Plasma Cells Of Multiple Myeloma Patients Research Articles

Ex Vivo Evaluation of VLA-4 Expression in Primary Human Multiple Myeloma Bone Marrow Samples and In Vivo Mobilization of Murine Multiple Myeloma Cells with Small Molecule VLA-4 Inhibitors

Background: The interaction of multiple myeloma (MM) cells with their microenvironment in the bone marrow (BM) affects disease progression and provides resistance to therapeutic agents. Very-late-antigen 4 (VLA-4, α4β1 integrin, CD49d/CD29) is a noncovalent, heterodimeric transmembrane receptor that is strongly implicated in the pathogenesis of MM via altering cell trafficking, proliferation and drug resistance. LLP2A is a high-affinity peptidomimetic ligand for activated VLA-4. We recently reported (Soodgupta et al. J. Nucl. Med 2016) the sensitive and specific molecular imaging of activated VLA-4 in mouse MM tumors using 64Cu-LLP2A and LLP2A-Cy5. Here we extended these studies by further characterizing VLA-4 expression in primary human MM samples and malignant plasma cells in mouse models of MM.Methods: We evaluated VLA-4 expression in 5 human MM cell lines (U266, OPM2, H929, RPMI-8226 and MM1.S), one mouse MM cell line (5TGM1) and seventeen primary human MM bone marrow samples by flow cytometry using LLP2A-Cy5, soluble VCAM-1/Fc recombinant protein and CD49d (α4) and CD29 (β1) antibodies. The relative mean fluorescence intensity (RMFI) of LLP2A-Cy5 binding was calculated by dividing the MFI of LLP2A-Cy5 binding in the absence of BIO5192 (small molecule VLA-4 inhibitor) by the MFI of LLP2A-Cy5 binding in the presence of excess BIO5192. The 5TGM1/KaLwRij immunocompetent mouse model of MM was used for in vivo study.Results: The expression of activated VLA-4 on MM cell lines as measured by LLP2A-Cy5+ mean fluorescent intensity (MFI) varied 10-fold as follows (LLP2A-Cy5 MFI in parentheses): 5TGM1 (23.7) > U266 (16.1) > OPM2 (4.6) > H929 (3.4) > RPMI-8226 (3.2) > MM1.S (2.1). We observed similar variable expression of LLP2A-Cy5 binding to primary human CD138+CD38+ MM plasma cells (PCs), with 76.47% (13/17) of MM patients exhibiting greater than 20% LLP2A-Cy5+ PCs. expressing VLA-4 on CD138+CD38+ cells. Overall, the mean percentage of positive cells and LLP2A-Cy5 relative MFI (RMFI) on malignant CD138+ PCs from these 13 patients were 78.2% (43.8-98.3%) and 4.3 (1.7-10.8), respectively. Other hematopoietic cells within the BM samples expressed less VLA-4 in descending order as follows; monocytes (58.2%, RMFI 3.0), T-lymphocytes (34.4%, RMFI 2.1) and B-lymphocytes (21.6%, RMFI 1.6). These levels of VLA-4 expression on normal cell subsets within MM patients were comparable to normal blood donors. In general, there was good correlation between LLP2A-Cy5 binding and expression of CD49d and CD29 on CD138+ PCs in MM patients. To our surprise, the four MM patients with <20% LLP2A-Cy5 binding demonstrated high expression of CD49d (92.1%) but very low percentages of CD29 positive cells (17.3%). Using BIO5192 (VLA-4 inhibitor), we found that the LLP2A-Cy5 reagent allowed more accurate detection of activated VLA-4 than the soluble VCAM-1 binding assay as determined by the magnitude of inhibition of binding in the presence of inhibitor. We next evaluated targeting VLA-4 molecule in murine MM model. Preliminary mouse mobilization studies demonstrated that VLA-4 inhibitors effectively and rapidly mobilized murine 5TGM1 MM cells from the bone marrow to the blood (2.49-fold increase in circulating GFP+CD138+ cells) within 1 hour of injection.Summary:This study is the first demonstration that activated VLA4 can be detected on primary human MM cells using LLP2A. These data support the continued development of LLP2A as a molecular diagnostic imaging reagent for MM and as a potential therapeutic target of VLA-4 in MM. Ongoing studies are testing whether small molecule VLA-4 inhibitors can sensitize MM cells to cytotoxic therapy in vivo. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Cannabinoid derivatives exert a potent anti-myeloma activity both in vitro and in vivo.

Although hematopoietic and immune system show high levels of the cannabinoid receptor CB2, the potential effect of cannabinoids on hematologic malignancies has been poorly determined. Here we have investigated their anti-tumor effect in multiple myeloma (MM). We demonstrate that cannabinoids induce a selective apoptosis in MM cell lines and in primary plasma cells of MM patients, while sparing normal cells from healthy donors, including hematopoietic stem cells. This effect was mediated by caspase activation, mainly caspase-2, and was partially prevented by a pan-caspase inhibitor. Their pro-apoptotic effect was correlated with an increased expression of Bax and Bak, a decrease of Bcl-xL and Mcl-1, a biphasic response of Akt/PKB and an increase in the levels of ceramide in MM cells. Inhibition of ceramide synthesis partially prevented apoptosis, indicating that these sphingolipids play a key role in the pro-apoptotic effect of cannabinoids in MM cells. Remarkably, blockage of the CB2 receptor also inhibited cannabinoid-induced apoptosis. Cannabinoid derivative WIN-55 enhanced the anti-myeloma activity of dexamethasone and melphalan overcoming resistance to melphalan in vitro. Finally, administration of cannabinoid WIN-55 to plasmacytoma-bearing mice significantly suppressed tumor growth in vivo. Together, our data suggest that cannabinoids may be considered as potential therapeutic agents in the treatment of MM.

Abstract 1077: Targeting oncogenic miR-17-92 primary transcripts by LNA gapmeRs in multiple myeloma: Molecular findings and therapeutic potential

Abstract There is emerging evidence that miR-17-92 plays a crucial role in c-Myc driven tumorigenesis of multiple myeloma (MM). We attempted to antagonize its full-oncogenic activity by targeting primary transcripts (pri-miR-17-92) with RNase H-triggering antisense oligonucleotides (LNA gapmeRs). Specifically, 7 different molecules were generated and screened for their ability to inhibit pri-miR-17-92 expression. The most effective molecule, henceforth named miR17-92-i-PT, was selected for furher investigation. As assessed by qRT-PCR, transfection of MM cells with miR17-92-i-PT resulted in downregulation of both pri-miR-17-92 and all 6 mature miRNA transcripts. Importantly, miR17-92-i-PT inhibited MM cell proliferation more effectively than inhibitors targeting each cluster's miRNA. Lack of relevant off-target effects was demonstrated by specifically-designed negative (LNA mixmeRs) and positive (not-phosphorothioated LNA gapmeRs) control oligos, and a dominant-negative genome-edited cell line by CRISPR/CAS9 technology is currently under development. Importantly, treatment of MM cells with naked miR17-92-i-PT resulted in miR-17-92 downregulation. Low micromolar concentrations of miR17-92-i-PT significantly affected survival of MM patient plasma cells (n = 14) and MM cell lines (n = 15), while the viability of CD138+ cells from MGUS patients (n = 3) or PBMCs from healthy donors (n = 3) was not impaired. Further, a synergistic in vitro activity with Bortezomib,Carfilzomib or Melphalan was demonstrated. Molecular perturbations in MM cells exposed to miR17-92-i-PT were firstly investigated by gene expression profiling (HTA 2.0, Affimetrix). Ingenuity pathway analysis (IPA) of differentially expressed genes highlighted alteration of relevant molecular functions, including “cell cycle”, “DNA replication, recombination, and repair”, and “cell death and survival”, which were validated by functional assays. Moreover, impairment of BRD4 activity was indicated by upstream regulator analysis (Zscore&amp;lt;2). Indeed, Hexim1 -the endogenous antagonist of BRD4- was upregulated upon treatment with miR17-92-i-PT and is currently being validated as a direct target of miR-18a, miR-19a and miR-19b cluster members. Consistent with a BET bromodomain inhibitor-like activity, miR17-92-i-PT downregulated c-Myc, thus indicating a novel feedback loop between miR-17-92 and c-Myc. Finally, we found a significant in vivo anti-tumor activity of systemically delivered naked miR17-92-i-PT against human MM xenografts in SCID/NOD mice. Different animal models were utilized, including SCID-hu and mouse orthotopic models, besides classic subcutaneous xenografts. Lack of systemic toxicity was demonstrated both in mice (balb/c) and in monkeys (cynomolgus monkeys). Overall, our results provide the rational framework for development of miR17-92-i-PT-based therapies in MM. Citation Format: Eugenio Morelli, Lavinia Biamonte, Cinzia Federico, Maria Teresa Di Martino, Nicola Amodio, Maria Eugenia Gallo Cantafio, Niels M. Frandsen, Maria Rita Pitari, Daniele Caracciolo, Annamaria Gullà, Marco Rossi, Pierosandro Tagliaferri, Pierfrancesco Tassone. Targeting oncogenic miR-17-92 primary transcripts by LNA gapmeRs in multiple myeloma: Molecular findings and therapeutic potential. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1077.

