Response Of Multiple Myeloma Cells Research Articles

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

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

The in vivo Transcriptional Response Towards Epigenetic Modulating Agents in Multiple Myeloma

Multiple myeloma (MM) is a hematological malignancy characterized by a plasma cell accumulation in the bone marrow (BM), for which novel treatment options are urgently needed. Epigenetic modulating agents such as histone deacetylase inhibitors (HDACi) and DNA methyltransferase inhibitors (DNMTi) are under intense investigation for cancer therapy. As shown in numerous functional in vitro studies, HDACi and DNMTi affect various biological processes important for tumor control including tumor cell survival, proliferation, differentiation and DNA repair. Given the broad mechanisms of action of these agents, it remains important to continue pre-clinical evaluation to identify in vivo relevant mechanisms of action. This may lead to the identification of novel biologically relevant targets and predictive biomarkers allowing clinical trial optimization.Recently, we developed gene expression based risk scores after treating human MM cell lines with TSA (HA-score) or decitabine (DM-score). These scores were predictive for MM patient survival using two independent cohorts (Heidelberg-Montpellier (HM) and University of Arkansas for Medical Sciences-Total Therapy 2) and identified potential biomarkers predictive for drug sensitivity. However, the transcriptional response of MM cells in vivo may be influenced by the close contact with the BM-environment. Therefore, we here aimed to characterize the transcriptional response of MM cells after in vivo treatment with the HDACi JNJ-26481585 (quisinostat) or the DNMTi decitabine using the syngeneic immunocompetent murine 5T33MM model.5T33MM mice (n=4/group) with established disease were treated with quisinostat or decitabine for 5 days after which tumor cells were isolated from the BM and subjected to microarray analysis. Using Significance Analysis of Microarray, we identified 574 and 180 probesets deregulated by respectively quisinostat and decitabine (of which 111 are in common). To assess the prognostic value of the deregulated genes, we performed MaxStat analysis in the HM cohort. JNJ-585 deregulated 31 genes associated with good prognosis and 31 associated with bad prognosis. Decitabine altered expression of 20 genes linked with poor prognosis while 5 genes were linked with good prognosis. The prognostic value of these genes was then implemented in a murine (Mu)-DM and Mu-HA score. The score values were significant higher in MM patients and human myeloma cell lines compared to MGUS and healthy bone marrow plasma cells. In addition, the scores were useful to separate patients of the 2 cohorts into a low risk and high risk group. Patients from the proliferation subgroup had a higher score compared to all other subgroups. In concordance, the scores were highest in patients with a high gene-expression based proliferation index. Using gene ontology (GO) tools (DAVID) and pathway tools (Reactome, STRING and Pathway-guide), we next explored the association of in vivo deregulated genes with biological processes and pathways. GO analysis showed that quisinostat-deregulated genes were mainly involved in immune modulation. Pathway analysis revealed associations with lymphocyte activation and proliferation, immune effector mechanisms and T-helper-1 development through processes like cytokine interactions, chemokine signaling and T-cell receptor- and NK-cell-signaling. In concordance, the signature represented elevated presence and signaling of interferon, tumor necrosis factor, interleukin-1 (IL-1), IL-2 and IL-12. The second most prominent alterations were genes linked with transcriptional misregulation in cancer. Pathways predicted to be affected by these alterations are linked with differentiation, resistance and cell survival. For decitabine, the gene list was substantially smaller and for more than half shared by JNJ-585. The pathway analysis also identified genes linked to immune system, gene expression regulation and metabolism pathways.In conclusion, in vivo treatment with epigenetic modulating agents identified a prognostic gene signature. In addition, HDACi (and to a lesser extent DNMTi) deregulated immunomodulatory genes and genes involved in transcriptional regulation. This indicates that immune regulation is an important in vivo anti-tumor property of HDACi and supports the rational to combine HDACi with immunomodulatory therapies such lenalidomide or cellular/peptide vaccination strategies. DisclosuresHose:Novartis: Research Funding. Seckinger:Novartis: Research Funding.

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

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

A Peptide Nucleic Acid Targeting Nuclear Rad51 Sensitizes Myeloma Cells to Melphalan Chemotoxicity Both in Vitro and in Vivo

Multiple myeloma (MM) cells are characterized by extensive genomic heterogeneity, which contributes to patient differences in prognosis and response to treatment. We previously reported that MM cells have elevated homologous recombination (HR) rates and expression of RAD51 and its paralogs, promoting genomic instability and disease progression that are reversed by RAD51 siRNA. We now examine the roles of HR and RAD51 in resistance to melphalan, one of the most widely used drugs for MM chemotherapy. The drug induces a variety of DNA lesions, with DNA interstrand crosslinks (ICL) accounting for most of the drug’s cytotoxicity. RAD51 is a central protein in the HR pathway and its overexpression may contribute to chemoresistance by enabling repair of DNA lesions induced by DNA damaging agents such as melphalan. MM cell sensitivity to melphalan correlates directly with melphalan-induced RAD51 foci, and high RAD51 expression predicts poor event-free and overall survival of MM patients. Activity of the Rad51 promoter increases >850-fold in cancer cells compared to normal cells, and tumor cells are selectively killed by a construct in which PRad51 drives expression of diphtheria toxin. In this study, we tested whether inhibiting RAD51 expression with a peptide nucleic acid (PNA) would inhibit MM cell growth and/or sensitize MM cells to melphalan. PNAs are DNA or RNA mimics in which a polymer of (2-amino­ethyl) glycine replaces the nucleic acid’s sugar-phosphate backbone. PNAs are highly specific, binding DNA with higher affinity than RNA or DNA, and they are quite stable to degradation both in vitro and in vivo.We designed a PNA to target the promoter region of the RAD51 gene (PNArad51), encompassing the transcription start site. To enhance cellular uptake and nuclear delivery without transfection, we conjugated the PNA to a nuclear localization signal rich in basic residues (PKKKRKVR). As a control we employed a scrambled PNA (PNAmt) with the same nucleotide composition but not targeting any genomic sequences. We used qRT-PCR to assess the effect of PNA on RAD51 mRNA expression and that of melphalan on mRNA levels of RAD51 and its paralogs (RAD51B, RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3) and BRCA1. Propidium iodide staining and flow cytometry were used to examine the cell-cycle effects of melphalan. γH2AX and RAD51 foci were quantitated using confocal immuno­fluorescence microscopy, and MM cell viability was assessed with the WST-1 assay. To examine the in vivo consequences of PNA ± melphalan for tumor growth, we injected H929 MM cells expressing luciferase into rabbit bone fragments implanted in SCID-rab mice, as previously described by us,. Total RNA extracted from cells recovered from the rabbit bones was analyzed by qRT-PCR to determine the in vivo effect of PNA on expression of RAD51.Melphalan treatment (10 µM) significantly induced expression of RAD51 and its paralogs, particularly RAD51 and XRCC3 (p≤0.01). Melphalan caused cell-cycle arrest, predominantly in the S-phase (55%, significantly elevated over vehicle alone, 17%; p<0.0001), the period in which HR is most active, and during which ICLs are converted into double strand breaks (DSBs) on encountering DNA replication forks. PNArad51 (10 µM) significantly reduced expression of RAD51 (~60%, p<0.001) relative to PNAmt. Pretreatment with PNArad51 inhibited melphalan-induced RAD51 focus formation, far more than PNAmt pretreatment (21% compared to 66%, p<0.0001) whereas the number of γH2AX foci increased (66%) relative to PNAmt (39%; p<0.0001). Consequently, pretreatment with PNArad51 produced synergistic synthetic lethality with melphalan, reducing the IC50 of melphalan by 4.5-fold. PNArad51 alone caused significant cytotoxicity compared to PNAmt (p<0.05). In the SCID-rab mouse model, a two-week treatment with PNArad51 alone or in combination with melphalan resulted in significant inhibition of tumor volume (p≈0.01 and p<0.05, respectively) compared to PNAmt, although the combination of PNAmt plus melphalan was ineffectual. Prolonged treatment (4 weeks) with PNArad51 ± melphalan (but not PNAmt + melphalan) reduced tumor growth compared to PNAmt treatment, although this was not statistically significant (p>0.05).These results highlight the importance of RAD51 in the response of MM cells to melphalan, and indicate for the first time the potential for RAD51-targeted PNA in tumor chemosensitization. DisclosuresNo relevant conflicts of interest to declare.

