Sensitivity Of Myeloma Cells Research Articles

Molecular Basis of Differential Sensitivity of Myeloma Cells to Clinically Relevant Bolus Treatment with Bortezomib

The proteasome inhibitor bortezomib (Velcade) is prescribed for the treatment of multiple myeloma. Clinically achievable concentrations of bortezomib cause less than 85% inhibition of the chymotrypsin-like activity of the proteasome, but little attention has been paid as to whether in vitro studies are representative of this level of inhibition. Patients receive bortezomib as an intravenous or subcutaneous bolus injection, resulting in maximum proteasome inhibition within one hour followed by a gradual recovery of activity. In contrast, most in vitro studies use continuous treatment so that activity never recovers. Replacing continuous treatment with 1 h-pulse treatment increases differences in sensitivity in a panel of 7 multiple myeloma cell lines from 5.3-fold to 18-fold, and reveals that the more sensitive cell lines undergo apoptosis at faster rates. Clinically achievable inhibition of active sites was sufficient to induce cytotoxicity only in one cell line. At concentrations of bortezomib that produced similar inhibition of peptidase activities a different extent of inhibition of protein degradation was observed, providing an explanation for the differential sensitivity. The amount of protein degraded per number of active proteasomes correlated with sensitivity to bortezomib. Thus, (i) in vitro studies of proteasome inhibitors should be conducted at pharmacologically achievable concentrations and duration of treatment; (ii) a similar level of inhibition of active sites results in a different extent of inhibition of protein breakdown in different cell lines, and hence a difference in sensitivity.

In Vivo Targeting of Stromal-Derived Factor-1 As a Strategy to Prevent Myeloma Cell Dissemination to Distant Bone Marrow Niches

AbstractAbstract 440 Background.Multiple myeloma (MM) patients present with multiple lytic lesions at diagnosis, indicating the presence of continuous dissemination of MM cells from the primary site of tumor development to multiple distant bone marrow (BM) niches. We hypothesized that stromal-derived factor-1 (SDF-1) may represent a target for preventing transition from MGUS (micrometastatic stage) to active-MM (macrometastatic stage); thus resulting in inhibition of MM progression. We therefore evaluated SDF-1 expression in the BM of patients with MGUS, MM, compared to healthy individuals; and tested NOX-A12, a high affinity l-oligonucleotide (Spiegelmer) binder to SDF-1 in MM, looking at its ability to modulate MM cell tumor growth and MM cell homing to the BM in vitro and in vivo . Methods.SDF-1 levels were evaluated by immunohistochemistry on BM specimens obtained from patients with MGUS, active-MM, or healthy individuals; and confirmed by ELISA, using conditioned-medium of BM-mesenchymal stromal cells obtained from MGUS, active-MM and healthy individuals. BM metastatic lesions from primary epithelial tumors were also considered. Co-localization of MM tumor cells (MM.1S-GFP+) with SDF-1 was tested in vivo by in vivo confocal microscopy, using both AlexaFluor633-conjugated-anti-SDF-1 monoclonal antibody and AlexaFluor647-conjugated-NOX-A12 oligonucleotide. Effect of NOX-A12 on modulating MM cell dissemination was tested in vivo, by using in vivo confocal microscopy. In vivo homing and in vivo tumor growth of MM cells (MM.1S-GFP+/luc+) were assessed by using in vivo confocal microscopy and in vivo bioluminescence, in SCID mice treated with 1) vehicle; 2) NOX-A12; 3) bortezomib; 4) NOX-A12+bortezomib. Detection of mobilized MM-GFP+ cells ex vivo was performed by flow cytometry. Effects of drug combination on dissemination of MM cells to distant BM niches was evaluated ex vivo by immunofluorescence on femurs obtained from each cohort of mice. DNA synthesis and adhesion of MM cells in the context of NOX-A12 (50–100nM) treated primary MM BM stromal cells (BMSCs) in presence or absence of bortezomib (2.5–5nM) were tested by thymidine uptake and adhesion in vitro assay, respectively. Synergism was calculated by using CalcuSyn software. NOX-A12-dependent-modulation of signaling was evaluated by western blot on MM cells exposed or not to primary BM-MSCs. Results.Patients with active-MM present with higher BM SDF-1 expression vs. MGUS patients and healthy individuals. Similarly, BM presenting with metastasis from epithelial primary malignancies had higher SDF-1 levels compared to healthy subjects, thus suggesting the importance of SDF-1 in favoring tumor cell metastasis to BM niches. SDF-1 co-localized at BM level with MM tumor cells in vivo. In vitro, NOX-A12 induced a dose-dependent de-adhesion of MM cells from the BMSCs supported by inhibition of BM-MSC-mediated phosphorylation of ERK1/2 and cofilin. These findings were corroborated and validated in vivo: NOX-A12 induced MM cell mobilization from the BM to the peripheral blood as shown ex vivo, by reduced percentage of MM cells in the BM and increased number of MM cells within the peripheral blood of mice treated with NOX-A12 vs. control (BM: 57% vs. 45%; PB: 2.7% vs. 15%). This was supported by inhibited homing of MM cells to the BM of those mice pre-treated with NOX-A12. We next showed that NOX-A12-dependent de-adhesion of MM cells from BMSCs lead to enhanced MM cell sensitivity to bortezomib, as shown in vitro, where a synergistic effect between NOX-A12 and bortezomib was observed (C.I.: .57-.76). These findings were validated in vivo: tumor burden was similar between NOX-A12- and control mice whereas bortezomib-treated mice showed significant reduction in tumor progression compared to the control (P<.05); importantly, significant reduction of tumor burden in those mice treated with sequential administration of NOX-A12 and bortezomib was observed, compared to bortezomib alone-treated mice (P <.05). Similarly, NOX-A12+bortezomib combination induced significant inhibition of MM cell homing, as shown by in vivo confocal microscopy. Conclusion.SDF-1 represents a valid target for inhibiting MM cell dissemination to distant BM niches, thus providing the evidence for using the SDF-1 inhibiting Spiegelmer NOX-A12 to target MM cells at the stage of micrometastasis (MGUS), thus preventing development of symptomatic macrometastatic MM. Disclosures:Zboralski:NOXXON Pharma AG, Berlin, Germany: Employment. Kruschinski:NOXXON Pharma AG, Berlin, Germany: Employment. Ghobrial:Novartis: Advisory Board Other; Onyx: Advisory Board, Advisory Board Other; Millennium: Advisory Board, Advisory Board Other; Bristol Myers Squibb: Advisory Board, Advisory Board Other.

