Bortezomib Sensitivity Research Articles

Down-Regulation of CD9 by Methylation Decreased Bortezomib Sensitivity in Multiple Myeloma

Bortezomib therapy has been proven successful for the treatment of relapsed and/or refractory multiple myeloma (MM). However, both intrinsic and acquired resistance has already been observed. In this study, we explored the relationship between CD9 expression and bortezomib sensitivity in MM. We found that down-regulation of CD9 by methylation decreased bortezomib sensitivity in multiple myeloma. CD9 expression obviously increased bortezomib sensitivity through inducing apoptosis, significantly inhibiting U266 cells' adhesion to HS-5 and primary bone marrow stromal cells, but increasing U266 cells' adhesion to fibronectin. CD9 expression also significantly inhibited U266 cell migration. The mechanisms may include: the endoplasmic reticulum stress pathway, cell adhesion related signaling pathway and osteoclast differentiation related signaling pathway. Combination therapy with de-methylation reagent 5-Aza-2-deoxycytidine may prove useful to the development of novel strategies for the treatment of bortezomib-resistant MM patients.

Expression Of Truncated Protein Tyrosine Phosphatase, Receptor Type, O (PTPROt) Influences Multiple Myeloma Sensitivity To Bortezomib Through Akt Signaling, and Is a Favorable Clinical Prognostic Factor

BackgroundProteasome inhibitors such as bortezomib and carfilzomib are an important part of our current chemotherapeutic armamentarium against multiple myeloma, and have improved outcomes in the up-front, relapsed, and relapsed/refractory settings. Their efficacy has been demonstrated both as single agents, and as part of rationally designed combination regimens, but they are at this time used empirically, since biomarkers to identify patients who would most or least benefit from their application have not been clinically validated. Moreover, the vast majority of patients eventually develop drug-resistant disease which precludes further proteasome inhibitor use through mechanisms that have not been fully elucidated. MethodsWe compared gene expression profiles (GEPs) of a panel of bortezomib-resistant myeloma cell lines and their vehicle-treated, drug-naïve counterparts to identify significant changes associated with drug resistance. The list of genes whose expression was changed by at least 2-fold was compared with independent RNA interference studies whose goal was to identify genes whose suppression conferred drug resistance. Further validation of genes of interest was pursued in a panel of myeloma cell lines, and in clinically annotated GEP databases. ResultsSuppression of PTPROt expression was noted in bortezomib-resistant RPMI 8226 and ANBL-6 myeloma cells compared to isogenic, drug-naïve controls, and this was confirmed by quantitative PCR. Overexpression of PTRPOt in RPMI 8226, ANBL-6 and other myeloma cell lines was by itself sufficient to increase the level of apoptotic, sub-G0/G1 cells compared to vector controls, or cells expressing a phosphatase-dead PTPROt mutant. Moreover, PTPROt enhanced the ability of bortezomib to reduce myeloma cell viability, in association with increased activation of caspases 8 and 9. Exogenous over-expression of PTPROt was found to reduce the activation status of Akt, a known anti-apoptotic pathway that reduces bortezomib activity, based on Western blotting with antibodies to phospho-Akt (Ser473), and Akt kinase activity assays. Notably, we also found that exogenous over-expression of PTPROt resulted in increased expression levels of p27Kip1. Interestingly, array CGH data from studies of myeloma cell lines and primary cells showed that the PTPROt gene was located in a genomic region with a high propensity for loss. Analysis of the Total Therapy databases of GEP and patient outcomes available on the Multiple Myeloma Genomics Portal showed that higher than median expression of PTPROt was associated with better long-term survival (P=0.0175). Finally, analysis of the Millennium Pharmaceuticals database of studies of bortezomib in the relapsed and relapsed/refractory setting showed high PTRPOt expression was more frequently seen in patients who achieved complete remission (P<0.01), and was associated with a better median overall survival (P=0.0003). ConclusionsTaken together, the data support the possibility that high expression of PTPROt is a good prognostic factor for response to bortezomib-containing therapies, and that this may occur through modulation by PTPROt of the Akt pathway. Moreover, they suggest that strategies to enhance the expression of PTPROt should be investigated to restore bortezomib sensitivity in patients with proteasome inhibitor-resistant disease. Disclosures:Orlowski:Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Millennium: The Takeda Oncology Company: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Onyx: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Resverlogix: Research Funding; Array Biopharma: Honoraria, Membership on an entity's Board of Directors or advisory committees; Genentech: Honoraria, Membership on an entity's Board of Directors or advisory committees; Merck: Membership on an entity's Board of Directors or advisory committees.

