Treatment Of Multiple Myeloma Cells Research Articles

CD26 Is a Novel Target for the Treatment of Tumor Progression and Its Related Osteolytic Bone Disease in Multiple Myeloma

Background: CD26, a 110-kDa transmembrane glycoprotein with DPPIV activity, has been implicated in tumorigenesis and shown to be expressed in several tumor cells including malignant lymphoma, whereas its role has not been characterized in plasma cell malignancies, yet. We have recently shown that CD26 is intensely expressed in activated osteoclasts (OCs) in osteolytic bone tumors including multiple myeloma (MM). CD26 is not expressed in hematopoietic stem cells, but M-CSF and sRANKL induced human OC differentiation with the upregulation of CD26 expression in monocyte-macrophage lineage cells, OC precursor cells and OCs. Blockade of CD26 signaling with humanized anti-CD26 monoclonal antibody (YS110) impaired OC differentiation via inhibiting RANKL-induced MKK3/6-p38MAPK-mi/Mitf phosphorylation in OC precursor cells (Nishida et al., 2014). In the present study, we characterize the biological function of CD26 in MM cells in the bone marrow (BM) and investigate the therapeutic potential of YS110 to treat MM cell growth and reduce MM-induced osteolytic lesions.Methods and Results: Although CD26 expression was not detected any of 11 MM cell lines in mono-culture, CD26 expression levels were upregulated in all MM cell lines when co-cultured with OCs for 72 hours by flow cytometry and immunohistochemistry analysis. CD26 protein levels in MM cell lines were also enhanced in co-culture with OCs by immunoblotting. To determine the factors responsible for CD26 upregulation in MM cells, we examined CD26 expression in MM cell lines under transwell co-culture conditions with OCs or under stimulation with anti-apoptotic cytokines produced by OCs. CD26 expression in MM cell lines was reduced under transwell co-culture conditions with OCs. TNFα, BAFF, APRIL and SDF-1 slightly upregulated CD26 expression in MM cell lines. To further explore CD26 expression in BM of MM patients, we performed immunohistochemical stainig on decalcified bone sections of biopsy specimens of MM cases. CD26/CD138 positive plasma cells were detected around CD26 positive OCs and certain endothelial vascular cells in several cases. Next, to clarify the role of CD26 in MM cell survival, we examined the effects of YS110 on MM cell growth and related osteolytic bone lesions in vitro and in vivo. YS110 had no significant effects on viability of MM cell lines in mono-culture, but dose-dependently inhibited growth of MM cell lines in co-culture with OCs. YS110 immediately inhibited p38 activation and its downstream Hsp27 phosphorylation in MM cells. Contitnued treatment of MM cells with YS110 also led to the reduction of total Hsp27 protein at 12 hours and 16 hours after the treatment of YS110, with commensurate decrease of phospho-Hsp27 and to increased MM apoptosis correlated with the induction of p53, increased activation of caspases and PARP, as well as the reduction of Mcl-1 and c-Myc. In parallel, we constructed a xenograft of murine model of human MM. A total of 5x106 CD26 positive MM cell lines by co-culture with OCs were inoculated to mice (NOD/SCID-s.c. mice) or directly injected into the implanted human bone chips in mice (NOD/SCID-hu mice). Mice treated with YS110 (500μg/dose) showed a significant effect in inhibiting MM tumor weight compared with control mice 4 weeks after tumor cell inoculation. To further investigate whether YS110 could reduce MM-induced osteolysis as well as suppress MM cell growth, we used NOD/SCID-hu mice model. Mice were treated with YS110 after the first detection of tumor growth. Continuous YS110 treatment significantly suppressed MM cell growth after 4 weeks. Immunohistochemistry for CD138 and TRAP staining showed decreased numbers of MM cells and TRAP positive OCs, with decreased bone resorption activity in human bones from YS110-treated versus control mice. Lastly, we examined the effects of targeting CD26 with YS110 in CD26 positive myeloma stem-like cells (SP cells). CD26 expression was demonstrated in SP cells of several MM cell lines in co-culture with OCs. YS110 dose-dependently inhibited cell viability of CD26 positive SP cells co-cultured with OCs, indicating targeting CD26 might reduce chemoresistance in MM.Conclusions: Theses results suggest that CD26 may be a promising novel target in MM, strongly supporting YS110 to inhibit MM cell growth in the BM as well as its related osteolytic bone lesions. Our results provide the framework for clinical development of YS110 as a novel therapeutic option in MM. DisclosuresMorimoto:Y's Therapeutics: Consultancy, Research Funding. Yamada:Y's Therapeutics: Consultancy, Research Funding.

Analysis of the anti-proliferative and the pro-apoptotic efficacy of Syk inhibition in multiple myeloma.

BackgroundMultiple myeloma (MM) is a clonal B cell malignancy characterized by proliferation of malignant plasma cells in the bone marrow. Despite high-dose melphalan therapy with autologous stem cell transplantation (ASCT) and the introduction of immunomodulatory drugs like bortezomib or lenalidomide, that have been associated with improved survival, MM is still incurable and new treatment options are needed. In B cell malignancies such as chronic lymphocytic leukaemia (CLL) or diffuse large B cell lymphoma (DLBCL), Syk (spleen tyrosine kinase) inhibitors have shown promising in vitro and first clinical results. In our study, we analyzed the potential of Syk as a target in MM.MethodsThe MM cell lines AMO-1, U266 and RPMI8226 and primary MM cells were treated with the Syk inhibitors BAY61-3606, R406 or Piceatannol and proliferation, migration and apoptosis induction were analyzed. Effects on involved intracellular signaling cascades were determined by Western blotting. Furthermore, we analyzed synergistic and additive effects of Syk inhibitors in combination with established anti-myeloma drugs and experimental inhibitors (e.g. PI-3-Kinase inhibitor NVP-BEZ235).ResultsIncubation of MM cell lines as well as primary MM cells with Syk inhibitors resulted in a reduced proliferation and stromal cell-derived factor-1 alpha (SDF-1 alpha) induced migration that was accompanied by a concentration dependent inhibition of the MAP-Kinase, characterized by reduced phosphorylation of ERK an p38 molecules, and NF-kappaB signalling pathways. Furthermore, Syk inhibition induced apoptosis in MM cells in a dose-dependent manner, characterized by reduced expression of pro-caspase 3, increased PARP-1 cleavage and enhanced release of cytochrome c. In addition combined treatment of MM cells with Syk inhibitors and NVP-BEZ235 (dual PI3-kinase/mTOR inhibitor) or MAPK inhibitors (PD98059, SP600125, U0126, SB203580) resulted in increased apoptotic activity of the drugs.ConclusionsOur results show that Syk inhibition might represent a promising new treatment option in MM with an increased efficacy when combined with MAP kinase inhibitors. Furthermore, our study strongly underlines the potency of Syk inhibitors as a potential therapeutic treatment option for MM patients.Electronic supplementary materialThe online version of this article (doi:10.1186/s40164-015-0016-z) contains supplementary material, which is available to authorized users.