Long Non-Coding RNA MALAT1 Is Associated with the Progression of Multiple Myeloma and Induced By Cellular Stress

Background: Recent transcriptome-wide analyses have revealed an overwhelming amount of transcribed, but not translated, non-coding RNAs capable of influencing diverse cellular processes such as proliferation, apoptosis, and motility. Long non-coding RNA (lncRNAs), which are commonly defined as transcripts >200 nt in length, have emerged as a class of key regulatory RNA. LncRNAs are deregulated in diverse human cancers and associated with disease progression; however, little is known about its role in multiple myeloma (MM). To elucidate the role of lncRNAs in MM, we studied the expression patterns of several well-known lncRNAs in the plasma cells of MM, MGUS and plasmacytoma patients and the function in MM cell lines in vitro. Moreover, to reveal the distinct lncRNA signature comprehensively, we performed next-generation sequencing-based RNA sequencing.Methods: CD138+ plasma cells from bone marrow (BM) mononuclear cells were obtained from 110 MM patients, 48 MGUS patients, 19 control subjects and 1 patient with extramedullary plasmacytoma of the liver and analyzed after obtaining informed consent from all the patients. The expression levels of lncRNAs MALAT1, ANRIL, HOTAIR, HOTTIP, and XIST were determined by a RQ-PCR analysis. RNase H-activating LNA™ GapmeR antisense oligonucleotides were used to knockdown lncRNA in vitro in MM cell lines. The cell lines were then treated with bortezomib, MG132, doxorubicin and hypoxic conditions to evaluate the effects of cytotoxic stress on the lncRNA expression. This study was approved by the IRB of Gunma University Hospital in accordance with the Declaration of Helsinki.Results: A significant higher level of MALAT1 expression was observed in BM plasma cells of MM patients (4.49) compared to MGUS patients (1.51) and control subjects (0.55) (p<0.001). Strikingly, MALAT1 expression in extramedullary plasmacytoma of the liver was 140-fold higher compared with BM plasma cells obtained at the same time of sampling (433.7 vs 3.21). MALAT1 expression was higher in MM patients with t(4;14) and del 17p (10.05 vs 3.90, p=0.049; 5.22 vs 2.76, p=0.03, respectively), but no difference was observed between stages according to the International Staging System (ISS) (p=0.87). Neither the overall survival nor the progression-free survival differed between patients with high and low MALAT1 expression. ANRIL expression levels were diverse according to the patients (range, 0 to 294.3), however, the median expression was significantly higher in MM patients (p<0.001). HOTAIR and HOTTIP expression levels were not detected in most samples, and XIST expression was found only in female patient samples as expected. Interestingly, the MM cell lines KMS12PE, OPM2, KMS11 treated with bortezomib showed elevated MALAT1 expression by 4.3 -21.8 fold and ANRIL by 2.2-4.7 fold; however, this increase was not observed in bortezomib-resistant cell lines. Another proteasome inhibitor, MG132, and a low dose of the cytotoxic drug doxorubicin also elevated both lncRNAs in the cell lines. Hypoxic stress, which has been shown to induce MALAT1 in vascular cells, did not increase either lncRNA. MALAT1 knockdown by GapmeR did not affect cell proliferation. It has been shown that MALAT1 enhances cell motility of lung adenocarcinoma cells by influencing cell motility associated genes; however, the expression of previously reported affected genes, such as HMMR, CTHRC1 and ROD1, was not altered in the MALAT1 knockdown MM cell lines. Although t(4;14) was associated with a high MALAT1 expression in the patient samples, MMSET knockdown by siRNA did not change the MALAT1 expression in the cell lines, thus MMSET was not a regulator of MALAT1. RNA sequencing of MM and MGUS samples revealed a distinct lncRNA expression signature as well as protein coding genes.Conclusion: Significant upregulation of lncRNAs MALAT1 and ANRIL might be associated with MM progression. Given that MALAT1 is associated with lung cancer metastasis, MALAT1 might be strongly associated with extramedullary plasmacytoma formation due to its high expression in liver plasmacytoma. Genotoxic and ER stress induced by therapeutic drugs might upregulate MALAT1 expression, leading to extramedullary extension, which is a recent problem in MM treatment. Determining the distinct lncRNA signature of MM is a current important issue to clarify the molecular mechanisms underlying MM progression for the development of novel therapies. DisclosuresNo relevant conflicts of interest to declare.

CD69 Expression Predicts Favorable Outcome in Multiple Myeloma Patients Treated with VTD

