ITF2357, to promote cell death in multiple myeloma (MM) cells. HDAC inhibitors, which promote histone hyperacetylation and increase gene expression, have been evaluated as candidate agents for combating malignancies because they impact the expression of genes related to proliferation, differentiation, and survival. Exposure of MM cell lines to 1 micromolar ITF2357 led to dramatically increased levels of histone acetylation at 4 hours and 8 hours by Western analysis. Sub-micromolar concentrations of ITF2357 promoted time- and concentration-dependent cell death in MM cell lines. Using 500 nM ITF2357, a concentration potentially achievable in vivo, viability of KAS-6/1 IL-6 dependent myeloma cells was reduced to 28% of control at 24 hrs and 2% of control at 48 hours (Figure 1). In contrast, viability of normal PBMCs was 100% at 24 hours and 80% at 48 hours (Figure 2). U266 and 8226 myeloma cells were found to be sensitive to ITF-2357 in a similar fashion with U266 being least sensitive. Cell death proceeded via apoptosis as measured using Annexin V/propidium iodide staining. ITF 2357 was superior to suberoylanilide hydroxamic acid (SAHA) at inhibition of stromal cell IL-6 production. IL-1beta (10 pg/ml) was used to stimulate bone marrow stromal cell IL-6 production (105 ng/ml) after 48 hours. Concentration of ITF2357:Stromal Cell IL-6 production after 48 hours were as follows - 10 nM: 78 ng/ml; 100 nM: 79 ng/ml; 1000 nM; 32 ng/ml. SAHA at similar concentrations showed no significant decrease in stromal cell IL-6 production compared with the no drug control. In summary, ITF2357 induces significant myeloma cell apoptosis and can inhibit stromal cell IL-6 production. It represents an attractive therapeutic candidate for MM clinical trials. Figure Figure Figure Figure

Biochemical Basis for Interactions Between Thalidomide and Inhibitors of the Isoprenoid Biosynthetic Pathway in Multiple Myeloma Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2635-2635
Author(s):  
Sarah A. Holstein ◽  
Huaxiang Tong ◽  
Raymond J. Hohl
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Annexin V ◽  
Differential Sensitivity ◽  
Parp Cleavage ◽  
Myeloma Cells ◽  
Protein Isoprenylation ◽  
Geranylgeranyl Transferase ◽  
Synthase Inhibitor ◽  
Rpmi 8226