Abstract 4434: Selinexor and melphalan combination therapy for the treatment of multiple myeloma

Abstract Introduction High-dose melphalan (MEL) followed by autologous stem cell transplant remains the standard of care for the treatment of multiple myeloma (MM). However, patients eventually develop drug resistance and die from progressive disease despite newer therapies using proteasome inhibitors and immunomodulatory drugs. The incurable nature of MM demonstrates the need for novel treatments. Our aim was to investigate whether MEL therapy could be improved by the addition of the XPO1 inhibitor selinexor (SEL) in drug-resistant and parental MM cells both in vitro and ex vivo. Materials and Methods SEL/MEL-treated human MM cells were assayed for cell viability (CT-Blue) and apoptosis by flow cytometry (activated caspase 3). Proximity ligation assays (PLD) were used to assess if XPO1-p53 binding was inhibited by SEL. Western blots of SEL-treated MM cells were performed for nuclear and total p53. MEL-resistant U266 (LR6) and 8226 (LR5) MM cell lines were developed by incremental exposure to MEL. MEL resistant and parental MM cells were treated in vitro with SEL +/- MEL and assayed for apoptosis and cell viability. Cells isolated from patients with newly diagnosed or relapsed MM, were treated with SEL +/- MEL and assayed for apoptosis. Results MM cell viability was decreased synergistically by SEL when used in combination with MEL, as shown by combination index (CI) values. Drug sequencing assays showed that concurrent treatment with MEL (10 μM) and SEL (300 nM) for 48 hours produced synergistic results in human H929 MM cells (CI value 0.079, n = 3). Sequential treatment, SEL for 24 hours followed by MEL for an additional 24 hours or the reverse sequence, also demonstrated synergy with CI = 0.208 (n = 3) and 0.142 (n = 3), respectively. Normal PBMCs (control) were unaffected by SEL/MEL treatment as shown by viability and apoptotic assays. PLD demonstrated that SEL blocks XPO1/p53 binding. Western blot data showed that the SEL treatment of MM cells increased nuclear and total p53. Drug-resistant LR5 and LR6 MM cells were found to be resistant to MEL when compared to parental cell lines. Both resistant MM cell lines were sensitized by SEL to MEL as shown by apoptosis assay (20-fold). CD138+/light chain+ MM cells derived from newly diagnosed and relapsed MM patients were also sensitized (5 to 10-fold) by SEL to MEL as demonstrated by apoptosis assay. Conclusions SEL synergistically improved the response of drug-resistant and parental MM cells to MEL in vitro and ex vivo. It is possible that this synergy may be due to an increase of nuclear p53 by SEL and the reported activation of p53 by MEL. Ongoing studies include in vitro experiments to investigate whether this drug combination reverses MEL resistance by the Fanconi Anemia/BRCA pathway, in vivo treatment of MM in NSG mice with SEL/MEL and a clinical trial using high-dose MEL in combination with SEL. Combination therapies using SEL and MEL may significantly improve the treatment outcomes of MM. Citation Format: Joel G. Turner, Jana L. Dawson, Christopher Cubitt, Taiga Nishihori, Claudio Anasetti, Melissa Alsina, Rachid C. Baz, Michael Kauffman, Sharon Shacham, William S. Dalton, Daniel M. Sullivan. Selinexor and melphalan combination therapy for the treatment of multiple myeloma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4434. doi:10.1158/1538-7445.AM2015-4434

Rational combination treatment with histone deacetylase inhibitors and immunomodulatory drugs in multiple myeloma.

Immunomodulatory drugs (IMiDs) thalidomide, lenalidomide (Len) and pomalidomide trigger anti-tumor activities in multiple myeloma (MM) by targetting cereblon and thereby impacting IZF1/3, c-Myc and IRF4. Histone deacetylase inhibitors (HDACi) also downregulate c-Myc. We therefore determined whether IMiDs with HDACi trigger significant MM cell growth inhibition by inhibiting or downregulating c-Myc. Combination treatment of Len with non-selective HDACi suberoylanilide hydroxamic acid or class-I HDAC-selective inhibitor MS275 induces synergic cytotoxicity, associated with downregulation of c-Myc. Unexpectedly, we observed that decreased levels of cereblon (CRBN), a primary target protein of IMiDs, was triggered by these agents. Indeed, sequential treatment of MM cells with MS275 followed by Len shows less efficacy than simultaneous treatment with this combination. Importantly ACY1215, an HDAC6 inhibitor with minimal effects on class-I HDACs, together with Len induces synergistic MM cytotoxicity without alteration of CRBN expression. Our results showed that only modest class-I HDAC inhibition is able to induce synergistic MM cytotoxicity in combination with Len. These studies may provide the framework for utilizing HDACi in combination with Len to both avoid CRBN downregulation and enhance anti-MM activities.

PGC-1α is responsible for survival of multiple myeloma cells under hyperglycemia and chemotherapy.

The association between hyperglycemia and outcomes during chemotherapy has been reported in several tumors, including multiple myeloma (MM). However, the underlying mechanism of how hyperglycemia affects the survival of MM cells during chemotherapy remain to be elucidated. MM cells were cultured in 10mM glucose with or without chemotherapeutic agents. Following treatment of MM cells with dexamethasone or bortezomib, an MTT assay was used to evaluate the toxicity of dexamethasone or bortezomib on cell proliferation, and changes of reactive oxygen species (ROS) level were detected by flow cytometry (FCM) analysis. Small interference RNA (siRNA) was applied to inhibit the expression of peroxisome proliferator-activated receptorγ coactivator-1α (PGC-1α). Expressions of PGC-1α and antioxidant factors such as superoxide dismutase2 (SOD‑2), glutathione peroxidase1 (GPX-1), and catalase (CAT) were measured by RT-PCR prior to and following treatment. The results showed that the level of PGC-1α in MM cells cultured in high-glucose medium was upregulated prior to and following treatment of chemotherapeutic agents, and these cells showed less fold-change of ROS after insult of drugs, when compared to the control. Genes encoding antioxidant factors such as SOD-2 and CAT were also upregulated. Inhibition of PGC-1α enhanced the toxicity of antitumor agents, associated with reduced expression of antioxidant factors, and elevated level of ROS. The present findings suggested that hyperglycemia may influence the anticancer effect of chemotherapeutic agents in MM by upregulating the expression of PGC-1α and associated antioxidant factors. Inhibition of PGC-1α or control of hyperglycemia may be beneficial in improving the efficacy of chemotherapy in MM patients with diabetes.

Nitric oxide donors increase PVR/CD155 DNAM-1 ligand expression in multiple myeloma cells: role of DNA damage response activation.