▪Multiple myeloma is an incurable disease characterized by proliferation of clonal malignant plasma cells (PC). Molecular characterization of malignant plasma cells is increasingly important for diagnostic and therapeutic stratification but the clinical and prognostic value of immunophenotyping in MM remains questionable. We have analyzed the prognostic impact of a relatively new marker as CD69. CD69 is a type II membrane protein. T cells express CD69 rapidly upon stimulation of the T-cell receptor (TCR), which is why CD69 has been mostly regarded as an activation marker. The precise role of CD69 in immunity has not been determined because its ligand is unknown, but an emerging role of CD69 in Multiple Myeloma (MM) has been postulated. Previous laboratoristic data, using tumor lines derived from murine model with genotypic and immunophenotypic features of resistance to bortezomib, showed that as the neoplastic plasma cells (PC) develop bortezomib resistance, they have a germinal center B cell like immunophenotype, including decreased to absent expression of CD69. Interestingly the activation antigen CD69 associates with and inhibits the function of Sphingosine 1-phosphate (S1P). S1P is a bioactive lysophospholipid which is known to induce diverse cellular responses through at least five G-protein-coupled receptors on various cell types. Other data showed that MM cells express the S1P receptors, S1P1, S1P2 and S1P3. Furthermore, S1P protects MM cells against dexametason-induced apoptosis. Importantly, S1P upregulates Mcl-1 expression in a time and concentration-dependent manner in human MM cell lines. In a previous abstract, we described for the first time in a clinical report, the CD69 expression on pathological PCs of MM patients. Our preliminary data also suggested an intriguing role of CD69 in patients treated with chemotherapy in different stages of disease. In this study, we report a larger setting of 97 patients where we confirmed the expression of CD 69 in 48 of them (49%) (see table I). Immunophenotyping was carried out by a 6-color method, using a FacsCanto II cytometer and the FacsDiva software. PCs were identified as CD138+/CD38+ events after an initial gate which included events with low SSC in the CD45/SSC cytogram. The MoAb panel also included CD19, CD20, CD117, CD56, cytoplasmic light chains K and Lambda. PerCP-Cy5.5-conjugated CD69 was evaluated on phenotypically abnormal plasma cells (i.e. CD19-, CD45- or dim), which were resulted to be clonally restricted. Results were considered positive when the percentage of positive cells was > 20%.After an induction regimen of treatment with four cycles of VDT (bortezomib, dexametasone, thalidomide), 69 patients were evaluable. 40/69 (65%) of patients obtained at least of a very good partial response or better (Responding pts). In this subgroup of patients 30/45 (66.6%) showed the expression of CD 69. On the contrary only in a little part of partial or less responding patients (NR pts) 9/24 (37.5%) CD69 was detected (see table II) (p=0.02 using a chi squared test and p=0.019 using a Fisher's exact test). Data on cytogenetic abnormalities, including del(13q), t(4;14) and del(17p), detected by fluorescence in situ hybridization, were available in >90% of patients. Clinical data were available in all patients and CD69 maintained its association with different response, independently of other prognostic variables.In conclusion CD69 is often expressed in PCM cases, and the expression of this marker is useful to reveal poor prognostic categories and delineate a risk stratification. This molecule could represent an emerging clinical factor to identify different outcomes in patients affected by MM and treated with the modern drugs.Table IPts CharacteristicsTotalCD69+9748/97SexMale51(52%)Female46(48%)Clinical statusMM non evaluableMM after VTD28/9769/97in VGPR/CR 45/69in PR/SD/PD 24/69Table IIPts treated with VTDResponding ptsNRpts4524CD69+ 39/6930/459/24CD69- 30/6915/4515/24 DisclosuresNo relevant conflicts of interest to declare.

Comparative Proteomic Profiling of Refractory/Relapsed Multiple Myeloma Patient Plasma Cells Reveals Biomarkers and Pathways Involved in Bortezomib-Based-Therapy Resistance

Introduction: Despite the new drugs introduced to the treatment of multiple myeloma (MM) the disease remains incurable due to increasing resistance to therapy. Understanding biology of disease and identification of biomarkers of resistance is a key research challenge and may enable the individualization of the treatment.Objective: The goal of this study was comparative proteomic profiling of pre-treatment plasma cells (PCs) from patients with refractory/relapsed MM (RRMM) subsequently treated with PAD (Bortezomib, Adriamycin, Dexamethasone) to identify biomarkers and signaling pathways differing PCs from patients who achieved CR and those with lower response. We wanted to expand recently published (Dytfeld et al, 2015) catalog of proteins and pathways involved in resistance to bortezomib-based therapy in newly-diagnosed multiple myeloma (NDMM).Material and Methods: Comparative proteome analysis was performed on purified by negative selection (EasySep) pre-treatment PCs from 24 patients with RRMM qualified for PAD. PCs acquired from bone marrow specimens afterobtaining informed consent from patients were lysed, and protein was isolated. Trypsin digested peptides were labeled with iTRAQ (4-plex) reagents and subsequently analyzed using Q-Exactive hybrid quadrupole-Orbitrap mass spectrometer coupled to the chromatograph Dionex 3000 Ultimate nanoLC (Thermo Scientific). Proteins which accumulation in the analyzed subgroups differed by at least 50% were considered to differentiate. Potential biomarkers were validated using ELISA in the patients' sera. The dysregulated proteins were subjected to two bioinformatic tools: PANTHER and DAVID, for identifying enriched signaling pathways or networks. Only these suggested by both tools were considered significant.Results and discussion: Out of 3821 proteins identified (FDR <1%), 325 showed significant differences in accumulation between patients who after treatment with PAD achieved CR and patients with worse response. TXNDC5 (peroxiredoxin activity) and PSME1 (proteasome component) found elevated in PCs from refractory patients were confirmed by ELISA in patients' sera (TXNDC5: 110.1pg/mL vs. 40.47, p =0.03; PSME1: 222.02 pg/ml vs. 141.99 pg/mL, p =0.03). Based on the log of p-value from Fisher's test and number of identified differential proteins, the most affected pathways constitute: proteasome pathway (55 proteins; p =3.5x10-19), oxidative phosphorylation (43 proteins; p =4.5x10-11), ribosome proteins (68 proteins; p =1.1x10-47), spliceosome proteins (46 proteins; p =1,5x10-13), translation initiation factor activity (24 proteins; p =1.16x10-11) and peroxiredoxin activity (7 proteins; p =3.7x10-6). All of them were up-regulated in resistant patients. The most affected pathways were previously identified in NDMM treated subsequently with VDD and RVD. Elevated proteasome activity in resistant patients may be a result of affected expression of proteins related to oxidative phosphorylation and enhanced production of reactive oxygen species connected with up-regulation of peroxisome activity proteins. As a consequence bortezomib may not inhibit proteasome sufficiently, thus prevents apoptosis. An alteration in the stoichiometry of components of the proteasome, ribosome, and spliceosome is the effect of genomic instability and thereby most cancers exhibit loss of genes responsible for tumor suppressor (Nijhawan et al, 2012).Conclusion: We indicated biomarkers of resistance which may be a potential therapeutic targets and basis for individualization of MM therapy. We also showed pathways involved in resistance to bortezomib-based therapy in RRMM that confirms our previous finding in NDMM. Further studies are necessary to explore these pathways and define their role in multiple myeloma treatment.Study was supported by research grant funded by Janssen Cilag. DisclosuresDytfeld:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Jakubowiak:Sanofi-Aventis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Millennium: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; SkylineDx: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: institutional funding for support of clinical trial conduct, Speakers Bureau; Sanofi-Aventis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Karyopharm: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Onyx: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; SkylineDx: Membership on an entity's Board of Directors or advisory committees; Onyx: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Karyopharm: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Millennium: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Selective G-Protein Estrogen Receptor (GPER) Activation Triggers Anti-Multiple Myeloma Activity and Synergizes with MiR-29b-Inducing Drugs

