FoxO1 promotes ovarian cancer by increasing transcription and METTL14-mediated m6A modification of SMC4.

The transcription factor forkhead box protein O1 (FoxO1) is closely related to the occurrence and development of ovarian cancer (OC), however its role and molecular mechanisms remain unclear. Herein, we found that FoxO1 was highly expressed in clinical samples of OC patients and was significantly correlated with poor prognosis. FoxO1 knockdown inhibited the proliferation of OC cells in vitro and in vivo. ChIP-seq combined with GEPIA2 and Kaplan-Meier database analysis showed that structural maintenance of chromosome 4 (SMC4) is a downstream target of FoxO1, and FoxO1 promotes SMC4 transcription by binding to its -1400/-1390 bp promoter. The high expression of SMC4 significantly blocked the tumor inhibition effect of FoxO1 knockdown. Furtherly, FoxO1 increased SMC4 mRNA abundance by transcriptionally activating methyltransferase-like 14 (METTL14) and increasing SMC4 m6A methylation on its coding sequence region. The Cancer Genome Atlas dataset analysis confirmed a significant positive correlation between FoxO1, SMC4, and METTL14 expression in OC. In summary, this study revealed the molecular mechanisms of FoxO1 regulating SMC4 and established a clinical link between the expression of FoxO1/METTL14/SMC4 in the occurrence of OC, thus providing a potential diagnostic target and therapeutic strategy.

Deciphering D4Z4 CpG methylation gradients in fascioscapulohumeral muscular dystrophy using nanopore sequencing.

Fascioscapulohumeral muscular dystrophy (FSHD) is caused by a unique genetic mechanism that relies on contraction and hypomethylation of the D4Z4 macrosatellite array on the Chromosome 4q telomere allowing ectopic expression of the DUX4 gene in skeletal muscle. Genetic analysis is difficult because of the large size and repetitive nature of the array, a nearly identical array on the 10q telomere, and the presence of divergent D4Z4 arrays scattered throughout the genome. Here, we combine nanopore long-read sequencing with Cas9-targeted enrichment of 4q and 10q D4Z4 arrays for comprehensive genetic analysis including determination of the length of the 4q and 10q D4Z4 arrays with base-pair resolution. In the same assay, we differentiate 4q from 10q telomeric sequences, determine A/B haplotype, identify paralogous D4Z4 sequences elsewhere in the genome, and estimate methylation for all CpGs in the array. Asymmetric, length-dependent methylation gradients were observed in the 4q and 10q D4Z4 arrays that reach a hypermethylation point at approximately 10 D4Z4 repeat units, consistent with the known threshold of pathogenic D4Z4 contractions. High resolution analysis of individual D4Z4 repeat methylation revealed areas of low methylation near the CTCF/insulator region and areas of high methylation immediately preceding the DUX4 transcriptional start site. Within the DUX4 exons, we observed a waxing/waning methylation pattern with a 180-nucleotide periodicity, consistent with phased nucleosomes. Targeted nanopore sequencing complements recently developed molecular combing and optical mapping approaches to genetic analysis for FSHD by adding precision of the length measurement, base-pair resolution sequencing, and quantitative methylation analysis.

A Scalable Computational Approach for Simulating Complexes of Multiple Chromosomes.

Significant efforts have been recently made to obtain the three-dimensional (3D) structure of the genome with the goal of understanding how structures may affect gene regulation and expression. Chromosome conformational capture techniques such as Hi-C, have been key in uncovering the quantitative information needed to determine chromatin organization. Complementing these experimental tools, co-polymers theoretical methods are necessary to determine the ensemble of three-dimensional structures associated to the experimental data provided by Hi-C maps. Going beyond just structural information, these theoretical advances also start to provide an understanding of the underlying mechanisms governing genome assembly and function. Recent theoretical work, however, has been focused on single chromosome structures, missing the fact that, in the full nucleus, interactions between chromosomes play a central role in their organization. To overcome this limitation, MiChroM (Minimal Chromatin Model) has been modified to become capable of performing these multi-chromosome simulations. It has been upgraded into a fast and scalable software version, which is able to perform chromosome simulations using GPUs via OpenMM Python API, called Open-MiChroM. To validate the efficiency of this new version, analyses for GM12878 individual autosomes were performed and compared to earlier studies. This validation was followed by multi-chain simulations including the four largest human chromosomes (C1-C4). These simulations demonstrated the full power of this new approach. Comparison to Hi-C data shows that these multiple chromosome interactions are essential for a more accurate agreement with experimental results. Without any changes to the original MiChroM potential, it is now possible to predict experimentally observed inter-chromosome contacts. This scalability of Open-MiChroM allow for more audacious investigations, looking at interactions of multiple chains as well as moving towards higher resolution chromosomes models.