Genome-Scale Crispr-Cas9 Knockout Studies Reveal Mutifactorial and Functionally Overlapping Mechanisms of Myeloma Cell Resistance to Proteasome Inhibition

Acquired or de novo resistance to established and investigational therapies represents a major clinical challenge for multiple myeloma (MM) and other neoplasias. Despite extensive efforts, clinically-validated molecular markers that predict for proteasome inhibitor (PSI) resistance in most MM patients remain elusive. This challenge is partly due to limited availability so far of molecular data on MM patients before the start of PSI treatment vs. immediately after resistance to it develops; this challenge may also reflect the heterogeneity of the complex molecular mechanisms regulating MM cell response to PSIs. We hypothesized that resistance to PSIs can be mediated by disruption of several functionally overlapping genes, and that the prevalence of any of these lesions may be too low to detect in datasets available thus far. To examine this latter hypothesis, we performed a genome-wide screen for genes whose loss confers to MM cells resistance against bortezomib, through the use of the CRISPR (clustered regularly interspaced short palindromic repeats)–associated nuclease Cas9 system. Specifically RPMI-8226 MM cells were transduced with lentiviral construct for Cas9 nuclease, followed by lentiviral delivery of a genome-scale pooled library of 123,411 single-guide RNAs (sgRNAs), which selectively align to target sequences at the 5′ constitutive exons of 18,080 genes and direct the Cas9 nuclease to cause double-stranded cleavage and loss of function of the respective gene. From the pool of MM cells transduced with the sgRNA library and treated with bortezomib, treatment-resistant cells were processed for deep sequencing, to identify enriched sgRNAs and their corresponding genes. We identified that loss-of-function of 33 candidate genes is associated with bortezomib resistance. We observed a high level of consistency between independent sgRNAs targeting the same gene, as well as a high rate of hit confirmation across different biological replicates. Notably, this set of candidate bortezomib-resistance genes was distinct from the “hits” we identified through a parallel CRISPR screen on the same cell line for resistance to a different targeted therapy (namely the bromodomain inhibitor JQ1), supporting the ability of this approach to identify treatment-specific resistance genes. These candidate bortezomib-resistance genes have documented or presumed roles in the regulation of extrinsic and intrinsic apoptotic cascades, autophagy, Toll-like receptor and NF-kappaB signaling, aggresome function, heat shock protein expression, chromatin remodeling, nutrient sensing, and tumor suppressor gene networks. Importantly, information from several publically available molecular profiling datasets converge to support the putative clinical relevance of these genes. For instance, gene expression data from tumor cells of bortezomib-naive patients with advanced MM revealed several transcriptional signatures of these candidate genes (defined by low transcript levels for any of the genes in the signature) which correlated with shorter time to disease progression after treatment with bortezomib (p<0.01, log-rank test), but not dexamethasone (p>0.426). Congruent with these findings, the highly bortezomib-responsive clinical setting of newly-diagnosed MM is associated with low cumulative frequency of mutations of these bortezomib-resistance genes (e.g. cumulative mutation rate of 3.9%, 95% confidence interval [CI] 1.25-6.55%). Notably, in other malignancies that are typically PSI-resistant, a higher cumulative frequency of such lesions is observed (average of ~28%, range 0-76%, 95% CI 22.46-32.70%; 57 datasets from 20+ neoplasias examined). In summary, this first application of the CRISPR/Cas9-based technology in MM illustrates its power to interrogate gene function on a genome-wide scale. This approach identifies bortezomib-resistance genes that are associated with pathways linked with the regulation of proteasome inhibitor response. Results from molecularly-annotated clinical samples converge to support a possible role for these genes in bortezomib resistance. This experience supports the value of CRISPR/Cas9-based studies to dissect the molecular mechanisms of treatment resistance in MM and other hematologic neoplasias (* equal contribution of M.S. and Y.H.). DisclosuresShalem:Broad Institute: Patent application for CRISPR technology Patents & Royalties. Sanjana:Broad Institute: Patent application for CRISPR technology Patents & Royalties. Zhang:Broad Institute: Patent application for CRISPR technology Patents & Royalties. Mitsiades:Johnson & Johnson: Research Funding; Amgen: Research Funding; Celgene: Consultancy, Honoraria; Millennium Pharmaceuticals: Consultancy, Honoraria.