Multiple myeloma (MM) is a plasma cell malignancy which remains incurable despite novel therapeutic approaches targeting both myeloma cells and their bone marrow milieu (BMM). MM cells express estrogen receptors (ER) belonging to both α and β isotypes and selective ER modulators or pure anti-estrogens have demonstrated therapeutic activity against this malignancy. GPER, formerly known as GPR30, is an orphan membrane-associated ER previously described to mediate non-genomic effects of estrogens and whose involvement in the pathophysiology of solid tumors is currently emerging. Here, we studied the expression pattern of GPER and the biological effects triggered by GPER activation using the synthetic compound G-1 ((±)-1-[(3aR*,4S*,9bS*)-4-(6-Bromo-1,3-benzodioxol-5-yl)-3a,4,5,9b-tetrahydro-3H cyclopenta[c]quinolin-8-yl] ethanone), a selective GPER agonist (Tocris). We detected GPER expression in 9 out of 9 MM cell lines either at mRNA and protein level, as assessed by qRT-PCR and western blotting, respectively. By analysis of our microarray dataset based on plasma cells from 4 normal donors, 11 MGUS, 133 MM and 9 plasma cell leukemias (PCLs), we observed that GPER mRNA levels progressively declined during MM progression, since lower levels were found in PCL and MM samples as compared to healthy controls or MGUS. Interestingly, adhesion of MM cells to bone marrow stromal cells (BMSCs) reduced GPER mRNA levels, supporting a potential role of the BMM in regulating GPER expression. To address the relevance of GPER in modulating MM cell proliferation and/or death mechanisms, first we tested the GPER agonist G-1 in vitro. We found that G-1 inhibited, in a dose-dependent manner, proliferation of IL-6 dependent (INA-6) and independent (MM1R, MM1S, U266, RPMI-8226, NCI-H929, OPM2) MM cell lines, with an IC50 ranging from 2 to 5 microM, while did not affect the survival of peripheral blood mononuclear cells from healthy donors. G-1 treatment caused cell cycle arrest by increasing cells in G0 phase; moreover, it induced a significant and dose-dependent apoptotic cell death in all MM cell lines tested, as assessed by Annexin V/7AAD staining and western blot analysis of active caspases 3, 7 and 9. G-1 promoted the expression of autophagic markers like Beclin-1 and LC3A/B, the cytosolic punctate pattern of LC3B and down-regulated p62/SQSTM-1 expression, indicating functional involvement of GPER in autophagy. Moreover, GPER transduced rapid non-genomic signaling through MAPKs, since G-1-mediated GPER activation triggered phosphorylation of ERK1/2 already after 15’ treatment in MM1S and U266 cells. Importantly, i.p. injection of G-1 (2mg/kg) in SCID mice significantly reduced the growth of subcutaneous MM1S xenografts, as compared to vehicle-treated animals.We next evaluated whether G-1-induced effects could be associated to modulation of miRNA levels in MM cells. Indeed, we found that G-1 up-regulated the tumor suppressor miR-29b; this effect was likely a consequence of the down-regulation of the miR-29b transcriptional inhibitor Sp1, whose mRNA and protein levels were reduced after G-1 treatment. Consistently, an inverse correlation between GPER and Sp1 mRNA levels in MM patient plasma cells could be gathered from our microarray dataset. In addition, miR-29b canonical targets, like CDK6 and MCL-1, were down-regulated at protein level in G-1-treated MM cell lines.Finally, we demonstrated that G-1 synergizes with established miR-29b-inducing compounds, like bortezomib and vorinostat, in the inhibition of MM cell survival.Taken together, our results indicate that the GPER agonist G-1 is a novel powerful anti-tumor compound enriching the repertoire of investigative anti-MM agents, and further strengthen the role of miR-29b as effector of anti-MM drugs. DisclosuresNo relevant conflicts of interest to declare.

CXCR3 Binding Chemokines MIG, IP-10 and ITAC Are Predictors of Overall Survival in Newly Diagnosed Multiple Myeloma

Background. The chemokine receptor CXCR3 and its binding molecules MIG, IP-10 and ITAC have been associated with tumor progression, immune escape and angiogenesis in several human malignancies. In multiple myeloma (MM), CXCR3 binding molecules were shown to induce migration of MM cells without effecting proliferation. More recent results suggest a tumor suppressive activity of IP-10. Presently, information about the precise role of CXCR3 binding chemokines in MM is limited and evidence for their clinical significance is lacking. Therefore we aimed to evaluate the prognostic relevance of CXCR3 binding chemokines in patients with MM.Patients and Methods. Serum levels of MIG, IP-10 and ITAC were analyzed by FACS-CBA array in 65 newly diagnosed MM patients. Expression of CXCR3 and its binding molecules was also analyzed by quantitative PCR in 7 human MM cell lines (HMCLs) and in a publically available gene expression dataset (GSE2658). Further analysis of MIG serum levels was performed by ELISA in an extended cohort of MM (n=105) and MGUS patients (n=17), and in healthy volunteers (n=37).Results. Determination of serum levels by FACS-CBA revealed significant expression of MIG (range: 33.4 – 157 960 pg/ml) and IP-10 (12 - 4418.8 pg/ml), while ITAC (0 - 351.5 pg/ml) was only detectable in a subset (20 of 65) of patients. Interestingly, serum levels of all three molecules showed a positive correlation with each other (MIG vs. IP-10, R=0.38, P=0.002; MIG vs. ITAC, R=0.62, P<0.0001; ITAC vs. IP-10, R=0.41, P=0.0007). We also observed a significant correlation with beta 2 microglobulin (B2M) (MIG: R=0.45, P<0.0001; IP-10: R=0.36, P=0.003; ITAC: R=0.3, P=0.016) and a trend regarding ISS stage (MIG: R=0.23, P=0.06; IP-10: R=0.24, P=0.05; ITAC: R=0.11, P=0.39). Importantly, a significant association with overall survival (OS) was observed as well. Survival was significantly worse in patients with high compared to low MIG (median OS 25.3 months vs. not reached, P=0.003) and IP-10 (19.97 months vs. not reached, P=0.0006) as well as in patients with detectable compared to absent ITAC serum levels (19.97 vs. 65.8 months, P=0.019). In multivariate analysis, MIG (P=0.03) and ITAC (P=0.013) along LDH and calcium were revealed as independent predictors of survival.Expression of CXCR3 binding chemokines was rarely detected in HMCLs (1 of 7 expressed MIG, 3 of 7 IP-10 and 2 of 7 ITAC, respectively). In line with this, in-silico analysis of previously published primary MM cell samples (n=414) (GSE2658), showed a present detection call of MIG, IP-10 and ITAC in 51 (12.3%), 11 (2.7%) and 0 (0%) patients, respectively. In contrast, all three cytokines were detectable in 100% of bone marrow plasma cells of healthy donors, MGUS and smoldering MM patients in this dataset. Hence, CXCR3 binding chemokines are silenced in myeloma cells indicating that the increased serum levels of CXCR3 binding chemokines are derived from other cell types.As MIG serum concentration was identified as one of the most important predictors for OS, we studied the prognostic relevance of this molecule in an extended cohort (n=105) of MM patients by ELISA. Median MIG levels (161.3 pg/ml, range: 9.4-1966) were significantly elevated in newly diagnosed MM patients compared to MGUS (92.7 pg/ml, range: 6.29-1303.1) and healthy volunteers (106.2, range: 51–390.6 pg/ml). MIG levels were significantly correlated with B2M, ISS stage, calcium, albumin, LDH, hemoglobin and with age (R=0.466, P<0.001). Importantly, high MIG levels predicted adverse survival (17.0 months vs. not reached, P<0.001), which was upheld when age-adjusted cut-off levels were used. In accordance with our findings, in-silico analysis of MIG expression in purified plasma cells of MM patients (n=559) treated within the total therapy 2 and 3 protocol (GSE2658) revealed shorter OS in patients with a present compared to those with an absent detection call for MIG (P=0.004).Conclusion. Our findings depict MIG, IP-10 and ITAC as novel prognostic markers for shorter survival in newly diagnosed MM patients. High serum levels of CXCR3 binding chemokines in conjunction with silenced expression in MM cells may shield myeloma cells from immune attack as previously shown for T cell lymphomas. Further experiments will aim to confirm these initial results by extending our patient cohort and define the source as well as functional role of CXCR3 chemokines in MM. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