Dll1/Notch Interaction Contributes to a Decreased Sensitivity of Myeloma Cells to Bortezomib

AbstractAbstract 1840One of the greatest challenges in multiple myeloma (MM) treatment is to overcome drug resistance. More and more evidence showed that not only the MM tumor cells should be targeted but also the bone marrow (BM) micro-environment. Interactions of MM cells with the BM micro-environment have a pivotal role in MM cell proliferation, survival, migration, angiogenesis as well as drug resistance. Many pathways are involved including the conserved Notch signaling pathway. The interaction of Notch receptors and ligands between adjacent cells induces proteolytic cleavage and release of the intracellular domain of the Notch receptor, also called Notch intracellular domains (NICD). NICD will then enter the nucleus and modify the expression of downstream target genes. Notch receptors are expressed by MM cells and Notch ligand Dll1 is present on bone marrow (BM) stromal cells. We investigated whether Notch activation in myeloma cells by the interaction with Dll1 on stromal cells contributes to bortezomib resistance. We analyzed Notch1 and Notch2 surface expression by flow cytometry on MM cells after Dll1 interaction using a stromal cell line modified to overexpress Dll1. Notch1 surface expression was not disturbed on mouse 5T33MMvt and human MMS1 and LP-1 cells while Notch2 expression on MM cells was significantly decreased after Dll1 interaction for 2 days. Next, we investigated NICD1 and NICD2 expression by western blot after Dll1/Notch interaction. NICD1 did not change in murine 5T33MMvt and human LP-1 and MMS-1 cells, while NICD2 is increased after Dll1 interaction. These results suggest that Dll1 can activate Notch signaling likely through the Notch2 receptor. We investigated whether Dll1/Notch activation could contribute to MM bortezomib resistance. MM cells were cocultured on immobilized recombinant Dll1 ligand and treated with 5 nM bortezomib for 48h. Compared to control, MM cells cocultured with Dll1 ligand were less sensitive to bortezomib. Furthermore, blocking the Notch pathway by DAPT (a gamma secretase inhibitor, GSI) could reverse this effect and increased the sensitivity to bortezomib. To delineate the molecular mechanism of Dll1-induced bortezomib resistance, we performed a drug resistance and metabolism gene array and found that CYP1A1 was significantly upregulated by Dll1/Notch interaction. CYP1A1 is a member of the cytochrome P450 family and regulates drug metabolism. We further demonstrated that inhibiting CYP1A1 by either α-Naphthoflavone (inhibitor) or CYP1A1-siRNA increases the sensitivity of MM cells to bortezomib, suggesting that CYP1A1 is involved in bortezomib resistance. As also previously demonstrated, CD138- 5T33MM cells are less sensitive to bortezomib than CD138+ 5T33MM cells. We analyzed CYP1A1 expression and activity and observed a higher CYP1A1 amount in CD138- cells compared to CD138+ MM cells. The higher CYP1A1 expression in CD138- cells might be a possible mechanism for their decreased bortezomib sensitivity compared to CD138+ cells. In addition, an in vivo experiment was performed. Combination treatment of DAPT with bortezomib was able to increase bortezomib sensitivity and prolonged overall survival in the 5T33MM mouse model.In conclusion, our results suggest that Dll1/Notch activation contributes to bortezomib resistance by upregulating CYP1A1, a molecule involved in drug metabolism. Our data provide a potential strategy to overcome bortezomib resistance by combination with a Notch pathway inhibitor. Disclosures:No relevant conflicts of interest to declare.

The Increased 1,25-(OH)2D3 Sensitivity of Myeloma Cells and Marrow Stromal Cells Enhances RANKL Production and Tumor Cell Growth

Abstract 3946Vitamin D plays multiple roles in normal and malignant cell function, regulating cell differentiation and proliferation as well as bone homeostasis. Epidemiologic studies suggest that low levels of vitamin D contribute to the progression of lung cancer, breast cancer, colorectal and prostate cancer as well as lymphoma and melanoma. However, the role of vitamin D in multiple myeloma (MM) is unclear. In contrast to its growth inhibition of solid tumors, vitamin D has little anti-proliferative effects on MM cells. The physiological responses of myeloma cells to vitamin D are unknown, as are its effects on the marrow microenvironment in myeloma bone disease. Vitamin D insufficiency or deficiency has been documented in the majority of myeloma patients. Vitamin D receptor (VDR) is expressed by RPMI8226 cells, but it is unknown if this is a common finding in MM. Further, the functional consequences of VDR expression in myeloma cells are not well characterized. We reported osteoclast (OCL) precursors from patients with Paget’s disease (PD) of bone were hypersensitive to 1,25-(OH)2D3 (1,25-D3) and formed OCL at physiologic concentrations of 1,25-D3 rather than the pharmacologic concentrations of 1,25-D3 required for normal OCL formation in vitro. This enhanced sensitivity to 1,25-D3 was due to increased expression of a novel VDR co-activator, TAF12, a member of the TFIID transcription complex. We found TAF12 expression was increased in marrow stromal cells (BMSC) by increased NFκB signaling and enhanced the capacity of BMSC to produce RANKL in response to low levels of 1,25-D3. Because the marrow microenvironment in MM and PD has many similarities in terms of increased OCL activity and enhanced NFκB signaling, we determined if MM cells induced TAF12 expression in BMSC of MM patients and if 1,25-D3 could enhanced RANKL production in BMSC of MM patients, even in patients with low levels of 1,25-D3. We found that both BMSC and CD138+ primary myeloma cells from MM patients expressed increased TAF12 levels compared to normal BMSC and CD138+ bone marrow cells. Four of four human MM cell lines (MM1.S, ANBL6, JJN3 and RPMI8266) expressed VDR, TAF12 and ATF7, which potentiates TAF12-mediated gene transcription. MM1.S and JJN3 but not RPMI8266 produced increased amounts of RANKL in response to very low levels of 1,25-D3. Further, 1,25-D3 increased VEGF, DKK1 and α4β1 integrin expression by MM1.S, JJN3 and RPMI8266 cells and enhanced adhesive interactions between MM cells and BMSC that increase MM growth. To confirm the role of TAF12 in the increased RANKL expression by MM cells treated with 1,25-D3, we established a stable TAF12 anti-sense JJN3 cell line (AS-TAF12-JJN3). AS-TAF12-JJN3 cells had markedly decreased RANKL production, VDR content and CYP24A1 accumulation in response to 1,25-D3. MM1.S and JJN3 myeloma cells treated with a VDR antagonist (TEI-9647) decreased RANKL production and α4β1 integrin expression in response to low levels of 1,25-D3. Further, 1,25-D3 induced VCAM-1 expression on normal human BMSC. Co-culture of JJN3 cells with BMSC treated with 1,25-D3 induced both MM cell growth and cell adhesion. In contrast, co-culture with AS-TAF12-JJN3 cells resulted in decreased cell growth and cell adhesion. Further, 1,25-D3 treatment of mouse OCL precursors co-cultured with JJN3 cells, but not AS-TAF12-JJN3 cells, increased OCL formation. These results suggest that increased TAF12 levels in MM cells and BMSC allow low levels of 1,25-D3 significantly to increase RANKL production by both MM cells and BMSC, and enhance adhesive interactions between MM cells and BMSC, thus increasing MM cell growth and OCL formation. Disclosures:No relevant conflicts of interest to declare.

