ALBERT

Description: Abstract 618 Introduction: Despite an increased number of new treatments, multiple myeloma (MM) remains mostly incurable. There is emerging evidence that achieving complete or near complete response (CR/nCR), or at least a 90% reduction of the disease (≥VGPR), in response to initial treatment when MM is most sensitive to chemotherapy is associated with improved progression-free survival (PFS) and possibly overall survival (OS). However, even with the most active regimens, a majority of patients (pts) with newly diagnosed MM achieve less than CR/nCR to initial therapy. The objective of this study is to establish predictors of response and drug resistance by applying proteomic profiling of MM. Here we present the analysis of differential proteomic profiling of baseline plasma cells (PCs) from pts with MM predicting achievement of CR/nCR after completion of a course of first line treatment with lenalidomide (Revlimid®), bortezomib (Velcade®), and dexamethasone (RVD) regimen. Methods: After obtaining informed consent from pts, we performed quantitative proteomic analysis of PCs isolated from bone marrow of 16 pts with previously untreated MM enrolled at the University of Michigan site in the Phase II portion of the multi-site frontline RVD clinical trial. Eight of the analyzed pts achieved CR/nCR, while the remaining had a lesser response (6 VGPR, 2 PR i.e ≥50% but 〈 90% reduction of disease). We used two independent proteomic platforms: iTRAQ (Isobaric Tags for Relative and Absolute Quantitation) technique in 8-plex variant, as well as a label-free approach (LF) based on spectra counting. PCs were acquired from bone marrow aspiration and, thereafter, were enriched with a RosetteSep negative selection kit (StemCell Technologies). In iTRAQ experiments, proteins were processed with reagents according to the manufacturer's protocol (Applied Biosystems) followed by SCX fractionation and LC-MS/MS analysis (4800 Plus MALDI TOF/TOF). Peptides from the MM1S cell line were used as an internal reference. The data were analyzed using ProteinPilot software. For LF analysis, proteins were pre-fractionated before trypsin digestion on NuPage Bis-Tris-Gel and subsequently run on LC-ESI-MS/MS on a linear trap mass spectrometer (LTQ Orbitrap). Database search was carried out using X!Tandem followed by Trans-proteomic Pipeline (TPP). A 1.5-fold difference in expression in both platforms was used as a cut-off value. Results: A total of 926 proteins were identified with high confidence (FDR

Description: Abstract 1902 Introduction: Multiple myeloma (MM) remains mostly incurable. Novel therapies have improved response rates, which are now reaching 100%. More importantly, number of recent studies showed that the depth of response, e.g. achievement of at least 90% reduction of the disease (≥VGPR) is associated with longer disease control. Therefore, improving VGPR rates and establishing predictors of VGPR to a given regimen may be an important clinical goal. High throughput quantitative proteomics may offer greater insight into the actual biology of the malignant cell than genome analysis and therefore, may be more useful in the development of personalized therapy. The objective of this study is to establish a proteomic signature predicting achievement of at least VGPR to initial treatment with bortezomib (Velcade®), pegylated liposomal doxorubicin, and dexamethasone (VDD). We previously reported preliminary proteomic profile of malignant plasma cells (PCs) obtained from a set of naïve MM pts enrolled in the VDD trial (Dytfeld et al., ASH 2009). Here we present the results of differential proteomic analysis of MM PCs of all available samples from the frontline VDD study (≥VGPR vs.

Description: Abstract 4034 Introduction: Multiple myeloma (MM) is a monoclonal gammopathy characterized by the uncontrolled proliferation of plasma cells (PCs). The lack of knowledge about MM cell biology compared to normal PCs is hindering the discovery of myeloma specific targeted therapeutics. Current therapeutics target broad cellular functions such as suppression of the bone marrow environment, myeloma cell proliferation and induction of apoptosis. The objective of our study was to determine biomarkers of the disease and identify new potential targets for future therapeutics, and therefore increase treatment options for MM. We utilized quantitative proteomics using an iTRAQ-based approach to identify biomarkers that can distinguish between MM and normal PCs. Methods: Tonsil tissues, removed from patients suffering from sleep apnea syndrome who consented for tissue repository, were the source of normal PCs. First, the tonsil cells were depleted of T-cells, granulocytes and macrophages using RosetteSep® antibody cocktail and, subsequently, CD138+ PCs were isolated by EasySep® magnetic bead selection. Bone marrow aspirates from MM patients who consented for IRB-approved MM repository protocol, were enriched for PCs with RosetteSep® antibody cocktail. PC percentage for purity assessment was performed by Wright-Giemsa staining of cytospin preparations. PCs (250,000) were lysed and proteomic profiles were generated by iTRAQ 4-plex methods where 2 tonsil PCs (TPC) and 2 MM plasma cells (MMPC) were in each 4-plex. After labeling with iTRAQ tags, the proteins were fractionated by cation exchange chromatography followed by LC-MS/MS analysis on a MALDI-TOF/TOF™ analyzer. The data were analyzed and quantification performed using ProteinPilot™ software. Real time PCR of cDNA from TPC and two independent MMPC samples was performed to validate the results. Results: We consistently obtained 100–250,000 normal PCs from each tonsil sample, at a purity of 〉80%. To obtain reliable data from proteomics we required 〉200,000 cells and therefore tonsil pools were utilized wherever necessary. Three types of MM patient samples were studied: newly diagnosed MM, relapsed MM and plasma cell leukemia. We detected and quantified 848 proteins with high confidence from three 4-plex iTRAQ experiments (FDR

Description: Introduction Exportin 1 (XPO1/CRM1) is the sole transporter of most tumor suppressor proteins (TSP) from the nucleus to the cytoplasm. Small molecule selective inhibitors of nuclear export (SINE) block XPO1-mediated nuclear export, leading to nuclear retention of TSP, inducing cancer cell death and sensitizing cancer cells to other cytotoxic drugs. Although the cytotoxic and apoptotic effects of SINE on different cancer cells have now been established, the mechanism of cell death is still not fully understood. Recently, autophagy emerged as a possible cell death pathway and alternate to the ubiquitin-proteasome pathway (UPP) by which excess and/or dysfunctional proteins and organelles are degraded and recycled. MM cells require basal autophagy for survival and caspase 10 protease activity is required to limit autophagic cell death. The possibility that autophagy may be involved in the mechanism of action of SINE is supported by observations that knockdown of XPO1 can promote autophagy and that cytoplasmic p53 can repress autophagy. In this study, we evaluated the contribution of autophagy to the effects of Selinexor (KPT-330), a SINE currently in two phase I clinical trials, on MM cell cytotoxicity. Because proteasome inhibition can also induce autophagy, we hypothesized that the combination of Selinexor and CFZ, a next generation irreversible proteasome inhibitor approved for treatment of MM, may synergistically augment cytotoxicity in MM cells. Methods Plasma cells (PC) were purified from consented MM patient bone marrow aspirates using EasySep (Stem Cell Technology). PC purity (〉80%) was determined by Wright-Giemsa staining of cytospins. Human myeloma cell lines (HMCL) NCI-H929, RPMI-8226, MM1.S and MM1.R were cultured in RPMI1640 with 10% FBS. IC50 values were determined using GraphPad Prism. Drug combination efficacy was determined using CalcuSyn (Biosoft). Combination index (CI) values

Description: 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