BackgroundDNAX accessory molecule-1 (DNAM-1) is an activating receptor constitutively expressed by macrophages/dendritic cells and by T lymphocytes and Natural Killer (NK) cells, having an important role in anticancer responses; in this regard, combination therapies able to enhance the expression of DNAM-1 ligands on tumor cells are of therapeutic interest. In this study, we investigated the effect of different nitric oxide (NO) donors on the expression of the DNAM-1 ligand Poliovirus Receptor/CD155 (PVR/CD155) in multiple myeloma (MM) cells.MethodsSix MM cell lines, SKO-007(J3), U266, OPM-2, RPMI-8226, ARK and LP1 were used to investigate the activity of different nitric oxide donors [DETA-NO and the NO-releasing prodrugs NCX4040 (NO-aspirin) and JS-K] on the expression of PVR/CD155, using Flow Cytometry and Real-Time PCR. Western-blot and specific inhibitors were employed to investigate the role of soluble guanylyl cyclase/cGMP and activation of the DNA damage response (DDR).ResultsOur results indicate that increased levels of nitric oxide can upregulate PVR/CD155 cell surface and mRNA expression in MM cells; in addition, exposure to nitric oxide donors renders myeloma cells more efficient to activate NK cell degranulation and enhances their ability to trigger NK cell-mediated cytotoxicity. We found that activation of the soluble guanylyl cyclase and increased cGMP concentrations by nitric oxide is not involved in the up-regulation of ligand expression. On the contrary, treatment of MM cells with nitric oxide donors correlated with the activation of a DNA damage response pathway and inhibition of the ATM /ATR/Chk1/2 kinase activities by specific inhibitors significantly abrogates up-regulation.ConclusionsThe present study provides evidence that regulation of the PVR/CD155 DNAM-1 ligand expression by nitric oxide may represent an additional immune-mediated mechanism and supports the anti-myeloma activity of nitric oxide donors.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1023-5) contains supplementary material, which is available to authorized users.

Disruption of the Ikaros-Mediated Gene Expression Program in Multiple Myeloma with Immunomodulatory Agents

The close chemical analogues lenalidomide and pomalidomide are immunomodulating drugs (IMiDs) that possess antineoplastic activity in multiple myeloma (MM) and other hematologic malignancies. IMiDs exert intrinsic antiproliferative effects on MM cells at least in part through direct interaction with intracellular cereblon. Cereblon (CRBN) is a component of the E3 ubiquitin ligase complex that also includes CUL4, RBX1, and DDB1 – together referred to as CRL4CRBN. Direct binding of IMiDs to a small hydrophobic pocket of the CRBN carboxy-terminal domain induces altered ubiquitinylation activity, including disrupted autoubiquitinylation of CRL4CRBN (Ito et al. Science, 2010; Fischer et al. Nature, 2014). Recently, we and others described the zinc finger transcription factors Ikaros (IKZF1) and Aiolos (IKZF3) as endogenous targets of CRL4CRBN ubiquitinylation in MM cells (Lu et al. Science, 2014; Kronke et al. Science, 2014). We found that treatment of MM cells with lenalidomide (LEN) causes increased ubiquitinylation of IKZF1 and IKZF3 by CRL4CRBN, and targets them for degradation by the proteasome. This loss of IKZF1/3 was both necessary and sufficient to observe the cellular antiproliferative effects of LEN, suggesting that this a primary mechanism of IMiD activity in MM. However, it remains unclear how depletion of these transcription factors ultimately leads to reduced proliferation of myeloma cells. Several gene products are known to be perturbed in MM cells upon IMiD treatment, including transcriptional downregulation of the master regulator transcription factor IRF4 (Zhu et al. Blood, 2011; Lu et al. Science, 2014). Yet rescue experiments with exogenous expression of IRF4 cannot fully rescue effects of IMiD treatment in MM cells, suggesting that other IKZF1/3 target genes play a role in the antiproliferative effects of IMiDs. Here we use complementary gene expression and genomic approaches to discern the global effects of IMiD treatment on MM cells. Using chromatin-immunoprecipitation followed by high-throughput sequencing (ChIP-seq), we have determined the genome-wide binding profile of both IKZF1 and IKZF3 in MM cells. We find both IKZF1 and IKZF3 occupy genomic regions including promoters, gene bodies, and distal enhancer elements. In MM cells, IKZF1 and IKZF3 enrichment almost always co-occurs, corresponding to reports of IKZF1/3 heterodimers facilitating transcriptional programs in lymphoid cells (Morgan et al. EMBO, 1997). Treatment of MM cells with LEN results in a dramatic decrease of both IKZF1 and IKZF3 binding to the genome at promoters and enhancers. Additionally, we performed ChIP-seq on RNA polymerase II (RNAPII) after LEN treatment in order to determine how IMiD-mediated IKZF1/3 depletion affects cellular transcriptional activity. Loss of IKZF1/3 binding to target genes most often correlates with increased density of RNAPII in gene bodies, suggesting transcriptional derepression of IKZF1/3 target genes. This observation was confirmed with genome-wide expression analysis by microarray. Among the derepressed target genes were genes with known tumor suppressor activity including CDKN1A, KLF6, and TXNIP. The IRF4 locus was also found to be a direct target of IKZF1/3, including binding to a large distal enhancer region upstream of the IRF4 coding region. Yet unlike most other IKZF1/3 target genes, RNAPII density within the IRF4 gene body is significantly decreased upon LEN treatment, suggesting that unique transcriptional regulatory mechanisms function at this locus that are distinct from other IKZF1/3 targets. Knockdown of IKZF1 and IKZF3 expression by shRNAs results in increased TXNIP and decreased IRF4 mRNA and protein expression, further suggesting that IMiD-mediated degradation of IKZF1/3 leads to opposite effects on these genes. TXNIP encodes the thioredoxin binding protein, which increases cellular reactive oxygen species and promotes G0/G1 cell cycle arrest. Forced exogenous overexpression of TXNIP inhibits MM cell growth, indicating a potential additional mechanism of IMiD activity. These studies define the IKZF1/3-mediated transcription program in MM cells and detail its perturbation by IMiDs. Ultimately these data will be informative for understanding the downstream effectors of intrinsic IMiD activity in hematologic malignancies, and to further understand mechanisms for acquired or innate resistance to these therapies. DisclosuresNo relevant conflicts of interest to declare.

