Citations

Abstract Background: For most patients with MDS, the disease-associated molecular abnormalities are unknown, which contributes to diagnostic uncertainty and has limited development of effective therapies. Chromosomal deletions such as del(5q) or monosomy 7 are more common than translocations in MDS, but balanced translocations are more likely to be pathobiologically informative. Here we report cloning of an MDS-associated translocation that revealed a novel recurrent molecular abnormality. Methods: We first studied a 71-year old man who presented with MDS and t(6;9)(p21.3;q34) as an isolated chromosomal abnormality in 18 of 20 metaphases. FISH assays for DEK/CAN and ABL rearrangements were unrevealing. We generated somatic hybrid murine/human cell lines from a buffy coat; microdissected and amplified derivative chromosomes; and hybridized to 6p/9q custom comparative genomic hybridization (CGH) arrays. The CGH results considerably narrowed the 6p and 9q breakpoints, which were then amplified by long-range PCR and sequenced to define the rearrangement. Results: The IER3 gene (Immediate Early Response 3; also known as IEX-1) at 6p21.3 was found to be separated from its upstream regulatory elements in this rearrangement and translocated to a transcript-poor region of 9q. RT-PCR confirmed marked downregulation of IER3 expression in the patient compared to healthy controls. IER3 is a plausible candidate for involvement in MDS because of its known role in regulating death receptor-induced apoptosis, interaction with NF-κb pathways, and its importance in response to genotoxic stresses such as ionizing irradiation. We then identified archival bone marrow cell pellets from 204 additional patients with various clonal hematological disorders and chromosomal rearrangements involving 6p21 or 6p22 (i.e., translocations, inversions, deletions, or additions), and designed a splitsignal FISH probe set to assay IER3. FISH results were abnormal in 8 additional patients (9/205 pts total abnormal, 4.4%): 3 split signals and 6 amplifications (one patient had both abnormalities); all 8 patients had MDS and 6p rearrangements as part of a more complex karyotype. FISH studies in MDS patients without 6p rearrangements are ongoing. RTPCR in MDS patients without 6p rearrangements demonstrated down-regulation of IER3 by >2-fold in 38% of patients (67% lower-risk) and up-regulation in 12% of patients (50% higher-risk) when compared to the mean for healthy controls. Direct sequencing of the IER3 coding region and the promoter region in 30 patients without 6p rearrangements revealed no point mutations, but the prevalence of non-synonymous single nucleotide polymorphism rs3094124 (p.A127P) was higher than expected in patients (variant in 28.3 % of MDS alleles vs. 8.6% of 116 HAP-CEU controls, p=0.0015). IER3−/− mice are known to have hypertension, cardiac hypertrophy, and epithelial proliferation, but are not anemic (Sommer SL et al J Appl Physiol 2006). We performed methylcellulose hematopoietic colony growth assays using bone marrow mononuclear cells from IER3−/− mice and wildtype controls, and noted no differences in BFU-E, CFU-E, and CFU-GM growth under unstressed conditions. Studies of IER3 expression induction under stressed conditions in primary MDS cells are ongoing. Conclusion: IER3 rearrangements represent a novel clonally-restricted recurrent genetic abnormality in MDS. Dysregulation of IER3 expression is common in MDS, even in patients without 6p rearrangements. This is the first time IER3 has been linked to human disease. Clarification of the role of IER3 in MDS is likely to yield new insights into MDS pathobiology.