The Bcl-2—IP3 Receptor Interaction As a Novel Target for Therapeutics in Myeloma

Abstract 4011The anti-apoptotic Bcl-2 (B-cell lymphoma 2) protein was first identified in follicular lymphoma, where the Bcl-2 gene was dysregulated as a result of chromosomal translocation. The anti-apoptotic activity of Bcl-2 can be attributed to two mechanisms conferred by structurally distinct domains. First, the BH domains 1–3 of Bcl-2 dimerize with pro-apoptotic members of the Bcl-2 family, such as Bax and Bak, and maintain mitochondrial integrity. Small molecules targeting these domains on Bcl-2 (e.g. ABT-737 and ABT-263/Navitoclax®) are under clinical evaluation. A less explored activity of Bcl-2 is that its BH4 domain functionally interacts with the IP3 receptor located on the endoplasmic reticulum (ER) membrane, the major organelle involved in regulating calcium homeostasis. Through this interaction, Bcl-2 inhibits IP3 receptor channel opening and prevents excessive calcium release from ER that can be harmful to cells. Our strategy is to disrupt the Bcl-2—IP3 receptor interaction to induce cell death in lymphoid malignant cells.Previously, our group has shown that an IP3 receptor derived peptide, or IDP, blocks the Bcl-2—IP3 receptor interaction and thereby induces elevation of cytosolic calcium in lymphoma cell lines. A modified version of the peptide—IDP-DD/AA, with an asparaginase cleavage site removed, is more potent in inducing calcium elevation as well as apoptosis in primary chronic lymphocytic leukemia (CLL) cells. Concentrations of the IDP-DD/AA peptide that killed CLL cells did not affect the viability of normal lymphocytes, suggesting a therapeutic window. We further exploited the cytotoxic activities in a panel of leukemia/lymphoma/myeloma cell lines and found that over 60% of myeloma cell lines tested were sensitive to the IDP-DD/AA peptide but not to the control peptide. Furthermore, in the sensitive myeloma cell lines treated with the IDP-DD/AA peptide, high amplitude cytosolic calcium elevation preceded caspase-3 activation and apoptotic morphology, suggesting the involvement of a calcium-mediated intrinsic apoptotic pathway.The in vivo anti-tumor activity of the IDP peptide was assessed in a myeloma xenograft model. Subcutaneous tumors were established in nude mice using the human myeloma cell line, NCI-H929. Randomly grouped mice treated by in-tumor injection of the IDP-DD/AA peptide showed prominent inhibition of tumor growth compared to saline-injected control. One of three mice treated with the IDP-DD/AA peptide showed no visible sign of tumor at the end of treatment. The data proffer promise of therapeutic use of the IDP peptide in myeloma disease.The effect of combinatory treatment of the IDP-DD/AA peptide and current anti-myeloma drugs (Bortezomib/Valcade®, doxorubicin/Adriamycin®, melphalan) were evaluated. Co-treatment of the myeloma cell line NCI-H929 cells with the IDP-DD/AA peptide and each one of the three anti-myeloma drugs induced synergistic cytotoxicity with combinatory indices (CI) less than 1. The data suggest that the IDP-DD/AA peptide augments cell killing activity of current chemotherapy drugs.In conclusion, the IDP-DD/AA peptide represents a novel anti-cancer agent that targets the anti-apoptotic Bcl-2 protein. The peptide exhibits both in vitro and in vivo anti-myeloma activity. Moreover, the IDP-DD/AA peptide can function as a single agent (in myeloma or CLL) as well as in combination with current anti-myeloma chemotherapy drugs. Disclosures:No relevant conflicts of interest to declare.

Casein Kinase II Modulates Topoisomerase II Alpha Nuclear Export and Drug Sensitivity in Multiple Myeloma

Abstract 4032Nuclear export of topoisomerase II alpha (topo IIα) to the cytoplasm results in de novo resistance to topo IIα inhibitors in hematological malignancies. We have previously demonstrated that topo IIα is exported from the nucleus of human multiple myeloma cells by a CRM1-dependent mechanism at densities similar to those in myeloma patient bone marrow (Engel et al, Exp. Cell Res. 295:421-31, 2004). We have also identified the nuclear export signals for topo IIα at amino acids 1017–1028 and 1054–1066 using mutated full-length FLAG-topo IIα protein and immunofluorescence microscopy, (Turner et al, J. Cell Sci. 117:3061-71, 2004). In addition, blocking nuclear export with a CRM1 inhibitor or by using CRM1-siRNA sensitizes myeloma cells to topo II poisons (Turner et al. Cancer Res. 69:6899-905, 2009). Therefore, preventing nuclear export may sensitize myeloma cells to topo II inhibitors.To determine what signals CRM1-mediated nuclear export of topo IIα we investigated the phosphorylation status of topo IIα, both in the nucleus and in the cytoplasm, using proteomic technologies. Topo IIα was isolated from the nuclei and cytoplasm of human myeloma cells by immunoprecipitation. Purified topo IIα fractions were analyzed by Orbitrap mass spectroscopy. We found that serine 1524 was highly phosphorylated in the cytoplasmic fraction. Using site-directed mutagenesis we converted serine 1524 to an alanine to determine if mutated FLAG-tagged topo IIα export was reduced as compared to wild-type FLAG-tagged topo IIα. Serine 1524 is a CK2 phosphorylation site, therefore, we tested a recently published and highly specific inhibitor of CK2; CX-4945 (Ferguson et al, FEBS Letters 585:104–110, 2011). This drug was used in combination with the topo IIα inhibitor doxorubicin and assayed for apoptosis by caspase 3 cleavage and flow cytometry both in vitro using myeloma cell lines and ex vivo using patient bone marrow mononuclear cells.Using mass spectroscopy we found that cytoplasmic but not nuclear topo IIα was phosphorylated at serine 1524, a published CK2 motif. Using site-directed mutagenesis we converted serine 1524 to an alanine and found that mutated FLAG-topo IIα export was reduced, as compared to wild-type FLAG- topo IIα. The CK2 inhibitor CX-4945 induced apoptosis in both myeloma cell lines and in myeloma patient bone marrow aspirates. In addition, we found that CX-4945 prevented nuclear export of topo IIα in high-density cells. These data were duplicated using a CK2 specific siRNA to knockdown CK2 expression (90% knockdown). Blocking nuclear export of topo IIα with the CK2 inhibitor CX-4945 or CK2-specific siRNA sensitized drug-resistant myeloma cells to the topo II poison doxorubicin. These data may have potential clinical implications in the treatment of multiple myeloma. Disclosures:No relevant conflicts of interest to declare.

Inhibition of Heat Shock Factor 1 (HSF1) Is More Effective At Sensitizing Myeloma Cells to Bortezomib Than Inhibition of Individual HSF1 Targets.

Abstract 2953Multiple myeloma is a plasma cell disorder with an average incidence of 21,000 new cases per year in the United States. Recent advances in therapeutic approaches such as the use of proteasome inhibitors have resulted in a significant increase in the overall survival of myeloma patients. Myeloma cells maintain many of the characteristics of normal plasma cells, including constitutive immunoglobulin production and secretion, therefore management of ER stress plays a role in myeloma cell sensitivity to proteasome inhibition. However, myeloma cells also upregulate protective genes in response to the proteotoxic stress that can limit the therapeutic response. Previous groups have published on the importance of the heat shock response and the heat shock protein (HSP) family, supporting preclinical and clinical exploration of HSP inhibition in myeloma.Our group had interest in regulation of the HSP response and has evaluated the master regulator HSF1 as a potential therapeutic target. We found that siRNA-mediated silencing of HSF1 enhances bortezomib-induced apoptosis in a myeloma cell line. To define the effectors of the heat shock response important in regulating bortezomib response, we determined which heat shock response genes are induced by bortezomib in an HSF1-dependent manner. From a realtime PCR array of 84 HSP family genes, we found 21 genes that were induced greater than 2-fold by bortezomib. Of these 21 genes, 10 genes showed >50% reduction in HSF1-silenced cells. 7/10 genes were confirmed by independent qRT-PCR and western blot analysis. These genes include: CRYAB (alpha-crystallin B chain), DNAJB1 (HSP40 subfamily B), HSPA1A (HSP70-1A), HSPA1B (HSP70-1B), HSPB1 (HSP27), HSPH1 (HSP105/110), and HSP90AB1 (HSP90b1).To begin to determine which of these genes was important for the HSF1-dependent protective response we silenced the 7 genes individually and subsequently treated the cells with bortezomib. Surprisingly only 1 of the 7 genes silenced individually, DNAJB1, had an observable effect on bortezomib-induced death. However DNAJB1 silencing does not account for all the HSF1 activity as the increase in cell death due to bortezomib is only 48% of that observed with HSF1 silencing. Thus targeting HSF1 is more effective at sensitizing multiple myeloma cells to bortezomib-induced apoptosis than targeting individual HSPs. Moreover these data suggest that HSP90 inhibitors are functioning by inhibiting at least two members of this family to be effective as single agents. Therefore, while clinical trials for individual HSP and HSP in combination with bortezomib are being conducted, a more effective strategy for apoptosis induction is achieved through inhibition of HSP regulators such as HSF1 in combination with bortezomib. These results provide support for investigating HSP regulation in response to PI to increase the efficacy of myeloma therapy. Disclosures:No relevant conflicts of interest to declare.

