Citations

Abstract BCMA/CD3-targeting bispecific antibodies (BsAb) are a recently developed immunotherapy class that shows potent tumor killing activity in multiple myeloma. Here, we investigated a murine BCMA/CD3-targeting BsAb in the immunocompetent Vk*MYC model and its immunomodulatory imide drug (IMiD)–sensitive derivative Vk*MYChCRBN model of multiple myeloma. The BCMA/CD3 BsAb was safe and efficacious in a subset of mice but failed in those with high tumor burden, consistent with clinical reports of BsAb in leukemia. The combination of BCMA/CD3 BsAb with pomalidomide expanded lytic T cells and improved activity even in IMiD-resistant high–tumor burden cases. Yet, survival was only marginally extended due to acute toxicity and T-cell exhaustion, which impaired T-cell persistence. In contrast, the combination with cyclophosphamide was safe and allowed for a tempered proinflammatory response associated with long-lasting complete remission. Concurrent cytotoxic therapy with BsAb actually improved T-cell persistence and function, offering a promising approach to patients with a large tumor burden. Significance: BCMA-targeted therapy induces deep but transient clinical responses. We developed an immunocompetent, IMiD-sensitive genetically engineered mouse model and show that IMiDs potentiate T-cell activation, increasing short-term efficacy of anti-BCMA/CD3 BsAb, but exacerbate T-cell exhaustion. Surprisingly, by reducing tumor burden and depleting regulatory T cells, cyclophosphamide prevents BsAb-induced T-cell exhaustion and promotes long-term multiple myeloma control. See related commentary by Louvet et al., p. 297.

Deregulated transcription and cell cycle control are hallmarks of cancer that are especially frequent in multiple myeloma (MM). Largely non-overlapping sets of cyclin-dependent kinases (CDKs) regulate cell division and RNA polymerase II (Pol II)-dependent transcription; and targeting of cell cycle CDKs has been long pursued as an attractive therapeutic strategy. Among CDKs, CDK7 presents a unique therapeutic opportunity as it functions as a CDK activating kinase (CAK), licensing the activity of cell cycle CDKs, and also serves as a core component of the general transcription factor TFIIH. Here we elucidated the biological role of CDK7 and its transcriptional regulatory landscape in MM, using genetic as well chemical approaches, including tools for CDK7 rapid protein degradation (dTAG) and the selective covalent inhibitor YKL-5-124 that targets a cysteine residue (C312) located outside of the kinase domain. We have observed that CDK7 inhibition via YKL-5-124 robustly inhibited the phosphorylation of the CDK1, 2 and 4 activation loops in a representative panel of MM cell lines at concentrations as low as 50 nM. This reduction was not observed in MM cells expressing a resistant mutation in the reactive cysteine (C312S). Consistent with decrease of CAK activity, we observed G1 arrest and S phase loss after CDK7 inhibition, which was also associated with a rapid and transient loss of Ser2 and Ser5 phosphorylation of the RNA Pol2 C-terminal domain. To understand the effect of CDK7 inhibition on MM cell growth and viability, we evaluated activity of YKL-5-124 across a large panel of 25 MM cell lines and observed a significant inhibition of MM cell proliferation, with a significantly lower IC50 compared to PHA-activated normal donor peripheral blood mononuclear cells (PBMCs), suggesting a specific sensitivity of MM cells to CDK7 inhibition. Longer exposure to YKL-5-124 caused apoptotic cell death in MM cells; however treatment with an inactive analog or in cells expressing the C312S mutation failed to inhibit MM cell proliferation, confirming that the antiproliferative potency of YKL-5-124 resides in its unique characteristic to covalently bind to C312 domain. Importantly, CDK7 inhibition impaired primary MM cells proliferation alone and when cultured in the presence of BM microenvironment. Selective pharmacological degradation of endogenously tagged CDK7 confirmed impact of CDK7 inhibition on MM cell proliferation via inhibition of CDK7 transcriptional and cell cycle activities. To complement the pharmacological studies, we have established MM cells to express inducible CRISPR/Cas9 constructs