Abstract Introduction: The isoprenoid biosynthetic pathway (IBP) is responsible for the production of key sterol and nonsterol species, including farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) which serve as substrates for protein isoprenylation reactions. Several agents known to target the IBP have been observed to have cytotoxic effects in multiple myeloma cells. Thalidomide (Thal) has emerged as an effective agent for treating multiple myeloma. While Thal has been noted to have a variety of direct and indirect effects on myeloma cells, the precise mechanism of action remains unknown. Aim: We examined interactions between inhibitors of the IBP and Thal in multiple myeloma cells. The mechanisms underlying the observed differential sensitivity to these agents were explored. Methods: Studies were performed in three human multiple myeloma cell lines (RPMI-8226, U266, H929). Cytotoxicity was assessed via MTT assays, while apoptosis induction was determined by Annexin V staining and evaluation of PARP cleavage. Western blot analysis was used to evaluate inhibition of protein isoprenylation. Intracellular FPP and GGPP levels were measured via enzymatic coupling to fluorescently-tagged peptides, HPLC fractionation and fluorescence detection. Pharmacologic manipulation of the IBP was achieved with the following agents: lovastatin (Lov) as an HMG-CoA reductase inhibitor, zoledronic acid (ZA) as a FPP synthase inhibitor, digeranyl bisphosphonate (DGBP) as a GGPP synthase inhibitor, FTI-277 as a farnesyl transferase inhibitor (FTI), and GGTI-286 as a geranylgeranyl transferase I inhibitor (GGTI). Results: Addition of Thal to Lov (at both 24 & 48h), zoledronic acid (at 48h), or DGBP (at 24 & 48h) in RPMI-8266 cells results in marked enhancement in cytotoxicity. Isobologram analysis could not be performed as Thal by itself does induce cytotoxicity in MTT assays. Although Lov induces cytotoxicity in a concentration- and time-dependent manner in the U266 and H929 cells, the addition of Thal did not result in increased cytotoxicity. Neither ZA nor DGBP induced cytotoxicity in the U266 cell line, while the H929 cell line showed effects only at 48 hours. Addition of Thal to FTI or GGTI did not result in enhanced cytotoxicity in tested cell lines. Annexin V experiments confirmed enhanced induction of apoptosis in RPMI-8226 cells incubated with the combination of Thal/Lov or Thal/DGBP. Add-back experiments revealed that the enhanced cytotoxicity/induction of apoptosis observed with the addition of Thal could be prevented with the addition of mevalonate or GGPP in Lov-treated cells or GGPP in DGBP-treated cells. PARP cleavage was demonstrated in RPMI-8226 and H929 cells treated with Lov or DGBP (with or without Thal) and in U266 cells treated with Lov. As expected, Lov resulted in the accumulation of unmodified forms of proteins normally farnesylated (Ras) and geranylgeranylated (Rap1a and Rab6) in these cells. Interestingly however, while DGBP led to accumulation of unmodified Rap1a and Rab6 in RPMI-8226 and H929 cells, no effect was seen in the U266 line. Examination of intracellular levels of FPP and GGPP revealed that the U266 line has markedly larger pools of FPP (8.5-fold) and GGPP (2.7-fold) compared to RPMI-8226 and that treatment with DGBP only partially depletes U266 cells of GGPP. Conclusions: These studies demonstrate an interaction between thalidomide and IBP inhibitors in multiple myeloma cells. These effects appear dependent on depletion of GGPP. Since treatment with a geranylgeranyl transferase-I inhibitor does not produce similar results, this suggests that substrates of geranylgeranyl transferase-II, such as the Rab proteins, may play critical roles in myeloma pathophysiology. The finding that intracellular levels of FPP and GGPP vary markedly amongst cell lines explains differential sensitivity of these cells to pharmacologic manipulation of the IBP and may also influence sensitivity to chemotherapeutic agents. Further studies will determine the extent to which isoprenoid pool sizes vary in primary samples and may ultimately allow for the identification of multiple myeloma patients who would benefit from the addition of an IBP inhibitor to their treatment plan. Figure Figure

Kinome-Wide RNAi Studies in Human Multiple Myeloma Identify a Lymphoid Restricted Kinase GRK6 as a Selectively Vulnerable Target That Regulates STAT3/MCL1.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 601-601
Author(s):  
Rodger E. Tiedemann ◽  
Yuan Xiao Zhu ◽  
Jessica Schmidt ◽  
Hongwei Yin ◽  
Quick Que ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Epithelial Cells ◽  
Tumor Cells ◽  
Research Funding ◽  
Kinase Inhibitors ◽  
Lymphoid Tissues ◽  
Myeloma Cells ◽  
Stat3 Phosphorylation ◽  
Human Multiple Myeloma