Interchromosomal Contacts of rDNA Clusters with DUX Genes in Human Chromosome 4 Are Very Sensitive to Heat Shock Treatment.

In order to study the effects of heat shock treatment on the distribution of rDNA contacts at the region possessing DUX genes inside chromosome 4 we used 4C approach. Our data indicate that the treatment removes the frequent rDNA contacts in this region. The recent data on involvement of superenhancers that are decorated by broad H3K27ac marks in the phase separation mechanisms and the previous data demonstrating that these broad marks are the favorite sites of rDNA contacts taken together with our data on sensitivity of the contacts to the heat shock treatment suggest that the phase separation mechanisms are involved in the reversible rDNA-mediated regulation of gene expression via the contacts.

"Cyst" on the forearm of a 28-year-old female: Case report of a CIC-rearranged sarcoma.

Capicua transcriptional repressor (CIC)-rearranged sarcomas are part of the group of Ewing-like sarcomas or atypical Ewing sarcomas which, thanks to the progress in molecular diagnosis, are being defined by particular genetic abnormalities separating this group into distinct entities with their own particular histological and immunohistochemical features, as well as different survival outcomes. We report the case of a healthy 28-year-old female presenting with a tender lesion on her forearm which after ultrasound examination was clinically favored to represent an infected sebaceous cyst. Hematoxylin-eosin staining showed a lobulated neoplasm within the subcutis composed of poorly differentiated epithelioid to round cells with a small amount of amphophilic cytoplasm. Frequent mitotic figures and tumor necrosis were present. Immunohistochemical studies showed patchy focal CD99 membranous positivity, negative WT1 and TLE1 staining and diffuse nuclear positivity for ETV4 (performed at outside laboratory). FISH analysis showed significant CIC rearrangement enabling a final diagnosis of an undifferentiated small round cell sarcoma harboring the t(4;19)(q35;q13.1) and CIC-DUX4 fusion. This case shows the importance of awareness of this entity as, unlike Ewing sarcoma, these lesions present in the soft tissues rather than bone and may, as in this case, arise in the superficial soft tissues and be submitted to a dermatopathology practice.

Enhancer hijacking activates oncogenic transcription factor NR4A3 in acinic cell carcinomas of the salivary glands.

The molecular pathogenesis of salivary gland acinic cell carcinoma (AciCC) is poorly understood. The secretory Ca-binding phosphoprotein (SCPP) gene cluster at 4q13 encodes structurally related phosphoproteins of which some are specifically expressed at high levels in the salivary glands and constitute major components of saliva. Here we report on recurrent rearrangements [t(4;9)(q13;q31)] in AciCC that translocate active enhancer regions from the SCPP gene cluster to the region upstream of Nuclear Receptor Subfamily 4 Group A Member 3 (NR4A3) at 9q31. We show that NR4A3 is specifically upregulated in AciCCs, and that active chromatin regions and gene expression signatures in AciCCs are highly correlated with the NR4A3 transcription factor binding motif. Overexpression of NR4A3 in mouse salivary gland cells increases expression of known NR4A3 target genes and has a stimulatory functional effect on cell proliferation. We conclude that NR4A3 is upregulated through enhancer hijacking and has important oncogenic functions in AciCC.

Aberrant activity of NKL homeobox gene NKX3-2 in a T-ALL subset.

T-cell acute lymphoblastic leukemia (T-ALL) is a hematopoietic malignancy originating from T-cell progenitors in which differentiation is blocked at early stages. Physiological expression of specific NKL homeobox genes obeys a hematopoietic NKL-code implicated in the process of lymphopoiesis while in differentiated T-cells these genes are silenced. We propose that this developmental expression pattern underlies the observation that NKL homeobox genes are the most ubiquitous group of transcription factors deregulated in T-ALL, including TLX1, TLX3, NKX2-5 and NKX3-1. Here, we describe a novel member of the NKL homeobox gene subclass, NKX3-2 (BAPX1), which is aberrantly activated in 18% of pediatric T-ALL patients analyzed while being normally expressed in developing spleen. Identification of NKX3-2 expression in T-ALL cell line CCRF-CEM qualified these cells to model its deregulation and function in a leukemic context. Genomic and chromosomal analyses demonstrated normal configuration of the NKX3-2 locus at chromosome 4p15, thus excluding cytogenetic dysregulation. Comparative expression profiling analysis of NKX3-2 patient data revealed deregulated activity of BMP- and MAPK-signalling. These candidate pathways were experimentally confirmed to mediate aberrant NKX3-2 expression. We also show that homeobox gene SIX6, plus MIR17HG and GATA3 are downstream targets of NKX3-2 and plausibly contribute to the pathogenesis of this malignancy by suppressing T-cell differentiation. Finally, NKL homeobox gene NKX2-5 was activated by NKX3-2 in CCRF-CEM and by FOXG1 in PEER, representing mutually inhibitory activators of this translocated oncogene. Together, our findings reveal a novel oncogenic NKL homeobox gene subclass member which is aberrantly expressed in a large subset of T-ALL patients and participates in a deregulated gene network likely to arise in developing spleen.