CD69, a New Potential Clinical Marker in Multiple Myeloma

CD69 is a type II membrane protein. T cells express CD69 rapidly upon stimulation of the T-cell receptor (TCR), which is why CD69 has been mostly regarded as an activation marker. The precise role of CD69 in immunity has not been determined because its ligand is unknown, but an emerging role of CD69 in Multiple Myeloma (MM) has been postulated. Previous data, using tumor lines derived from murine model with genotypic and immunophenotypic features of resistance to bortezomib, showed that as the neoplastic plasma cells (PC) develop bortezomib resistance, they have a germinal center B cell like immunophenotype, including decreased to absent expression of CD69. CD69 has not been yet studied in human multiple myeloma, though it has been shown that human chronic lymphocytic lymphoma cells, when induced toward a plasma cell phenotype with tetradecanoyl phorbol acetate (TPA) have increased CD69 expression. Interestingly the activation antigen CD69 associates with and inhibits the function of Sphingosine 1-phosphate (S1P). S1P is a bioactive lysophospholipid which is known to induce diverse cellular responses through at least five G-protein-coupled receptors on various cell types. Other data showed that MM cells express the S1P receptors, S1P1, S1P2 and S1P3. Furthermore, S1P protects MM cells against dexametason-induced apoptosis. Importantly, S1P upregulates Mcl-1 expression in a time and concentration-dependent manner in human MM cell lines. Therefore, we analyzed the CD69 expression on pathological PCs, from bone marrow samples of 43 patients, by flow cytometry with two aims: to evaluate the real expression of CD69 on pathological PCs and to determine the clinico-pathological significance of this molecule. Immunophenotyping was carried out by a 6-color method, using a FacsCanto II cytometer and the FacsDiva software. PCs were identified as CD138+/CD38+ events after an initial gate which included events with low SSC in the CD45/SSC cytogram. The MoAb panel also included CD19, CD20, CD117, CD56, cytoplasmic light chains K and Lambda. PerCP-Cy5.5-conjugated CD69 was evaluated on phenotypically abnormal plasma cells (i.e. CD19-, CD45- or dim), which were resulted to be clonally restricted. Results were considered positive when the percentage of positive cells was > 20%. 22 of 43 pts (see table I, group A) were MM resistant/refractory to at least two different chemotherapy regimens (including bortezomib in all patients). 21 patients (table I, group B) were smouldering multiple myeloma (SMM) or MM in at least very good partial response (VGPR) after first line treatment. CD69 was detected on bone marrow PCs in 19 of the 43 patients evaluated (44%). Of the 19 patients with CD69+ (see table II) only 6 (27%) were in the group of refractory/resistant MM, while the majority of these advanced patients, 16/22 (73%), had an absent expression of CD69. On the contrary in the group of SMM/VGPR/CR MM 13 patients (62%) were CD69+ (p=0.04, using a Chi squared test with Yates correction). At the best of our Knowledge this is the first clinical report that confirms CD69 expression on pathological PCs of MM patients. Our preliminary data also suggest an intriguing role of CD69, this molecule could represent an emerging clinical factor to identify different outcomes in patients affected by MM and treated with the modern drugs. Table IPts CharacteristicsGroup AGroup B2221SexMale8(36%)11(52%)Female14(64%)10 (48%)Clinical statusSMMMM inVGPR/CR9 (43%)12 (57%)Relapsed/refractory22(100%)Number of Previous Therapy (range)3,5 (2-6)1 (0-1)Previous Bor regimenSMM0MM inVGPR/CR12(100%)Relapsed/refractory22(100%)Previous Lena regimenSMM0MM inVGPR/CR0Relapsed/refractory17(77%)Table IIPts ResultsGroup AGroup B2221CD69+ 19/43 (44%)6 (27%)13 (62%)CD69-24/43 (56%)16 (73%)8 (38%) DisclosuresNo relevant conflicts of interest to declare.

Prognostic Value Of Quantifying Circulating Plasma Cells By Multiparametric Flow Cytometry In Patients With Relapsed Multiple Myeloma

BackgroundThe presence of circulating plasma cells (PCs) in multiple myeloma (MM) is a known poor prognostic marker. Prior studies have utilized a technically challenging slide-based immunofluorescence technique to detect and quantify the presence of circulating PCs which was not widely adapted in clinical practice. More recently, the routine use of flow cytometry has provided the opportunity to quantitatively assess circulating PCs in MM patients with relative ease. We report the prognostic value of quantifying circulating clonal PCs using multiparametric flow cytometry in MM patients with relapsed disease. MethodsWe evaluated all MM patients seen at the Mayo Clinic, Rochester from 2009 to 2011 with previous or ongoing relapsed disease and who had their peripheral blood samples evaluated by flow cytometry. Each blood sample had its peripheral blood mononuclear cells isolated by ficoll gradient and stained with antibodies to CD45, CD19, CD38, CD138 and cytoplasmic Kappa and Lambda Ig light chains. A six-color multi-parameter flow cytometer (Becton Dickinson FacsCantos II) was used to examine each sample with a target of detecting 150,000 events (cells) that was then analyzed using the Facs Diva Software. Plasma cells were selectively analyzed through combinatorial gating using light scatter properties and CD38, CD138, CD19, and CD45. Normal PC's were then separated from clonal plasma cells based on the differential expression of CD45, CD19 and polytypic Ig light chains. The clonal plasma cells detected were reported out as the number of clonal events/150,000 collected total events. For those samples where less than 150,000 events were gated or examined, the number of final clonal events was adjusted to 150,000 events. Survival analysis was performed by the Kaplan-Meier method and differences in survival assessed using the log rank test. ResultsThere were 647 consecutive patients with a history of treated MM who had a peripheral blood flow cytometry as part of their routine clinical evaluation. The median patient age was 62 years (29-88) and 55% were male. The median time since diagnosis was 12 months (range: 1-363) and the median number of lines of treatment received was 2 (1-11). There were 81 (13%) patients with clonal circulating PCs with a median of 368 cells (4 – 133,464). The 2-year overall survival (OS) for the 81 (13%) patients with any circulating PCs was 17% compared with 65% for those with none (P<0.001). The presence of circulating clonal PCs was associated with high-risk disease by FISH (P <0.001) as well as higher PCLI (P <0.001). We then correlated the presence of circulating clonal PCs with the disease status at flow cytometry assessment. Among the study patients, only 145 (22%) had actively relapsing disease with the remaining 502 (78%) in a plateau including patients in CR. Circulating clonal PCs were more likely to be detected in patients with actively relapsing disease compared with plateau phase (43% vs. 4%, P < 0.001); none of the CR patients and <5% of the remaining plateau phase patients had any detectable circulating clonal PCs.We then restricted the analysis to the actively relapsing patients; the best cutoff predicting 1-year mortality by ROC analysis was 100 events. Based on this, we defined >=100 events as a cutoff for defining the prognostic role of circulating clonal PCs in actively relapsing patients. The median OS for those with >=100 clonal PC events was 12 months compared with not reached for those with <100 events (p<0.001; Figure 1 [Display omitted] ). Among patients with actively relapsing disease, >= 100 circulating PCs was associated with a higher ISS stage, plasma cell labeling index and bone marrow PC% compared with those with <100 circulating PCs. In a multivariable model, only LDH > 222 (HR: 2.08, P = 0.045) and >= 100 circulating PCs (HR: 2.48, P = 0.048) were found to adversely affect OS in actively relapsing patients. ConclusionThe utilization of flow cytometry to quantify circulating clonal PCs in patients with relapsed MM appears to have significant prognostic relevance. In patients with actively relapsing disease, >= 100 circulating PCs predicted for worse OS with a median of 12 months. Future studies are needed to determine if this would allow an opportunity to develop a more risk adapted approach for patients with relapsed disease.▪ Disclosures:Kumar:Celgene: Consultancy, Research Funding; Millennium: Consultancy, Research Funding; Onyx: Consultancy, Research Funding. [Display omitted]

Expression and Clinical Significance of Notch1 On the Membrane of Bone Marrow CD38+CD138+Plasma Cells in the Patients with Multiple Myeloma