Abstract Abstract 601 A paucity of validated kinase targets in human multiple myeloma (MM) has delayed clinical deployment of kinase inhibitors in treatment strategies. We therefore conducted a kinome-wide small interfering RNA (siRNA) lethality study in MM tumor lines bearing common t(4;14), t(14;16) and t(11;14) translocations to identify critically vulnerable kinases in MM tumor cells without regard to preconceived mechanistic notions. Primary screening was performed in duplicate using an 1800-oligo siRNA library in a single-siRNA-per-well format. siRNA were transfected at low concentration (13nM) to minimize off-target effects using conditions that resulted in transfection of >95% cells and <5% background cytotoxicity. After 96 hours, viability was measured by ATP-dependent luminescence. Fifteen kinases were consistently vulnerable in MM cells, including AKT1, AK3L1, AURKA, AURKB, CDC2L1, CDK5R2, FES, FLT4, GAK, GRK6, HK1, PKN1, PLK1, SMG1, and TNK2. While several kinases (PLK1, HK1) were equally vulnerable in epithelial cells, others and particularly the G-protein coupled receptor kinase, GRK6, appeared selectively vulnerable in MM. GRK6 inhibition is selectively lethal to KMS11, OPM1, H929, KMS18 and OCI-MY5 myeloma cells and has minimal effect on 293, MCF7, SF767, A375 or A549 epithelial cells. Persistent expression of FLAG-GRK6 via cDNA rescued KMS11 cells from the lethal effect of a 3'UTR-targeted GRK6 siRNA, but not from control siRNA, validating identification of GRK6 as an essential myeloma survival kinase. Furthermore, concordant results were obtained using four different exon-based GRK6 siRNA, all of which induced GRK6 silencing and similar inhibition of KMS11 proliferation and viability. Significantly, GRK6 is ubiquitously expressed in lymphoid tissues and myeloma, but by comparison appears absent or only weakly expressed in most primary human somatic tissues. From co-immunoprecipitation experiments we demonstrate that GRK6 is highly expressed in myeloma cells via direct association with the HSP90 chaperone. Inhibition of HSP90 with geldanamycin blocks GRK6 protein expression. Importantly, direct GRK6 silencing causes rapid and selective suppression of STAT3 phosphorylation that is associated with sustained reductions in total MCL1 protein levels and MCL1 phosphorylation (within 24 hours), providing a potent mechanism for the cytotoxicity of GRK6 inhibition in MM tumor cells. GF109203X is an inhibitor of both protein kinase C and of GRK6 that causes near total inhibition of these kinases in vitro at distinct concentrations of 0.1μM and 1-10μM respectively. Notably, GF109203X was substantially cytotoxic to 10/14 myeloma tumor lines at concentrations most consistent with GRK6 inhibition (5-20μM), and was selectively more cytotoxic to myeloma tumor cells than to non-myeloma cell lines (P=0.01), highlighting the potential of GRK6 as a pharmaceutical target for selective therapeutic intervention in myeloma. As mice that lack GRK6 are healthy, inhibition of GRK6 represents a uniquely targeted novel therapeutic strategy in human multiple myeloma. Disclosures: Perkins: MMRC: Employment. Reeder:Celgene: Research Funding; Millennium: Research Funding. Fonseca:Otsuka: Consultancy; BMS: Consultancy; Amgen: Consultancy; Medtronic: Consultancy; Genzyme: Consultancy.

The Proteasome Inhibitor Bortezomib Stimulates Osteoblastic Differentiation of Human Osteoblast Precursor Cells Via Upregulation of Vitamin D Receptor Signaling

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1899-1899
Author(s):  
Martin Kaiser ◽  
Maren Mieth ◽  
Orhan Sezer ◽  
Ulrike Heider
Keyword(s):  
Multiple Myeloma ◽  
Vitamin D ◽  
Proteasome Inhibitor ◽  
Research Funding ◽  
Fold Increase ◽  
Osteoblastic Differentiation ◽  
Luciferase Reporter ◽  
Human Osteoblast ◽  
Myeloma Cells ◽  
Osteoblast Precursors