Small molecule MIRA-1 induces in vitro and in vivo anti-myeloma activity and synergizes with current anti-myeloma agents

Background:Small molecule MIRA-1 induced mutant p53-dependent apoptosis in several types of solid tumours. However, anti-tumour activity of MIRA-1 in haematological malignancies including multiple myeloma (MM) is unknown. In this study, we evaluated the effect of MIRA-1 in MM.Methods:We examined the anti-tumour activity of MIRA-1 alone or in combination with current anti-myeloma agents in a panel of MM cell lines, primary MM samples, and in a mouse xenograft model of MM.Results:MIRA-1 treatment resulted in the inhibition of viability, colony formation, and migration and increase in apoptosis of MM cells irrespective of p53 status accompanied by upregulation of Puma and Bax and downregulation of Mcl-1 and c-Myc. Genetic knockdown of p53 did not abrogate apoptotic response of MIRA-1. MIRA-1 triggered activation of PERK and IRE-α leading to splicing of XBP1 indicating an association of endoplasmic reticulum stress response. Furthermore, combined treatment of MIRA-1 with dexamethasone, doxorubicin or velcade displayed synergistic response in MM cells. Importantly, MIRA-1 alone or in combination with dexamethasone retarded tumour growth and prolonged survival without showing any untoward toxicity in the mice bearing MM tumour.Conclusions:Our data provide the preclinical framework for clinical evaluation of MIRA-1 as a novel therapeutic agent to improve patient outcome in MM.

Abstract A260: BP-4-018, a novel inhibitor of STATs demonstrates anti-myeloma activity in preclinical models: Therapeutic implications of targeting aberrant STAT3 signaling in multiple myeloma.

Abstract As a master regulator of the malignant process, the oncogenic transcription factor STAT3 has become the focus of therapeutic targeting in human tumors that harbor constitutive STAT3 activity, including Multiple Myeloma (MM). In the present studies, we employ genetic and chemical tools to further validate STAT3 as a therapeutic target in MM and investigate a novel small molecule STAT3 inhibitor, BP-4-018, with promising in vitro and in vivo anti-MM activity. Using three molecularly heterogeneous human myeloma cell lines (HMCLs), we show that shRNA-mediated knockdown (KD) of STAT3 induces apoptosis, as evidenced by increased Annexin V+ staining and PARP cleavage. Consistent with this biological readout, we observed a decrease in protein expression of STAT3-regulated survival genes including c-Myc, Mcl-1, Bcl-2 and Bcl-xL in STAT3 KD cells. These findings are therapeutically relevant as treatment of HMCLs with BP-4-018 displays broad anti-myeloma activity with IC50 values in the low μM range (1.91-6.48 μM). This rationally designed STAT3 SH2 domain-binding compound induces apoptotic responses in HMCLs and primary MM cells. Mechanistic studies reveal that BP-4-018 dose-dependently inhibits constitutive STAT3 phosphorylation (pSTAT3). Moreover, in selected HMCLs the dose of BP-4-018 required to inhibit pSTAT3 correlated precisely with their respective sensitivity to BP-4-018. HMCLs expressing a STAT3-driven luciferase reporter construct confirmed inhibition of STAT3 transcriptional activity following BP-4-018 treatment, which was associated with a decrease in protein expression of STAT3 target genes. In cell-based assays, BP-4-018 did not demonstrate selectivity for inhibiting pSTAT3 over that of STATs 1 or 5. Importantly however, BP-4-018 did not inhibit phosphorylation of other SH2-dependent proteins including AKT, ERK, SRC and LYN. In vitro combinatorial studies reveal that BP-4-018 is highly synergistic with bortezomib (Btz; CI=0.37). Preliminary data suggests that this interaction may be related to Btz-induced STAT3 activation and induction of Mcl-1 accumulation as concomitant treatment with BP-4-018 inhibited Btz-induced Mcl-1 accumulation. Supporting these findings, cells expressing a dominant negative STAT3 mutant demonstrate enhanced sensitivity to Btz. Finally, we report findings from in vivo efficacy studies using a novel STAT3-driven luciferase xenograft model of MM. We show that BP-4-018 (15 mg/kg/day, PO) reduces STAT3-driven luciferase expression in vivo, significantly delays tumor growth when administered as a single agent, and in combination with Btz, promotes tumor regression without significant toxicities. Taken together, our results highlight the validity of STAT3 as a therapeutic target in MM and support continued study of BP-4-018 and its analogs for the treatment of MM. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A260. Citation Format: Danielle C. Croucher, Zhi Hua Li, Brent DG Page, Ellen Wei, Patrick T. Gunning, Suzanne Trudel. BP-4-018, a novel inhibitor of STATs demonstrates anti-myeloma activity in preclinical models: Therapeutic implications of targeting aberrant STAT3 signaling in multiple myeloma. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A260.

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.

Small Molecule MIRA-1 Induces p53-Independent Apoptosis in Multiple Myeloma Cells Through Activation of the p38 MAPK Signaling Pathway.