Abstract Multiple Myeloma (MM) is the second most prevalent haematological disorder in the US with great recent progress propelled by various technological and therapeutic advances. Nevertheless, the complexity of MM genomes remains inadequately characterized. We have combined high-resolution (44K Agilent) array comparative genomic hybridization (aCGH) and gene expression data with clinical outcomes in an initial attempt at indexing genomic complexity in MM and its clinical implication in 100 MM patients. We enumerated the Number of Aberrations per Tumor (NAPT) as defined by the ADM-1 algorithm available within CGH Analytics. There appear to be no difference in degree of genetic complexity between hyperdiploid and non-hyperdiploid myeloma. Generally, t(11;14) have low NAPT whereas D2 tumors have high NAPT. Amongst the high-risk genetic subtypes, tumors with maf translocations have relatively low NAPT whereas t(4;14) have high NAPT. Whilst a Multivariate Cox Proportional Hazards regression of survival outcome showed little statistical support for popular indicators such as ploidy and TC classification, results for NAPT were considerably more encouraging (p~ 0.07, median survival of 40 months in low complexity vs 18 months in high). Log-rank tests further supported prognosis based on NAPT independent of clinical status and ploidy. To characterize complexity, we clustered aCGH profiles by clinical status, ploidy and TC classification. Clear patterns of aggregation and recurrent aberration were observed suggesting that they were possibly instrument to aberrant gene expression. Indeed, expression of a large proportion of genes approximately 2Mb of the recurrent breakpoints varied significantly between patients carrying aberrations vis-a-viz others that did not. Comparison against known gene sets showed an enrichment for cell-cycle and apoptosis genes. We are presently characterizing several such breakpoint-associated genes for their functional significance.

Abstract Background: The combination of lenalidomide and dexamethasone (Rev/Dex) has been shown to be a highly effective therapy for multiple myeloma (MM). Over half of the patients with newly diagnosed MM achieve a complete response with prolonged therapy. More recently, a phase III trial demonstrated improved survival with use of lenalidomide and low dose dexamethasone with one year survival in excess of 90%. We report the initial results from a phase II trial combining lenalidomide and low dose dexamethasone with cyclophosphamide (CRd) as initial therapy of newly diagnosed MM. Methods: The trial was initiated in July 2006 and completed the target accrual of 33 patients by July 2007. The treatment protocol consisted of lenalidomide given orally at a dose of 25 mg daily on days 1–21 of a 28-day cycle. Dexamethasone (dex) was given orally at a dose of 40 mg on days 1, 8, 15, and 22 of each cycle. Cyclophosphamide at a dose of 300 mg/m2 was given on days 1, 8, and 15 of each cycle. Patients also received an aspirin once daily as thromboprophylaxis. Response was defined as a decrease in serum monoclonal (M) protein by 50% or higher and a decrease in urine M protein by at least 90% or to a level less than 200 mg/24 hours, confirmed at least 4 weeks apart. Results: The median age was 63 years (range, 44–79). Eight patients (24%) had ISS stage III disease. At this time, 19 of the 33 patients are evaluable for confirmed responses (i.e., off-study or completed at least 4 cycles of therapy). Of these, 2 achieved VGPR and 13 had a partial response giving an overall response rate of 79%. The response rate was affected by 5 of 19 patients who went off study within three cycles due to toxicities [interstitial nephritis (1 pt), multiple grade 3 toxicities including infection (1 pt) atrial fibrillation and infection (1 pt)] or alternative treatment [no response and possible renal toxicity (1 pt) and progression at 4 cycles (1 pt)]. Overall, hematological toxicity was the most common with grade 4 toxicity seen in 6 patients (20%). Non-hematological grade 3 or higher toxicities included fatigue (4 pts), thrombosis (3 pts) and renal failure (2 pts). One patient with thrombosis came off study for other toxicities, and the other two continued on study with anticoagulation. Thirteen patients (43%) had dose reductions of both cyclophosphamide and lenalidomide, most commonly due to hematological toxicity. So far, 12 patients have gone off study, 6 went to stem cell transplant, 3 due to adverse events, 1 due to disease progression and 2 patients went to alternate treatment. Conclusions: CRd has excellent activity in newly diagnosed myeloma with an estimated overall response rate of 79%. Response evaluation of the entire 33 patients will be presented at the meeting. We believe that the initial use of a 300 mg/m2 dose of cyclophosphamide resulted in 5 of the first 19 pts experiencing early toxicity and withdrawal. As a result, an expansion of the current trial is evaluating lower doses of cyclophosphamide (300 mg fixed dose). Full analysis of this study will be needed to determine if CRd can improve outcome compared to Rd.