Abstract Abstract 1899 Introduction In multiple myeloma (MM), interactions of the malignant plasma cell clone with the bone marrow microenvironment lead to an enhanced osteoclast recruitment and impaired osteoblast activity. The proteasome inhibitor bortezomib has been shown to suppress osteoclast activity, and there is recent evidence that bortezomib enhances osteoblast differentiation. The aim of this study was to investigate the effects of bortezomib on human osteoblast precursors, focusing on vitamin D (VD) dependent osteoblastic differentiation. Since vitamin D receptor (VDR) is degraded by the proteasome, we hypothesized that bortezomib could influence its signaling and hence vitamin D induced osteoblastic differentiation. This might be of clinical importance, since an increased rate of vitamin D deficiency has recently been reported in patients with MM. Methods Primary human mesenchymal stem cells (hMSC) and primary human osteoblasts (hOB) were isolated from bone marrow aspirates or from bone fragments of healthy donors undergoing orthopedic surgery, respectively. Ascorbic acid and β-glycerolphosphate were used for osteoblastic stimulation (OS), either in combination with or without vitamin D. In order to analyze the effects of proteasome inhibition on osteoblastic differentiation and activity, hMSC and hOB were incubated with bortezomib at subapoptotic doses (1 - 5 nM). In addition, coculture experiments of hMSC, hOB and myeloma cells were performed. Expression of osteocalcin and osteopontin (OPN) were quantified by real-time PCR as markers of osteoblastic lineage differentiation. Expression of VDR was analyzed by western blot in subcellular fractions and VDR signaling was investigated using luciferase reporter assays. Results In coculture experiments, myeloma cells inhibited the vitamin D dependent differentiation and activity of osteoblast precursors, e.g. coculture of hMSC with the myeloma cell line LP-1 for 4 days decreased their osteocalcin expression by 58%. Treatment with bortezomib led to an increased osteoblastic differentiation of hMSC and hOB by OS, represented by an enhanced expression of osteoblast markers osteocalcin and OPN. Importantly, this effect could be further increased, when vitamin D was added. In hMSC stimulated with OS only, addition of 5 nM bortezomib led to an 18.3 fold increase in OPN mRNA expression. In comparison, hMSC stimulated with OS + vitamin D showed a 27.5 fold increase in OPN mRNA with the addition of bortezomib. Osteocalcin expression was increased 1.9 fold by bortezomib in the presence of OS and vitamin D, but not with OS alone. Similar results were obtained with osteoblasts: Incubation with bortezomib slightly increased osteocalcin and OPN mRNA expression in cells stimulated with OS only (1.3 fold and 2.4 fold, respectively). In comparison, in cells stimulated with OS and vitamin D, bortezomib elevated osteocalcin and OPN expression 2.9 fold and 5.5 fold, respectively. Bortezomib led to an increase in nuclear VDR levels in hMSC in western blot analyses. Primary hMSC transfected with a VDR luciferase reporter construct showed a 3.7 fold increase in VDR signaling with bortezomib. Conclusion Our data show that bortezomib stimulates osteoblastic differentiation of hMSCs and hOBs and acts, at least in part, through VDR signaling. Substitution of vitamin D in multiple myeloma patients treated with bortezomib may be beneficial for bone turnover and needs clinical evaluation. Disclosures: Kaiser: Johnson & Johnson: Research Funding. Mieth:Johnson & Johnson: Research Funding. Sezer:Johnson & Johnson: Research Funding. Heider:Johnson & Johnson: Research Funding.

Investigational Agent MLN9708 Target Tumor Suppressor MicroRNA-33b in Multiple Myeloma Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 136-136
Author(s):  
Ze Tian ◽  
Jian-Jun Zhao ◽  
Jianhong Lin ◽  
Dharminder Chauhan ◽  
Kenneth C. Anderson
Keyword(s):  
Multiple Myeloma ◽  
Tumor Suppressor ◽  
Board Of Directors ◽  
Expression Profiling ◽  
Research Funding ◽  
Advisory Committees ◽  
Myeloma Cells ◽  
Investigational Agent ◽  
Reporter Activity