Antitumor Activity of Phosphoinositide 3-Kinases Inhibitor, Copanlisib in Multiple Myeloma Cells

Multiple myeloma (MM) is one of the common hematological malignancies and is a uniformly fatal disorder of B cells characterized by accumulation of abnormal plasma cells in the bone marrow. Proteasome inhibitor, bortezomib, and immunomodulatory drugs such as thalidomide and lenalidomide play important roles in the treatment of MM patients. Although novel agents including, e.g. bortezomib, have significantly improved the response and survival of patients with MM, a large number of patients eventually have relapsed. For the patients who relapse after treatment with novel agents, the prognosis is still poor. Thus circumstanced, alternative strategies are required for continued disease control. Phosphoinositide 3-kinases (PI3Ks) are a family of proteins involved in the regulator of cell growth, metabolism and proliferation. PI3K signaling pathway also plays a critical regulatory role in MM pathology, including survival, cellular proliferation, migration and angiogenesis. Therefore, PI3K signaling pathway may present attractive targets for MM treatment. Copanlisib also known as BAY80-6946 is a potent and highly selective reversible PI3K inhibitor. Copnalisib is currently investigated in a pivotal phase 2 clinical trial against hematological malignancy such as malignant lymphoma.We hypothesized that treatment with PI3K inhibitor and proteasome inhibitors together would result in enhanced therapeutic activity in MM cells. In this study, we investigated the efficacy of copanlisib by using the MM cell lines, RPMI8226, MM1.S and MM1.R and primary sample.72 h treatment of copanlisib exhibits cell growth inhibition of MM cell lines in a dose dependent manner. The treatment of proteasome inhibitors, bortezomib and carfilzomib exhibits cell growth inhibition partially against RPMI8226 cells in the presence of feeder cell line, HS-5. We examined the intracellular signaling in the presence of HS-5. Phosphorylation of Akt and activation of caspase 3 and poly (ADP-ribose) polymerase (PARP) was partially reduced by carfilzomib or bortezomib in the presence of HS-5. We found that the treatment of copanlisib abrogated the protective effects of HS-5 in RPMI8226 cells. We examined the intracellular signaling after treatment of copanlisib. Activity of caspase 3 and poly (ADP-ribose) polymerase (PARP) was increased after copnlisib treatment in a dose dependent manner. Because PI3K signaling pathway regulates MM cell migration, we next evaluated the chemotactic response of MM cells to stromal cell-derived factor 1α (SDF-1α). We found that 4 h treatment of SDF-1α significantly induced the migration of MM cells compared to control medium. Treatment of copanlisib inhibited SDF-1α-stimulated chemotaxis in a dose dependent manner. We found that phosphorylation of Akt was reduced after copanlisib treatment suggesting that intracellular PI3K signaling pathway may play the important role in SDF-1α induced chemotaxis of MM cells. We investigated the copanlisib activity against MM cells. Combined treatment of MM cells with proteasome inhibitor, carfilzomib or bortezomib, and copanlisib caused significantly more cytotoxicity than each drugs alone. Phosphorylation of Akt was reduced and cleaved PARP was increased after copanlisib with or without proteasome inhibitor. We also found that copanlisib which was combinaed with carfilzomib or borteomib exhibited cell growth inhibition against MM primary sample.Data from this study suggested that administration of the PI3K inhibitor, copanlisib may be a powerful strategy against stroma-associated drug resistance of MM cells and enhance cytotoxic effects of proteasome inhibitors in those residual MM cells. DisclosuresNo relevant conflicts of interest to declare.

Pyk2 promotes tumor progression in multiple myeloma

AbstractProline-rich tyrosine kinase 2 (Pyk2) is a member of the focal adhesion kinase family that has been recently linked to tumor development. However, its role in modulating multiple myeloma (MM) biology and disease progression remains unexplored. We first demonstrated that patients with MM present with higher expression of Pyk2 compared with healthy individuals. By using loss-of-function approaches, we found that Pyk2 inhibition led to reduction of MM tumor growth in vivo as well as decreased cell proliferation, cell-cycle progression, and adhesion ability in vitro. In turn, overexpression of Pyk2 promoted the malignant phenotype, substantiated by enhanced tumor growth and reduced survival. Mechanistically, inhibition of Pyk2 reduced activation of Wnt/β-catenin signaling by destabilizing β-catenin, leading to downregulation of c-Myc and Cyclin D1. Furthermore, treatment of MM cells with the FAK/Pyk2 inhibitor VS-4718 effectively inhibited MM cell growth both in vitro and in vivo. Collectively, our findings describe the tumor-promoting role of Pyk2 in MM, thus providing molecular evidence for a novel tyrosine kinase inhibitor as a new therapeutic option in MM.

Abstract 3383: JunB/AP-1 controls MM cell proliferation, survival and drug resistance in the bone marrow microenvironment

Abstract The activator protein-1 (AP-1) transcription factor is a dimeric complex that comprises members of the JUN, FOS, activating transcription factor (ATF) and musculoaponeurotic fibrosarcoma (MAF) protein families. Resultant heterodimers and homodimers determine the genes that are regulated by AP-1. Functionally, AP-1 has been implicated in a multitude of biological processes but also tumorigenesis, dependent on the cell type and the pathophysiologic context. In multiple myeloma (MM), the role of AP-1 is largely unknown. The present study focuses on the impact of JunB, a central member of the AP-1 family, in MM growth, survival and drug resistance. First, our data demonstrate rapid and strong induction of AP-1/JunB expression in MM cell lines and primary tumor cells upon co-culture with bone marrow stromal cells (BMSCs), i.e. isotypic primary BMSCs as well as BMSC lines KM-105 and HS-27A. Importantly, utilizing a non-contact co-culture system, we show that this effect is predominantly mediated by soluble factors secreted by BMSCs rather than direct MM-BMSC contact. Consistent with gene expression profiling data, JunB mRNA levels detected by quantitative real-time PCR are increased in MM cells when co-cultured with BMSCs. Specifically, we found that upregulation of JunB occurs predominantly at the translational level, since cycloheximide, but not actinomycin D or MG-132 significantly blocked BMSC- induced JunB expression. Pharmacologic inhibition was used next in order to identify upstream signaling pathways, which mediate BMSC- induced JunB upregulation in MM cells. Our data demonstrate that activation of MEK/MAPK or NF-kB, but not PI3K/Akt is required for induction of JunB expression and AP-1 transcriptional activity. To delineate the specific functional role of JunB in MM pathogenesis, we subsequently generated stable MM cell lines carrying the pLKO.1-puro-JunB shRNA vector or the pLKO.1-puro control vector, respectively. Our preliminary data show significantly decreased proliferation and resistance to chemotherapy in MM/ pLKO.1-puro-JunB shRNA but not MM/ pLKO.1-puro control cells when co-cultured with BMSCs. Consistently, 4-hydroxytamoxifen (4-HT) treatment of MM cells stably transduced with pBabe-puro-JunB-ER-IRES-GFP but not of MM cells transduced with pBabe-puro-IRES-GFP promotes tumor cell proliferation, survival and drug resistance, independent of BMSC co-culture. Ongoing studies aim to identify downstream effectors of JunB, using gene array profiling of MM/ pLKO.1-puro-JunB shRNA cells versus MM/ pLKO.1-puro control cells. Furthermore, we are conducting a survey of published and unpublished datasets to uncover the clinical and prognostic relevance of JunB expression in MM patient samples. In summary, our data demonstrate for the first time an important role of AP-1/JunB in MM tumorigenesis and strongly propose it as a novel therapeutic target in MM. Citation Format: Fengjuan Fan, Sonia Vallet, Martin Sattler, Giovanni Tonon, Muhammad Hasan Bashari, Latifa Bakiri, Hartmut Goldschmidt, Erwin F. Wagner, Dirk Jaeger, Klaus Podar. JunB/AP-1 controls MM cell proliferation, survival and drug resistance in the bone marrow microenvironment. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3383. doi:10.1158/1538-7445.AM2014-3383