Abstract 4981 Objective:To investigate the expression of bone marrow CD38+CD138+, CD38+CD138-plasma cells and the expression of Notch1 on the membrane of them in the patients with multiple myeloma(MM), and explore the importance of Notch signaling pathway in the formation and progression of MM further. Methods:Thirty-three MM patients and 15 healthy controls were enrolled in this study. The expression of bone marrow CD38+CD138+, CD38+CD138-plasma cells and the expression of Notch1 on the membrane of them were analyzed by flow cytometry. The expression of Notch1 mRNA of bone marrow mononuclear cells were analyzed by RT-PCR. Results:The ratio of CD38+CD138+ plasma cells from 24 newly diagnosed MM patients was (51. 50%±12. 48%) which was significantly higher than CD38+CD138- plasma cells of MM patients (42. 88%±11. 41%)(P=0. 016)and controls 20. 13%±5. 8(P=0. 000). The expression of CD38+CD138+ plasma cells from 24 newly diagnosed MM patients was correlated to the level of malignant plasma cells in there bone marrow(r=0. 546, p=0. 006), serum level of lactate dehydrogenase(LDH)(r=0. 567, p=0. 004), and β2-MG(r=0. 431, p=0. 035). The ratio of Notch1 on the membrane of CD38+CD138+ plasma cells of MM patients was (60. 21%±25. 06%) which was significantly higher than those of CD38+CD138- plasma cells of MM patients 39. 84%±18. 94%(P=0. 000)and controls (38. 34%±19. 39%)(P=0. 004). There was no statistical difference between the two latter groups(P>0. 05). The expression of Notch1 on CD38+CD138+ plasma cells from 24 newly diagnosed MM patients was correlated to the level of malignant plasma cells in there brone marrow(r=0. 914, p=0. 000), serum level of lactate dehydrogenase(LDH) (r=0. 604, p=0. 002), and β2-MG(r=0. 455, p=0. 026). The ratio of Notch1 on the membrane of CD38+CD138+ plasma cells of MM patients who had renal dysfunction was correlated to their abnormal serum creatinine levels. The expression of Notch1 on CD38+CD138+ plasma cells from 17 MM patients who received VD chemotherapy was correlated to the ratio of plasma cell reduction after the first VD chemotherapy(r=0. 842, p=0. 000). The expression of Notch1 mRNA of bone marrow mononuclear from 10 MM patients was (0. 8252±0. 4079) which was significantly higher than those of controls (0. 3759±0. 0813)(p=0. 032). Conclusion:Notch1 over expressed on CD38+CD138+ plasma cells with relation to the effects of early VD therapy and long term outcome of MM. Disclosures:No relevant conflicts of interest to declare.

Epigenetic induction of adaptive immune response in multiple myeloma: sequential azacitidine and lenalidomide generate cancer testis antigen‐specific cellular immunity

Patients with multiple myeloma (MM) undergoing high dose therapy and autologous stem cell transplantation (SCT) remain at risk for disease progression. Induction of the expression of highly immunogenic cancer testis antigens (CTA) in malignant plasma cells in MM patients may trigger a protective immune response following SCT. We initiated a phase II clinical trial of the DNA hypomethylating agent, azacitidine (Aza) administered sequentially with lenalidomide (Rev) in patients with MM. Three cycles of Aza and Rev were administered and autologous lymphocytes were collected following the 2nd and 3rd cycles of Aza-Rev and cryopreserved. Subsequent stem cell mobilization was followed by high-dose melphalan and SCT. Autologous lymphocyte infusion (ALI) was performed in the second month following transplantation. Fourteen patients have completed the investigational therapy; autologous lymphocytes were collected from all of the patients. Thirteen patients have successfully completed SCT and 11 have undergone ALI. Six patients tested have demonstrated CTA up-regulation in either unfractionated bone marrow (n = 4) or CD138+ cells (n = 2). CTA (CTAG1B)-specific T cell response has been observed in all three patients tested and persists following SCT. Epigenetic induction of an adaptive immune response to cancer testis antigens is safe and feasible in MM patients undergoing SCT.

Increased expression of extracellular matrix metalloproteinase inducer (CD147) in multiple myeloma: role in regulation of myeloma cell proliferation

Multiple myeloma (MM) is preceded by the asymptomatic pre-malignant state, monoclonal gammopathy of undetermined significance (MGUS). Although MGUS patients may remain stable for years, they are at increased risk of progressing to MM. A better understanding of the relevant molecular changes underlying the transition from an asymptomatic to symptomatic disease state is urgently needed. Our studies show for the first time that the CD147 molecule (extracellular matrix metalloproteinase inducer) may be having an important biological role in MM. We first demonstrate that CD147 is overexpressed in MM plasma cells (PCs) vs normal and pre-malignant PCs. Next, functional studies revealed that the natural CD147 ligand, cyclophilin B, stimulates MM cell growth. Moreover, when MM patient PCs displaying bimodal CD147 expression were separated into CD147(bright) and CD147(dim) populations and analyzed for proliferation potential, we discovered that CD147(bright) PCs displayed significantly higher levels of cell proliferation than did CD147(dim) PCs. Lastly, CD147-silencing significantly attenuated MM cell proliferation. Taken together, these data suggest that the CD147 molecule has a key role in MM cell proliferation and may serve as an attractive target for reducing the proliferative compartment of this disease.

CRM1 Is Highly Expressed in Myeloma Plasma Cells and Its Inhibition by KPT-SINE Induces Cytotoxicity by Increasing p53 in the Nucleus of Multiple Myeloma (MM) Cells

Abstract 1852 Background:CRM1 (XPO1, exportin) is a nuclear export protein which controls the nuclear-cytoplasmic localization of multiple tumor suppressor proteins and cell proliferation pathways including p53, p21, PI3K/AKT/FOXO, Wnt/ß-catenin/APC, topoisomerase II, and NF-κB/I-κB. Transport of nuclear proteins to the cytoplasm can render them ineffective as tumor suppressors or as targets for chemotherapy. Small molecule, selective inhibitors of nuclear export (SINE) that block CRM1-dependent nuclear export can force the nuclear retention of tumor suppressor proteins, thus rendering cancer cells more susceptible to apoptosis and responsive to other chemotherapy. In this study we evaluated CRM1 as a potential target in MM and the effect of SINE on the activity of established anti-myeloma agents currently in use in treatment of MM. KPT-276 is the lead CRM1 inhibitor being investigated which will be submitted for IND in 2012. Methods:To evaluate expression of CRM1, bone marrow aspirates from MM patients and tonsil tissue from normal patients were enriched for plasma cells (PC) and proteins from cell lysates were separated by SDS-PAGE followed by immunoblotting with CRM1 antibodies. In functional experiments, isolated fresh MM PCs from patients, and NCI-H929, MM1.S, MM1.R and RPMI-8226 cell lines were cultured in RPMI-1640 with 10–15% serum. Cells were treated for 24–72 hrs with CRM1 inhibitors KPT-SINE compounds with or without bortezomib and dexamethasone and were analyzed for cytotoxicity by MTT assay. Drug concentrations for combination experiments were chosen to be at or below IC50 for each individual drug. Apoptosis induction in primary MM cells and cell lines was studied by Annexin V labeling and flow cytometry. Cell lysates from primary MM PCs and cell lines were prepared after treatment with KPT-SINE and were used to determine the expression of p53 and CRM1. Results:Primary MM plasma cells derived from naïve, previously untreated patients show 4–20 fold higher CRM1 protein expression, compared to normal peripheral blood mononuclear cells (PBMCs) and normal tonsilar PCs. Dose response analysis of KPT-SINE compounds in myeloma cell lines showed potent activity with IC50s in the range of 10–100nM. The lead compound KPT-276 had an IC50 of <100 nM in NCI-H929, MM1.S, MM1.R and RPMI-8226 cells. Functional studies in MM patient plasma cells showed that in vitro inhibition of CRM1 with related SINEs KPT-185, −225 or −276 increase apoptosis induction as measured by Annexin V assay. In addition, the inhibition of CRM1 with KPT-SINE results in a dose-dependent increase in levels of nuclear as well as total p53 in MM patient plasma cells within 48 hrs. When combined with proteasome inhibitors like bortezomib and/or dexamethasone, KPT-SINE compounds potently increase the cellular cytotoxicity of these drugs in MM cell lines. Mechanism of activity of drug combinations is under investigation in MM cell lines and MM patient plasma cells. Conclusions:MM plasma cells express CRM1 that is functionally active and therefore is a valid target in the treatment of myeloma. Moreover, higher expression of CRM1 in malignant plasma cells compared to normal PBMCs and normal PCs suggests possibility of therapeutic index. Early mechanistic studies indicate that CRM1 inhibition can lead to an increased expression of p53 (and other tumor suppressors) and its nuclear localization in myeloma cells and therefore might serve as a mechanism for the activity of CRM1 inhibitors in MM. Potentiation of cytotoxicity of bortezomib and dexamethasone by KPT-SINE suggests that these drugs might be useful in treating MM refractory to currently used agents and provide rationale for combining inhibitors of nuclear transport with other drugs. Disclosures:Off Label Use: KPT-SINE family of drugs are not approved for the treatment of multiple myeloma. These drugs have a novel mechanism and are in pre-clinical development for the treatment of several malignancies. McCauley:Karyopharm Therapeutic Inc.: Employment. Shacham:Karyopharm Therapeutics Inc.: Employment. Kauffman:Karyopharm Therapeutics Inc.: Employment. Jakubowiak:Exelixis: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Onyx Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Millennium Pharmaceuticals, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Ortho Biotech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Adaptive Immunotherapy In Multiple Myeloma: Epigenetic Modification and Immunomodulation by Sequential Azacitidine and Lenalidomide to Generate Cancer Testis Antigen Specific Cellular Immune Response