encoding 4 independent small guide RNAs targeting CDK7, resulting in the reduction of the abundance of CDK7 protein by 20-60% which was sufficient to inhibit MM cell viability over time, phenocopying pharmacologic inhibition of CDK7. These results support the view that CDK7 is a pharmacologically relevant target for MM. Gene expression analysis after CDK7 inhibition in MM1S and H929 cells revealed that transcripts for only a subset of genes were substantially affected by treatment with low dose of YKL-5-124, showing a strong leading-edge enrichment for downregulation of E2F expression program, cell cycle, DNA damage, and MYC targets. We have indeed confirmed a potent reduction in phosphorylation of RB protein, with consequent decrease of E2F activity in MM cells confirmed using E2F-driven luciferase reporter. These data suggest significant role for CDK7 in the CDK-pRB-E2F pathway in MM, which was strengthened by the observation of a positive correlation between expression of CDK7 and expression of E2F target genes in primary MM cells (n=409). Finally, we have evaluated the in vivo effect of CDK7 inhibition in several murine models of human MM. In the localized subcutaneous model, and the disseminated MM model where treatment with YKL-5-124 decreased tumor burden and improved survival. The effect of CDK7 inhibition explored in an aggressive, genetically engineered model of Myc-dependent MM, revealed evidence of response by decline in measurement of monotypic serum immunoglobulins. In conclusion, our study demonstrates that CDK7 contributes to the 'transcriptional addiction' and the cell cycle deregulation frequently observed in MM and represents an attractive molecular vulnerability to be exploited therapeutically. Disclosures Anderson: Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Oncopep and C4 Therapeutics.: Other: Scientific Founder of Oncopep and C4 Therapeutics.; Celgene: Membership on an entity's Board of Directors or advisory committees. Munshi:Takeda: Consultancy; Karyopharm: Consultancy; AbbVie: Consultancy; Amgen: Consultancy; Legend: Consultancy; Adaptive: Consultancy; Janssen: Consultancy; C4: Current equity holder in private company; OncoPep: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; BMS: Consultancy. Fulciniti:NIH: Research Funding.

Abstract BCMA is an ideal target antigen for myeloma immunotherapy as it is specifically expressed by plasma cells and T cell-engaging, BCMA-bispecific antibodies show potent tumor killing activity in human multiple myeloma (MM). However, a lack of longevity, potential immunotoxicity, and efficacy in high tumor burden situations are limitations to effective clinical utility. In order to optimize this therapeutic approach, we performed in vitro and in vivo assessments of a BCMA bispecific tool using the Vk*MYC immunocompetent mouse model of MM. Analysis at gene and protein level revealed variable levels of BCMA in Vk*MYC tumors, mirroring the heterogeneity of BCMA observed in human MM. In addition, we found that treatment with a gamma secretase inhibitor enhanced BCMA expression in Vk*MYC MM cells, similar to observations from patients. Having found the Vk*MYC model as a suitable model for BCMA targeted therapy, we treated Vk*MYC de novo and transplant mice with the BCMA targeting tool and established that the treatment was safe and efficacious (significant reduction in M-spike levels compared to control antibody). However, the response was transient and modest activity was noted in high tumor burden setting, likely because of poor T cell infiltration. Therefore, we hypothesized that combining BCMA therapy with agents capable of debulking the tumors would prolong and deepen therapeutic benefits and thus selected IMiDs for their anti-tumor and immunomodulatory properties. Because mice are resistant to therapy with IMiDs, we generated a humanized transgenic mouse strain with robust human CRBN expression and crossed it with the Vk*MYC mouse to obtain a novel immunocompetent, IMiD-responsive model, Vk*MYChCRBN. When combined with the IMiD pomalidomide (POM), bispecific-directed T cell control of MM was enhanced relative to bispecific alone. M-spike levels in two different transplantable models were reduced regardless of tumor burden. In