Inhibition Of PI3K Classia Kinases Using GDC0941 Overcomes Protection Of Multiple Myeloma Cells In The Bone Marrow Microenvironment

Despite of newly developed and more efficacious therapies, multiple myeloma (MM) remains incurable as most patient will eventually relapse and become refractory. The bone marrow (BM) microenvironment provides niches that are advantageous for drug resistance. Effective therapies against MM should ideally target the various protective BM niches that promote MM cell survival and relapse. In addition to stromal mesenchymal/myofibroblastic cells, osteoclasts play a key supportive role in MM cell viability. Additionally, 80% of patients develop osteolytic lesions, which is a major cause of morbidity. Increased osteoclast activity is characteristic in these patients and targeting osteoclast function is desirable to improve therapies against MM.Osteoclasts need to form an F-actin containing ring along the cell margin that defines a resorbing compartment where protons and degradative enzymes are secreted for dissolution of bone mineral. Remodelling of F-actin and vesicle secretion are regulated by the class IA PI3K pathway during osteoclastic bone resorption. Additionally, it has recently been shown that inhibition of the class IA PI3K pathway in MM cells with GDC0941 induces apoptosis-mediated killing. We hypothesised that GDC0941 could be used as a therapeutic agent to overcome MM-induced osteoclast activation.GDC0941 inhibited maturation of osteoclasts derived from BM aspirates from MM patients in a dose dependent manner. This correlated with decreased bone resorption of osteoclasts cultured on dentine discs. Exposure of mature osteoclasts to GC0941 resulted in abnormal organisation of larger F-actin rings, suggesting a negative effect on the dynamics of the actin cytoskeleton required for bone resorption. We also found that GDC-0941 can prevent protection of the MM cell lines MM1.S and MM1.R by osteoclasts against killing. GDC-0941 alone blocked MM cell proliferation independently of the presence of BM stromal cells and synergised with other therapeutic agents including Lenalidomide, Pomalidomide, Bortezomid and Dexamethasone.We also found that in the presence of MM cells, Dexamethasone (a drug commonly used alone or in combination with new drugs against MM) induced the proliferation of BM stromal cells and adhesion of MM cells on this protective stroma in a dose dependent manner. Dexamethasone is highly effective at MM cell killing when cells are cultured alone. However, we found that at low doses (below 1 uM) and in the presence of BM stromal cells, Dexamethasone could induce MM cell proliferation. GDC0941 enhanced Dexamethasone killing even in the presence of BM stromal cells by blocking Dexamethasone-induced stromal cell proliferation and adhesion of MM cells on the stroma. Targeting individual the PI3K Class IA isoforms alpha, beta, delta or gamma proved to be a less efficient strategy to enhance Dexamethasone killing. Previous work has shown that efficacy of targeting individual PI3K Class I A isoforms would be low for activation of caspases in MM cells as it would be dependent on relative amounts of isoforms expressed by the MM patient. GDC-0941 also inhibited the proliferation of MM1.R and RPMI8266 MM cell lines, which are less sensitive to treatment to Dexamethasone.Co-culture of MM cells with BM stromal cells induced the secretion of IL-10, IL-6, IL-8, MCP-1 and MIP1-alpha. The dose-dependant increased proliferation of Dexamethasone-treated MM cells in the presence of the BM stroma correlated with the pattern of secretion of IL-10 (a cytokine that can induce B-cell proliferation) and this was blocked by the combination of Dexamethasone with GDC0941.GDC-0941 alone or in combination with Dexamethasone was more efficacious at inducing MM cell apoptosis in the presence of the BM stroma cells vs treatment of MM cells alone. These are very encouraging results as they suggest that GDC-0941 in combination with Dexamethasone would be potentially highly efficacious for targeting MM cells in the BM microenvironment. We are currently performing in vivo data using C57BL/KaLwRij mice injected with 5T33-eGFP MM cells that will be discussed at the meeting.We propose that MM patients with active bony disease may benefit from treatment with GDC0941 alone or in combination with currently used therapeutic drugs against MM. Disclosures:No relevant conflicts of interest to declare.

Lactate Is a Crucial Energy Source For Multiple Myeloma (MM) Cells In Bone Marrow Microenvironment

IntroductionIt has been reported that the growth of certain cancer cells depends on the Warburg effect (aerobic glycolysis) for such cells to adapt to hypoxic environment and obtain ATP efficiently via glycolysis. We have previously reported that genes related to aerobic glycolysis are up-regulated in MM cells, and MM cells produce large amounts of lactate (Br J Cancer 2013, 108, 170-8). In the last ASH meeting, we reported that the expressions of lactate transporters were critical for the survival of MM cells. In this study, we further investigated the roles of lactate transporters in MM cells, focusing on the role of stromal cells as the supplier of lactate in microenvironment. MethodsSix MM cell lines, RPMI8226, U266, KMS12BM, KMS12PE, KHM11, and KMM1 were utilized. Bone marrow samples were obtained from newly diagnosed MM patients. Bone marrow mononuclear cells were isolated by Ficoll-Hypaque density sedimentation. To obtain stromal cells, bone marrow mononuclear cells were cultured in RPMI1640 medium supplemented with 10% FCS and allowed to attach to the bottom of plastic wells. After 3-4 weeks incubation, adherent cells were harvested and utilized as stromal cells. The expression of three components of the lactate transporter complex [monocarboxyl transporter 1 (MCT1), MCT4, and CD147] were analyzed with western blotting or flow cytometry. Lactate uptake was quantified using [14C]-Lactate. Cellular ATP production was quanatified with the ATP Determination Kit (Molecular Probes). An inhibitor of MCT1, a-cyano-4 hydroxycinnamic acid (CHC), was utilized to analyze cytotoxic effects on MM cells under both normoxia ( O2 20% ) and hypoxia (O2 1% ). AnnexinV/PI staining was performed to quantify cytotoxicity. Knockdown of MCT1 was achieved using siRNA. ResultsKnockdown of MCT1, a molecule responsible for lactate incorporation into cytoplasm, induced apoptosis in MM cells, while lactate uptake and ATP production of MM cells were reduced, suggesting that MCT1 inhibition might induce apoptosis in MM cells by decreasing the lactate-derived ATP production. On the other hand, we found significant lactate production by stromal cells. We also found an abundant amount of MCT4, a molecule responsible for lactate excretion, in bone marrow-derived stromal cells. By contrast, lactate amount in the culture supernatant of stromal cells was significantly decreased when they were co-cultured with MM cells. Therefore, it appears that lactate produced by stromal cells is incorporated into MM cells as an energy source. Interestingly, we found that the expression of MCT1 is up-regulated in MM cells under an aerobic condition, while that of MCT4 is up-regulated under a hypoxic condition. The treatment of MM cells with a-cyano-4 hydroxycinnamic acid (CHC), a MCT1 inhibitor, induced apoptosis in MM cells, suggesting that lactate may be more preferentially incorporated into MM cells under a normoxic than hypoxic condition (Figure A). [Display omitted] ConclusionOur results suggest that the growth of MM cells, at least in part, depends on lactate under a normoxic condition. The data also suggest that lactate may play a role as an energy interplay shuttle between stromal cells and MM cells (Figure B). Therefore, regulating lactate incorporation by MM cells may provide a new avenue for a therapeutic strategy for multiple myeloma. Disclosures:No relevant conflicts of interest to declare.