Abstract 2937Mutation of p53, a tumor suppressor protein, is relatively rare (∼10% in newly diagnosed patients) in multiple myeloma (MM). However, p53 mutations/deletions are important risk factors for predicting the resistant to chemotherapy and no treatment is currently available for this subgroup of patients. MIRA-1, a novel class of small molecules with the ability to restore wild type conformation and function to mutant p53, induces apoptosis in different types of solid tumors harboring mutant p53. However, its effect on MM cells is not known. In this study we examined the ability of MIRA-1 to induce cytotoxic and apoptotic response in MM cells and inhibit tumor growth in MM mouse xenograft model. In addition, we explored the molecular mechanisms of MIRA-1-induced apoptosis in MM cells.Treatment of MM cells with MIRA-1 resulted in a time- and dose-dependent decrease in survival and increase in apoptosis of MM cells harboring either wild type (MM.1S, H929) or mutant (U266, 8226, and LP1) p53 suggesting that MIRA-induced apoptosis in MM cells is independent of p53 status. The IC50 of MIRA-1 observed in these cells was ranged between 10 and 15 μM. In addition, MIRA-1 elicited a dose-dependent inhibition of myeloma cell growth in seven primary MM samples with an average IC50of 10 μM. Two of the seven patient samples harbors p53 mutations/deletions. In contrast, MIRA-1 did not have a significant inhibitory effect on the survival of bone marrow or peripheral blood mononuclear cells obtained from three healthy donors at the concentrations (10–20 μM) that induced apoptosis of MM cells, indicating a preferential killing of myeloma cells by this drug. Apoptosis induced by MIRA-1 in MM cells harbouring either wild type or mutant p53 was associated with time- and dose-dependent activation of caspas-8, caspase-3 and PARP with subsequent up-regulation of a pro-apoptotic protein, Noxa and down-regulation of an anti-apoptotic protein, Mcl-1. Interestingly, MIRA-1 did not significantly modulate the level of p53 expression, although immunoprecipitation studies confirmed the restoration of wild type conformation of mutant p53 in LP1 and 8226 cells. Importantly, genetic knockdown of p53 using siRNA against wild type or mutant p53 had only a little effect on apoptosis induction by MIRA-1 in MM.1S or LP1 cells, respectively, confirming that apoptosis induction by MIRA-1 in MM cells is independent of p53. Furthermore, the combination of MIRA-1 with current anti-myeloma agents, dexamethasone or doxorubicin displayed synergistic cytotoxic response in MM.1S or LP1 cells (CI<1; p<0.05).To delineate the molecular mechanisms of apoptosis in MM cells induced by MIRA-1, we performed RT2 profiler PCR array analysis for the differential expression of 84 genes related to mitogen activated protein kinase (MAPK) signaling pathway. A significant number of genes of the MAPK family including MAP3K: MAP3K2 (MEKK2), MAP3K4 (MEKK4), PAK1; MAP2K: MAP2K5 (MEK5); and MAPK: MAPK11 (p38bMAPK) as well as transcription factors such as c-Jun, c-FOS, EGR1, and MKNK1, whose expression is induced by MAPK signaling, were up-regulated by more than 2-fold in MIRA-1-treated 8226 cells. On the other hand, expression of the scaffolding/anchoring genes, MAPK8IP2 (JIP-1) was down-regulated by ∼2-fold. Up-regulations of c-Jun, c-Fos, and EGR1 at their protein levels were further confirmed by Western blot analysis of MM.1S and 8226 cells treated with MIRA-1. Importantly, Western blot analysis revealed that treatment of MIRA-1 resulted in a time- and dose-dependent increase of phosphorylated p38 MAPK level in both MM.1S and 8226 cells. Taken together, our data indicates that activation of the MAPK signaling pathway is, at least in part, associated with MIRA-1-induced apoptosis of MM cells.Finally, we evaluated anti-tumorigenic potential of MIRA-1 in MM xenograft SCID mouse models. 8266 cells were inoculated into SCID mice and the mice received i.p. injections of either 100 μL PBS (control) or 10 mg/kg MIRA-1 once daily for 18 days after tumor formation was evident. Administration of MIRA-1 resulted in significant inhibition of tumor growth (p<0.05) and increase in survival (p=0.007) of the mice with no apparent toxicity.Our study for the first time demonstrates potent in vitro and in vivo anti-myeloma activity of MIRA-1 and thus providing a framework for clinical evaluation of MIRA-1 either alone or in combination with current anti-myeloma agents. Disclosures:Reece:Celgene: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Merck: Consultancy, Honoraria, Research Funding; Bristol Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Otsuka: Honoraria, Research Funding; Millennium Pharmaceuticals: Research Funding.

Identification of Toyocamycin, an Agent Cytotoxic for Multiple Myeloma Cells, As a Potent Inhibitor of ER Stress-Induced XBP1 mRNA Splicing

AbstractAbstract 5034 Introduction:The IRE1α-XBP1 pathway, a key component of the endoplasmic reticulum (ER) stress response, is considered to be a critical regulator for survival of multiple myeloma (MM) cells. Because of the production of abundant immunoglobulins and cytokines, MM cells need to survive under chronic ER stress. In addition, MM cells are located in the bone marrow milieu, which is usually considered hypoxic compared to other organs. Therefore, MM cells need to possess mechanisms to protect against ER stress. Among the unfolded protein responses in MM cells, the IRE1α-XBP1 pathway has been implicated in the proliferation and survival of MM cells to a greater extent than in those of monoclonal gammopathy of undetermined significance or normal plasma cells. It has been reported to be a prognostic factor and could be a target for immunotherapy or chemotherapy. Based on previous reports, it is proposed that an inhibitor of IRE1α-XBP1 activation should be a potent therapeutic agent for MM. Therefore, the availability of small molecule inhibitors targeting this pathway would offer a new therapeutic strategy for MM. Here, we screened small molecule inhibitors of ER stress-induced XBP1 activation, and identified toyocamycin from a culture broth of an Actinomycete strain. Materials & Methods:First, we evaluated the mechanism of toyocamycin-induced inhibition of IREα activity, with focused on its kinase activity, endonuclease activity, and other unfolded protein responses. Next, the activity of toyocamycin was evaluated on MM cell lines and other tumor cells about IRE1α activity and cytotoxicity. Similarly, 9 primary MM cells were tested. Finally, the in vivo efficacy of toyocamycin was evaluated in a human MM xenograft model. Results & Discussion:Toyocamycin was shown to suppress thapsigargin-, tunicamycin- and 2-deoxyglucose-induced XBP1 mRNA splicing in HeLa cells without affecting ATF6 and PERK activation. Furthermore, although toyocamycin was unable to inhibit IRE1 a phosphorylation, it prevented IRE1α-induced XBP1 mRNA cleavage in vitro. Thus, toyocamycin is an inhibitor of IRE1α-induced XBP1 mRNA cleavage. Next, we examined the effect of toyocamycin on MM cells. Most MM cell lines have activated XBP1 protein expression, represented as the overexpression of spliced XBP1 isoform, whereas non-MM cells including other hematological and solid tumor cells have little activation of XBP1. Toyocamycin inhibited constitutive activation of XBP1 in MM cell lines without affecting IRE1α phosphorylation. This inhibition occurred within 6 hours after exposure to 30 nM toyocamycin. We then evaluated the growth inhibitory effect of toyocamycin on 7 MM cell lines with high spliced-XBP1 expression, 3 MM cell lines with low spliced-XBP1 expression, and 4 non-MM cell lines as assessed by MTS assay. All MM cells with high spliced-XBP1 expression showed remarkable decline in cellular viability at 30 nM or higher concentrations of toyocamycin than other MM cells with low spliced-XBP1 expression, and non-MM cell lines showed little reduction in cellular viability. MM cell lines expressing high spliced-XBP1 showed robust dose-dependent apoptosis after exposure to various concentrations of toyocamycin for 24 hours, as assessed by the number of Annexin V-positive cells. Toyocamycin also induces marked apoptosis on two bortezomib (BTZ)-resistant MM cells at nM concentration. It also inhibited constitutive activation of XBP1 expression in primary MM cells derived from patients, showing dose-dependent reduced viability without any cytotoxicity to PBMCs from healthy donors. Toyocamycin also showed synergistic effects with bortezomib, and induced apoptosis of primary MM cells from patients including bortezomib-resistant cases at nano-molar levels in a dose-dependent manner. It also inhibited growth of xenografts in an in vivo model of human MM, and showed enhanced growth inhibition when combined with bortezomib. Taken together, we found that adenosine analog toyocamycin has a potent IRE1α-XBP1 inhibitory effect on MM cells with excessive ER-stress. It triggers dose-dependent apoptosis in MM cells. These results suggest toyocamycin can be a lead compound for developing novel anti-MM therapy, and also provide a preclinical rationale for conducting clinical trials using toyocamycin or other adenosine analog alone or in combination with BTZ for treating MM. Disclosures:No relevant conflicts of interest to declare.