Interleukin-6 Enhances the Survival of Myeloma Cells by Regulating Bim Binding to Anti-Apoptotic Bcl-2 Proteins

AbstractAbstract 4024The survival of multiple myeloma cells in the bone marrow is largely dependent on its interactions with cellular components of the microenvironment. These interactions, in the form of cell-cell interactions and soluble factors support myeloma cell survival and allow for the mutations associated with transformation to drive plasma cell growth and proliferation. Moreover survival signals from the bone marrow microenvironment influence the response to therapeutic agents, therefore understanding the molecular basis of these interactions is necessary to improve the depth of responses to therapy in myeloma patients.Cell survival is regulated by the Bcl-2 family of proteins and we previously reported that in myeloma cells one can map which Bcl-2 protein is necessary for cell survival by determining which protein is sequestering the pro-apoptotic molecule, Bim. This could also be used to predict myeloma cell sensitivity to th Bcl-2/Bcl-xL antagonist ABT-737. While studying the response of freshly isolated patient samples to ABT-737, we found that in 40% of the samples tested (N=10) as well as in 5 myeloma cell lines, inclusion of stromal components significantly inhibited ABT-737-induced apoptosis. Thus the microenvironment can influence survival by altering the pattern of Bcl-2 dependence in cells. We have determined that a soluble factor secreted by stromal cells is necessary for this activity and now report that this factor is interleukin-6.We found that addition of conditioned medium from the HS-5 stromal line or freshly isolated bone marrow stromal cells (N=4) from myeloma patients or normal donors (N=1) to human myeloma cell lines all resulted significant resistance to ABT-737. Since all of these cells are a rich source of IL-6, we tested if the addition of recombinant IL-6 mimicked the addition of stromal cell supernatant. Indeed rIL-6 (10 ng/ml) was as potent as co-culture with HS-5 cells or addition of HS-5 or stromal cell conditioned medium at blocking ABT-737-induced apoptosis in MM.1s cells. Thus IL-6 is sufficient to block ABT-737-induced apoptosis. To demonstrate that IL-6 was necessary we determined the effect of the addition of an IL-6 neutralizing antibody on stromal cell-induced resistance to ABT-737. We found that addition of the antibody to HS-5 conditioned medium blocked resistance to ABT-737 in a dose-dependent fashion. Moreover we found that addition of anti-IL6 to a patient sample had a similar effect as removing the stromal cells on the response to ABT-737-induced death. Taken together these data suggest that IL-6 is both necessary and sufficient for regulating Bcl-2 dependence in multiple myeloma.To define the molecular basis for these findings we initially investigated the expression of the key Bcl-2 proteins involved in Bcl-2 dependence and myeloma cell survival. Despite their profound effects on ABT-737-induced apoptosis we found that neither co-culture of MM.1s cells with HS-5 cells, HS-5 conditioned medium or rIL-6 had any effect on the expression of Mcl-1, Bcl-xL or Bim. We then investigated the patterns of interaction between these proteins and found that the addition of stromal components or IL-6 resulted in a shift of Bim binding from Bcl-xL to Mcl-1. This is consistent with our previous findings that when Bim is bound to Mcl-1, myeloma cells are less sensitive to ABT-737. Finally we set out to determine how IL-6 signaling results in a change in ABT-737 sensitivity. We treated MM.1s cells with ABT-737 and IL-6 in the presence of inhibitors of the MEK/ERK, p38, PI3K/AKT, and JAK/STAT pathways. We found that only the MEK inhibitor, U0126 could re-sensitize IL-6-treated cells to ABT-737-induced death at concentrations that did not kill cells or have additive effects with ABT-737 in the absence of IL-6 (P<0.05 2-way ANOVA). Taken together, these data indicate that stromal cells regulate myeloma cell survival through IL-6 by altering Bim binding in a MEK-dependent fashion. This results in a cell that is highly dependent on Mcl-1. These data offer an explanation for why proteasome inhibitors are so effective in this disease. While the bone marrow microenvironment is enforcing Mcl-1 dependence, proteasome inhibitors induce the Mcl-1 inhibitor Noxa, which overcomes this activity of IL-6. Moreover these data point to potential ways of combining the targeting of Bcl-2 proteins and IL-6 signaling as a means to effectively kill myeloma cells in the bone marrow microenvironment. Disclosures:Kaufman:Millenium: Consultancy; Celgene: Consultancy; Novartis: Consultancy; Onyx Pharmaceuticals: Consultancy.

Cell Death via DR5, but not DR4, Is Regulated by p53 in Myeloma Cells

Myeloma cells are sensitive to TRAIL through the two death receptors DR4 and DR5. Because p53 directly modulates expression of death receptors, we investigated here whether p53 can modulate myeloma sensitivity to TRAIL. We found that p53 affects the sensitivity of myeloma cells to the DR5 agonistic human antibody lexatumumab but not the DR4 antibody mapatumumab. TP53 wild-type myeloma cells overexpressed DR5 in correlation with sensitivity to lexatumumab. Both nongenotoxic (nutlin-3a) and genotoxic (melphalan) p53-inducing stresses increased DR5 expression only in TP53 wild-type cells and synergistically increased lexatumumab efficiency yet did not increase DR4 expression, nor sensitivity to mapatumumab. Silencing of p53 strongly decreased DR5 expression and induced resistance to nutlin-3a and lexatumumab but did not modulate DR4 expression or sensitivity to mapatumumab. Increase of lexatumumab efficiency induced by nutlin-3a was related to a p53-dependent increase of DR5 expression. In primary myeloma cells, nutlin-3a increased DR5 expression and lexatumumab efficiency but did not increase mapatumumab efficiency. Taken together, our findings indicate that p53 controls the sensitivity of myeloma through DR5 but not DR4 and suggest that a subset of patients with multiple myeloma may benefit from DR5 therapy.

Prolonged early G1 arrest by selective CDK4/CDK6 inhibition sensitizes myeloma cells to cytotoxic killing through cell cycle–coupled loss of IRF4

AbstractDysregulation of cyclin-dependent kinase 4 (CDK4) and CDK6 by gain of function or loss of inhibition is common in human cancer, including multiple myeloma, but success in targeting CDK with broad-spectrum inhibitors has been modest. By selective and reversible inhibition of CDK4/CDK6, we have developed a strategy to both inhibit proliferation and enhance cytotoxic killing of cancer cells. We show that induction of prolonged early-G1 arrest (pG1) by CDK4/CDK6 inhibition halts gene expression in early-G1 and prevents expression of genes programmed for other cell-cycle phases. Removal of the early-G1 block leads to S-phase synchronization (pG1-S) but fails to completely restore scheduled gene expression. Consequently, the IRF4 protein required to protect myeloma cells from apoptosis is markedly reduced in pG1 and further in pG1-S in response to cytotoxic agents, such as the proteasome inhibitor bortezomib. The coordinated loss of IRF4 and gain of Bim sensitize myeloma tumor cells to bortezomib-induced apoptosis in pG1 in the absence of Noxa and more profoundly in pG1-S in cooperation with Noxa in vitro. Induction of pG1 and pG1-S by reversible CDK4/CDK6 inhibition further augments tumor-specific bortezomib killing in myeloma xenografts. Reversible inhibition of CDK4/CDK6 in sequential combination therapy thus represents a novel mechanism-based cancer therapy.