PRIMA-1Met/APR-246 Displays High Antitumor Activity in Multiple Myeloma By Induction of p73 and Noxa

Targeting p53 by the small-molecule PRIMA-1(Met)/APR-246 has shown promising preclinical activity in various cancer types. However, the mechanism of PRIMA-1(Met)-induced apoptosis is not completely understood and its effect on multiple myeloma cells is unknown. In this study, we evaluated antitumor effect of PRIMA-1(Met) alone or its combination with current antimyeloma agents in multiple myeloma cell lines, patient samples, and a mouse xenograft model. Results of our study showed that PRIMA-1(Met) decreased the viability of multiple myeloma cells irrespective of p53 status, with limited cytotoxicity toward normal hematopoietic cells. Treatment of multiple myeloma cells with PRIMA-1(Met) resulted in induction of apoptosis, inhibition of colony formation, and migration. PRIMA-1(Met) restored wild-type conformation of mutant p53 and induced activation of p73 upregulating Noxa and downregulating Mcl-1 without significant modulation of p53 level. siRNA-mediated silencing of p53 showed a little effect on apoptotic response of PRIMA-1(Met), whereas knockdown of p73 led to substantial attenuation of apoptotic activity in multiple myeloma cells, indicating that PRIMA-1(Met)-induced apoptosis is, at least in part, p73-dependent. Importantly, PRIMA-1(Met) delayed tumor growth and prolonged survival of mice bearing multiple myeloma tumor. Furthermore, combined treatment of PRIMA-1(Met) with dexamethasone or doxorubicin displayed synergistic effects in both multiple myeloma cell lines and primary multiple myeloma samples. Consistent with our in vitro observations, cotreatment with PRIMA-1(Met) and dexamethasone resulted in enhanced antitumor activity in vivo. Our study for the first time shows antimyeloma activity of PRIMA-1(Met) and provides the rationale for its clinical evaluation in patients with multiple myeloma, including the high-risk group with p53 mutation/deletion.

ADAM10 CONTRIBUTES TO MICA/B SHEDDING FROM MULTIPLE MYELOMA CELLS IN RESPONSE TO GENOTOXIC STRESS

Event Abstract Back to Event ADAM10 CONTRIBUTES TO MICA/B SHEDDING FROM MULTIPLE MYELOMA CELLS IN RESPONSE TO GENOTOXIC STRESS Francesca Cecere1*, Alessandra Zingoni1*, Elisabetta Vulpis1, Rosa Molfetta1, Alessandra Soriani1, Cinzia Fionda1, Maria Teresa Petrucci2, Maria Rosaria Ricciardi2, Rossella Paolini1, Marco Cippitelli1 and Angela Santoni1 1 "Sapienza" University of Rome, Molecular Medicine, Italy 2 "Sapienza" University of Rome, Hematology, Italy The MHC class I-related chain MICA/B ligands for the activating receptor NKG2D can be shed from tumor cells and their presence in sera is associated with impaired NK and CTL effector functions and disease progression. We showed that multiple myeloma (MM) cells up-regulate surface expression of NKG2D ligands after treatment with low doses of chemotherapeutic drugs and become more susceptible to NK cell lysis. Here, we investigated whether drug treatment of MM cells could result in MICA/B release. Soluble MICB was up-regulated after drug treatment and the ADAM10 specific inhibitor GI254023X dramatically reduced MICB shedding in a dose-dependent manner and was accompanied by a concomitant increase of cell-surface MICB. Similar results were obtained by shRNA-mediated gene silencing. Remarkably, chemotherapeutic agents increased ADAM10 expression either in MM cell lines or in ex-vivo malignant CD138+/CD38+ plasma cells. Regarding MICA, since its polymorphism can affect the shedding process, we investigated the effect of chemotherapeutic agents on the release of two allelic variants of MICA: MICA*019 and MICA*008 which represent the prototype of the long and short form of the MICA alleles, respectively. Similarly to MICB, drug treatment stimulated MICA*019 shedding with an ADAM10 dependent mechanism while didn’t affect MICA*008 release. Our findings indicate that chemotherapeutic drugs not only enhance MICA/B surface expression on MM cells, but also promote their shedding. The MICA genotype and the relative contribution of membrane associated vs soluble MICA/B in NKG2D dependent mechanisms of NK cell recognition of MM cells, should be considered to design NK cell-based immunochemotherapeutic approaches. References Soriani, A., Zingoni, A., Cerboni, C., Iannitto, M.L., Ricciardi, M.R., Di Gialleonardo, V., Cippitelli, M., Fionda, C., Petrucci, M.T., Guarini, A., Foa’, R., Santoni, A. (2009). ATM-ATR dependent up-regulation of DNAM-1 and NKG2D ligands on multiple myeloma cells by therapeutic agents results in enhanced NK-cell susceptibility and is associated with a senescent phenotype. Blood, 113, 3503-3511. Keywords: Natural Killer cells, NKG2D, Metalloproteinases, Multiple Myeloma, MICA polymorphism Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Innate immunity Citation: Cecere F, Zingoni A, Vulpis E, Molfetta R, Soriani A, Fionda C, Petrucci M, Ricciardi M, Paolini R, Cippitelli M and Santoni A (2013). ADAM10 CONTRIBUTES TO MICA/B SHEDDING FROM MULTIPLE MYELOMA CELLS IN RESPONSE TO GENOTOXIC STRESS. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00115 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 11 Mar 2013; Published Online: 22 Aug 2013. * Correspondence: Dr. Francesca Cecere, "Sapienza" University of Rome, Molecular Medicine, Rome, Italy, francesca.cecere@uniroma1.it Dr. Alessandra Zingoni, "Sapienza" University of Rome, Molecular Medicine, Rome, Italy, alessandra.zingoni@uniroma1.it Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Francesca Cecere Alessandra Zingoni Elisabetta Vulpis Rosa Molfetta Alessandra Soriani Cinzia Fionda Maria Teresa Petrucci Maria Rosaria Ricciardi Rossella Paolini Marco Cippitelli Angela Santoni Google Francesca Cecere Alessandra Zingoni Elisabetta Vulpis Rosa Molfetta Alessandra Soriani Cinzia Fionda Maria Teresa Petrucci Maria Rosaria Ricciardi Rossella Paolini Marco Cippitelli Angela Santoni Google Scholar Francesca Cecere Alessandra Zingoni Elisabetta Vulpis Rosa Molfetta Alessandra Soriani Cinzia Fionda Maria Teresa Petrucci Maria Rosaria Ricciardi Rossella Paolini Marco Cippitelli Angela Santoni PubMed Francesca Cecere Alessandra Zingoni Elisabetta Vulpis Rosa Molfetta Alessandra Soriani Cinzia Fionda Maria Teresa Petrucci Maria Rosaria Ricciardi Rossella Paolini Marco Cippitelli Angela Santoni Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. 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MiR-29b Exerts Anti-Multiple Myeloma Activity by Targeting Key Oncogenic Pathways and Modulating DNA Methylation Profile.

