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Toward our goal of personalized medicine, we comprehensively profiled pre-treatment malignant plasma cells from multiple myeloma patients and prospectively identified pathways predictive of favourable response to bortezomib-based treatment regimens. We utilized two complementary quantitative proteomics platforms to identify differentially-regulated proteins indicative of at least a very good partial response (VGPR) or complete response/near complete response (CR/nCR) to two treatment regimens containing either bortezomib, liposomal doxorubicin and dexamethasone (VDD), or lenalidomide, bortezomib and dexamethasone (RVD). Our results suggest enrichment of 'universal response' pathways that are common to both treatment regimens and are probable predictors of favourable response to bortezomib, including a subset of endoplasmic reticulum stress pathways. The data also implicate pathways unique to each regimen that may predict sensitivity to DNA-damaging agents, such as mitochondrial dysfunction, and immunomodulatory drugs, which was associated with acute phase response signalling. Overall, we identified patterns of tumour characteristics that may predict response to bortezomib-based regimens and their components. These results provide a rationale for further evaluation of the protein profiles identified herein for targeted selection of anti-myeloma therapy to increase the likelihood of improved treatment outcome of patients with newly-diagnosed myeloma.

Metastasis represents an end stage in the evolution of cancer progression and has been related to specific genetic pathways. Overexpression of mutant RAS in particular appears to promote invasion and metastasis, although exactly how this occurs has not been well characterized. It was previously showed that activation of the WASF3 protein regulates actin cytoskeleton dynamics that promote invasion. In this report, how WASF3 overexpression interacts with mutant RAS to increase invasion and metastasis was investigated. The ability of RAS to promote invasion and metastasis was shown to be dependent on WASF3 activation in a PI3K and AKT dependent manner. Proteomics analysis demonstrates the presence of AKT in the WASF3 immunocomplex which is enhanced by overexpression of mutant RAS. During these processes activation of ERK1/2 is not affected by loss of WASF3 expression. Analysis of the relative involvement of p85 and p110 in the WASF3 complex demonstrates that mutant RAS promotes dissociation of p85 promoting activation of p110. These studies provide a deeper understanding of the critical role for WASF3 in facilitating increased invasion potential in cancer cells expressing mutant RAS and supports the idea that targeting WASF3 in metastatic cells overexpressing RAS may be used to suppress invasion and metastasis.

Background Cellular differentiation is characterized by the acquisition of specialized structures and functions, cell cycle exit, and global attenuation of the DNA damage response. It is largely unknown how these diverse cellular events are coordinated at the molecular level during differentiation. We addressed this question in a model system of neuroblastoma cell differentiation induced by HOXC9. Results We conducted a genome-wide analysis of the HOXC9-induced neuronal differentiation program. Microarray gene expression profiling revealed that HOXC9-induced differentiation was associated with transcriptional regulation of 2,370 genes, characterized by global upregulation of neuronal genes and downregulation of cell cycle and DNA repair genes. Remarkably, genome-wide mapping by ChIP-seq demonstrated that HOXC9 bound to 40% of these genes, including a large number of genes involved in neuronal differentiation, cell cycle progression and the DNA damage response. Moreover, we showed that HOXC9 interacted with the transcriptional repressor E2F6 and recruited it to the promoters of cell cycle genes for repressing their expression. Conclusions Our results demonstrate that HOXC9 coordinates diverse cellular processes associated with differentiation by directly activating and repressing the transcription of distinct sets of genes.

WASF3 has been shown to be required for invasion and metastasis in different cancer cell types and knockdown of WASF3 leads to suppression of invasion/metastasis. Aberrant signaling through the interleukin 6/Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) axis in cancer cells has emerged as a major mechanism for cancer progression. In this study, we demonstrate that interleukin 6 induces both WASF3 expression and phosphoactivation in breast and prostate cancer cell lines through the JAK2/STAT3 pathway in two different ways. First, we show that STAT3 binds directly to the WASF3 promoter and increases transcription levels, which correlates with increased migration potential. Inactivation of STAT3 with short hairpin RNA, dominant negative constructs or S3I-201 leads to reduced WASF3 levels and reduced migration. Second, we have shown that JAK2, while activating STAT3, also interacts with and activates WASF3. Inhibition of JAK2 with short hairpin RNA or AG490 leads to loss of migration due to reduced WASF3 activation levels and prevention of its membrane localization. Together, these results define a novel signaling network whereby JAK2/STAT3 signaling creates a feed-forward loop to raise activated WASF3 levels that promote cancer cell motility.