GABRA2 Alcohol Dependence Risk Allele is Associated with Reduced Expression of Chromosome 4p12 GABAA Subunit Genes in Human Neural Cultures.

BACKGROUND: Genetic variation in a region of chromosome 4p12 that includes the GABAA subunit gene GABRA2 has been reproducibly associated with alcohol dependence (AD). However, the molecular mechanisms underlying the association are unknown. This study examined correlates of in vitro gene expression of the AD-associated GABRA2 rs279858*C-allele in human neural cells using an induced pluripotent stem cell (iPSC) model system. METHODS: We examined mRNA expression of chromosome 4p12 GABAA subunit genes (GABRG1, GABRA2, GABRA4, and GABRB1) in 36 human neural cell lines differentiated from iPSCs using quantitative polymerase chain reaction and next-generation RNA sequencing. mRNA expression in adult human brain was examined using the BrainCloud and BRAINEAC data sets. RESULTS: We found significantly lower levels of GABRA2 mRNA in neural cell cultures derived from rs279858*C-allele carriers. Levels of GABRA2 RNA were correlated with those of the other 3 chromosome 4p12 GABAA genes, but not other neural genes. Cluster analysis based on the relative RNA levels of the 4 chromosome 4p12 GABAA genes identified 2 distinct clusters of cell lines, a low-expression cluster associated with rs279858*C-allele carriers and a high-expression cluster enriched for the rs279858*T/T genotype. In contrast, there was no association of genotype with chromosome 4p12 GABAA gene expression in postmortem adult cortex in either the BrainCloud or BRAINEAC data sets. CONCLUSIONS: AD-associated variation in GABRA2 is associated with differential expression of the entire cluster of GABAA subunit genes on chromosome 4p12 in human iPSC-derived neural cell cultures. The absence of a parallel effect in postmortem human adult brain samples suggests that AD-associated genotype effects on GABAA expression, although not present in mature cortex, could have effects on regulation of the chromosome 4p12 GABAA cluster during neural development.

A review of the JR blood group system.

The JR blood group system (ISBT 032) consists of one antigen,Jra, which is of high prevalence in all populations. The rare Jr(a-) phenotype has been found mostly in Japanese and other Asian populations, but also in people of northern European ancestry, in Bedouin Arabs, and in one Mexican. Anti-Jra has caused transfusion reactions and is involved in hemolytic disease of the fetus and newborn. The Jra antigen is located on ABCG2 transporter, a multipass membrane glycoprotein (also known as the breast cancer resistance protein, BCRP), which is encoded by the ABCG2 gene on chromosome 4q22.1. The Jr(a-) phenotype mostly results from recessive inheritance of ABCG2 null alleles caused by frameshift or nonsense changes.

Distinct phenotypic differences associated with differential amplification of receptor tyrosine kinase genes at 4q12 in glioblastoma.

Gene amplification at chromosome 4q12 is a common alteration in human high grade gliomas including glioblastoma, a CNS tumour with consistently poor prognosis. This locus harbours the known oncogenes encoding the receptor tyrosine kinases PDGFRA, KIT, and VEGFR2. These receptors are potential targets for novel therapeutic intervention in these diseases, with expression noted in tumour cells and/or associated vasculature. Despite this, a detailed assessment of their relative contributions to different high grade glioma histologies and the underlying heterogeneity within glioblastoma has been lacking. We studied 342 primary high grade gliomas for individual gene amplification using specific FISH probes, as well as receptor expression in the tumour and endothelial cells by immunohistochemistry, and correlated our findings with specific tumour cell morphological types and patterns of vasculature. We identified amplicons which encompassed PDGFRA only, PDGFRA/KIT, and PDGFRA/KIT/VEGFR2, with distinct phenotypic correlates. Within glioblastoma specimens, PDGFRA amplification alone was linked to oligodendroglial, small cell and sarcomatous tumour cell morphologies, and rare MGMT promoter methylation. A younger age at diagnosis and better clinical outcome in glioblastoma patients is only seen when PDGFRA and KIT are co-amplified. IDH1 mutation was only found when all three genes are amplified; this is a subgroup which also harbours extensive MGMT promoter methylation. Whilst PDGFRA amplification was tightly linked to tumour expression of the receptor, this was not the case for KIT or VEGFR2. Thus we have identified differential patterns of gene amplification and expression of RTKs at the 4q12 locus to be associated with specific phenotypes which may reflect their distinct underlying mechanisms.