Cells of the Osteoblast Lineage Confer Myeloma Cell Resistance to Established and Investigational Therapeutic Agents

AbstractAbstract 3995 Introduction:Multiple myeloma (MM) cells suppress osteoblast (OB) maturation and function and induce osteoclast-mediated bone resorption. However, immature cells of the osteoblast lineage (e.g. pre-osteoblasts) remain present in the MM bone microenvironment and their impact on MM cell response to treatment has not been fully characterized. We therefore studied human immortalized cells of the osteoblast lineage as surrogate models for in vitro studies on the effects of pre-osteoblasts on MM cell proliferation, survival and drug resistance. Methods:Immortalized hFOB 1.19 (here referred to as hFOB) and HOBIT cells were utilized in our studies, because, in the absence of Dexamethasone or vitamin D, they express markers consistent with pre-osteoblast stage of differentiation (high type I collagen levels; and low alkaline phosphatase, osteocalcin and osteopontin expression). Monolayer cultures of hFOB cells were performed to determine drug concentrations that did not exhibit cytotoxic effects on these pre-osteoblasts. Next, co-cultures of hFOB and HOBIT cells were performed using a panel of tumor cell lines from MM (n=13), breast (n=1) and lung (n=1) cancer. Compartment-specific bioluminescence imaging (CS-BLI) assays were undertaken to quantify the proliferative responses of the tumor cells in the presence of pre-osteoblasts, and if any changes are observed in tumor cell responses to established or novel therapeutic agents. Assays with Transwell insert chambers were conducted to allow for culture of tumor cells proximate to pre-osteoblasts, but preclude their direct cell-to-cell contact, in order to determine whether paracrine factors released by pre-osteoblasts are sufficient to recapitulate the effect(s) of their co-culture with tumor cells. Finally, cytokine or cytokine receptor neutralization studies with monoclonal antibodies or selective kinase inhibitors were performed to investigate the functional contribution of IL-6 or IGF1R-mediated signalling in tumor-pre-osteoblast interactions. Results:Pharmacologically relevant doses of conventional and novel therapies did not exhibit substantial cytotoxic effects on hFOB cells. Within 48hrs of co-culture, pre-osteoblasts stimulated proliferation of several tumor cell lines, including MM.1S, MM.1R, RPMI8226 and Dox40 in co-culture with hFOB; and NCI-H929, OPM2, H23 and Dox40 with HOBIT co-culture. hFOB cells decreased the sensitivity of 8 MM cell lines to at least one established (doxorubicin, dexamethasone, lenalidomide) or novel (vorinostat, pomalidomide) therapy tested. These observations were particularly pronounced in MM.1S and KMS34 cell lines. Subsequent Transwell-based co-culture assays showed that lack of direct cell-to-cell contact significantly decreases or even completely abrogates the resistance observed in respect to doxorubicin, lenalidomide or vorinostat in mono-cultures of tumor cells with pre-osteoblasts. Finally, IL-6 neutralization with monoclonal antibody did not overcome the proliferative effect of hFOB on MM cells. The anti-MM effect of IGF1R kinase inhibitors was also suppressed by co-culture with hFOB cells. These 2 latter results taken together indicate that the protection provided by hFOB cells is independent of IL-6 and IGF-1R-signaling in tumor or pre-osteoblasts. Conclusion:Our results suggest that cells of the osteoblast lineage may play a role as promoters of MM cell survival and resistance to diverse established and investigational therapeutics. Ongoing mechanistic and functional validation studies aim to delineate new therapeutic strategies to overcome pre-osteoblast-mediated drug resistance in MM and, potentially, other neoplasias. Disclosures:Richardson:Johnson & Johnson: Membership on an entity’s Board of Directors or advisory committees; Novartis Pharmaceuticals: Membership on an entity’s Board of Directors or advisory committees; Celgene Corporation: Membership on an entity’s Board of Directors or advisory committees; Millenium Pharmaceuticals: 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. Anderson:Millennium Pharmaceuticals: Consultancy; Celgene: Consultancy; Onyx: Consultancy; Merck: Consultancy; Bristol-Myers Squibb: Consultancy; Acetylon: Membership on an entity’s Board of Directors or advisory committees; Oncopep: Membership on an entity’s Board of Directors or advisory committees. 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 Other.

WS2-5 - How to Overcome Acquired Resistance against Novel Agents in Multiple Myeloma: Strategies based on the Responsible Mechanisms for Bortezomib Resistance

ABSTRACT Multiple myeloma (MM) is a hematological malignancy characterized by the accumulation of tumorigenic plasma cells in bone marrow, and has several unique biological abnormalities compared with normal plasma cells or lymphocytes. Recently, several novel agents targeting MM cells have been clinically developed and some of them have become approved for the patients with MM. Although these novel agents such as bortezomib, thalidomide, and lenalidomide are effective for MM, the duration of response is not long enough and the acquired resistance suddenly occurring during the treatment has become a serious problem in clinical practice. From this view point, understanding the underlying mechanisms responsible for the acquired resistance against each novel agent is crucial for developing new therapies to overcome this resistance. We are currently focusing not only on the mechanisms of action of bortezomib in MM cells but also on those responsible for the resistance against it. In this presentation, we introduce several mechanisms demonstrated in the bortezomib-resistant cells including the specific mutation in the beta5 subunit of 26S proteasome that we have recently discovered. We also demonstrate several strategies used in ex vivo and in clinical trials to overcome bortezomib resistance. A novel compound targeting the endoplasmic reticulum (ER) stress response in MM cells, which has the potential ability to overcome this resistance, is also presented.