Inhibition of Inducible Heat Shock Protein-70 (Hsp72) Enhances Bortezomib-Induced Cell Death in Human Bladder Cancer Cells

The proteasome inhibitor bortezomib (Velcade) is a promising new agent for bladder cancer therapy, but inducible cytoprotective mechanisms may limit its potential efficacy. We used whole genome mRNA expression profiling to study the effects of bortezomib on stress-induced gene expression in a panel of human bladder cancer cell lines. Bortezomib induced strong upregulation of the inducible HSP70 isoforms HSPA1A and HSPA1B isoforms of Hsp72 in 253J B-V and SW780 (HSPA1Ahigh) cells, but only induced the HSPA1B isoform in UM-UC10 and UM-UC13 (HSPA1Alow) cells. Bortezomib stimulated the binding of heat shock factor-1 (HSF1) to the HSPA1A promoter in 253JB-V but not in UM-UC13 cells. Methylation-specific PCR revealed that the HSPA1A promoter was methylated in the HSPA1Alow cell lines (UM-UC10 and UM-UC13), and exposure to the chromatin demethylating agent 5-aza-2′-deoxycytidine restored HSPA1A expression. Overexpression of Hsp72 promoted bortezomib resistance in the UM-UC10 and UM-UC13 cells, whereas transient knockdown of HSPA1B further sensitized these cells to bortezomib, and exposure to the chemical HSF1 inhibitor KNK-437 promoted bortezomib sensitivity in the 253J B-V cells. Finally, shRNA-mediated stable knockdown of Hsp72 in 253J B–V promoted sensitivity to bortezomib in vitro and in tumor xenografts in vivo. Together, our results provide proof-of-concept for using Hsp72 inhibitors to promote bortezomib sensitivity in bladder cancers and suggest that selective targeting of HSPA1B could produce synthetic lethality in tumors that display HSPA1A promoter methylation.

Cap dependent translation contributes to resistance of myeloma cells to bortezomib.

Multiple myeloma (MM) is the second most predominant blood malignancy. Proteasome inhibitors like bortezomib have increased life expectancy, but eventually patients develop resistance to therapy. It was proposed that bortezomib acts through the induction of the Unfolded Protein Response (UPR), i.e., accumulation of misfolded proteins causing a lethal stress response. By this theory, increasing the proteasome load by the stimulation of translation may worsen the UPR. Here we evaluated the crosstalk between translation and bortezomib toxicity in both bortezomib sensitive and resistant cells. We found that bortezomib toxicity does not correlate with induction of proapoptotic eIF2α phosphorylation, but rather caused a late reduction in initiation of translation. This effect was accompanied by dephosphorylation of the mTORC1 target 4E-BP1. Infection of myeloma cells with constitutively dephosphorylated 4E-BP1, worsened bortezomib induced cell death. Since mTORC1 inhibitors cause pharmacological inhibition of 4E-BP1 phosphorylation, we tested whether they could act synergistically with bortezomib. We found that both rapamycin, a specific mTORC1 blocker, and PP242 a mTOR antagonist induce the arrest of myeloma cells irrespective of bortezomib sensitivity. Sensitivity to mTOR inhibitors has been associated to the levels of eIF4E/4E-BPs. We found that levels of eIF4E and 4E-BPs are variable among patients, and that 15% of myeloma patients have increased levels of 4E-BP1/2. Primary cells of myeloma retain sensitivity to mTOR inhibition, when plated on stromal cells. We propose that translational load does not contribute to bortezomib-induced death, but rather mTOR targeting may be successful in bortezomib resistant patients, stratified for eIF4E/4EBPs.

BAK and NOXA Are Critical Determinants of Mitochondrial Apoptosis Induced by Bortezomib in Mesothelioma

Based on promising preclinical efficacy associated with the 20S proteasome inhibitor bortezomib in malignant pleural mesothelioma (MPM), two phase II clinical trials have been initiated (EORTC 08052 and ICORG 05–10). However, the potential mechanisms underlying resistance to this targeted drug in MPM are still unknown. Functional genetic analyses were conducted to determine the key mitochondrial apoptotic regulators required for bortezomib sensitivity and to establish how their dysregulation may confer resistance. The multidomain proapoptotic protein BAK, but not its orthologue BAX, was found to be essential for bortezomib-induced apoptosis in MPM cell lines. Immunohistochemistry was performed on tissues from the ICORG-05 phase II trial and a TMA of archived mesotheliomas. Loss of BAK was found in 39% of specimens and loss of both BAX/BAK in 37% of samples. However, MPM tissues from patients who failed to respond to bortezomib and MPM cell lines selected for resistance to bortezomib conserved BAK expression. In contrast, c-Myc dependent transactivation of NOXA was abrogated in the resistant cell lines. In summary, the block of mitochondrial apoptosis is a limiting factor for achieving efficacy of bortezomib in MPM, and the observed loss of BAK expression or NOXA transactivation may be relevant mechanisms of resistance in the clinic.