Abstract The retinoblastoma 1 (RB1) gene is a frequent target of mutation or inactivation in neoplastic diseases. However, the current model for RB1-mediated malignancy requires inactivation of both alleles for tumor progression. Single–copy loss of RB1 has been shown to correlate with an approximate 50% reduction in RB1 mRNA expression in patients diagnosed with multiple myeloma and survival in this cohort of patients is significantly reduced. Using siRNA to reduce Rb protein levels to 50% and Rb-expressing adenovirus to restore RB1, variations in proliferation and cell cycle were observed. The KMS–11 adherent cell line with normal Rb protein expression was labeled with carboxyfluorescein succinimidyl ester (CFSE) for tracking of cellular division and RB1 siRNA added to silence 50% of protein expression. Transfection efficiency for siRNA was measured at 95% or greater. Flow cytometric analysis was performed at 72 hours to identify changes in proliferation between controls and Rb knock–down. Using CFSE proliferation analysis software, reduction of Rb protein levels by 50% caused an average increase in the proliferation index from 2.05 to 2.50. Additionally, reduction of Rb protein levels caused an increase in the percentage of cells in S–phase, from 24 to 29%. CFSE and cell cycle experiments were performed in triplicate. Conversely, addition of Rb–expressing adenovirus to MM.1R and U266 cell lines, with mono-allelic and bi-allelic loss of RB1 respectively, resulted in a decrease in proliferation assayed by CFSE and an average decrease in the percentage of cells in S-phase from 29 to 23% compared to controls. No changes in proliferation or cell cycle compared to untreated controls were observed following null adenovirus infection. Western blot and quantitative RT–PCR (qPCR) were used to confirm reduction or addition of RB1 to cell lines. Further, qPCR was used to identify potential activation of the interferon response following addition of siRNA and adenovirus. No changes in annexin V expression were observed following reduction or replacement of Rb as compared to controls. These results suggest the addition of proliferation advantages to tumors with single copy loss of RB1. For malignancies such as multiple myeloma characterized by deletion of 13q14 coupled with an extended period of development, the proliferation advantage associated with RB1 haploinsufficiency may contribute to decreased survival in the deletion 13 cohort.

Abstract Background: We previously reported an 85% response rate with 54% CR/nCR using a combination of cyclophosphamide (Cy), prednisone, and bortezomib (BZ) in relapsed myeloma (MM). We now report a Phase II clinical trial using a combination of Cy, BZ, and dexamethasone (Dex) [Cybor-D] in newly-diagnosed MM. We then compare the activity of this combination with historical controls treated with our current induction standard of Lenalidomide-Dexamethasone (L-Dex). Methods: 30 patients have been enrolled on Cybor-D. Treatment consists of Cy 300mg/m2 p.o. days 1, 8, 15, and 22; BZ 1.3 mg/m2 days 1, 4, 8, and 11, and Dex 40mg p.o. days 1–4, 9–12, 17–20. Prophylactic use of acyclovir, a quinolone and antifungal prophylaxis was highly recommended for all patients on study. Growth factors were allowed. As a relevant contemporaneous control for speed and depth of response, we used 33 patients treated on a recent Mayo Clinic trial of L-Dex (Rajkumar. Blood: 2005 106;4050–3). Response was defined according to IMWG criteria although in the current trial, bone marrows for confirmation of CR are not yet available on all patients. Results: At time of current analysis, 17 patients on Cybor-D were evaluable through at least one cycle for response and toxicity. Baseline characteristics included mean age of 57, 29% female, 41/24% ISS stage II/III. There were statistically more early stage patients on L-Dex; otherwise, baseline patient characteristics were similar. The mean % decline in monoclonal (M) protein from baseline following cycles 1–4 of Cybor-D were 71%, 90%, 93%, 96%. This compares favorably with L-Dex where equivalent % decline in M proteins were 66%, 77%, 80%, and 79%. Thus, in preliminary results, Cybor-D produces a more rapid initial decline and mean % reduction in M protein than L-Dex. Overall response rate (≥ PR) after one cycle of Cybor-D was 77% with 36% obtaining ≥VGPR (L-Dex 73% and 18% respectively). The % of patients obtaining ≥VGPR after each cycle was 36%, 64%, 77%, and 100% for Cybor-D as compared to 18%, 36%, 42%, and 33% for L-Dex. The overall ≥VGPR at the end of four cycles of Cybor-D is currently100% (≥ 66%nCR using older nomeclature). Major toxicities ≥ grade 3 for Cybor-D included hematologic (37%), hyperglycemia (15%) and neuropathy (11%). Conclusions: In summary, for newly-diagnosed MM patients, four cycles of Cybor-D produces more frequent, rapid and deep responses when contrasted with historical L-Dex controls. Further confirmation of response rates and effect on TTP will be required.