Stat1 activation attenuates IL-6 induced Stat3 activity but does not alter apoptosis sensitivity in multiple myeloma

BackgroundMultiple myeloma (MM) is at present an incurable malignancy, characterized by apoptosis-resistant tumor cells. Interferon (IFN) treatment sensitizes MM cells to Fas-induced apoptosis and is associated with an increased activation of Signal transducer and activator of transcription (Stat)1. The role of Stat1 in MM has not been elucidated, but Stat1 has in several studies been ascribed a pro-apoptotic role. Conversely, IL-6 induction of Stat3 is known to confer resistance to apoptosis in MM.MethodsTo delineate the role of Stat1 in IFN mediated sensitization to apoptosis, sub-lines of the U-266-1970 MM cell line with a stable expression of the active mutant Stat1C were utilized. The influence of Stat1C constitutive transcriptional activation on endogenous Stat3 expression and activation, and the expression of apoptosis-related genes were analyzed. To determine whether Stat1 alone would be an important determinant in sensitizing MM cells to apoptosis, the U-266-1970-Stat1C cell line and control cells were exposed to high throughput compound screening (HTS).ResultsTo explore the role of Stat1 in IFN mediated apoptosis sensitization of MM, we established sublines of the MM cell line U-266-1970 constitutively expressing the active mutant Stat1C. We found that constitutive nuclear localization and transcriptional activity of Stat1 was associated with an attenuation of IL-6-induced Stat3 activation and up-regulation of mRNA for the pro-apoptotic Bcl-2 protein family genes Harakiri, the short form of Mcl-1 and Noxa. However, Stat1 activation alone was not sufficient to sensitize cells to Fas-induced apoptosis. In a screening of > 3000 compounds including bortezomib, dexamethasone, etoposide, suberoylanilide hydroxamic acid (SAHA), geldanamycin (17-AAG), doxorubicin and thalidomide, we found that the drug response and IC50 in cells constitutively expressing active Stat1 was mainly unaltered.ConclusionWe conclude that Stat1 alters IL-6 induced Stat3 activity and the expression of pro-apoptotic genes. However, this shift alone is not sufficient to alter apoptosis sensitivity in MM cells, suggesting that Stat1 independent pathways are operative in IFN mediated apoptosis sensitization.

Modulation of eIF5A Expression Using SNS01 Nanoparticles Inhibits NF-κB Activity and Tumor Growth in Murine Models of Multiple Myeloma

Despite recent advances in the first-line treatment of multiple myeloma, almost all patients eventually experience relapse with drug-resistant disease. New therapeutic modalities are needed, and to this end, SNS01, a therapeutic nanoparticle, is being investigated for treatment of multiple myeloma. The antitumoral activity of SNS01 is based upon modulation of eukaryotic translation initiation factor 5A (eIF5A), a highly conserved protein that is involved in many cellular processes including proliferation, apoptosis, differentiation and inflammation. eIF5A is regulated by post-translational hypusine modification, and overexpression of hypusination-resistant mutants of eIF5A induces apoptosis in many types of cancer cells. SNS01 is a polyethylenimine (PEI)-based nanoparticle that contains both a B-cell-specific expression plasmid expressing a non-hypusinable mutant of eIF5A and a small interfering RNA (siRNA) which depletes endogenous hypusinated eIF5A. Reducing hypusine-modified eIF5A levels was found to inhibit phosphorylation and activity of ERK MAPK and nuclear factor-κB (NF-κB), and thus sensitize myeloma cells to apoptosis resulting from transfection of a plasmid expressing eIF5A(K50R). SNS01 exhibited significant antitumoral activity in both KAS-6/1 (95% inhibition; P < 0.05) and RPMI 8226 (59% inhibition; P < 0.05) multiple myeloma xenograft models following systemic administration. These results highlight the potential of using this approach as a new therapeutic strategy for multiple myeloma.

Deubiquitinase Usp9X Regulates Cell Survival and Metabolic Signal Transduction in Multiple Myeloma Cells

Abstract 1835Multiple myeloma (MM) therapy has significantly improved with the addition of a number of new agents that target the proteasome and other pathways. However, most patients relapse and eventually become resistant to any drug therapy, leaving limited or no treatment options. As a result, there is tremendous interest in understanding drug resistance and regulation of MM cell survival. Toward this goal, a number of studies have assessed differential gene expression and proteome profiles between normal cells, primary myeloma and drug-resistant disease. Recent reports suggest that increased expression of the deubiquitinase (DUB) Usp9x is associated with poor prognosis of MM patients. Additional analyses indicate that Usp9x regulates a number of survival and signaling proteins including Mcl-1, a Bcl-2-family pro-survival protein previously associated with drug and apoptotic resistance in myeloma patients, and 5’-AMP kinase (AMPK), a negative regulator of the mTOR cascade. We previously described WP1130, a small molecule with Usp9x DUB inhibitory activity. This compound induced apoptosis in MM cell lines and primary cells from drug-resistant MM patients (IC50 ∼1 μM) but not in normal CD34 cells even at 5-fold higher concentrations, suggestive of a favorable therapeutic index. To determine the role of Usp9x in MM cell survival and signaling, we silenced Usp9x expression in MM cells and assessed changes in Usp9x DUB activity, Mcl-1 levels, cell survival and mTOR signaling. We also compared these results with the effects of WP1130 treatment. Constitutive knockdown of Usp9x by shRNA in H929, MM1.S and RPMI-8226 cells reduced Usp9x protein levels by >90%, but reduced Usp9x DUB activity by no more than 40%. Importantly, although Usp9x silencing reduced MM cell survival by 5–40%, major changes in Mcl-1 protein levels were not detected. In contrast, treatment of control or Usp9x-silenced cells with WP1130 resulted in a rapid reduction in both Usp9x DUB activity and Mcl-1 protein levels. These results suggest that Usp9x DUB activity was not solely dependent on Usp9x protein levels and that cells with shRNA-mediated Usp9x knockdown may activate other compensatory mechanisms to stabilize Usp9x activity. We examined the effects of Usp9x knockdown on the mTOR negative regulator AMPK, whose activation requires deubiquitination by Usp9x. Both Usp9x shRNA and WP1130 treatment stabilized ubiquitination of AMPK, resulting in inhibition of AMPK kinase activity. Such AMPK inhibition led to activation of the mTOR cascade, thereby increasing phosphorylation of Akt, S6 and GSK3β. These observations suggested that activation of mTOR through chronic Usp9x knockdown suppresses apoptosis in MM cells. Inhibition of mTOR activity (with Torin-2) in Usp9x-silenced cells rapidly induced apoptosis with a corresponding reduction in Mcl-1 protein levels. This Mcl-1 reduction was associated with decreased GSK3β activity and suppression of Mcl-1 phosphorylation at Thr159, a GSK3β phosphorylation site that negatively regulates Mcl-1/Usp9x association. Dissociation of Mcl-1/Usp9x complexes increased ubiquitination of Mcl-1 and its destruction by the proteasome. Together, these results suggest that Usp9x regulates multiple substrates with differential effects on mTOR signal transduction and apoptotic proteins. This compound impact of Usp9x on Mcl-1 and mTOR regulation may complicate the assessment Usp9x as a potential therapeutic target in MM, but may help explain the sensitivity of MM cells to WP1130. Specifically, WP1130 increases the formation of Mcl-1/Usp9x complexes by inhibiting AMPK and thereby activating mTOR/Akt/GSK3β. The Mcl-1 in complex with Usp9x becomes ubiquitinated due to rapid inhibition of Usp9x activity by WP1130. Our results predict that Usp9x inhibitors will have therapeutic activity in MM cells by modulating at least two separate pathways that regulate metabolic signaling and MM cell survival. Considering that increased Usp9x expression has been associated with short survival and poor prognosis in MM patients, Usp9x inhibitors like WP1130 could show therapeutic promise for this patient population with limited treatment options. Disclosures:Talpaz:ARIAD: Research Funding. Jakubowiak:Ortho Biotech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Millennium Pharmaceuticals, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Onyx Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Exelixis: Consultancy, Honoraria.