Profiling Bortezomib Resistance Identifies Secondary Therapies in a Mouse Myeloma Model

Multiple myeloma is a hematologic malignancy characterized by the proliferation of neoplastic plasma cells in the bone marrow. Although the first-to-market proteasome inhibitor bortezomib (Velcade) has been successfully used to treat patients with myeloma, drug resistance remains an emerging problem. In this study, we identify signatures of bortezomib sensitivity and resistance by gene expression profiling (GEP) using pairs of bortezomib-sensitive (BzS) and bortezomib-resistant (BzR) cell lines created from the Bcl-XL/Myc double-transgenic mouse model of multiple myeloma. Notably, these BzR cell lines show cross-resistance to the next-generation proteasome inhibitors, MLN2238 and carfilzomib (Kyprolis) but not to other antimyeloma drugs. We further characterized the response to bortezomib using the Connectivity Map database, revealing a differential response between these cell lines to histone deacetylase (HDAC) inhibitors. Furthermore, in vivo experiments using the HDAC inhibitor panobinostat confirmed that the predicted responder showed increased sensitivity to HDAC inhibitors in the BzR line. These findings show that GEP may be used to document bortezomib resistance in myeloma cells and predict individual sensitivity to other drug classes. Finally, these data reveal complex heterogeneity within multiple myeloma and suggest that resistance to one drug class reprograms resistant clones for increased sensitivity to a distinct class of drugs. This study represents an important next step in translating pharmacogenomic profiling and may be useful for understanding personalized pharmacotherapy for patients with multiple myeloma.

Nelfinavir augments proteasome inhibition by bortezomib in myeloma cells and overcomes bortezomib and carfilzomib resistance

HIV protease inhibitors (HIV-PI) are oral drugs for HIV treatment. HIV-PI have antitumor activity via induction of ER-stress, inhibition of phospho-AKT (p-AKT) and the proteasome, suggesting antimyeloma activity. We characterize the effects of all approved HIV-PI on myeloma cells. HIV-PI were compared regarding cytotoxicity, proteasome activity, ER-stress induction and AKT phosphorylation using myeloma cells in vitro. Nelfinavir is the HIV-PI with highest cytotoxic activity against primary myeloma cells and with an IC50 near therapeutic drug blood levels (8–14 μM), irrespective of bortezomib sensitivity. Only nelfinavir inhibited intracellular proteasome activity in situ at drug concentrations <40 μℳ. Ritonavir, saquinavir and lopinavir inhibited p-AKT comparable to nelfinavir, and showed similar synergistic cytotoxicity with bortezomib against bortezomib-sensitive cells. Nelfinavir had superior synergistic activity with bortezomib/carfilzomib in particular against bortezomib/carfilzomib-resistant myeloma cells. It inhibited not only the proteasomal β1/β5 active sites, similar to bortezomib/carfilzomib, but in addition the β2 proteasome activity not targeted by bortezomib/carfilzomib. Additional inhibition of β2 proteasome activity is known to sensitize cells for bortezomib and carfilzomib. Nelfinavir has unique proteasome inhibiting activity in particular on the bortezomib/carfilzomib-insensitive tryptic (β2) proteasome activity in intact myeloma cells, and is active against bortezomib/carfilzomib-resistant myeloma cells in vitro.

Bortezomib sensitivity of acute myeloid leukemia CD34+ cells can be enhanced by targeting the persisting activity of NF-κB and the accumulation of MCL-1

Sustained NF-κB activation is often observed in acute myeloid leukemia (AML); therefore, proteasome inhibition has been proposed to efficiently target AML cells. In this study, we questioned whether leukemic stem cell-enriched CD34(+) cells are sensitive to the proteasome inhibitor bortezomib. Surprisingly, we observed in short-term and long-term culture assays that CD34(-) AML cells were more sensitive to bortezomib treatment compared with the CD34(+) AML cells at a clinical relevant dosage. Cotreatment with the apoptosis-inducing cytokine TRAIL did not enhance cell death in CD34(+) AML cells, in contrast to the effects in AML cell lines. The better survival of CD34(+) AML cells upon bortezomib treatment was due to a persisting NF-κB activity that could be overcome by the IKK inhibitor BMS-345541. This difference in sensitivity might be related to differences in NF-κB activation in AML CD34(+) versus CD34(-) cells, as suggested by a gene expression profiling study. Besides NF-κB, MCL-1 strongly determines the effectiveness of bortezomib. MCL-1 accumulated in CD34(+) AML cells upon bortezomib treatment and inhibition of MCL-1 by shRNA, or Obatoclax, significantly improved the sensitivity of CD34(+) AML cells to bortezomib. These results demonstrate that combining bortezomib with specific NF-κB or MCL-1 inhibitors might potentially target the leukemic stem cells.