Abstract Abstract 136 Investigational Agent MLN9708 Target Tumor Suppressor MicroRNA-33b in Multiple Myeloma Cells Ze Tian, Jianjun Zhao, Jianhong Lin, Dharminder Chauhan, Kenneth C. Anderson Medical Oncology, Dana Farber Cancer Institute and Harvard Medical School, Boston, MA, 02115 MicroRNAs (miRNAs) are 19–25 nucleotide-long noncoding RNA molecules that regulate gene expression both at the level of messenger RNA degradation and translation. Emerging evidence shows that miRNAs play a critical role in tumor pathogenesis by functioning as either oncogene or tumor suppressor genes. The role of miRNA and their regulation in response to proteasome inhibitors treatment in Multiple Myeloma (MM) is unclear. Here, we utilized MLN9708, a selective orally bio-available proteasome inhibitor to examine its effects on miRNA alterations in MM.1S MM cells. Upon exposure to aqueous solutions or plasma, MLN9708 rapidly hydrolyzes to its biologically active form MLN2238. Our previous study using both in vitro and in vivo models showed that MLN2238 inhibits tumor growth and triggers apoptosis via activation of caspases. Moreover, MLN2238 triggered apoptosis in bortezomib-resistant MM cells, and induced synergistic anti-MM activity when combined with HDAC inhibitor SAHA, dexamethasone, and lenalidomide. In the current study, we treated MM.1S cells with MLN2238 (12 nM) for 3 hours and harvested; total RNA was subjected to miRNA profiling using TaqMan® Array Human miRNA A-Card Set v3.0 and the data was analyzed using dChip analysis. Results showed that MLN2238 modulates miRNA expression with a total of 36 miRNA changing their expression profiling (δδCT>1.5 or δδCT <-1.5; 19 were upregulated and 17 showed a downregulation). Among all miRNA, miR-33b was highly (δδCT>7) upregulated in response to MLN2238 treatment. We therefore hypothesized that miR-33b may play a role in MM pathogenesis as well as during MLN2238-induced proteasome inhibition in MM cells. We first utilized quantitative polymerase chain reaction (q-PCR) to validate the changes in miRNA expression profiling. Results confirmed that MLN2238 treatment triggers significant increase in the miR-33b expression in MM.1S cells (2.1 and 2.2 folds at 3h and 6h, respectively; P<0.001). Examination of normal PBMCs and plasma cells showed higher expression of miR-33b than patient MM cells (P<0.001). We further investigated the functional role of miR-33b in MM cells at baseline and during MLN2238 treatment. Drug sensitivity, cell viability, apoptosis, colony formation, and migration assays were performed using cell TilTer-Glo, Annexin V-FITC/PI staining, MTT staining, and Transwell assays, respectively. Signaling pathways modulated post miR-33b overexpression were evaluated by q-PCR, immunoblot, and reporter assays. Our findings show that overexpression of miR-33b significantly decreased cell viability, cell migration, colony formation, as well as increased apoptosis and sensitivity of MM cells to MLN2238 treatment. Targetscan analysis predicted pim-1 as a putative downstream target of miR-33b. Overexpression of miR-33b downregulated pim-1 mRNA and protein expression. To further corroborate these data, we co-tranfected miR-33b and Pim-1-wt or Pim-1-mt in 293T and MM.1S cell lines. In concert with our earlier findings, miR-33b decreases pim-1-wt, but not pim-1-mt reporter activity in both cell lines. Reflecting the overexpression study results, MLN2238 treatment also decreases pim-1-wt, but not pim1-mt reporter activity. Moreover, a biochemical inhibitor of pim1/2 triggered apoptosis in MM cells. Finally, overexpression of miR-33b inhibits tumor growth (P<0.001) and prolongs survival (P<0.001) in both subcutaneous and disseminated human MM xenograft models. In summary, our study suggests that miR-33b is a tumor suppressor, which plays a role during MLN2238-induced apoptotic signaling in MM cells, and provide the basis for novel therapeutic strategies targeting miR-33b in MM. Disclosures: Anderson: Millennium: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: 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; Acetylon: Equity Ownership.

MAF Protein Elicits Innate Resistance To Bortezomib In Multiple Myeloma

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 281-281 ◽  
Author(s):  
Shiqiao Ye ◽  
Wei Qiang ◽  
Yu Chen ◽  
Bo Hu ◽  
Qing Zhang ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Research Funding ◽  
Molecular Heterogeneity ◽  
Molecular Subgroups ◽  
Immunoblotting Analysis ◽  
Primary Resistance ◽  
Myeloma Cells ◽  
Innate Resistance ◽  
E Cadherin