Potentiation of bortezomib's Anti-Myeloma Effects in Combination with Anti-DR5 Agonistic Antibody: The Role of ER Stress in DR5 Editing and DR-Mediated Apoptotic Signaling

Abstract 5104Bortezomib (BTZ) is widely used in the treatment of myeloma (MM) with marked response rates in both relapsed/refractory as well as newly diagnosed MM. However, significant numbers of patients still remain outside benefit of the BTZ treatment; and various combinatory treatments with BTZ have been implemented to improve BTZ's anti-MM effects. On the other hand, immunotherapies seem attractive for yet incurable malignancies by chemotherapeutic agents such as MM and their clinical application has been studied. One such approach is a TNF-related apoptosis-inducing ligand (TRAIL)-mediated immunotherapy. In the present study, we therefore explored the role of BTZ on TRAIL receptor editing and its downstream signaling with special reference to endoplasmic reticulum (ER) stress and the cytotoxic effects of BTZ and anti-TRAIL receptor agonistic antibody in combination on MM cells. Most MM cells expressed DR4 but weakly DR5, while normal peripheral blood mononuclear cells expressed neither DR4 nor DR5. BTZ at 10 nM markedly up-regulated the surface levels of DR5 and its mRNA expression but not those of DR4 in MM cell lines and primary MM cells. Furthermore, BTZ decreased the levels of c-FLIP, an inhibitor of DISC, along with activation of caspase-8 and caspase-3, suggesting potentiation of the DR-mediated extrinsic apoptotic pathway. Consistently, BTZ and anti-DR5 agonistic antibody cooperatively enhanced the cytotoxicity against MM cells. BTZ induced phosphorylation of eIF2alpha, ATF4 and CHOP, along with disappearance of anti-apoptotic proteins including Mcl-1 in MM cells, suggesting the enhancement of ER stress and subsequent suppression of protein translation by BTZ. However, such induction of ER stress by BTZ was not observed in BTZ-resistant MM cell lines, KMS-11/BTZ and OPM-2/BTZ, with a point mutation in BTZ-binding proteasome beta5 subunit (Ri et al. Leukemia 2010). In KMS-11/BTZ and OPM-2/BTZ, surface protein as well as mRNA levels of DR5 were not up-regulated by BTZ, suggesting a critical role of ER stress in up-regulation of DR5 expression by BTZ. Because DR5 expression has been shown to be transcriptionally up-regulated by CHOP, the up-regulation of DR5 mRNA and protein in MM cells by BTZ is suggested to be at least in part due to CHOP induced by BTZ-mediated ER stress. Although BTZ exerts its anti-MM effects through induction of ER stress, the present study demonstrates that induction of ER stress by BTZ is also able to sensitize MM cells to TRAIL-mediated immunotherapy. Therefore, the combination of BTZ and TRAIL-mediated immunotherapy is warranted for further study. Disclosures:No relevant conflicts of interest to declare.

ANKHD1 Silencing Induces S-Phase Arrest and Modulates Cell Cycle Gene Expression in Human Myeloma Cells

Abstract 1828ANKHD1 is a multiple ankyrin repeats containing protein with a single KH domain. It is a large protein (∼ 280 kDa) derived from an 8 kb transcript. The ANKHD1 gene, present in human chromosome 5q31.3 as a single copy is ubiquitously expressed in normal human tissues and reported to be highly expressed in cancers, such as acute leukemia. Previous study showed higher expression of ANKHD1 in bone marrow plasma cells (CD138+) from Multiple Myeloma patients as compared to control (1) and it is also over expressed in multiple myeloma cell lines such as MM1S, MM1R, U266 and RPMI 8266 at both mRNA and protein level (2). However, the functional role of ANKHD1 in myeloma cells is unknown. In the present study, by silencing ANKHD1 gene expression in glucocorticoid resistant (U266) and sensitive (MM1S) myeloma cell lines, we studied its effect on cell cycle, proliferation and apoptosis. For gene silencing, specific shRNA-expressing lentiviral vector targeting the ANKHD1 gene and as negative control, sequence specific to Lac z gene were used. Cell growth was measured using the MTT colorimetric assay, whereas for apoptosis and cell cycle analysis Flow cytometry was used. Western blot and RTPCR were used for studying gene expression and protein levels, respectively. The results showed that lentiviral vector containing coding sequences for shRNA significantly downregulated ANKHD1 gene expression in Multiple Myeloma cells at the mRNA and the protein levels (p<0.05). Furthermore, we found that the cell cycle was arrested at S phase and the cell proliferation was significantly inhibited in both cell lines studied (p<0.05). However, ANKHD1 suppression did not induce apoptosis in myeloma cells, as evidenced by annexin V binding assay and flow cytometric detection of sub-G1 DNA content. To address the mechanism of the antiproliferative effect of ANKHD1 silencing, we examined the effect of ANKHD1 inhibition on cell cycle-related gene expression and proteins. ANKHD1 suppression caused downregulation of CDKN1B (p27), CCNB1 (cyclin B1), CDC25, CCNE1 (cyclin E1) and WEE 1 gene expression. There was no significant change in CCNA2 (Cyclin A2), CDC20 expression at mRNA levels. On the other hand, expression of CDKN1A (p21),which inhibits cyclin dependent kinases (CDKs) and plays role in preventing proliferation, was highly upregulated in both the cell lines. At protein levels, expression of Cdk2,Cdk4, p27 (CDKN1B) and E2F1 was decreased in both the cell lines with almost complete inhibition of expression in U266 cells. Taken together, the above results suggest that accumulation of cells in S phase (S phase arrest) can be due to inhibition of CDKs which binds with cyclins and are responsible for progression of cell cycle. Further, this inhibition of CDKs could be associated to increased induction of (CDKN1A) p21 in both cell lines.In conclusion, the present study demonstrates that the suppression of ANKHD1 potently inhibits proliferation and promotes cell cycle arrest without affecting rate of apoptosis in both glucocorticoid resistant as well as sensitive multiple myeloma cells. Also, as ANKHD1 suppression prevents S to G2/M progression, ANKHD1 protein might have role in cell cycle control by modulating cell cycle gene expression in intra S phase check point. The mechanisms modulating expression of these genes are under investigation. Further studies with combination of drugs that induce apoptosis and suppression of ANKHD1 may be an effective strategy for treatment of cancers, and therefore needed to be explored. Disclosures:No relevant conflicts of interest to declare.