addition, POM increased T cell effector activation, proliferation, and cytokine production. We also monitored transient weight loss unique to the bispecific-POM combination. Following this promising combinatorial result in vivo, we studied the relative roles of POM on target vs. effector cells by an in vitro killing assay using Vk*MYC tumor and splenocytes with or without expression of CRBN. Separately from the direct tumoricidal effects of POM on the tumor, we observed T cell-intrinsic effects of POM on the enhancement of BCMA-bispecific directed tumor lysis that correlated with T cell activation and proliferation. In summary, POM potentiated BCMA-CD3 bispecific directed T cell cytolysis of MM in the Vk*MYChCRBN animal model by enhancing effector cell activation, proliferation, cytokine production and directly reducing tumor burden. These results support ongoing preclinical studies of the benefits of combining standard of care therapy with potent T cell immunotherapy for MM. Citation Format: Erin W. Meermeier, Meaghen E. Sharik, Seth J. Welsh, Caleb K. Stein, Victoria M. Garbitt, Sochilt Brown, Kennedi T. Todd, Marco Bergsagel, P. Leif Bergsagel, Marta Chesi. Anti-BCMA bispecific tool therapy against Vk*MYC multiple myeloma is enhanced by IMiDs [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5630.

Abstract Previously, the IAP antagonist LCL161 was shown to have robust anti-myeloma activity in a transgenic mouse model where an acute inflammatory response accompanied a reduction in tumor burden. In order to fully examine alterations to the immune microenvironment, murine splenocytes from twenty mice transplanted with Vk12598 myeloma and administered single-agent treatment regimens of LCL161, cyclophosphamide, or bortezomib were harvested after 24 hours and subjected to single cell RNA-seq sequencing. After initial removal of erythrocytic and mitochondrial expressing cells, count matrices of over 50,000 total cells span twenty murine whole spleen samples. In an examination of the myeloid component, we identified 13 clusters of immune cells with unique compositions across samples due to differing treatment regimens and initial tumor burden. While minimally present in all seven non-transplanted mice, macrophages with elevated expression of complement activation system genes (C1q) were the most abundant myeloid cell population in each of our untreated, Vk12598 transplanted mice. Alternatively, populations of cDC2, pDC, moDC, and monocytes lacking expression of MHC class II markers were fixtures for healthy controls while markedly less abundant in tumor-bearing mice. Tumor burdens in three samples subjected to LCL161 therapy were significantly reduced 24-hours post treatment and accompanied by an expansion of late-stage neutrophils and migratory DCs with expression features mirroring those of DCs from mesenteric lymph nodes. This composition of immune cells was distinct from all other sample including those treated with cyclophosphamide or bortezomib. In addition, relative proportions of B, T, and NK cells were nearly fully reconstituted, matching levels observed in healthy controls, in two of these samples. In a direct functional assessment, expression levels of Fscn1 and Ccr7 (key over-expressed genes in these migratory DCs) markedly increased following treatment of bone marrow derived DCs with LCL161. Overall, the transcriptional profile of these migratory DCs indicates their pivotal role in maturation of DCs (Fscn1, Tmem123) as well as the recruitment of T and NKT cells (Cxcl16, Ccr7) and increase in antigen presentation (Cd63)–all of which may serve to ‘warm' tumors, increasing T cell infiltration necessary for immunotherapy approaches. With the increased resolution of scRNA-seq, we observe unique alterations in the composition of the immune microenvironment of MM induced by treatment with LCL161 that further detail its unique mechanism of anti-MM activity. Citation Format: Caleb K. Stein, Erin Meermeier, Seth Welsh, Victoria Garbitt, Meaghen Sharik, P. Leif Bergsagel, Nicholas Banovich, Jonathan Keats, Marta Chesi. scRNA-seq reveals restructuring of the immune microenvironment of multiple myeloma following treatment with an IAP antagonist [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5622.