Iron increases the susceptibility of multiple myeloma cells to bortezomib

Multiple myeloma is a malignant still incurable plasma cell disorder. Pharmacological treatment based on proteasome inhibition has improved patient outcome; however, bortezomib-resistance remains a major clinical problem. Inhibition of proteasome functionality affects cellular iron homeostasis and iron is a potent inducer of reactive oxygen species and cell death, unless safely stored in ferritin. We explored the potential role of iron in bortezomib-resistance. We analyzed iron proteins, oxidative status and cell viability in 7 multiple myeloma cell lines and in plasma cells from 5 patients. Cells were treated with increasing bortezomib concentrations with or without iron supplementation. We reduced ferritin levels by both shRNA technology and by drug-induced iron starvation. Multiple myeloma cell lines are characterized by distinct ferritin levels, which directly correlate with bortezomib resistance. We observed that iron supplementation upon bortezomib promotes protein oxidation and cell death, and that iron toxicity inversely correlates with basal ferritin levels. Bortezomib prevents ferritin upregulation in response to iron, thus limiting the ability to buffer reactive oxygen species. Consequently, reduction of basal ferritin levels increases both bortezomib sensitivity and iron toxicity. In patients' cells, we confirmed that bortezomib prevents ferritin increase, that iron supplementation upon bortezomib increases cell death and that ferritin reduction overcomes bortezomib resistance. Bortezomib affects iron homeostasis, sensitizing cells to oxidative damage. Modulation of iron status is a strategy worth exploring to improve the efficacy of proteasome inhibition therapies.

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 Role of 14-3-3ζ in Regulation of Proteasome Inhibitor Bortezomib Sensitivity in Multiple Myeloma

Abstract 1845The 14-3-3 proteins are a highly conserved protein family which includes β, γ, ε, η, σ, τ and ζ isoforms in mammalian cells. Through interacting with a number of protein clients, such as Cdc25, Bad, Raf1, and Foxo, 14-3-3 proteins act as integrators of diverse cell signaling pathways and participate in metabolism, cell cycle regulation, survival and apoptosis. We have previously demonstrated that expression of 14-3-3ζ is increased in the transition from MGUS to multiple myeloma (MM), and among all MM subsets, is expressed highest in the high risk genetic subsets [Gu et al. Blood (ASH Annual Meeting Abstracts), Nov 2011; 118: 1369]Using our 14-3-3ζ knockdown and overexpression stable MM cell lines, we found 14-3-3ζ played important roles in MM cell proliferation and growth. We also found that silencing 14-3-3 appeared to impair bortezomib-induced cell apoptosis in LP1 cells as well as to another proteasome inhibitor MG132. To further explore the function of 14-3-3ζ in MM cell biology, we performed co-immunoprecipitation followed by mass spectrometry to identify the potential 14-3-3ζ binding partners in the LP1 14-3-3ζ overexpressed cell lines. Analysis using IPA (Ingenuity Pathway Analysis) revealed critical functions and pathways related to EIF2, eIF4, p70S6K and mTOR signaling, protein ubiquitination pathway and aminoacyl-tRNA biosynthesis. Among all the identified binding candidates, we also found proteasome subunits, which could explain the impact of overexpression on sensitivity to bortezomib.To verify whether 14-3-3ζ can directly interact with the proteasome, we performed a His pull down assay using purified His-14-3-3ζ and LP1 cell lysate. We found that 14-3-3ζ can bind the 11S proteasome activator, PA28α. Using our LP1 14-3-3ζ overexpression stable cell line, we also confirmed the interaction between 14-3-3ζ and PA28α. Further experiments using native gel electrophoresis and western blot revealed more proteasome complexes formation in LP1 14-3-3ζ knockdown cells, suggesting 14-3-3ζ may regulate the function of proteasome through modulating proteasome assembly.In summary, 14-3-3ζ expression appeared to be critical for MM cell proliferation, and may influence sensitivity to proteasome inhibition by its interaction with proteasome subunits. Our data provides invaluable information on the molecular mechanism of resistance to proteasome inhibitor in MM and also helps to develop new strategies to overcome proteasome resistance. Given the importance of 14-3-3ζ overexpression among the high risk cohort of MM patients, 14-3-3ζ represents an important therapeutic target in high risk MM. Disclosures:Kaufman:Millenium: Consultancy; Celgene: Consultancy; Novartis: Consultancy; Onyx: Consultancy. Lonial:Millennium: Consultancy; Celgene: Consultancy; Onyx: Consultancy; Merck: Consultancy; Novartis: Consultancy; BMS: Consultancy.

Tumor Cell-Dependent Differences in Stroma-Induced Changes to Bortezomib Sensitivity.