Abstract A49: Combination of bendamustine and the HDAC inhibitor entinostat enhances apoptosis and DNA damage response in multiple myeloma cells

Abstract Bendamustine (Cephalon, Inc., West Chester, PA), a hybrid molecule of purine analog and alkylator, induces cell death by activation of DNA damage response and apoptosis, inhibition of mitotic checkpoints, and induction of mitotic catastrophe. Entinostat (also known as MS-275 or SNDX-275, Syndax Pharmaceuticals, Inc., Waltham, MA), a selective class I inhibitor of histone deacetylase (HDAC), induces apoptosis in multiple myeloma (MM) cells. We sought to determine whether the combination of bendamustine with entinostat enhances cell death of MM cells, and the potential mechanisms. Cell growth assay showed that bendamustine inhibited proliferation of MM cells in a dose-dependent manner. The IC50 of bendamustine for RPMI8226, U266 and MM1.S cells was approximately 662.6, 295.5, and 159.6 umol/L. Apoptotic-ELISA indicated that either bendamustine or entinostat was able to induce apoptosis in all three myeloma cells, and their combinations exhibited a much more potent activity to promote the MM cells undergoing apoptosis. Further studies with western blot analyses revealed that bendamustine in combination with entinostat dramatically induced PARP cleavage and activation of caspase-3 in all three MM cell lines, and also reduced the expression levels of cyclin D1, phospho-Stat3 (P-Stat3) and Stat3 in U266 cells. Moreover, studies on DNA damage response showed that phosphohistone H2A.X, a hall marker of DNA double strand break, was significantly induced by the combinations of bendamustine and entinostat as compared to either agent alone in all three MM cell lines. Our data suggest that bendamustine and its combination with entinostat possess anti-MM activity in vitro. Regimens consisting of bendamustine and/or entinostat may represent novel therapeutic strategies against MM.

MARCKS Confers Resistance to Velcade Through Skp2/p27-Mediated Pathway in Multiple Myeloma

Abstract 985▪▪This icon denotes a clinically relevant abstractDespite advances in clinical care, multiple myeloma (MM) remains an almost universally fatal disease. Velcade, a novel proteasome inhibitor represents a promising new clinical strategy for relapsed and refractory MM. However, only 30–40% of MM patients respond to this treatment. In order to gain insight into the mechanism of velcade resistance, we examined the differentially expressed proteins between 8226S (velcade sensitive) and 8226R5 (velcade resistant) myeloma cell lines using iTRAQ mass spectrometry (MS). The MS based multiple-reaction-monitoring technique (MRM) was then used to independently verify the quantitative differences of the protein expression levels between the two cell lines. We identified several cancer specific proteins to be highly differentially expressed between 8226S and 8226R5 by iTRAQ mass spectrometry. Of particular interest is Myristoylated Alanine-Rich C-Kinase Substrate (MARCKS), a PKC substrate that has been implicated in conferring drug resistance to ovarian carcinoma cells. Differential expression of MARCKS between sensitive and resistant cells was verified by MRM and then confirmed by RT-PCR and Western blot analysis. MARCKS over-expression was also found in additional MM cell lines as well as patient samples that were resistant to velcade. Knockdown of MARCKS through siRNA strategies enhanced velcade sensitivity by increasing apoptosis in the velcade treated resistant cell line 8226R5. Moreover, inhibition of PKC, the upstream activator of MARCKS by enzastaurin significantly blocked phosphorylation of MARCKS leading to a caspase dependent apoptosis in the 8226R5 cells. In addition, enzastaurin enhanced velcade proteosome inhibitory effect and rendered the 8226R5 cells more sensitive to velcade treatment. Combination of velcade with enzastaurin showed a synergistic cytotoxic response in MM cells compared to either drug used alone. Furthermore, knockdown of MARCKS or inhibition of MARCKS phosphorylation by enzastaurin led to a decrease in Skp2 expression and an increase in p27 and p21, as well as a reduction of cyclin E and CDK2, resulting in G1/S cell cycle arrest and an increase in apoptosis in MM cells. Taken together our results suggest that phosphorylated MARCKS plays an important role in velcade resistance in MM and as such may be used as an important therapeutic target. In addition, MARCKS may be a biomarker to select patients that would most benefit from patient specific treatment. Disclosures:No relevant conflicts of interest to declare.

Adaptation to Survival in Germinal Center Is the Initial Step in Onset of Indolent Stage of Multiple Myeloma

Aberrant mutations of centrocytes in germinal centers (GC) can generate two completely different diseases: B-cell lymphomas and monoclonal gammopathy of undetermined significance (MGUS). In this article we use computational models to examine the evolutionary dynamics by which initial adaptation to survival in the GC allows naive MGUS cells to proliferate in the bone marrow and initiate the evolutionary process that will lead to aggressive multiple myeloma (MM). Our simulations show that MGUS cells may generate bone marrow tumors ranging from indolent to aggressive, depending on the original adaptation in the GC. All these tumors, however, are limited to approximately 15% of the marrow cellularity due to hypoxia-induced quiescence (this correlates with the cellularity that separates MGUS and MM, ∼10%). Resistance to hypoxia-induced quiescence and cell death was one of the two major bone marrow adaptations that allowed continued tumor growth and establishment of paracrine cytokine loops, known to increase MM cell replication and de novo multidrug resistance. The second major adaptation was an increase in IL-6-independent growth rate, which correlates with the mutations observed in advanced stage patients. Even though there was an increase in the microvessel density in all simulations, the "angiogenic switch" was not due to a MM angiogenic phenotype, but rather the response of MM cells to the regional hypoxia caused by the increased tumor burden. These results indicate that treatments targeting the adaptation to survival and proliferation in hypoxia, in conjunction with currently available therapies, may have synergistic effects, by delaying tumor growth and reducing cytokine paracrine loops mediated by angiogenic factors.