Abstract NRAS Q61 mutations are prevalent in advanced/relapsed multiple myeloma (MM) and correlate with poor patient outcomes. Thus, we generated a novel MM model by conditionally activating expression of endogenous NrasQ61R and an MYC transgene in germinal center (GC) B cells (VQ mice). VQ mice developed a highly malignant MM characterized by a high proliferation index, hyperactivation of extracellular signal-regulated kinase and AKT signaling, impaired hematopoiesis, widespread extramedullary disease, bone lesions, kidney abnormalities, preserved programmed cell death protein 1 and T-cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibition motif domain immune-checkpoint pathways, and expression of human high-risk MM gene signatures. VQ MM mice recapitulate most of the biological and clinical features of human advanced/high-risk MM. These MM phenotypes are serially transplantable in syngeneic recipients. Two MM cell lines were also derived to facilitate future genetic manipulations. Combination therapies based on MEK inhibition significantly prolonged the survival of VQ mice with advanced-stage MM. Our study provides a strong rationale to develop MEK inhibition–based therapies for treating advanced/relapsed MM.

Abstract Over 40% of cases of multiple myeloma (MM) are associated with translocations of the immunoglobulin heavy (IgH) chain gene gene. The t(4;14) translocation, present in ca. 20% of myeloma cases, results in the overexpression of two potential oncogenes, MMSET and FGFR3, via juxtaposition of their endogenous promoters to regulatory elements of the IgH locus. The presence of t(4;14), and MMSET overexpression, is an adverse prognostic factor in MM irrespective of FGFR3 expression. MMSET contains several conserved motifs found in proteins involved in chromatin function (PWWP, HMG, PHD domains) and in the epigenetic control of transcription (SET domain). Accordingly, we found that the two main isoforms of the MMSET protein exhibit exclusive nuclear localization in both transfected fibroblasts and myeloma cells carrying t(4;14). Towards our goal of defining the ability of MMSET to affect gene regulation and contribute to the disease pathogenesis, we found that the SET domain of MMSET possesses in vitro methyltransferase activity specific for core histones H3 and H4. Using a computational approach and theoretical extrapolation from the solved NMR structure of vSET, we identified residues in the active site of MMSET essential for catalysis, whose mutation drastically reduces enzymatic activity. Reporter assays using Gal4 fusion constructs showed that both the amino terminus of MMSET, containing the PWWP and HMG domains, as well as the SET-containing carboxy terminus act as transcriptional repressors. MMSET interacts physically and functionally with a number of known co-repressor molecules, such as HDAC1, HDAC2, Sin3a, and SIRT1, but not HDAC4 or HDAC6. As such, MMSET co-expression enhances HDAC1 and HDAC2-mediated repression in transcriptional reporter assays, and MMSET repression is partially relieved by the addition of an HDAC inhibitor. A yeast two hybrid screen identified a number of other functional partners of MMSET, including ZNF331/RITA (Rearranged in Thyroid Adenoma), a KRAB domain/zinc finger protein previously implicated in malignancy. MMSET and ZNF331 co-localize in the nuclei of transfected fibroblasts, co-immunoprecipitate, and display cooperative repression in reporter assays. Collectively, these data support the idea that MMSET is a biologically active, bifunctional transcriptional mediator acting as a HMT enzyme in chromatin remodeling and as a complex adaptor in the recruitment of repressor species. Presently we are modeling the biological effects of MMSET through a conditional overexpression system in a B cell line. While low levels of MMSET are ubiquitiously expressed, induction of high levels of MMSET expression in the B cell line is associated with growth suppression and G1 arrest. While paradoxical for a presumed oncoprotein, such actions have been observed for other disease-associated proteins such as Runx1/MTG8. In contrast, a myeloma cell line harboring t(4;14) proliferates in the presence of high level MMSET expression. RNAi-mediated knockdown of MMSET in these cells induces apoptotic cell death. This suggests that MMSET may be critical for growth and survival of myeloma cells. Profiling of gene expression changes in these systems should link the transcriptional and biological activities of MMSET.