Abstract 2468In multiple myeloma (MM) and other hematologic malignancies, bone marrow stromal cells (BMSCs) confer resistance to diverse conventional or investigational therapeutics. During the last decade, data from many groups have concurred that the in vitro anti-MM activity of the proteasome inhibitor bortezomib is very similar in the presence and absence of BMSCs, including primary and immortalized BMSCs. These well-validated observations have supported the notion that novel, more effective, therapies for the treatment of MM should ideally be, similarly to bortezomib, capable of overcoming the protective effect of BMSCs. Interestingly, however, we have observed that primary CD138+ MM tumor cells isolated from patients with clinical refractoriness to bortezomib occasionally exhibit substantial in vitro response to clinically achievable concentrations of this drug. We therefore hypothesized that, under certain previously under-explored experimental settings, BMSCs may alter the threshold of MM cell response to bortezomib-induced apoptosis. To address this hypothesis in conditions that better simulate the clinical context, we conducted compartment-specific bioluminescence imaging (CS-BLI) assays to evaluate the effect of bortezomib on tumor cells co-cultured with BMSCs for different time periods prior to bortezomib administration. We observed that prolonged tumor-stromal co-culture (48–96hrs) prior to initiation of bortezomib treatment did not affect drug sensitivity for several MM cell lines (OPM2, H929, UM9, KMS11, KMS18 and RPMI-8226) tested. Prolonged co-culture of OPM1, RPMI-8226-Dox40, OCI-My5, KMS12BM and KMS18 cells prior to bortezomib treatment enhanced its activity. Importantly, extended co-culture of MM cell lines MM.1S and MM.1R with BMSCs prior to drug treatment induced significant attenuation of their response to bortezomib, as evidenced by 2–3 fold increase of IC50 values in several independent replicate experiments and a mean % area under the bortezomib dose response curve (AUC) of 5.82% vs 14.10% in the absence vs. presence of BMSCs, respectively (p=0.0079). Consistent with these in vitro results, heterotypic s.c. xenografts of Luc+ MM.1S cells mixed with Luc- BMSCs did not show statistically significant reduction in MM burden with bortezomib treatment (0.5 mg/kg s.c. twice weekly for 5 weeks) compared to vehicle-treated controls (p=0.1320), as quantified by bioluminescence imaging. In contrast, the same dose and schedule of bortezomib treatment significantly suppressed tumor burden, compared to vehicle-treated controls, of monotypic s.c. xenografts of Luc+ MM.1S cells in SCID mice (p=0.0022), as in prior experience. To evaluate the molecular mechanisms of cell non-autonomous decrease in MM cell response to bortezomib, we compared the transcriptional profiles of MM.1S cells in extended co-cultures with HS-5 BMSCs vs. MM.1S cells cultured in isolation. These studies identified a distinct transcriptional signature of stroma-induced transcripts, including several (e.g. PSMC3, ITGB7, FOS, ALDH1L2) for which transcript expression higher than the median levels for refractory MM patients correlated with shorter overall survival (p<0.02, log-rank tests) after treatment with bortezomib. These observations highlight the notion that tumor cell responses to a given agent in the presence of non-malignant stromal cells can exhibit substantial qualitative and quantitative variation, depending on the specific tumor cell type tested, as well as the particular stromal cell population and conditions of the co-culture. Our findings highlight the need to apply combinatorial high-throughput scalable platforms, such as CS-BLI, to evaluate the different permutations of interactions between tumor cells, non-malignant accessory cells of the microenvironment and administered therapeutics. This study also provides a comprehensive functional oncogenomic framework to identify prognostically relevant molecular mediators of stroma-induced resistance to therapy in MM. Disclosures:Groen:Genmab BV: Research Funding. McMilllin:Axios Biosciences: Equity Ownership. Mitsiades:Millennium Pharmaceuticals: Honoraria; Celgene: Honoraria; Novartis Pharmaceuticals: Honoraria; Bristol-Myers Squibb: Honoraria; Merck &Co.: Honoraria; Centocor: Honoraria; Arno Therapeutics: Honoraria; Amgen: Research Funding; AVEO Pharma: Research Funding; OSI: Research Funding; EMD Serono: Research Funding; Sunesis: Research Funding; Johnson & Johnson: Research Funding; PharmaMar: Licensing royalties Other; Axios Biosciences: Uncompensated Role as advisor, Uncompensated Role as advisor Other.

Dll1/Notch activation contributes to bortezomib resistance by upregulating CYP1A1 in multiple myeloma

One of the greatest challenges in multiple myeloma (MM) treatment is to overcome drug resistance. Many pathways are involved including Notch signaling. Notch receptors are expressed by MM cells and Notch ligand Dll1 is present on bone marrow (BM) stromal cells. In this study, we demonstrate that Dll1 can activate Notch signaling mostly through Notch2 receptor and can contribute to drug resistance to bortezomib, both in murine and human MM cells. Blocking the Notch pathway by DAPT (gamma secretase inhibitor) could reverse this effect and increased sensitivity to bortezomib. We describe the upregulation of CYP1A1, a Cytochrome P450 enzyme involved in drug metabolism, as a possible mechanism of Dll1/Notch induced bortezomib resistance. This was confirmed by inhibition experiments using α-Naphthoflavone or CYP1A1-siRNA that resulted in an increased sensitivity to bortezomib. In addition, in vivo data showed that combination treatment of DAPT with bortezomib was able to increase bortezomib sensitivity and prolonged overall survival in the 5T33MM mouse model. Our data provide a potential strategy to overcome bortezomib resistance by Notch inhibition in MM therapy.