Abstract 2926Patients with multiple myeloma (MM) undergoing high dose therapy and autologous stem cell transplantation (SCT) remain at risk for disease progression. Maintenance therapy may delay recurrence but is associated with toxicities, highlighting the need for alternative strategies for long-term disease control. Malignant plasma cells in MM patients occasionally express highly immunogenic cancer testis antigens (CTA). CTA expression is regulated by DNA methylation, and may be increased by 5-azacitidine (Celgene corp., Summit, NJ) (Aza), a DNA hypomethylating agent. The addition of lenalidomide (Celgene corp.) (L) may augment any ensuing adoptive CTA-specific immune response. These immune effector cells may then be collected and adoptively transferred in a setting of lympho-depletion and minimal residual disease following SCT, serving a maintenance function. To demonstrate the feasibility of this approach, we initiated a phase II clinical trial of Aza administered sequentially with L in patients with residual disease following initial therapy (NCT01050790). Three cycles of Aza (75 mg/m2 day 1–5 SQ) and L (10 mg PO daily, day 6–21) were administered every 4 weeks; autologous lymphocytes (AL) were collected around day 21 of the 2nd and 3rd cycles of Aza-L and cryopreserved. Subsequent stem cell mobilization was followed by melphalan 200 mg/m2 and SCT. GM-CSF was used post-transplant for hematopoietic engraftment. Autologous lymphocyte infusion (ALI) was performed between day +30 to +60.Twelve patients have been enrolled; median age is 60 years (range 40–69). Eight are African American, 10 had disease in first partial remission (PR) and 2 in very good PR (VGPR) at the time of initiation of therapy. Median of 2 prior regimens had been administered (range 1–2) and 6 had prior therapy with L. Stage at diagnosis was II (n=6) and III (n=6) and 4 had abnormal cytogenetics. Eight patients have completed all three cycles of Aza-L; 2 developed grade 3 neutropenia, no other grade 3 to 4 toxicity has been observed. Eight patients have gone through both AL collections, 21 days following cycles 2 and 3 of Aza-L, yielding 0.90±0.41 and 0.81±0.36 ×108 CD3+ cells/kg (mean ± SD) with the first and second procedures. Three patients had further disease response, (1 near complete remission, 2 VGPR) and 5 had stable disease following three months of Aza-L, representing a median 18% decline in para-protein levels before and after therapy. So far 8 patients have undergone stem cell mobilization with either GCSF alone (n=6) or GCSF + plerixafor (n=2), and have collected 11.2±3.3 ×106 CD34+ cells/kg body weight. To date 6 patients have undergone SCT (tandem SCT in 1). Neutrophil engraftment was at median of 14 days (13–14), and no unexpected post transplant toxicities were observed. Four patients received ALI at a median 42 days following SCT with no immediate or remote infusional toxicities. With a median follow up of 9 months post-transplant, all four ALI recipients are progression free with either complete remission (n=1) or VGPR (n=3).Quantitative RT-PCR evaluating a panel of 10 CTA in unfractionated bone marrow specimens collected before and after Aza-L from five patients demonstrated 6–8 discrete CTA induced in each patient (Figure 1). This expression was seen in CD138+ plasma cells when tested. In one patient with an induced increase in NY-ESO 1 expression following Aza-L, an antigen specific immune response was recorded (IFN-g release assay) when blood mononuclear cells were co-cultured with recombinant NY-ESO 1 pulsed monocyte derived dendritic cells. This response was maintained for several months post-transplant (Figure 2). Consistent with this observation, CD3+ T cell counts before and after ALI demonstrated a marked increase in T cell counts at two (mean 959/μl; n=4) and eight (1277/μl) weeks, compared with baseline (414/μl; P=0.05); no difference was seen in the NK cell counts at these times. Further studies to confirm these observations in the remaining patients are ongoing. [Display omitted] In conclusion, we demonstrate the safety and feasibility of epigenetic modification resulting in over-expression of antigenic targets in MM. This may then be exploited in formulating adaptive immunotherapy protocols in these patients. Adoptively transferred cells may maintain long-term surveillance against malignant plasma cells in patients with MM. Disclosures:Toor:Celgene corporation: Research Funding. Off Label Use: azacitidine in multiple myeloma. Manjili:Celgene corporation: Research Funding.

SAGE analysis highlights the importance of p53csv, ddx5, mapkapk2 and ranbp2 to multiple myeloma tumorigenesis

Serial analysis of gene expression (SAGE) allows a comprehensive profiling of gene expression within a given tissue and also an assessment of transcript abundance. We generated SAGE libraries from normal and neoplastic plasma cells to identify genes differentially expressed in multiple myeloma (MM). Normal plasma cells were obtained from palatine tonsils and MM SAGE library was generated from bone marrow plasma cells of MM patients. We obtained 29,918 SAGE tags from normal and 10,340 tags from tumor libraries. Computer-generated genomic analysis identified 46 upregulated genes in the MM library. Ten upregulated genes were selected for further investigation. Differential expression was validated by quantitative real-time PCR in purified plasma cells of 31 patients and three controls. P53CSV, DDX5, MAPKAPK2 and RANBP2 were found to be upregulated in at least 50% of the MM cases tested. All of them were also found upregulated in MM when compared to normal plasma cells in a meta-analysis using ONCOMINE microarray database. Antibodies specific to DDX5, RANBP2 and MAPKAPK2 were used in a TMA containing 57 MM cases and confirmed the expression of these proteins in 74%, 96%, and 21% of the MM samples, respectively. Analysis of differential expression using SAGE could identify genes important for myeloma tumorigenesis ( P53CSV, DDX5, MAPKPK2 and RANBP2) and that could potentially be useful as therapeutic targets.