Proteasome Inhibitors Sensitize Myeloma Cells to T Cell-Mediated Killing

Abstract 1838 Introduction:Graft-versus-myeloma (GvM) effect following allogeneic stem cell transplantation (allo-SCT) is well established and often accompanied by graft-versus-host disease (GVHD). Unfortunately, most patients still relapse following allo-SCT, underscoring the need for new transplant strategies. Interestingly, studies evaluating the proteasome inhibitor bortezomib combined with donor lymphocyte infusions (DLI) in myeloma patients suggest enhanced efficacy of the combination. In contrast, other reports demonstrate that bortezomib has activity in preventing GVHD in various malignancies, including myeloma. Here, we investigate the effects of bortezomib on T cell-mediated myeloma cell killing in vitro. Methods:Tumor cell compartment-specific bioluminescence imaging (CS-BLI) technology was used to monitor the cytotoxic effects of T cells on myeloma cells in the presence or absence of antimyeloma agents. To this end, myeloma cell lines were stably transduced with a firefly luciferase construct. Two different T cell clones were used: 1) the alloreactive CD8+-T cell clone allo-A2, which kills all HLA-A2-positive cells, including the myeloma cell line U266; and 2) the CD4+ T-cell clone 3AB11, generated from a myeloma patient with clinical GvM/GVHD after allo-SCT. 3AB11 cells recognize a hematopoietically restricted minor antigen, also presented by the myeloma cell line UM9. Protein expression was determined by western blot analysis. Results:The CD4+-T cell clone 3AB11 and the allo-A2 specific CD8+- T cell clone displayed a time- and dose-dependent lysis of myeloma cell lines UM9 and U266, respectively. Concurrent treatment of myeloma cells with the T cell clones and bortezomib resulted in synergistic enhancement of myeloma cell death, compared to T cells alone or bortezomib alone. This synergism was observed at 24, 48, and 72 hours (but not after 4 hours) of co-treatment. Dexamethasone, which significantly inhibited myeloma cell death induced by T cells, functioned as control.When myeloma cells were treated for 12 hours with bortezomib and then extensively washed, there was still enhancement of T cell-mediated killing. However, pre-incubation of T cells with bortezomib significantly impaired their ability to induce myeloma cell lysis. These washout experiments indicate that bortezomib sensitizes myeloma cells to T cell-mediated killing, but does not enhance the intrinsic cytotoxic activity of T cells.Mechanistic studies demonstrated that antibodies blocking FAS ligand partially impaired myeloma cell lysis by 3AB11 and allo-A2 clones. More interestingly, however, blockade of FAS/FAS ligand interactions completely abrogated the synergism between bortezomib and T cells. Antibodies against TRAIL had no such effects. Furthermore, western blot analyses revealed that in UM9 and U266 cells bortezomib reduced protein levels of FLICE inhibitory protein (cFLIP), which is a potent inhibitor of FAS ligand-induced apoptosis. These results support the idea that bortezomib enhances the sensitivity of myeloma cells toward T cell-mediated lysis by restoring apoptosis signaling via the FAS/FAS ligand pathway. Conclusion:Proteasome inhibition sensitizes myeloma cells to T cell-mediated killing via a FAS/FAS ligand-dependent mechanism. Conversely, bortezomib impairs T cell function. However, the net effect is a synergistic killing of myeloma cells when T cells and bortezomib are combined. These data support further exploration of the use of bortezomib in preclinical models, with derived clinical trials in the allogeneic setting (e.g. in combination with DLI or following allo-SCT) or in the autologous setting (e.g. in combination with a tumor vaccine) potentially warranted. (CSM and TM are joint senior authors) Disclosures:van de Donk:Celgene: Research Funding. Lokhorst:Celgene: Research Funding; Jansen-Cilag: Research Funding. Hayes:Pfizer: Research Funding; Amgen: Research Funding. Munshi:Celgene: Consultancy; Millenium: Consultancy; Novartis: Consultancy; Onyx: Consultancy. Richardson:Millennium:; Celgene:; Johnson & Johnson:; Novartis:; Bristol Myers Squibb:. Anderson:Celgene: Membership on an entity’s Board of Directors or advisory committees; Millennium: Membership on an entity’s Board of Directors or advisory committees; Onyx: Membership on an entity’s Board of Directors or advisory committees; Merck: Membership on an entity’s Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity’s Board of Directors or advisory committees; Novartis: Membership on an entity’s Board of Directors or advisory committees; Acetylon: Membership on an entity’s Board of Directors or advisory committees. Mitsiades:Millennium Pharmaceuticals: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Novartis Pharmaceuticals: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Merck: Consultancy, Honoraria; Kosan: Consultancy, Honoraria; Pharmion: Consultancy, Honoraria; Centocor: Consultancy, Honoraria; Amnis Therapeutics: Consultancy, Honoraria; PharmaMar: Licencing royalties; OSI Pharmaceuticals: Research Funding; Amgen: Research Funding; AVEO Pharma: Research Funding; EMD Serono: Research Funding; Sunesis: Research Funding; Gloucester Pharmaceuticals: Research Funding; Genzyme: Research Funding; Johnson & Johnson: Research Funding.

Valproic Acid Upregulates NKG2D Ligand Expression and Potentially Enhances Natural Killer Cell–mediated Cytolysis of Myeloma

Abstract 5061 Objective:Multiple myeloma (MM) is a plasma cell malignancy. Although high-dose chemotherapy supported by autologous hematopoietic stem-cell transplantation can produce higher response rates and longer survival than standard chemotherapy, MM remains largely incurable by current therapeutic strategies. Clinical and preclinical data have been demonstrated that natural killer (NK) cells have an anti-myeloma effect. However, low NK cell activity affected the successful utilization of NK cell adoptive immunotherapy for myeloma. In this study, we investigated the effect of valproic acid (VPA), as a histone deacetylase inhibitor, on the expression of NKG2D ligands on human myeloma cell lines and primary myeloma cells derived from patients. We also evaluated the sensitivity of myeloma cells to NK cell-mediated lysis before and after treatment with VPA, and the possible underlying mechanisms. Method:ARK, OPM2 myeloma cell lines and primary myeloma cells (n = 4) from patients with myeloma were cultured with 0.5–3mM VPA for 24 to 72 hours. Cells cultured without VPA were as control groups. To evaluate the effect of VPA on NKG2D ligand expression of myeloma cells, mRNA and protein expression of NKG2D ligands (including MICA/B, ULBP1, ULBP2 and ULBP3) of myeloma cells were tested by Real time-PCR and flow cytometry. 51Cr release assay and NKG2D antibody blocking experiments were combined to examine the susceptibility of myeloma cells to NK cell-mediated lysis in response to VPA and the possible killing mechanisms. Results:ARK and OPM-2 myeloma cell lines were treated with various concentrations of VPA. The most prominent upregulations of mRNA expressions of MICA/B, ULBP2 and ULBP 3 were observed in the two tested myeloma cell lines. The enhancement of cell surface expression of MICA/B, ULBP2 and ULBP 3 was detected by flow cytometry after cells treatment with VPA. We also found that VPA upregulated MICA/B expression on OPM2 MM cell line in a time- and dose-dependent manner. These data suggest that VPA could upregulate expression of NKG2D ligands in myeloma cell line at both the mRNA and protein levels. A similar increase of surface expression of NKG2D ligands was obtained in 2–3 mM VPA treated primary MM cells (n=4). NK cell function was studied by 51Cr release assay. VPA treatment of OPM2 showed higher sensitivity to NK cell lysis than untreated cells (specific killing: 72.8±6.2% vs 32.1±3.7% at E/T 10:1, p <0.01). The killing of VPA treatment of patient myeloma cells by NK cells also significantly increased compared with control cells (specific killing: 46.7±5.6% vs 21.6±4.5% at E/T 10:1, n=4, p <0.01). The enhancing effect of VPA was blocked by pretreatment of OPM2 cells with a combination of anti-MICA/B, anti-ULBP1,2,3 mAbs or pretreatment of NK cells with anti-NKG2D mAbs, indicating the contribution of NKG2D-NKG2D ligands interations to VPA-modulated lysis of myeloma. We also found that constitutive phosphorylated extracellular signal-regulated kinase (ERK) but not AKT signaling pathway may be involved in VPA-induced higher NKG2D ligand expression. Conclusion:Histone deacetylase inhibitor valproic acid could upregulate cell surface expression of NKG2D ligands, rendering myeloma cells more sensitive to NK cells, and thereby enhance NK cell–mediated lysis of myeloma. Our findings imply that regulation of the expression of NKG2D ligands by treatment with histone deacetylase inhibitors may be an attractive strategy for immunotherapy of myeloma. Acknowledgments:This work was supported by grants from National Natural Science Foundation of China (81071856 and 30973450 to JS) and funds from Shanghai Tenth People's Hospital (10RD103 and 11SC103 to JS). Disclosures:No relevant conflicts of interest to declare.