Abstract Constitutive activation of NF-κB signaling pathways has been identified in a variety of malignancies, however the molecular basis has remained largely unknown. Recently, we have shown mutations in 10 genes encoding positive and negative regulators of NF-κB signaling pathways in 20% of multiple myeloma (MM) patients, with TRAF3 being the most commonly affected gene. TRAF3 is a negative regulator of NF-κB signal and its inactivation results in the constitutive activation of the alternative or noncanonical NF-κB signaling pathway. With the exception of two recent studies in MM, there is no available data regarding the mechanisms that dysregulate NF-κB in other B-cell malignancies. The aim of this study was to analyze the TRAF3 status in a cohort of Waldenström’s macroglobulinemia (WM) patients. Forty-nine WM were analyzed by using a combination of array-based comparative genomic hybridization (aCGH) and cIgM-FISH. Genome sequencing from 28 of these patients was performed on all coding exons of TRAF3 and the adjacent intron-exon junctions. A NF-κB index, as a measure of NF-κB transcription activity, was calculated from 21 patients with gene expression data. The index is the mean expression level of four probe sets differentially expressed between human myeloma cell lines with identified mutations compared to those without identified NF-κB pathway mutations (CD74, IL2RG and 2x TNFAIP3). By performing aCGH we identified a small monoallelic deletion (∼460 Kb), including the TRAF3 locus in one of 22 patients. Genome sequencing identified a missense mutation (D483N) in the remaining allele. This substitution affects a highly conserved amino acid throughout vertebrate evolution, suggesting its functional relevance. We used cIgM-FISH to screen 27 patients with no available aCGH data and we found one biallelic TRAF3 deletion and one chromosome 14 monosomy. In the later case, no DNA was available to sequence the remaining allele. Both samples with biallelic inactivation of TRAF3 possess the two highest NFKB index values of our WM cohort, suggesting NF-κB transcription activity. Moreover, the case with biallelic deletion showed the lowest level of TRAF3 gene expression. Additionally, the entire TRAF3 coding region was sequenced in 28 of these 49 patients and one homozygous nonsense mutation (K286X) was identified. No GEP was available in this sample to calculate the NF-κB index. TRAF3 was only recently implicated in a malignant process, when inactivating mutations and biallelic deletions were reported in approximately 13% of MM patients, making it the most frequently inactivated tumor suppressor in MM. Our study has identified biallelic TRAF3 inactivation in at least 3 of 49 WM patients, suggesting that the constitutive activation of NF-κB signaling pathways may be involved in the pathogenesis of WM. Recent findings showing an association between TRAF3 loss of function and good response to bortezomib in MM patients suggest that proteasome inhibitor-based therapy may be an attractive option in the treatment of the WM patients with constitutive activation of NF-κB signaling pathways. Additional mutational screening of TRAF3 and other regulators of these pathways are ongoing to determine the extent of constitutive activation present in WM and other B-cell malignancies.