AbstractAbstract 2941The enforced expression of tumor suppressor microRNAs (miRNAs) is a promising strategy for cancer treatment. Among miRNAs frequently deregulated in cancer, miR-29b is of great interest since its overexpression leads to tumor growth inhibition in solid tumors and leukemias. On these bases, we first investigated miR-29b expression pattern in multiple myeloma (MM) cells. miRNA profiling of primary CD138+ MM patient cells (n=55) revealed deregulated expression of miR-29b with almost 60% of cases showing strongly reduced levels. Moreover, as assessed by quantitative real time PCR (qRT-PCR), miR-29b expression was significantly downregulated in a panel of 11 MM cell lines and even further decreased when RPMI-8226 MM cells were co-cultured with bone marrow stromal cells (BMSCs), indicating that the human bone marrow microenvironment (huBMM) modulates miR-29b levels. Either transient expression of synthetic miR-29b mimics through electroporation or stable lentivirus-enforced miR-29b expression induced growth inhibition and apoptosis of MM cells in vitro. In vivo intratumoral or systemic delivery of neutral lipidic formulated-miR-29b mimics in different clinically relevant murine models of human MM, including our recent SCID-synth hu model which recapitulates the huBMM, led to significant tumor growth inhibition together with increased survival of miR-29b-treated animals. By Western blot and qRT-PCR analysis, we observed dramatic reduction of predicted targets of miR-29b including CDK6 and MCL-1, which can thus explain antiproliferative and pro-apoptotic effects triggered by miR-29b expression. Most importantly, we identified Sp1, a transcription factor endowed with oncogenic activity in MM, as a negative regulator of miR-29b expression providing evidence of a novel regulatory loop in MM cells: in fact, enforced expression of Sp1 was able to reduce miR-29b-promoter activity and miR-29b levels, whereas miR-29b expression decreased Sp1 mRNA and protein levels via 3'UTR binding, as assessed in luciferase reporter assays. Moreover, treatment of MM cells with the proteasome inhibitor bortezomib led to Sp1 downregulation and miR-29b upregulation. Strikingly, miR-29b transfection significantly strengthened the in vitro anti-proliferative and apoptotic effects induced by bortezomib, thus highlighting its role in the mechanism of anti-MM activity of this drug. Additional miR-29b targets include de novo DNA methyltransferase 3A (DNMT3A) and DNA methyltransferase 3B (DNMT3B). Microarray profiling revealed increased DNMT3A and DNMT3B mRNA levels in cancer cells as compared to normal plasma cells. The integrated analysis of miRNA/mRNA profiling in a panel of 18 MM cell lines highlighted an inverse correlation between miR-29b and DNMT3B levels. Finally, miR-29b was proven to target DNMT3A and DNMT3B and significantly reduced the global DNA methylation levels in MM cells, as assessed by performing an in vitro DNA methylation assay with genomic DNA extracted from MM cells after synthetic miR-29b electroporation. In conclusion, our findings indicate that miR-29b exerts anti-MM activity by targeting relevant oncogenic pathways and by restoring the aberrant methylation pattern of MM cells. These results support a potential therapeutic role for miR-29b mimics in MM as single agent or in combination with the proteasome inhibitor bortezomib and/or demethylating agents. Disclosures:Munshi:Celgene: Consultancy; Millenium: Consultancy; Merck: Consultancy; Onyx: Consultancy.

Fas apoptosis inhibitory molecule is upregulated by IGF-1 signaling and modulates Akt activation and IRF4 expression in multiple myeloma

Multiple myeloma (MM) is an incurable malignancy of terminally differentiated B-lymphoid cells. Here, we investigate the role of Fas apoptosis inhibitory molecule (FAIM) in MM. We demonstrate that insulin-like growth factor 1 (IGF-1) treatment upregulated FAIM expression in MM cells in a dose-dependent manner. Silencing of FAIM expression attenuates Akt signaling downstream of IGF-1 and compromises the viability of MM cells. We further showed that IGF-1 stimulation of MM cells leads to enhanced expression of IRF4, a known 'addictive' factor for MM. This upregulation of IRF4 expression by IGF-1 treatment of MM cells is abrogated when FAIM expression is silenced or Akt activation is inhibited. Thus, FAIM modulates IGF-1-induced Akt activation and IRF4 expression and has a role in MM cell survival. Consistent with these findings, FAIM expression is shown to be higher in plasma cells of symptomatic MM patients compared with normal individuals or patients with premalignant conditions. Moreover, a higher level of FAIM expression is shown to correlate with poorer survival outcomes of newly diagnosed MM patients treated with stem cell transplantation or relapsed MM patients treated in clinical trials with Bortezomib. Thus taken together, our study reveals a novel, as well as clinically relevant role for FAIM in MM.

Effect of matrix metallopeptidase 13 (MMP13) on multiple myeloma (MM) cells, osteoclast (OCL) activity, and bone resorption.