Rapid, Massively Parallel Single-Cell Drug Response Measurements via Live Cell Interferometry

A central question in cancer therapy is how individual cells within a population of tumor cells respond to drugs designed to arrest their growth. However, the absolute growth of cells, their change in physical mass, whether cancerous or physiologic, is difficult to measure directly with traditional techniques. Here, we develop live cell interferometry for rapid, real-time quantification of cell mass in cells exposed to a changing environment. We used tunicamycin induction of the unfolded protein stress response in multiple myeloma cells to generate a mass response that was temporally profiled for hundreds of cells simultaneously. Within 2 h, the treated cells were growth suppressed compared to controls, with a few cells in both populations showing a robust increase (+15%) or little change (<5%) in mass accumulation. Overall, live cell interferometry provides a conceptual advance for assessing cell populations to identify, monitor, and measure single cell responses, such as to therapeutic drugs.

Disruption of IκB Kinase (IKK)-mediated RelA Serine 536 Phosphorylation Sensitizes Human Multiple Myeloma Cells to Histone Deacetylase (HDAC) Inhibitors

Post-translational modifications of RelA play an important role in regulation of NF-κB activation. We previously demonstrated that in malignant hematopoietic cells, histone deacetylase inhibitors (HDACIs) induced RelA hyperacetylation and NF-κB activation, attenuating lethality. We now present evidence that IκB kinase (IKK) β-mediated RelA Ser-536 phosphorylation plays a significant functional role in promoting RelA acetylation, inducing NF-κB activation, and limiting HDACI lethality in human multiple myeloma (MM) cells. Immunoblot profiling revealed that although basal RelA phosphorylation varied in MM cells, Ser-536 phosphorylation correlated with IKK activity. Exposure to the pan-HDACIs vorinostat or LBH-589 induced phosphorylation of IKKα/β (Ser-180/Ser-181) and RelA (Ser-536) in MM cells, including cells expressing an IκBα "super-repressor," accompanied by increased RelA nuclear translocation, acetylation, DNA binding, and transactivation activity. These events were substantially blocked by either pan-IKK or IKKβ-selective inhibitors, resulting in marked apoptosis. Consistent with these events, inhibitory peptides targeting either the NF-κB essential modulator (NEMO) binding domain for IKK complex formation or RelA phosphorylation sites also significantly increased HDACI lethality. Moreover, IKKβ knockdown by shRNA prevented Ser-536 phosphorylation and significantly enhanced HDACI susceptibility. Finally, introduction of a nonphosphorylatable RelA mutant S536A, which failed to undergo acetylation in response to HDACIs, impaired NF-κB activation and increased cell death. These findings indicate that HDACIs induce Ser-536 phosphorylation of the NF-κB subunit RelA through an IKKβ-dependent mechanism, an action that is functionally involved in activation of the cytoprotective NF-κB signaling cascade primarily through facilitation of RelA acetylation rather than nuclear translocation.

Triggering of toll-like receptor-4 in human multiple myeloma cells promotes proliferation and alters cell responses to immune and chemotherapy drug attack

Multiple myeloma (MM) is an incurable B-cell malignancy characterized by accumulation of malignant plasma cells in the bone marrow and by recurrent or persistent infections. Toll-like receptors (TLRs) are essential in the host defense against infections. The aim of this study was to investigate TLR initiated responses in MM cells including proliferation, anti-apoptosis and immune escape. Myeloma cell lines gene transcription, cell cycle and protein expression were detected by RT-PCR, real-time PCR, western blot, ELISA and flow cytometry analysis. 3H-thymidine was used for measuring cell proliferation, and Annexin V-PI flow cytometry for the detection of cell apoptosis. We show that human myeloma cell lines expressed TLRs，and LPS induced the proliferation and partially protected MM.1S and ARP-1 cells from adriamycin-induced apoptosis. LPS appears to induce proliferation via MyD88 and MAPKs signaling. In addition, LPS treatment upregulated myeloma cell secretion of cytokine IL-18 and expression of immunoregulatory factors B7-H1, B7-H2 and CD40 mRNA and helped myeloma cells to escape immune surveillance. Our results show that TLRs are functional on myeloma tumor cells, and the ligands to these TLRs have a functional role in affecting myeloma cell proliferation, survival, and response to chemotherapy and immune attacks.See commentary: The TOLL of inflammation in multiple myeloma

Dynamic Regulation of the Level of Hypoxia In the Bone Marrow Regulates Cell Dissemination In Multiple Myeloma