Abstract p90 Ribosomal S6 Kinase 2 (p90RSK2) is a substrate of ERK and plays an active role in anti-apoptosis signaling and cell cycle regulation. Our proteomics studies revealed that p90RSK2 is not only activated but also tyrosine-phosphorylated in hematopoietic cells expressing constitutively activated leukemogenic FGFR3 mutants. FGFR3 is a receptor-tyrosine kinase that responds to fibroblast growth factor (FGF). Recent studies have suggested that FGFR3 may play a significant role in the pathogenesis and disease progression of some hematopoietic malignancies including t(4;14)(p16.3;q32.3) multiple myeloma, which is associated with dysregulation of FGFR3. We found that the MEK1 inhibitor U0126 effectively inhibited RSK2 activation induced by FGFR3, suggesting that FGFR3 activates RSK2 through the MEK/ERK pathway. Interestingly, FGFR3-dependent tyrosine phosphorylation at Y529 was required for phosphorylation and activation of the Ser/Thr kinase RSK2 by ERK. By using a specific phospho-RSK2 (Y529) antibody, we identified FGFR3 as the direct tyrosine kinase that phosphorylates RSK2 at Y529 in an in vitro kinase assay, which only contains purified recombinant active FGFR3 and RSK2 proteins. Substitution of Y529 in RSK2 attenuates inactive ERK binding to RSK2, which is prior to and required for ERK-dependent phosphorylation and activation of RSK2. In addition, reconstitution of RSK2 phosphorylation at Y529 consequently enhanced inactive ERK binding by a GST pull down, suggesting that phosphorylation at Y529 by FGFR3 facilitates ERK binding to RSK2, which is a sequential event and precedes inactive ERK binding. These observations support a novel two-step model that FGFR3 activates RSK2 by both assisting inactive ERK binding via tyrosine phosphorylation of RSK2 at Y529 and activating the MEK/ERK pathway. Moreover, we observed that RSK2 is specifically phosphorylated at Y529 and activated in FGFR3 expressing various human t(4;14) positive MM cell lines. Targeted down-regulation of RSK2 by siRNA induced significant apoptosis in FGFR3-expressing human myeloma cell lines including OPM1 and KMS11. In addition, a specific RSK inhibitor fmk induced significant apoptosis in t(4;14) positive human myeloma cells including KMS11, OPM1, LP1 and KMS18, but not in control t(4;14) negative myeloma cell lines including ANBL6 and U266. Similar results were obtained by using another RSK specific and potent inhibitor BI-D1870. Furthermore, we observed that fmk induced significant apoptosis in primary CD138-positive, FGFR3-expressing myeloma cells from a t(4;14) positive multiple myeloma patient, but not in the control CD138-negative cells from the same patient, nor primary samples from a t(4;14)-negative patient as a control. Together, these data not only support our model that RSK2 is a critical signaling effector of FGFR3 in hematopoietic transformation, but also provide “proof of principle” for the therapeutic potential of RSK2 inhibitors such as fmk in treatment of t(4;14) positive, FGFR3-expressing multiple myeloma with minimal non-specific cytotoxicity.