8099 Background: MM cells produce OCL-activating factors that induce excessive bone resorption resulting in lytic lesions. The role of MMPs in invasion/progression of solid tumors is well-known, but its function in MM has not been well elucidated. Our group has shown that MMP13 is highly expressed in primary MM cells and in sera of MM patients. Levels of MMP13 significantly correlate with the extent of bone disease. MMP13 is induced by IL-6 via AP-1 activation in MM cells and enhances fusion of OCL precursors resulting in excessive bone resorption. OCL formation using MNCs of mmp-13-/- mice resulted in a fusion defect, significantly decreased OCL size and activity, which could be reversed by exogenous MMP13 (ASH 2009, IMW 2011). Methods: Methods will be presented in the Results section. Results: RT-PCR and western blotting revealed that IL-6 treatment of MM cells induced MMP13 transcription (30-fold) and secretion (>1000-fold). Protein expression of the AP-1 members c-Jun and c-Fos was induced by IL-6, which correlated with MMP13 upregulation. Our data further indicate that the catalytic activity of MMP13 is not required to enhance OCL formation and bone resorption. To prove this, we generated the MMP13 activity-dead mutation MMP13-E223A construct by site-directed mutagenesis PCR-based cloning. The mutated protein was overexpressed in HEK293 cells and purified from the supernatant to confirm whether loss of catalytic activity blocks MMP13 function. To further investigate the in vivo role of MMP13 in MM bone disease, MMP13 expression was knocked down (KD) in murine 5TGM1-MM cells by pKLO. 1 puro lentiviral infection containing sh-RNA targeting mouse MMP13 sequence. MMP13-KD 5TGM1-MM cells or WT-5TGM1-MM cells were intratibially injected into RAG2-/- mice. Development of lytic bone lesions are monitored by micro-QCT and data will be available at the time of presentation. Conclusions: Our data suggest that MMP13, secreted by MM cells, plays a critical role in the development of lytic lesions. Targeting MMP13 represents a promising approach to treat or to prevent bone disease in MM.

Abstract 1534: Hes1 regulates hypoxia induced chemoresistance of myeloma cells

Abstract Bone marrow (BM) microenvironment plays a critical role in response of tumor cells to chemotherapeutic drugs. Hypoxia has been shown to contribute to pathogenesis not only solid but also hematological tumors. While normal BM environment is hypoxic, tumor growth is associated with increased hypoxia in BM niche. We have previously demonstrated that activation of Notch signaling could be one of the mechanisms responsible for survival of multiple myeloma (MM) cells from chemotherapeutic drugs. Here we investigated whether hypoxia could modulate Notch signaling and induce chemoresistance of MM cells. Three MM cell lines were treated with doxorubicin or melphalan in hypoxic (1% O2) or normoxic conditions. Using Annexin V binding assay we demonstrated that hypoxia induced chemoresistance in only one (H929) cell line studied. This effect was associated with significant up-regulation of expression of Notch target gene Hes1 and its protein level while Hes1 level was unchanged in other two MM cell lines. Inhibition of Hes1 with siRNA canceled the protective effect of hypoxia on MM cells. To further confirm the involvement of Notch pathway in regulation of drug sensitivity, Hes-1 was overexpressed in MM cells in normoxic condition using vector pIRES-AcGFP-Hes-1. MM cells with increased Hes1 level became less sensitive to chemotherapeutics as compared to cells transfected with control vector. Hes-1 is considered to be a Notch target gene. However, hypoxia did not affect Notch receptors expression level in MM cells. The effect of hypoxia on Hes1 was Notch-independent as was evident from Notch/CBF1 reporter assay. In addition, inhibition of Notch receptor cleavage with gamma-secretase inhibitor GSI-XII (Calbiochem) did not affect hypoxia induced MM cell chemoresistance. Up-regulation of Hes-1 was induced by HIF-1a as treatment of MM cells with hypoxia mimetic deferoxamine resulted in up-regulation of Hes1 and inhibition of HIF-1a with siRNA abrogated hypoxia-induced protection of MM cells. Thus, our study, for the first time, demonstrates that hypoxia could induce chemoresistance of MM cells through Notch-independent up-regulation of Hes-1. These results could be important not only for understanding the cancer biology but for designing therapeutic intervention in MM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1534. doi:1538-7445.AM2012-1534

Inhibition of STAT3 signaling and induction of SHP1 mediate antiangiogenic and antitumor activities of ergosterol peroxide in U266 multiple myeloma cells

BackgroundErgosterol peroxide (EP) derived from edible mushroom has been shown to exert anti-tumor activity in several cancer cells. In the present study, anti-angiogenic activity of EP was investigated with the underlying molecular mechanisms in human multiple myeloma U266 cells.ResultsDespite weak cytotoxicity against U266 cells, EP suppressed phosphorylation, DNA binding activity and nuclear translocalization of signal transducer and activator of transcription 3 (STAT3) in U266 cells at nontoxic concentrations. Also, EP inhibited phosphorylation of the upstream kinases Janus kinase 2 (JAK2) and Src in a time-dependent manner. Furthermore, EP increased the expression of protein tyrosine phosphatase SHP-1 at protein and mRNA levels, and conversely silencing of the SHP-1 gene clearly blocked EP-mediated STAT3 inactivation. In addition, EP significantly decreased vascular endothelial growth factor (VEGF), one of STAT3 target genes at cellular and protein levels as well as disrupted in vitro tube formation assay. Moreover, EP significantly suppressed the growth of U266 cells inoculated in female BALB/c athymic nude mice and immunohistochemistry revealed that EP effectively reduced the expression of STAT3 and CD34 in tumor sections compared to untreated control.ConclusionThese findings suggest that EP can exert antitumor activity in multiple myeloma U266 cells partly with antiangiogenic activity targeting JAK2/STAT3 signaling pathway as a potent cancer preventive agent for treatment of multiple myeloma cells.

Inhibition of the MUC1-C oncoprotein induces multiple myeloma cell death by down-regulating TIGAR expression and depleting NADPH

AbstractThe MUC1-C oncoprotein is aberrantly expressed in most multiple myeloma cells. However, the functional significance of MUC1-C expression in multiple myeloma is not known. The present studies demonstrate that treatment of multiple myeloma cells with a MUC1-C inhibitor is associated with increases in reactive oxygen species (ROS), oxidation of mitochondrial cardiolipin, and loss of the mitochondrial transmembrane potential. The MUC1-C inhibitor-induced increases in ROS were also associated with down-regulation of the p53-inducible regulator of glycolysis and apoptosis (TIGAR). In concert with the decrease in TIGAR expression, which regulates the pentose phosphate pathway, treatment with the MUC1-C inhibitor reduced production of NADPH, and in turn glutathione (GSH) levels. TIGAR protects against oxidative stress-induced apoptosis. The suppression of TIGAR and NADPH levels thus contributed to ROS-mediated late apoptosis/necrosis of multiple myeloma cells. These findings indicate that multiple myeloma cells are dependent on MUC1-C and TIGAR for maintenance of redox balance and that targeting MUC1-C activates a cascade involving TIGAR suppression that contributes to multiple myeloma cell death.