Rational Drug Design: Proteasome Inhibitor Mediated Down-Regulation of the FA/BRCA Pathway Is Synergistic with PARP Inhibition in Myeloma Cell Lines

Abstract 2922The development of new and biologically-based therapeutic regimens is critical for the successful control, if not cure, of multiple myeloma. Incorporation of the novel agents, including the proteasome inhibitor bortezomib, harbored large strides in disease modification. However, even with the success of bortezomib containing regimens, drug resistance and disease relapse remain inevitable. As such, it is critical that we use preclinical models to not only develop drugs, but also to consider strategies for co-development of novel drug combinations capitalizing on complementary biological activities. Our investigations in drug resistance recently revealed that increased homologous recombination (HR) potential, via over-expression of the FA/BRCA DNA repair pathway (FA/BRCA pathway), contributed to acquired melphalan-resistance in myeloma cell lines.(Yarde et al 2009) Drug resistance was causally linked to a novel transcriptional regulation of the FA/BRCA by NF-κB. Further examination demonstrated that bortezomib attenuated this component of the HR repair pathway and reversed melphalan resistance. To this end, we anticipated that bortezomib treatment may sensitize cells to inhibitors of complementary DNA repair pathways in a manner similar to the synthetic lethality elicited in by PARP1/2 inhibition in BRCA1 or FANCD1/ BRCA2 mutant cancers.(Farmer 2005, Bryant 2005) Consistent with this rationale, treatment of myeloma cells with bortezomib and the PARP inhibitor AZD2281/olaparib demonstrated synergism in specific myeloma cell lines. Pre-treatment of RPMI8226 myeloma cells with bortezomib for 6 hours greatly enhanced myeloma cell sensitivity to PARP inhibition with AZD2281/olaparib. The inhibitory concentration(IC)-50 was decreased by 17.7-fold (n=3; IC50 AZD2281 alone: 62.7 microM (39.0–84.0) and pretreated with bortezomib 3.54 microM (2.4–4.6)). Combination Index (CI) demonstrated a mean of 0.41 in 8226 and 0.43 in U266 myeloma cells, consistent with a synergistic relationship. Further analysis confirmed that synergism correlated with decreased expression of FANCD2 mRNA and protein by 6 hours. In contrast to sequential treatment, concomitant treatment with these agents did not elicit the synergistic phenotype. Interestingly, sequential treatment of NCIH929 myeloma cells did not demonstrate the same synergistic response (CI :0.89, slight synergism). Consistent with this, treatment of NCIH929 cells with bortezomib did not negatively regulate FANCD2 mRNA or protein expression, suggesting that FA/BRCA pathway can be differentially regulated in myeloma cells. To more specifically determine if FANCD2 was a key factor regulated by bortezomib, we targeted FANCD2 with siRNA. Pretreatment of myeloma cells with FANCD2 siRNA also sensitized cells to AZD2281/olaparib relative to siRNA control (IC50: 19.0 microM vs 35.0 microM n=4; p<0.05). These results show that bortezomib (or other proteosome inhibitors) and AZD2281/olaparib (or other PARP inhibitors) may represent an exciting new combination therapy for myeloma. We are currently examining the applicability of these studies to other proteosome inhibitors and the clinical relevance with ex vivo studies with myeloma patient samples. We believe that data presented here are innovative as they introduce a novel biological rationale, the abrogation complementary pathways in DNA damage repair, for the preclinical development of novel targeted drug combinations in myeloma. Further, we anticipate that although this study has focused on multiple myeloma, the results of the proposed research will be applicable to a wide range of hematologic and solid tumors. Disclosures:No relevant conflicts of interest to declare.

Suppressing miRNA-15a/-16 expression by interleukin-6 enhances drug-resistance in myeloma cells

The bone marrow microenvironment facilitates the survival, differentiation, and proliferation of myeloma (MM) cells. This study identified that microRNA-15a and -16 expressions tightly correlated with proliferation and drug sensitivity of MM cells. miRNA-15a/-16 expression in MM cells was significantly increased after treatment with cytotoxic agents. The interaction of bone marrow stromal cells (BMSC) with MM cells resulted in decreased miRNA-15a/-16 expression and promoted the survival of the MM cells. Interleukin-6 (IL-6) produced by BMSCs suppressed the expression of miRNA-15a and 16 in a time- and dose- dependent pattern, with the suppression on miRNA-15a being more significant than on miRNA-16. miRNA-15a-transfected MM cells were found to be arrested in G1/S checkpoint, and the transfected MM cells had decreased growth and survival. In conclusion, our data suggest that via suppressing miRNA-15a and -16 expressions, IL-6 secreted by BMSCs promotes drug-resistance in myeloma cells.

Bone marrow stromal cells protect myeloma cells from bortezomib induced apoptosis by suppressing microRNA-15a expression

Despite unsurpassed anti-tumor activity of bortezomib for multiple myeloma (MM), drug resistance has emerged as a challenge, especially when MM cells adhere to the stroma. This study aimed to determine whether bone marrow stromal cells (BMSCs) have a role in the development of chemoresistance in MM. Our data demonstrate that the secretion of interleukin-6 (IL-6), vascular endothelial growth factor (VEGF), and cell-to-cell contact with microenvironment-derived stromal cells from patients with multiple myeloma (MM-BMSCs) significantly decreased the sensitivity of myeloma cells to bortezomib treatment. Mechanistically, we found that microRNA (miRNA)- 15a expression was up-regulated in U266 and NCI-H929 cells treated by bortezomib, which was inhibited by MM-BMSCs. miRNA-15a transfected myeloma cells were arrested in G1/S checkpoint and secreted less VEGF compared to control transfected cells, although no significant difference was found in VEGF mRNA levels. In conclusion, our data suggest that via suppressing miRNA-15a expression, BMSCs provide survival support and protect myeloma cells from bortezomib induced apoptosis.