Targeting the insulin-like growth factor-1 receptor to overcome bortezomib resistance in preclinical models of multiple myeloma

AbstractProteasome inhibition with bortezomib is a validated approach to the treatment of multiple myeloma, but drug resistance often emerges and limits its utility in the retreatment setting. To begin to identify some of the mechanisms involved, we developed bortezomib-resistant myeloma cell lines that, unlike previously reported models, showed no β5 subunit mutations. Instead, up-regulation of the insulin-like growth factor (IGF)–1 axis was identified, with increased autocrine and paracrine secretion of IGF-1, leading to increased activation of the IGF-1 receptor (IGF-1R). Exogenous IGF-1 reduced cellular sensitivity to bortezomib, whereas pharmacologic or small hairpin RNA–mediated IGF-1R suppression enhanced bortezomib sensitivity in cell lines and patient samples. In vitro studies with OSI-906, a clinically relevant dual IGF-1R and insulin receptor inhibitor, showed it acted synergistically with bortezomib, and potently resensitized bortezomib-resistant cell lines and patient samples to bor-tezomib. Importantly, OSI-906 in combination with bortezomib also overcame bor-tezomib resistance in an in vivo model of myeloma. Taken together, these data support the hypothesis that signaling through the IGF-1/IGF-1R axis contributes to acquired bortezomib resistance, and provide a rationale for combining bortezomib with IGF-1R inhibitors like OSI-906 to overcome or possibly prevent the emergence of bortezomib-refractory disease in the clinic.

Rad knockdown induces mitochondrial apoptosis in bortezomib resistant leukemia and lymphoma cells

To understand the molecular mechanism(s) underlying bortezomib resistance, we sought to identify potential target genes that were differentially expressed in bortezomib-resistant leukemia cells versus parental controls. Microarray analysis revealed that the mRNA levels of Rad (Ras associated with diabetes) were higher in the bortezomib-resistant Jurkat (Jurkat-R) cells than in the parental control cells. The importance of Rad for bortezomib resistance was supported by three observations. First, Rad knockdown overcame bortezomib resistance and induced mitochondrial apoptosis via Noxa/Bcl-2 modulation. Second, Rad decreased cell death in response to bortezomib. Third, leukemia and lymphoma cell lines (K-562, Raji, IM-9 and Jurkat-R) with elevated Rad expression levels showed higher degrees of bortezomib resistance versus those (Sup-B15, JVM-2, U266 and Jurkat) with low Rad expression levels (r=0.48, P=0.0004). Thus, Rad over expression could be a molecular target to improve bortezomib sensitivity in human leukemia and lymphoma.

Abstract 4892: EBV infectivity determines sensitivity to bortezomib in hematopoietic malignancy cells

Abstract Epstein-Barr virus (EBV) is closely associated with several human malignancies where it expresses limited subsets of latent proteins. Of the latent proteins, latent membrane protein 1 (LMP1) is a potent transforming protein that constitutively induces multiple cell signaling pathways. C-terminal signaling domain of LMP1 (CTAR2) is known to engage the c-Jun N-terminal kinase (JNK) and NF-κB pathways, which contributes to EBV-associated oncogenesis. Bortezomib, proteasome inhibitor, blocks degradation of IκBα, thereby blocking not only NF-κB activation but also related cytokine production and the survival advantage for patients. However, to date, no study has examined that EBV infectivity is correlated with efficacy of bortezomib in the hematopoietic malignancies cells. Here, we used EBV-positive cell lines (Hank-1, Jijoye, Daudi and Granta519), EBV-negative cells (NK-92, Ramos, Jurkat and Rec1) and stable EBV-transfected cells (Ramos/EBV, Jurkat/EBV and Rec1/EBV) to evaluate the bortezomib sensitivity, highlighting the role of NF-κB. In the present study, we investigated that treatment with bortezomib significantly induced apoptosis and growth inhibition through blocking nuclear NF-κB signaling pathway in EBV-positive cells than EBV-negative cells. Thus, this data suggest that LMP1-induced NF-κB plays a critical role of survival and growth in EBV-positive cells. Additionally, treatment with bortezomib may provide a therapeutic advantage for patients with EBV-positive cells. 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 4892. doi:1538-7445.AM2012-4892

Truncation of NF-KB2 Is Associated with Poor Response to Bortezomib Treatment in Multiple Myeloma