Abstract Multiple myeloma (MM) is a malignancy of terminally differentiated clonal plasma cells displaying significant molecular heterogeneity with 7 subgroups defined by gene expression profiling (GEP). Our previous work showed that MS and MF subgroups have been associated with inferior survival (Zhan et al, blood 2006). Furthermore, clinical studies have demonstrated that the addition of the proteasome inhibitor (PI) bortezomib (Bzb) to high dose melphalan based regimens provided a major advantage to patients in MS subgroup (Barlogie NJE 2006, Blood 2008; Pineda-Roman et al BJH 2008), while patients in the MF subgroups did not benefit from Bzb (Nair, Blood 2010). These findings led us to hypothesize that overexpression of MAF protein confers innate resistance to Bzb. In the present study, we assessed the ability of MAF to influence the innate resistance to Bzb and identify the molecular mechanism underlying the resistance of Bzb in high MAF-expressing patients. To investigate association of the limited therapeutic effect of Bzb with molecular subgroup of myeloma, we established the IC50 of Bzb in 24 myeloma cell lines (MMCL) belonging to different GEP-based molecular subgroups. IC50 concentration were higher (>25nM) in 7 of 9 MAF and in all 5 MAFb MMCL, and >40 nM in 5 MAF and one MAFb MMCL, which expressed the highest levels of MAF protein, as determined by immunoblotting analysis. In contrast, Bzb IC50 levels were lower (7.5-20 nM) for the MMCL belonging to the other molecular subgroups. These results indicate that high MAF expression in myeloma cells may contribute to primary resistance to Bzb. Mechanistically, immunoblotting analysis demonstrated that exposure to Bzb resulted in increased MAF protein levels. These results suggested that Bzb prevents the degradation of MAF protein in myeloma cells. To further confirm that Bzb-induced stabilization of MAF protein confers resistance to Bzb, we overexpressed MAF cDNA in myeloma cells lacking MAF expression, and silenced MAF expression in myeloma cells expressing high level of MAF mRNA and protein. MM cells were infected with Lentiviral vector containing MAF cDNA or with empty vector, and stable clones selected with puromycin, designated as MMmaf and MMEV, respectively. qPCR and immunoblotting analysis showed that expression of MAF mRNA and protein in MMmaf cells were significantly higher (1.8x105-fold) than in MMEV cells. The functionality of ectopic MAF protein was confirmed by qRT-PCR analysis of downstream target genes; the mRNA level of E-cadherin was higher in MMmaf cells than those of MMEV (1.42-fold, p<0.01). MTT assay showed that the proliferation rate of MMmaf cells was 53% higher than that of MMEV cells (p <0.001). Similar results were observed in other two MM cell lines that transiently ectopic expressed MAF. Moreover, MMmaf showed higher IC50 of Bzb than that of MMEV , indicating that increase MAF protein in myeloma cells reduces sensitivity to Bzb. We further generated loss of functional MAF by silencing MAF expression in MAF positive myeloma cells using shRNA specific to maf mRNA (shMAF) by lentiviral expressing system. shMAF infected myeloma cells had 75% lower levels of MAF mRNA and protein level compared with the cells infected with scrambled shRNA. Additionally, significantly decreased integrin E-cadherin (9.1-fold), cyclin D2 (4.99x105-fold), and CCR1 (4.9-fold) levels were observed in these cells, compared with the cells infected with control viral vector. Silencing MAF expression significantly decreased proliferation of myeloma cells (81.9% decrease, p=2.5E-6). Moreover, Bzb treatment of the cells infected with shMAF lead to 53.1%, inhibition (P=3.3E-8) of proliferation compared with control cells. Taken together, our results indicate that high expression of MAF protein confers myeloma primary resistance to Bzb, and Bzb induces stabilization of MAF protein further increases resistance to Bzb, providing the molecular rational for therapeutic strategy for high-MAF expressing myeloma patients. Disclosures: van Rhee: Jansen & Jansen: Research Funding. Barlogie:Celgene: Consultancy, Honoraria, Research Funding; Internation Myeloma Foundation: Consultancy, Honoraria; Millennium: Consultancy, Honoraria, Research Funding; Novartis: Research Funding; National Cancer Institute: Research Funding; Johnson & Johnson: Research Funding; Centocor: Research Funding; Onyx: Research Funding; Icon: Research Funding; Myeloma Health, LLC: Patents & Royalties.