Abstract The availability of human myeloma cell lines (HMCLs) has drastically benefited out understanding of multiple myeloma (MM). They represent essential resources to the myeloma research community at large who use them to identify and more importantly validate oncogenic events in MM. Furthermore, they represent the front line tools in efforts to identify novel therapeutic agents for MM patients. However, the myeloma research community is not immune to problems of cell line contamination (mixed populations) or systemic errors where HMCLs are labeled incorrectly. As the community evolves towards the development of tailor medicine it is essential that our basic tools are both pure and correct. Otherwise, a potentially beneficial therapeutic agent designed for a specific patient subset maybe lost simply because of a simple laboratory mix-up at some point over the years. To that end we have developed a simple PCR based method that can be used to validate the purity and authenticity of HMCLs. First two reference collections of over 50 HMCL maintained at the NCI and Mayo Clinic Arizona were compared using microsatellite fingerprinting. This process identified several previously unnoticed discrepancies. First, in both labs several cell lines that reportedly originated from different patients had the same fingerprints. These include the following unexpected pairs; ANBL-6 and DP–6, CAG and ARP–1, KMS–11 and KMS–20, KMS–11 and KMS–21PE/BM, JIM3 and KPMM2. Second, both KP–6 and KAS6/1 showed a level of cross-contamination with ANBL–6. All of the discrepant cell lines were requested from the original contributor and those that were available were retested. After this step we had identified unique fingerprints for ANBL–6, DP–6, KMS–11, and KMS–20. Furthermore, pure populations of KP–6 and KAS6/1 were confirmed. However, CAG and ARP–1 still had an identical fingerprint. Since the use of microsatellite fingerprinting is an expensive task and the identification of cross-contamination is not very robust we set out to develop better method. We decided to use a simple qualitative PCR method, as the binomial presence or absence of a band makes the interpretation of the fingerprint extremely easy. Furthermore, the high sensitivity of a qualitative PCR can pick up a small amount of contamination in the culture that can be lost in microsatellite analysis. This method involves a multiplex PCR screen that simultaneously interrogates 10 regions of the human genome. The screen is divided into two reactions, one with a panel of highly polymorphic copy number variations (CNV) that are often completely absent in some individuals, and a second one based on known biologic events present in the cell lines. The integration of the results from the CNV and Biological Fingerprints will identify each HMCL as a unique entity. We validated the methodology by confirming that this qualitative screen could pick up the cross–contamination identified in the original KP–6 and KAS6/1 samples. As our understanding of myeloma increases and we strive to develop new therapies for specific biological entities it will be essential that groups confirm both the authenticity and purity of their test cultures. We hope that widespread use of this simple screening methodology will significantly improve the quality and reproducibility of assays carried out in labs around the world.

Abstract Genetic abnormalities are important in the pathobiology of myeloma and established prognostic factors. High-density aCGH allow global unbiased detection of genomic gains and losses. In this study, we attempt to identify survival critical genomic aberrations by correlating aCGH-defined abnormalities with survival in 2 different cohorts of patients. aCGH was performed in 64 MM patients treated with high dose therapy from the Mayo Clinic using Agilent’s 44k DNA microarray genechip. In addition, a publicly available aCGH dataset performed in 67 MM patients treated with total therapy from the University of Arkansas Medical School using the lower density Agilent 22k chip (Human 1A v2) was also analyzed. Base 2 logarithms of the ratios between red and green intensities (log-ratios) were treated as a primary measure of copy number ratio between tumor and normal samples. To detect the regions of genomic aberration, values of a sliding window of 15 consecutive log-ratios were compared with those of an artificial ‘reference’ chromosome using the Wilcoxon rank sum test. The reference chromosome, constructed for each sample, was assembled by collecting log-ratio values close to zero. For this, 15 randomly sampled log-ratios from autosomal regions whose p-value of Wilcoxon signed rank test against 0 was larger than 0.05 were recursively accepted until the number of log-ratios in the assembled reference chromosome reached 1000. The p-values and their corresponding false discovery rates (q-values) for the copy number gain or loss were calculated independently for each sliding window and assigned to the center position of corresponding sliding window. The survival