DNA Damage Induced Sumoylation of p53 in Multiple Myeloma: Implications for Cell Proliferation and Apoptosis.

The tumor suppressor protein p53 has a critical role in malignant transformation through its critical functions in the regulation of cell proliferation, DNA repair, and apoptosis. The level and activity of the p53 tumor suppressor protein is regulated by post-translational modifications such as phosphorylation and ubiquitination. To address the role of p53 in the pathogenesis of Multiple Myeloma (MM), a number of patient-derived MM cell lines were probed using a p53-specific antibody and immunoblotting indicated a significantly elevated level of the p53 protein in the lysate of a vast majority of cell lines relative to that of normal CD138+ plasma cells. Since p53 is regulated by a number of post-translational modifications, the MM cell line RPMI-8226 was then treated with g-radiation (5 Gy), total lysate prepared and probed with a p53 monoclonal antibody As early as 30 minutes following treatment with radiation, a dramatic induction in the steady-state levels of the p53 protein and, in addition, a new higher molecular weight (∼68kDa) immunoreactive form of p53 was observed. The 68 kDa form of p53 was immunoprecipitable from MM total cell lysates with an antibody to the small-ubiquitin-like modifier (Sumo-1) but importantly, was not immunoprecipitated by an antibody generated to ubiquitin. Sumo-1 is covalently conjugated to target proteins through a conjugating enzyme, Ubc9, and a substrate ligase, PIAS1. Both Ubc9 and PIAS1 were rapidly induced at the protein level upon treatment of MM cells with g-radiation. Sumoylation of p53 was detected following treatment of MM cells with a number of genotoxic stressors in addition to g-irradiation, such as etoposide, doxorubicin, methylethylsulfonate and Ni++ and was detected using cell lysate from a number of MM cell lines. MM cells were transformed with a plasmid that expressed a dominant negative mutant form of UBC9 that abolished sumoylation of p53. Transfectants displayed increased sensitivity to g-radiation. A second plasmid that over expressed a mutant form of p53 that had mutated attachment site for covalent linkage of Sumo-1 (p53-K386R) was transfected into MM cells. These cells displayed a greater proliferative capacity relative to mock or wild-type p53 transfected cells. We then examined CD138+ plasma cells that had been immunoaffinity purified from MM patient bone marrow samples. Similar to the MM cell lines, the steady-state level of p53 in MM patient samples was significantly elevated relative to that of normal CD138+ cells. Importantly, the sumoylated form of p53 was also detected in the cell lysate prepared from MM patient samples (but not MGUS samples). Our results indicate that p53 is rapidly sumoylated upon exposure of MM cells to genotoxic stressors. Importantly, the sumoylated form of p53 was readily detected in the purified plasma cells of MM patients and led to increased cell proliferation. The results indicate a critical role for the sumoylation pathway in the DNA damage response, the proliferative and apoptotic functions of p53 and the pathogenesis of MM.

Induction of UBC9 and the Sumoylation Pathway in Multiple Myeloma Promotes Plasma Cell Growth and Correlates with Decreased Patient Survival.

The pursuit of rationale, targeted therapies relies on a detailed understanding of the mechanisms that subvert normal growth control and lead to development of Multiple Myeloma (MM). To further define the mechanistic steps that contribute to MM pathogenesis, we examined mRNA expression profiles of CD138+ plasma cells obtained from normal, Monoclonal Gammopathy of Unknown Significance (MGUS), and MM patient samples. Using genomic results in combination with molecular and cellular-based assays, we demonstrate a critical role for UBC9 and the sumoylation pathway in myeloma cell growth and survival. Notably, Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) demonstrated a ten-fold induction in UBC9; a gene that encodes the sole Sumo-conjugating enzyme in human cells. We demonstrated an elevation of UBC9 in both MM primary patient cells and in a number of patient-derived MM cell lines. In addition, a number of other sumoylation pathway components were induced in primary MM cells at the gene and protein level relative to normal plasma cells. We believe that induction of UBC9 is an early genetic event in the pathogenesis of MM since the induction was observed at the gene and protein level in plasma cells from patients with MGUS. Importantly, UBC9 induction was functionally significant since a different pattern of sumoylation was observed in total cell lysate from MM patient plasma cells relative to that of normal plasma cells. Furthermore, overexpresion of a mutant form of UBC9 deficient in Sumo-conjugating activity increased the sensitivity of plasma cells to apoptosis by chemotherapeutic agents and these cells were impaired in other essential functions that included cellular proliferation, DNA synthesis, resistance to apoptosis and adhesion to bone marrow stroma. Immunoblotting of MM patient cell lysates also demonstrated a similar induction of the UBC9 gene product (Ubc9) as well as induction of the Sumo ligases Nse2 and PIAS1. These studies identify UBC9 as a target upregulated early in the pathogenesis of MM and indicate a critical role for sumoylation in disrupting the controls that govern normal plasma cell growth. To further develop prognostically relevant molecular signatures and classifications of MM subtypes, we analyzed the survival outcome of patients that expressed induced levels of UBC9, as well as other sumoylation components, and demonstrate significantly reduced survival of such patients following current treatment modalities. The results provide evidence for critical role of UBC9 and sumoylation in MM pathogenesis. Furthermore, sumoylation pattern may serve as a therapeutic target in MM, help stratify clinical management and provide a framework for the identification of sumoylation pathway targets that govern MM cell growth and progression.

ABCG2 expression, function, and promoter methylation in human multiple myeloma

AbstractWe investigated the role of the breast cancer resistance protein (BCRP/ABCG2) in drug resistance in multiple myeloma (MM). Human MM cell lines, and MM patient plasma cells isolated from bone marrow, were evaluated for ABCG2 mRNA expression by quantitative polymerase chain reaction (PCR) and ABCG2 protein, by Western blot analysis, immunofluorescence microscopy, and flow cytometry. ABCG2 function was determined by measuring topotecan and doxorubicin efflux using flow cytometry, in the presence and absence of the specific ABCG2 inhibitor, tryprostatin A. The methylation of the ABCG2 promoter was determined using bisulfite sequencing. We found that ABCG2 expression in myeloma cell lines increased after exposure to topotecan and doxorubicin, and was greater in logphase cells when compared with quiescent cells. Myeloma patients treated with topotecan had an increase in ABCG2 mRNA and protein expression after treatment with topotecan, and at relapse. Expression of ABCG2 is regulated, at least in part, by promoter methylation both in cell lines and in patient plasma cells. Demethylation of the promoter increased ABCG2 mRNA and protein expression. These findings suggest that ABCG2 is expressed and functional in human myeloma cells, regulated by promoter methylation, affected by cell density, up-regulated in response to chemotherapy, and may contribute to intrinsic drug resistance.

Cytokine and chemokine profiles in multiple myeloma; significance of stromal interaction and correlation of IL-8 production with disease progression

Multiple myeloma (MM) is a product of interactions between tumor plasma cells and multiple cell types native to the bone marrow (BM). We have used antibody array technology to examine the proteins produced by BM stromal cells in response to stimulation by BM taken from patients diagnosed with monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM), and MM. We observed increased production of the chemokine IL-8 by stromal cells co-cultured with supernatants from bone marrow cells of patients with active myeloma. IL-8 production is correlated with active disease and is dependent upon IL-1β and NF-κB signaling. Consistent with the pro-angiogenic activity of IL-8, increased BM microvessel density (MVD) correlated with stimulation of stromal cell IL-8 production. In addition, the majority of MM cell lines and MM patient plasma cells were found to express IL-8 receptors CXCR1 and CXCR2. We conclude that stromal cell IL-8 production parallels MM disease activity, is IL-1β induced, and correlates with bone marrow angiogenesis.