AbstractAbstract 4035 INTRODUCTION:The interaction of multiple myeloma (MM) cells with the bone marrow (BM) microenvironment plays a crucial role in MM pathogenesis, implying that progression of MM occurs through continuous interaction between the BM and MM cells, which controls the ability of MM cells to egress out of the BM and home into new BM niches. We have previously shown that the CXCR4/SDF1 axis as well as Rho GTPases downstream of the receptor was important for chemotaxis, adhesion, homing and egress of MM cells. However, the driving force for MM cells to leave the BM and metastasize to other BM sites is not well understood. Regions of severe oxygen deprivation (hypoxia) arise in tumors due to rapid cell division and are associated with poor patient prognosis, cell motility, associated angiogenesis and metastasis. In this study, we tested the role of hypoxia in the dissemination of MM cells in vivo, as well as regulation of the retention/egress of MM cells in and out of the BM. METHODS:To test the effect of hypoxia on induction of MM egress, MM1s-GFP+/Luc+ cells were injected into 12 SCID mice, and then mice with different stages of tumor development (based on the tumor size detected by bioluminescence) were treated with the hypoxia marker pimonidazole. Blood was drawn and BM was obtained from the femur. Mononuclear cells were then fixed, permeabilized, and stained with antibodies against pimonidazole, followed with an APC- secondary antibody, PE-mouse-anti-human CXCR4, and anti-cadherin antibody followed by an Alexa-Fluor-594 secondary antibody. MM cells in BM and peripheral blood were identified by gating on cells with high GFP signal. To confirm the effects of severe hypoxia found in vivo compared to physiologic mild hypoxia found in the BM, we tested the effect of mild hypoxic conditions (6% O2) and severe hypoxic conditions (0.5% O2) on MM expression of cadherins and CXCR4, as well on functional adhesion of MM cells to stromal cells and chemotaxis. RESULTS:Twelve mice with different stages of MM tumor progression were used. A bi-phasic correlation between tumor progression and the percent of hypoxic cells in BM was found, showing that severe hypoxic conditions in the BM correlated with tumor burden. The correlation between the tumor burden and the number of circulating cells was not linear; however, a direct linear correlation was observed between the number of circulating MM cells and hypoxia in the BM. Moreover, hypoxia in BM correlated directly with the expression of CXCR4 and negatively correlated with the expression of cadherins in MM cells isolated from the BM. To test the effect of the severe hypoxic conditions induced by tumor progression compared to mild hypoxic conditions found physiologically in the BM, we tested the effect of 0.5% O2 (severe hypoxia) and 6% O2 (mild hypoxia) compared to normoxia (21%) on MM cell adhesion to BMSCs, as well as on chemotaxis in response to SDF1, as well as expression of CXCR4 and cadherins. We found that severe hypoxic conditions decreased MM expression of cadherins and adhesion to BMSCs, as well as increased expression of CXCR4 and chemotaxis to SDF1 compared to cells in normoxia. In contrast, mild hypoxic conditions did not alter the expression of CXCR4 and cadherins, adhesion of MM cells to BMSCs, or chemotaxis of MM to SDF1 compared to normoxic cells. CONCLUSION:Hypoxia in the BM directly correlates with the number of circulating MM cells, and with changes in expression of cadherins and CXCR4 in vivo. Severe hypoxic conditions, but not mild hypoxic conditions, induce hypoxic responses in MM cells. Based on these findings, further studies to manipulate hypoxia in order to regulate tumor dissemination as a therapeutic strategy in MM are warranted. Disclosures:Anderson:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Ghobrial:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees.

Bortezomib Induced BRCAness Sensitizes Multiple Myeloma Cells to PARP Inhibitors

AbstractAbstract 789 Background:Poly-ADP-ribose-polymerase (PARP) inhibitors are cytotoxic to tumor cells with impaired DNA damage repair machinery (DRR), in particular those with a deficient homology directed repair (HR) of DNA double stranded breaks (DSB). Multiple Myeloma (MM) cells are characterized by a highly unstable genome and while the exact mechanisms for this karyotypic instability is largely unknown, their DDR machinery is thought to be highly stressed. The ubiquitin-proteasome system (UPS) is involved in the regulation of several cellular functions including DDR and in particular HR. In addition proteasome inhibitors are reported to induce an unfolded protein response (UPR) in MM cells resulting in their apoptotic death. We have postulated that inhibition of the 26S proteasome also alters the DNA-DSB repair machinery leading to a BRCAness state in MM cells, sensitizing them to PARP inhibitors. Methods and results:In order to biochemically inhibit PARP in MM cells, we used a novel selective inhibitor of PARP1 and PARP2, 2-(R)-2-methylpyrrolidin-2-yl]-1H-benzimidazole-4-carboxamide or ABT-888. We first demonstrated inhibition of PARP activity as measured by a reduction in poly-ADP-ribose (PAR) polymer levels (western blotting) in human MM cell lines (MM1S, U266, H929, RPMI8226, KMS-11, OPM2, INA-6) treated with ABT-888 (5 μM). PARP inhibition and the reduction of PAR levels resulted in DNA damage as evidenced by ATM phosphorylation and induced DNA-DSBs with increased γH2AX (phospho-Ser139-H2AX) levels within 6–12 hours of MM cells treatment with ABT-888. Increased γH2AX foci formation was also detected by immunofluorescent staining within 6–12 hours of ABT-888 treatment and nearly fully resolved by 24 hours, consistent with repair of resultant DNA-DSBs. As expected treatment with ABT-888 alone had no effect on the viability of MM cells consistent with their ability to repair DNA-DSBs resulting from PARP inhibition.We then examined the effect of bortezomib on HR-mediated repair of DNA-DSBs, in particular on the BRCA/FA pathway. A significant reduction of MM cells' FANCD2, BRCA1, BRCA2 and RAD51 mRNA levels (qRT-PCR) was observed within 6–12 hours of bortezomib treatment (10 nM). Similar results were observed at the protein level indicating that bortezomib impedes homology-directed DNA-DSBs repair and results in an operational BRCAness state in MM cells. Therefore, we next tested whether this bortezomib-induced BRCAness was sufficient to sensitize MM cells to PARP inhibition with ABT-888. Consistent with our hypothesis, we observed that co-treatment of MM cell lines with bortezomib and ABT-888 lead to persistent and increased γH2AX foci at 24 hours compared to treatment with ABT-888 alone. Co-treatment also significantly potentiated cell death (Annexin V/PI staining) compared to treatment with bortezomib alone. Similar results were observed in CD138+ primary MM cells (n=8) with strong synergistic effect (CI < 1) between bortezomib and ABT-888. Importantly, no impaired viability (Annexin/PI staining) or function (colony forming unit assay) was noted for CD138− cells or CD34+ peripheral blood stem cells after bortezomib and ABT-888 co-treatment. Mechanistic studies have also shown that apoptotic events (caspase 3, caspase 8 and PARP cleavage) are markedly enhanced by this combination.Based on our in vitro data, we evaluated in vivo the activity of ABT-888 in combination with bortezomib in a Scid murine xenograft model of human MM. Significant inhibition of tumour growth (p<0.005) was noted in mice treated with the combination of bortezomib and ABT-888 compared to bortezomib alone or control-treated mice. This tumour growth inhibition also resulted in a significant increase in survival (p<0.05) of the animals. No toxicity (e.g. weight loss, ruffled coats, paralysis, etc.) was observed in mice treated with the combination. Induction of DNA-DSBs was also confirmed in vivo as shown by an increase in 53BP1 and γH2AX foci formation in tumors of mice treated with the combination compared to bortezomib alone. Conclusion:Our studies indicate that bortezomib induces a BRCAness state in MM cells by impairing HR-mediated repair of DNA-DSBs and results in a contextual synthetic lethality when combined with the PARP inhibitor ABT-888. These data provide the scientific basis for the future clinical testing of PARP inhibitors in combination with proteasome inhibitors for the treatment of MM. Disclosures:No relevant conflicts of interest to declare.