Abstract Cancer/testis antigens (CTAs), comprising the MAGE, CTAG, SSX and GAGE families, are expressed in different tumor types and restricted in normal tissues to germ cells of the testis. Many CTAs contain epitopes recognized by cytotoxic T cells and are potential targets for active immunotherapy. In MM, different CTAs have been shown to be expressed in cell lines and patient cells, and are associated with more proliferative and advance stage tumors. However, correlation with survival has never been reported. We undertook a global survey of CTA expression using gene expression profiling of CD138-enriched plasma-cell (PC) RNA in 15 normal, 101 MM, 22 MGUS and 24 SMM samples using the Affymetrix U133A chip (Affymetrix, Santa Clara, CA). 22 CTA genes were differentially expressed across the samples and unsupervised clustering using these genes clearly identifies a group of patients with high CTA expression. Most CTAs have significantly higher expression in MM as compared to SMM and especially MGUS, which has similar expression as normal PCs. As these antigens are not expressed in normal PCs, an upper limit of normal expression (ULN) was derived based on mean+3SD of expression in normal PCs for each CTA. A strict cut-off of normalized expression value greater than 2xULN was used to assign positive expression for each CTA genes. Using this method, the most commonly expressed CTAs are MAGE-A3 (73% of MM), MAGE-C1 (45%), MAGE-A6 (35%), SSX-1 (21%), GAGE-5 (18%) and NY-ESO-1 (14%). We then assigned patients with expression of more than 2 of these genes as having a CTA signature and correlated with clinical parameters. 45 patents (41 MM and 4 SMM) have the CTA signature. These patients have a significantly higher PCLI (median 0.2 v 0.7, p=0.0003). In fact, there is a significant correlation between the number of positive CTAs and PCLI (r=0.59, p<0.0001). Interestingly, there is no association between patients with a CTA signature and any of the recurrent genetic abnormalities (IgH translocations, chromosome 13 deletion and TC classes), yet MM patients with this signature have a significantly shorter survival (median 459 days vs not reached, log-rank p=0.017). A prognostic hierarchy exists amongst these CTAs. Only MAGE-A6, MAGE-A12, SSX-1 and SSX-5 expression has significant prognostic implications with the SSX family conferring the worse prognosis. Using a restricted panel of 3 CTAs (SSX-1, -5 and MAGE-A12), about 28% of MM patients expressing one or more of these genes can be identified with poor survival (median less than 1 year) that is independent of genetic subtypes. The genes differentially expressed between patients with and without the CTA signature are mainly involved in cell cycle regulation. Since the expression of this large set of CTAs appears to be under epigenetic control, we speculate that the same epigenetic processes that lead to dysregulation of the cell cycle also cause ectopic expression of CTA genes. As CTA expression is rare in MGUS, epigenetic processes may therefore also mediate transformation from MGUS to MM. These poor prognosis patients represent good candidates for novel therapies targeting the epigenetic machinery.