Abstract▪2891▪This icon denotes a clinically relevant abstractThe proteasome inhibitor, bortezomib, is a powerful agent for the treatment of Multiple Myeloma (MM). However, many patients fail to achieve an optimal response. To identify biomarkers that could predict an optimal response to bortezomib, we assessed the role of the noncanonical NF-kB pathway in the response to bortezomib. Our in vitro studies identified that NF-kB2/p100 protein levels correlated directly with bortezomib sensitivity. Based on this finding, we measured at diagnosis the mRNA levels of different NF-kB2 regions in 64 patients schedule to receive bortezomib-based regimens. While no difference in expression of the 5'end regions was found between the CD138 (+) and CD138 (-) cells in these patients, half of them demonstrated low or absent 3'end expression levels in the CD138 (+) cells. Among forty-seven patients treated with four cycles of Bortezomib and dexamethasone, we found that low plasma cell NF-kB2 3'end levels were associated with a lower overall response rate (low: 47.8% vs. high: 96%, see table). In contrast, low plasma cell NF-kB2 3'end levels failed predict bortezomib response when lenalidomide or thalidome was added to the therapeutic regimen. To further understand the cause for NF-kB2 truncation, we are currently analyzing RNA sequencing in 3 non-responder patients and whole genome sequencing of 38 MM patients to determine the potential causative mechanisms. Preliminary results suggest that NF-kB2 truncation result from multiple genetic alterations, including: premature stop codons produced by ALU insertions, inversions and splicing variants. Overall, this study substantiates the basis of a more precise therapeutic algorithm according to NF-kB2 3'end plasma cell levels, which include the use of bortezomib and dexamethasone in patients with high NF-kB2 3'end plasma cell levels and immunomodulators or DNA damaging agents in patients with low NF-kB2 3'end plasma cell levels.BD+BTD or BRD++NF-kB2 3’ end mRNA (CD 138(+)/CD138(−)LowHighLowHigh> Very Good Partial Response5 (21.7)9 (37.5)1 (11)4 (50)Partial Response6 (26.1)14 (58.3)7 (78)4 (50)Stable Disease4 (17.4)1 (4.2)0 (0)0 (0)Progressive disease8 (34.8)0 (0)1 (12)0 (0)Overall Response11 (47.8)23 (95.8)8 (88)8 (100)*Bortezomib + Dexamethasone**Bortezomib+Lenalidomide/Thalidomide+Dexamethasone Disclosures:Kaufman:Millenium, Onxy, Novartis, Keryx: Consultancy; Merck, Celgene: Research Funding.

ERN1/IRE1 and XBP1 Are Dispensable for Myeloma Cell Survival but Are Required for the Anti-Myeloma Activity of Bortezomib

Abstract 986We have previously conducted kinome- (Tiedemann et al., Blood 2010) and genome- (Zhu et al., Blood 2011) wide small interfering RNA (siRNA) studies in human myeloma cells in the absence and presence of titrated bortezomib (BTZ) in order to identify genes that modulate BTZ sensitivity or resistance. From high throughput RNAi screening, endoplasmic reticulum-to-nucleus-signaling-1 (ERN1), the human homolog of yeast IRE-1, was identified with multiple siRNA as the kinase most potently required for BTZ activity, with genetic silencing associated with BTZ resistance. ERN1/IRE1 is an endoplasmic reticulum (ER)-resident transmembrane protein and a core component of the unfolded protein response (UPR). By gene expression profiling and western blot IRE1 is ubiquitously expressed across primary myeloma tumors and a panel of 14 human myeloma tumor lines (HMCL). Significantly, silencing of IRE1 with lentiviral shRNA induced BTZ resistance in 9/9 HMCL. IRE1 silencing in the absence of BTZ was surprisingly well tolerated in the majority of HMCL tested, with no loss of viability; however in 2/9 HMCL (JJN3, 8226) silencing of IRE1 resulted in reduced proliferation. Multiple RNAi yielded identical results. In response to unfolded protein accumulation within the ER lumen, IRE1 oligomerizes and auto-phosphorylates to reveal an endonuclease activity that splices X-box protein 1 (XBP1) mRNA to activate the potent bZIP transcription factor, XBP1s. XBP1 is essential for physiologic differentiation of B cells to plasma cells and its absence results in profound plasma cell deficiency (Reimold et al., 2001). Despite it's essential role in plasma cell differentiation, we find that XBP1, like IRE1, is redundant for myeloma cell survival under standard growth conditions. Lentiviral knockdown of XBP1 using multiple distinct RNAi failed to induce HMCL cytotoxicity; and resulted in reduced growth in only 2/9 HMCL, closely mirroring results observed with IRE1 inhibition. Significantly, XBP1 silencing also closely reproduced the BTZ resistance observed with IRE1 silencing. As plasmacytoma in patients grow under hypoxic conditions we next evaluated myeloma cell growth in response to hypoxia (0.2% O2) and concurrent UPR signaling disruption. At 48hrs, viability was comparable in IRE1 knockouts and parental cells. In contrast, HMCL with IRE1 silencing showed marked vulnerability to protein synthesis inhibition induced by puromycin, with selection against IRE1 silenced cells in mixed cultures. Notably, ER stress is signaled by 3 different transmembrane transducers (IRE1, PERK and ATF6), each contributing to distinct UPR pathways. As induced BTZ resistance from IRE1 or XBP1 inhibition might conceivably result either from loss of a death signal transmitted via IRE1-XBP1 following PI-induced ER stress, or from a compensatory increase in PERK or ATF6 homeostasis signaling in response to IRE1-XBP1 silencing, we generated single, double and triple knockdown HMCL with suppressed expression of 1, 2 or all 3 ER stress transducers (IRE1, PERK, ATF6). Individual silencing of either IRE1 or PERK in HMCL had no effect on levels of other ER stress transducers by western blot, however silencing of ATF6 caused compensatory increases in PERK while XBP1 silencing resulted in reduced IRE1. Importantly, HMCL lacking IRE1 and PERK or ATF6, showed no loss of BTZ resistance induced by either IRE1 or XBP1 knockdown, demonstrating that secondary increases in signaling via these alternative ER stress pathways do not account for the BTZ resistance induced by IRE1-XBP1 pathway inhibition. Consistent with the hypothesis that increased UPR signaling (via IRE1-XBP1) causes myeloma cell death, knock down of the ER luminal chaperone BiP (HSPA5), which negatively regulates all 3 stress transducers, proved cytotoxic in multiple HMCL. Overall, these data suggest that the anti-myeloma activity of BTZ involves a specific IRE1-XBP1 death signal generated in response to proteasome inhibition and unfolded protein ER stress, rather than ER failure from unfolded protein. As IRE1-XBP1 signaling is dispensable for myeloma cell survival under many growth conditions, targeted small molecule inhibitors of IRE1 currently under development may prove ineffective as single agent therapy for myeloma; moreover such agents may prove antagonist to PI therapy. Finally, disruption of the IRE1-XBP1 axis in primary tumors may mediate bortezomib resistance in patients. Disclosures:No relevant conflicts of interest to declare.