CD38-Targeted Immunochemotherapy Of Multiple Myeloma: Preclinical Evidence For Its Combinatorial Use In Lenalidomide and Bortezomib Refractory/Intolerant MM Patients

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 277-277 ◽  
Author(s):  
Inger S. Nijhof ◽  
Willy A. Noort ◽  
Jeroen Lammerts van Bueren ◽  
Berris van Kessel ◽  
Joost M. Bakker ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Research Funding ◽  
Plasma Cells ◽  
Cell Lysis ◽  
In Vitro Assays ◽  
Human Antibody ◽  
Myeloma Cells ◽  
Clinical Phase

Abstract Multiple myeloma (MM) remains an incurable malignancy of clonal plasma cells. Although the new generation of immunomodulatory agents, such as lenalidomide (LEN), and the potent proteasome inhibitor bortezomib (BORT) have significantly improved the overall survival of MM patients, all chemotherapy strategies are eventually hampered by the development of drug-resistance. The outcome of patients who are refractory to thalidomide, lenalidomide (LEN) and bortezomib (BORT) is very poor. Set out with the idea that targeted immunotherapy with human antibodies may offer new perspectives for MM patients, we have recently developed daratumumab (DARA), a CD38 human antibody with broad-spectrum killing activity, mainly via ADCC (antibody dependent cellular cytotoxicity) and CDC (complement dependent cytotoxicity). In our previous preclinical studies and in current clinical phase I/II trials, DARA induces marked anti-MM activity. Based on these encouraging results, we now explored the potential activity of DARA for patients who are refractory to LEN- and/or BORT. In a recently developed human-mouse hybrid model that allows the in vivo engraftment and outgrowth of patient-derived primary myeloma cells in immune deficient Rag2-/-gc-/- mice, single dose DARA treatment appeared to effectively inhibit the malignant expansion of primary MM cells derived from a LEN- and BORT-refractory patient, indicating the potential efficacy of DARA even in LEN/BORT refractory patients. To substantiate the conclusions of these in vivo data, we conducted in vitro assays, in which full BM-MNCs from LEN (n=11) and LEN/BORT (n=8) refractory patients were treated with DARA alone or the combination of DARA with LEN or BORT to induce MM cell lysis. As expected, LEN alone induced no or little lysis of MM cells in the LEN-refractory patients and also BORT was not able to induce any lysis in the BORT-refractory patients. On the contrary, DARA induced substantial levels of MM cell lysis in all LEN and LEN/BORT-refractory patients. This lysis was significantly enhanced by combination with LEN or BORT. The combination of DARA and BORT improved MM lysis by additive mechanisms. However, LEN improved DARA-mediated lysis of MM cells in a synergistic manner through the activation of effector cells involved in DARA-mediated ADCC. In conclusion, our results demonstrate that DARA is also effective against multiple myeloma cells derived from LEN- and BORT-refractory patients. Especially LEN seems to improve responses in a synergistic manner. Our results provide a rationale for clinical evaluation of DARA in combination with LEN to achieve more effective results in LEN- and BORT-refractory patients. Disclosures: Lammerts van Bueren: Genmab: Employment. Bakker:Genmab: Employment. Parren:Genmab: Employment. van de Donk:Celgene: Research Funding. Lokhorst:Genmab A/S: Consultancy, Research Funding; Celgene: Honoraria; Johnson-Cilag: Honoraria; Mudipharma: Honoraria.

Dual Antitumoral and Bone Antiresorptive Effect Of The Pan-Pim Kinase Inhibitor, LGH447, In Multiple Myeloma

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4435-4435
Author(s):  
Teresa Paíno ◽  
Antonio Garcia-Gomez ◽  
Lorena González-Méndez ◽  
Laura San-Segundo ◽  
Montserrat Martín-Sánchez ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Kinase Inhibitor ◽  
Annexin V ◽  
Myeloma Cells ◽  
Pim Kinases ◽  
Castilla Y Leon ◽  
Ic50 Values ◽  
Rpmi 8226 ◽  
Resorptive Activity