Abstract Hyperdiploid myeloma (H-MM) affects about 50% of MM. These patients exhibit significant karyotypic and clinical heterogeneity. In this study, we addressed possible biological heterogeneity within H-MM by gene expression profiling of CD138-enriched plasma-cell RNA from 53 H-MM patients using the Affymetrix U133A chip (Affymetrix, Santa Clara, CA). Analysis was performed using GeneSpring 7 (Agilent Technologies, Palo Alto, CA). Unsupervised hierarchical clustering performed using the top 100 most variable genes (generated by ANOVA) identifies 4 putative patient clusters. These clusters were reproduced using principal component analysis. Cluster 1 was characterized by high expression of a large group of cancer testis antigens and mitosis/proliferation related genes (TOP2A, NEK, ASPM, CENPA). Cluster 2 was characterized by high expression of genes involved in IL-6 and HGF signaling (MET, IL6R, SOCS3, SPINT1). Cluster 3 was characterized by high expression of genes involved in TNF/NF-KB signaling and anti-apoptosis (NFKB2, CFLAR, BIRC3, TNFAIP3, RRAS2). Cluster 4 was characterized by high expression of genes involve in angiogenesis (protocadherin, EDNRB). Unsupervised hierarchical clustering using the same set of genes in an independent cohort of H-MM patients (n=60) yielded the same 4 patient clusters over-expressing similar genes. The gene expression profile of these clusters were not driven by differences in trisomies (only about 25% of genes that define the clusters were located on the commonly trisomic chromosomes). Association between the 4 clusters and clinical parameters such as survival, chromosome 13 deletion (Δ13), presence of IgH translocations, PCLI, RAS mutation and p16 methylation was analyzed. Cluster 1 tumors were more proliferative (median PCLI 3.8, p<0.05). Cluster 2 was associated with presence of Δ13 (56%, p<0.05) although none of the genes that defined this cluster falls on chromosome 13. p16 methylation was also less common in cluster 2 (13%, p=0.06). Cluster 3 was associated with low PCLI (median 0.2, P<0.05). Most of the patients with p16 methylation also belonged to this cluster (67%, p=0.06). There was a complete absence of Δ13 in Cluster 4. Patients in the 4 clusters also had significantly different survival (log-rank p=0.002.). The difference in survival was independent of Δ13 since Δ13 has no impact on survival in this patient cohort. The molecular profiles of these subclasses may explain some of these associations. CTA expression is associated with proliferative tumors with poor prognosis. IL-6 is an important survival factor and HGF signaling has been associated with poor survival in MM which may explain the relative poor prognosis of this group. Cluster 3 may have better survival as it is associated with an anti-apoptotic signature rather than proliferative signature. In summary, gene expression profiling identified 4 sub-classes within H-MM (validated on an independent dataset and using different clustering techniques) with distinct clinical and biological associations. The gene signature associated with each sub-classes provide a molecular basis for the different clinical behaviour and further insights into deregulated pathways in H-MM.

Abstract Hyperdiploid (>48 chromosomes) multiple myeloma (H-MM) and high hyperdiploid (>50 chromosomes) acute lymphoblastic leukemia (H-ALL) are characterized by aneuploidy and multiple recurrent trisomies (chromosome 3,5,7,9,11,15,19 for H-MM and chromosomes X,4,6,10,14,17,18,21 for H-ALL). Little is known about the oncogenic events, consequences of the trisomies and reasons for the different recurrent trisomies. In an attempt to answer these questions, we undertook a combined gene expression and network/pathway analysis approach. Gene expression data was generated using the Affymetrix U133A chip (Affymetrix, Santa Clara, Ca) for 53 H-MM and 37 non-hyperdiploid MM (NH-MM) cases using CD138-enriched plasma-cell RNA. Gene expression data using the same chip for ALL was obtained from previous published data (Ross ME et al Blood2004; 104: 3679–3687). Analysis was performed using Genespring 7 (Agilent Technologies, Palo Alto, CA). By comparing the median expression of all genes on each chromosome between H-MM/H-ALL and their non-hyperdiploid counterparts (NH-MM and NH-ALL) for the 23 chromosomes (excluding Y), one can clearly identify the commonly trisomic chromosomes in H-ALL and H-MM. However, the relationship of gene expression was highly variable for H-MM and NH-MM as compared to H-ALL and NH-ALL which tended to have expression ratios close to 1 for the non-trisomic chromosomes. Sixty-nine percent of the differentially expressed genes generated by ANOVA analysis (p<0.001) in H-ALL were on the commonly trisomic chromosomes and were upregulated whereas the corresponding figure in H-MM is 40%. These similarities and differences probably reflect both an overall gene dosage effect and the different complexities of the karyotypes of H-MM and H-ALL compared to NH-MM and NH-ALL respectively (MM karyotypes are more complex, hence difference between H and NH-MM is greater and less confined to the trisomic chromosomes). We next performed network analysis using a curated web-based software (MetaCore, GeneGo Inc, St Joseph, MI) using the 2 sets of differentially expressed genes. Majority of genes differentially expressed in H-MM are involved in mRNA translation/protein synthesis whereas the genes differentially expressed in H-ALL were mainly involved in signal transduction. Therefore the transcriptional program that characterize the difference between H and NH-MM/ALL seem to recapitulate normal cellular function: protein synthesis in the mature secretory plasma cells and signal transduction in response to cytokines in a differentiating early-B cell. However, due to the concurrent deregulation of many genes on these trisomic chromosomes, these and other cellular programs are deregulated resulting in malignant transformation. We also intersected the 2 lists of differentially expressed genes to find genes that are up- or downregulated in both H-MM and H-ALL relative to the NH tumors. Thirteen genes including interferon response genes (TNFSF10, MX1, ZNF185) and transcription factors like RUNX1 were upregulated, whereas 13 genes including a cancer testis antigen gene (MAGED4) were downregulated in both H-MM and H-ALL. These genes may point to common oncogenic mechanisms.