difference was examined between samples that had gained or lost a chromosomal region (sliding window) and samples that had not using the method of Kaplan-Meier and the statistical significance was assessed by the log-rank test. The sliding window was identified as gained or lost if P-value < 10 −4 and Q-value < 10[suo]-3. Sliding windows whose gains or losses led to poor survival with log-rank test p-value < 0.5, corresponding q-value < 0.5, and happened to ≥ 10% of all samples were retained. Several regions of genomic gains and losses were significantly associated with poorer survival with 5 deletion hotspots common between the 2 datasets (Table). We validated the prognostic significance of these survival critical loci using FISH in an independent dataset of 169 patients treated with high dose therapy. Clustering of these survival critical loci revealed clusters of MM characterized by low, intermediate or high number of these abnormalities, which are independent of ploidy and translocations subtypes. These subgroups have significantly different survival. In addition, the clustering analysis suggests the chromosome 13 loss is an early event whereas chromosome 1 abnormalities are late events. Our strategy therefore identified novel regions of genomic aberration that are important prognostically. However, it is likely that these regions are manifestation of genomically unstable tumors with complex abnormalities that have poorer prognosis. Chr Start End Length Cytoband 1 74,137,824 75,972,544 1,834,720 p31.1 1 62,433,123 84,792,809 22,359,686 p31.3 – p22.3 20 4,100,452 6,189,410 2,088,958 p13 – p12.3 20 6,717,170 13,142,205 6,425,035 p12.3 – p12.1 20 4,708,668 10,566,333 5,857,665 p13 – p12.2

Abstract Events mediating transformation from the pre-malignant MGUS to MM is largely unknown. Previous studies have highlighted RAS mutation as a progression event. However, this abnormality is present in only a small subset of patients. Global gene expression differences between MGUS and MM have been reported but provided little coherent biological insights. To define progression events, we examined the differences in gene expression profiles (GEP, Affymetrix U133A genechip) between 101 MM and 22 MGUS using a powerful functional genomic tool, Gene Set Enrichment Analysis (Subramanium et al PNAS 2005), that allow extraction of biological information based on enrichment of genes associated with a phenotype within gene-sets derived experimentally or extracted from database. We found that a predominant theme relating to MM progression is the over-expression of proliferation related genes in MM highlighted by the enrichment of cell cycle related gene-sets. Another common theme is the enrichment of gene-sets relating to MYC activation. Seven of eight gene-sets related to MYC activation in the database are significantly enriched (p-value <0.05 and false detection rate <0.05). Leading edge (enriched genes) analysis of enriched MYC-related and proliferation-related gene-sets revealed a core, overlapping signature of 8 genes specific to MYC and not proliferation. These genes are further verified as MYC transcriptional targets using Metacore, a network analysis tool. In addition, 7 of the 8 genes had been validated as MYC target genes by chromatin immunoprecipitation previously (www.myccancergene.org). We validated this signature in independent publicly available cell lines and mouse gene expression experiments where this signature is present in mammary epithelial cell lines engineered to over-express MYC but not E2F3, SRC and BCAT, and mouse embryonic fibroblast over-expressing MYC but not HER2 or with RB knocked-out. In addition, analysis of proliferation index in these datasets showed that there is dissociation of proliferation from this signature. This showed conclusively that our signature is MYC specific and not related to proliferation. Of interest, the signature is also present in cell lines with activating RAS mutation, which also have high expression of MYC, suggesting that MYC activation is a downstream effect of activated RAS signaling. Indeed, in our patient cohort, most patients with RAS mutation express this signature. When applied to 2 large GEP MM patient cohorts, this signature was not present in normal plasma cells, rarely present in MGUS, and present in a significant proportion of MM and almost all the human myeloma cell lines. When analyzed in the context of other B-cell malignancies, the signature is very strong in Burkitt’s lymphoma, present in most myeloma but absent in chronic lymphocytic leukemia, Waldenstrom Macroglobulinemia, and normal B-cells. Our analysis therefore suggests that MYC activation may be a unifying pathological event in MGUS to MM progression. As MYC translocations occur in only about 13% of newly diagnosed MM patients, activating RAS mutations and other trans-mechanisms may mediate MYC activation. Our recent demonstration that sporadic MYC activation in a mouse strain prone to monoclonal gammopathy led to MM development provides important functional proof.