Introduction Multiple myeloma (MM) is characterized by the accumulation of malignant plasma cells in the bone marrow (BM) and is closely associated with osteolytic lesions, in part due to an increase in the bone-resorptive activity and number of osteoclasts (OCs). The activation of survival pathways in myeloma cells could be the cause of treatment failure rendering the disease incurable. Pim kinases are a family of survival serine/threonine kinases composed of three members (Pim1, Pim2 and Pim3) that are overexpressed in MM cells and may have a role in MM pathogenesis. However, little is known about the role of Pim kinases in OCs and its involvement in myeloma bone disease. Here, we have evaluated the preclinical activity of a new pan-Pim kinase inhibitor, LGH447, on MM cells and OCs. Cell lines, primary samples, material and methods LGH447 was provided by Novartis Pharmaceuticals. The human MM cell lines MM1S, MM1R, RPMI-8226 (or RPMI-8226-luc), RPMI-LR5, MM144, NCI-H929, OPM-2, U266, U266-Dox4 and U266-LR7 were employed. PBMCs from healthy volunteers were used to generate OCs, whereas primary mesenchymal stromal cells (MSCs) were obtained from bone marrow aspirates of MM patients. Cell viability was studied using MTT colorimetric assay or bioluminescence. Apoptosis was measured by annexin-V staining. For cell cycle analysis, propidium iodide staining was used. OC formation was assessed by enumeration of multinucleated (≥3) TRAP-positive cells and OC resorption was assessed on calcium-coated slides. Immunoblotting, quantitative PCR and immunofluorescence were used to further investigate the mechanism of action of LGH447. Results All MM cell lines expressed the three isoforms of Pim kinases with higher levels of Pim2. The dose-response curves to LGH447 after a 48 hour treatment revealed two groups of MM cell lines with regard to sensitivity to this drug: high sensitive, with IC50 values ranging from 0.2 to 3.3 µM (MM1S, MM1R, RPMI-8226, MM144, U266 and NCI-H929); and low sensitive, with IC50 values >7 µM (OPM-2, RPMI-LR5, U266-Dox4 and U266-LR7). Our results indicated that LGH447 promoted apoptosis in myeloma cells as shown by the increase in annexin-V positive cells and by the cleavage of initiator (caspases 8 and 9) and effector caspases (caspases 3 and 7) and of PARP. LGH447 also blocked the cell cycle in MM cells as demonstrated by the increase in G0-G1 and the decrease in S-G2-M phases. Importantly, LGH447 was also able to overcome the growth advantage conferred to RPMI-8226-luc cells by co-culture with MSCs or OCs. Regarding the mechanisms involved in these effects, LGH447 inhibited the mTOR pathway, demonstrated by a decreased phosphorylation of the downstream mTOR effectors, 4EBP1 and S6 in residues Thr37/46 and Ser235/236, respectively. Interestingly, LGH447 also inhibited OC formation and resorption activity. LGH447 treatment of human pre-OCs diminished the expression of key molecules involved in OC differentiation (p-Erk1/2 and NFATc1) and function [CAII (carbonic anhidrase II), CLCN7 (chloride channel 7), ATP6V1A (vacuolar-H+-ATPase catalytic subunit A1) and MMP9 (matrix metalloproteinase 9)] and also disrupted the F-actin ring necessary for OC effective resorption. Conclusion Overall, our results demonstrate that both MM cells and OCs are targets of the pan-Pim kinase inhibitor, LGH447. Therefore, the inhibition of Pim kinases could potentially provide a dual benefit in myeloma patients as a consequence of cytotoxic effects exerted on MM cells and an anti-resorptive activity on bone. This work was supported by funding from the Fundación Española de Hematología y Hemoterapia (AG-G), Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla y León, the RTICC-Hematology Group (RD12/0036/0058), Spanish FIS (PI12/02591) and the Junta de Castilla y León, Gerencia Regional de Salud (GRS 862/A/13). Disclosures: Off Label Use: LGH447 is a pan-Pim kinase inhibitor (Novartis Pharmaceuticals). It has been used for pre-clinical studies in multiple myeloma.

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