Abstract Hyperdiploid MM (H-MM), characterized by recurrent trisomies constitute about 50% of MM, yet very little is known about its pathogenesis and oncogenic mechanisms. Studies in leukemia and solid tumors have shown gene dosage effect of aneuploidy on gene expression. To determine the possible gene dosage effect and deregulated cellular program in H-MM we undertook a gene expression study of CD138-enriched plasma-cell RNA from 53 hyperdiploid and 37 non-hyperdiploid MM (NH-MM) patients using the Affymetrix U133A chip (Affymetrix, Santa Clara, CA). Gene expression data was analyzed using GeneSpring 7 (Agilent Technologies, Palo Alto, CA). Genes differentially expressed between H-MM and NH-MM were obtained by t-test (p<0.01). The majority of the differentially expressed genes (57%) were under-expressed in H-MM. Genes located on the commonly trisomic chromosomes were mostly (but not always) over-expressed in H-MM and constitute 76% of over-expressed genes. Chromosome 1 contained the most differentially expressed genes (17%) followed by chromosome 12 (9%), and 19 (8%). To examine the relationship of gene copy number to gene expression, we examined the expression of genes on chromosomes 9 and 15 in subjects with 2 copies (15 normal control and 20 NH-MM) and 3 copies (12 H-MM) of each chromosome as detected by interphase FISH. We then derived a ratio of the mean expression of each gene on these chromosomes between patients with 3 copies and 2 copies of the chromosome. If a simple relationship exists between gene expression and gene copy number, one would expect the ratio of expression of most genes on these two chromosomes to be about 1.5 in H-MM compared to NH-MM. However, many genes have ratios either higher than 2 or lower than 0.5. Furthermore, when the heterogeneity of cells with underlying trisomies is taken into consideration by correcting the ratio for the number of cells with trisomies, the actual ratio is always lower than the expected ratio. When the expression of genes on a chromosome was compressed to a median value, this value was always higher in the trisomic chromosomes for H-MM compared to NH-MM. The data suggests that although gene dosage influence gene expression, the relationship is complex and some genes are more gene dosage dependent than others. Amongst the differentially expressed genes with known function, 33% are involved in mRNA translation/protein synthesis. Of note, 37 of the top 100 differentially expressed genes are involved in these processes. In particular, 60 ribosomal protein (RP) genes are significantly (p<0.05) upregulated in H-MM. This signature in H-MM is not associated with increase proliferation as measured by PCLI. This predominant signature suggests that deregulated protein synthesis may be important for the biology of H-MM. Many of these RPs are involved in the synthesis of product of oncogenic pathways (e.g. MYC, NF-KB pathways) and may mediate the growth and survival of tumor cells. It is therefore possible that these tumor cells may be sensitive to the disruption of mRNA translation/protein synthesis. Targeting the mTOR pathway with rapamycin may therefore be useful for treatment of H-MM.