Inactivation of HSP90 and HSP70 leads to loss of invasion in a variety of cancer cell types, presumably as a result of destabilization of, as yet, undefined clients of these molecular chaperones that influence this phenotype. The WASF3 gene has been shown to be up-regulated in high-grade tumors and its down-regulation leads to loss of invasion and metastasis. WASF3 phosphorylation by ABL kinase is essential for its ability to regulate invasion. Mass spectroscopy analysis now shows that HSP90 is present in the WASF3 immunocomplex from prostate cancer cells. Inactivation of HSP90 in these and other cell types does not affect WASF3 stability but prevents its phosphoactivation as a result of destabilization of ABL. HSP70 was also found in the WASF3 immunocomplex and inactivation of HSP70 results in destabilization of WASF3 through proteasome degradation. Knockdown of WASF3, HSP90, and HSP70 individually, all lead to loss of invasion but as knockdown of WASF3 in the presence of robust expression of HSP90/70 has the same effect, it seems that the influence these chaperone proteins have on invasion is mediated, at least in part, by their control over the critical invasion promoting capacity of the WASF3 protein. Overexpression of HSP70 in WASF3 null cells does not enhance invasion. These observations suggest that targeting HSP90/70 may have efficacy in reducing cancer cell invasion.

The liquid secondary ionization mass spectrometry of oligosaccharide anions is investigated with an external source quadrupole Fourier transform mass spectrometer (FTMS). Selected linear and cyclic oligosaccharides are analyzed to determine the performance of the external source FTMS in the anion mode. Fragmentation behavior of disaccharides is characterized and related to the fragmentation behavior of larger oligomers. General correlation is found between the linkages and the fragment ions observed.

Metastable decay rates of alpha-cyclodextrin and maltohexaose coordinated to proton and alkali metal ions were determined from ions produced by liquid secondary ion mass spectrometry in an external source Fourier transform mass spectrometry instrument. For both oligosaccharide compounds the decay rates of the protonated species are faster than any alkali metal coordinated species. Decay rates of the metal cationized species decrease in the order Li+, Na+, K+, and Cs+. The anion of alpha-cyclodextrin has the slowest measurable decomposition rate. The relationships between cation affinities and rates are explored.

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 Conclusions: We analyzed proteomic characteristics of patient-derived MM cells using two independent proteomics platforms. As a proof of concept, analysis of PCs obtained from pts enrolled in the frontline RVD trial shows differential expression of 70 proteins in patients who achieved CR/nCR versus those with a lesser response. Consistent with our prior observations, differentially regulated proteins are involved in the c-Myc pathway, which has an established critical role in pathogenesis of MM. Validation studies of candidate proteins are in progress. This study was supported by a grant from the Multiple Myeloma Research Foundation. Disclosures: Off Label Use: Lenalidomide in combination with bortezomib and dexamethasone for the treatment of newly diagnosed Myeloma. Richardson:Millennium Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees. Jakubowiak:Millennium Pharmaceuticals, Inc.: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Centocor Ortho Biotech: Consultancy, Honoraria; Exelixis: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Research Funding.

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. Methods: PCs were acquired from pre-treatment bone marrow specimens after obtaining informed consent from patients (pts), and were thereafter enriched with a RosetteSep® negative selection kit. Quantitative proteomic analysis of PCs from 17 naïve pts with MM from the VDD study was performed using iTRAQ approach in 8-plex variant. To increase confidence of analysis, label-free quantitative proteomics (LF) based on spectra counting was conducted on PCs from 12 pts. In iTRAQ experiments, proteins were processed with reagents according to the manufacturer's protocol followed by SCX fractionation and LC-MS/MS analysis (4800 Plus MALDI TOF/TOF). Peptides from the MM1S cell line were used as a reference. The data were analyzed using ProteinPilot™. For LF analysis, proteins were fractionated before trypsin digestion on Bis-Tris-Gel and subsequently run on LC-ESI-MS/MS on a linear trap mass spectrometer (LTQ Orbitrap). A database search was carried out using X!Tandem followed by Trans-proteomic Pipeline. At least 1.5-fold difference in expression in both platforms was used as a cut-off value. To correlate proteomics with gene expression of dysregulated proteins of interest, mRNA levels were analyzed by quantitative real time PCR (RT-PCR). Validation of proteomic findings on proteins of interest was performed using Western Blot. Results: We identified a total of 894 proteins in 3 iTRAQ experiments with high confidence (FDR Conclusions: We present patient-derived proteomic characteristics of MM cells using two independent proteomic platforms. As a proof of concept, analysis of PCs obtained from pts enrolled in the frontline VDD study shows differential expression of 34 proteins in pts who achieved ≥VGPR vs. pts with This study was supported by a grant from the Multiple Myeloma Research Foundation. Disclosures: Jakubowiak: Millennium, Celgene, Bristol-Myers Squibb, Johnson & Johnson Ortho-Centocor: Honoraria; Millennium, Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Millennium, Celgene, Centocor-Ortho Biotech: Speakers Bureau.

Unimolecular metastable decomposition of α-cyclodextrin cations is observed in a fast-atom bombardment Fourier transform mass spectrometry instrument. Cleavage reactions occur at the glycosidic bonds to produce oligomeric fragment ions. The first-order rate constant for the decay of the protonated parent was measured and found to be (4.2 ± 1.0) x 102 s−1.