Response of myeloma to the proteasome inhibitor bortezomib is correlated with the unfolded protein response regulator XBP-1

Multiple myeloma, a malignancy of the antibody-secreting plasma cells, remains incurable by current therapy. However, the proteasome inhibitor bortezomib and other new drugs are revolutionizing its treatment. It remains unclear why myelomas are peculiarly sensitive to bortezomib, or what causes primary or acquired resistance. The 'unfolded protein response' is necessary for folding and assembly of immunoglobulin chains in both normal and malignant plasma cells, as well as for the disposal of incorrectly folded or unpaired chains via the ubiquitin-proteasome pathway. We tested the hypothesis that levels of transcription factor XBP-1, a major regulator of the unfolded protein response, predict response to bortezomib. Expression of XBP-1 and other regulators of the unfolded protein response were measured in myeloma and other cancer cell lines and two cohorts of patients with refractory myeloma and correlated with sensitivity/response to bortezomib. Bortezomib-resistant myeloma cell lines were derived and the effects on expression of unfolded protein response regulators, immunoglobulin secretion, proteasome activity and cross-resistance to cytotoxic drugs and tunicamycin determined. The consequences of manipulation of XBP-1 levels for sensitivity to bortezomib were tested. Low XBP-1 levels predicted poor response to bortezomib, both in vitro and in myeloma patients. Moreover, myeloma cell lines selected for resistance to bortezomib had down-regulated XBP-1 and immunoglobulin secretion. Expression of ATF6, another regulator of the unfolded protein response, also correlated with bortezomib sensitivity. Direct manipulation of XBP-1 levels had only modest effects on sensitivity to bortezomib, suggesting it is a surrogate marker of response to bortezomib rather than a target itself. The unfolded protein response may be a relevant target pathway for proteasome inhibitors in the treatment of myeloma and its regulator XBP-1 is a potential response marker. (The BIR study was registered with Australian Clinical Trial Registry Number 12605000770662).

Inhibition of HSP90 by triptolide (TPL) augments Bortezomib-induced U266 cells apoptosis

HSP90 has high expression in myeloma cells and very important for prognosis, and that triptolide (TPL), the main active component of the traditional Chinese herbal medicineTripterygium wilfordii Hook F (Celastraceae) has the inhibition effects in the growth of myeloma cells. In this study, we test the ability of TPL to induce apoptosis in U266 human myeloma cells by reducing HSP90 expression, as well as the effect of TPL on Bortezomib sensitivity. The MTT cell viability assay was used to assess growth inhibition of U266 human myeloma cells due to different TPL concentrations and the combined use of different concentration of TPL and 0.01 mM Bortezomib. Annexin-V and Propidium iodide (PI) labeling were used to detect cell death and determine the mode of death. TPL alone inhibited U266 cell growth and apoptosis inducement in a concentration-dependent manner. When TPL is combined with 0.01 mM Bortezomib, both effects increase. Western blotting revealed that caspase 3 and caspase 9 gene expression increased, while nuclear factor (NF)-KB and HSP 90 protein expression decreased, related with apoptosis and growth inhibition. TPL increases U266 cell sensitivity to Bortezomib, inducing apoptosis, which might be the result of suppress NF-KB and HSP90 activity. Key words: Bortezomib, multiple myeloma, heat shock protein gp90, triptolide.