A truncating mutation of CEP135 causes primary microcephaly and disturbed centrosomal function.

Autosomal-recessive primary microcephaly (MCPH) is a rare congenital disorder characterized by intellectual disability, reduced brain and head size, but usually without defects in cerebral cortical architecture, and other syndromic abnormalities. MCPH is heterogeneous. The underlying genes of the seven known loci code for centrosomal proteins. We studied a family from northern Pakistan with two microcephalic children using homozygosity mapping and found suggestive linkage for regions on chromosomes 2, 4, and 9. We sequenced two positional candidate genes and identified a homozygous frameshift mutation in the gene encoding the 135 kDa centrosomal protein (CEP135), located in the linkage interval on chromosome 4, in both affected children. Post hoc whole-exome sequencing corroborated this mutation's identification as the causal variant. Fibroblasts obtained from one of the patients showed multiple and fragmented centrosomes, disorganized microtubules, and reduced growth rate. Similar effects were reported after knockdown of CEP135 through RNA interference; we could provoke them also by ectopic overexpression of the mutant protein. Our findings suggest an additional locus for MCPH at HSA 4q12 (MCPH8), further strengthen the role of centrosomes in the development of MCPH, and place CEP135 among the essential components of this important organelle in particular for a normal neurogenesis.

Rapidly progressive renal disease as part of Wolfram syndrome in a large inbred Turkish family due to a novel WFS1 mutation (p.Leu511Pro).

Wolfram syndrome, also named "DIDMOAD" (diabetes insipidus, diabetes mellitus, optic atrophy, and deafness), is an inherited association of juvenile-onset diabetes mellitus and optic atrophy as key diagnostic criteria. Renal tract abnormalities and neurodegenerative disorder may occur in the third and fourth decade. The wolframin gene, WFS1, associated with this syndrome, is located on chromosome 4p16.1. Many mutations have been described since the identification of WFS1 as the cause of Wolfram syndrome. We identified a new homozygous WFS1 mutation (c.1532T>C; p.Leu511Pro) causing Wolfram syndrome in a large inbred Turkish family. The patients showed early onset of IDDM, diabetes insipidus, optic atrophy, sensorineural hearing impairment and very rapid progression to renal failure before age 12 in three females. Ectopic expression of the wolframin mutant in HEK cells results in greatly reduced levels of protein expression compared to wild-type wolframin, strongly supporting that this mutation is disease-causing. The mutation showed perfect segregation with disease in the family, characterized by early and severe clinical manifestations.

The Krüppel-like factor 15 as a molecular link between myogenic factors and a chromosome 4q transcriptional enhancer implicated in facioscapulohumeral dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD), a dominant hereditary disease with a prevalence of 7 per 100,000 individuals, is associated with a partial deletion in the subtelomeric D4Z4 repeat array on chromosome 4q. The D4Z4 repeat contains a strong transcriptional enhancer that activates promoters of several FSHD-related genes. We report here that the enhancer within the D4Z4 repeat binds the Krüppel-like factor KLF15. KLF15 was found to be up-regulated during myogenic differentiation induced by serum starvation or by overexpression of the myogenic differentiation factor MYOD. When overexpressed, KLF15 activated the D4Z4 enhancer and led to overexpression of DUX4c (Double homeobox 4, centromeric) and FRG2 (FSHD region gene 2) genes, whereas its silencing caused inactivation of the D4Z4 enhancer. In immortalized human myoblasts, the D4Z4 enhancer was activated by the myogenic factor MYOD, an effect that was abolished upon KLF15 silencing or when the KLF15-binding sites within the D4Z4 enhancer were mutated, indicating that the myogenesis-related activation of the D4Z4 enhancer was mediated by KLF15. KLF15 and several myogenesis-related factors were found to be expressed at higher levels in myoblasts, myotubes, and muscle biopsies from FSHD patients than in healthy controls. We propose that KLF15 serves as a molecular link between myogenic factors and the activity of the D4Z4 enhancer, and it thus contributes to the overexpression of the DUX4c and FRG2 genes during normal myogenic differentiation and in FSHD.

Induced pluripotent stem cells can be used to model the genomic imprinting disorder Prader-Willi syndrome.

The recent discovery of induced pluripotent stem cell (iPSC) technology provides an invaluable tool for creating in vitro representations of human genetic conditions. This is particularly relevant for those diseases that lack adequate animal models or where the species comparison is difficult, e.g. imprinting diseases such as the neurogenetic disorder Prader-Willi syndrome (PWS). However, recent reports have unveiled transcriptional and functional differences between iPSCs and embryonic stem cells that in cases are attributable to imprinting errors. This has suggested that human iPSCs may not be useful to model genetic imprinting diseases. Here, we describe the generation of iPSCs from a patient with PWS bearing a partial translocation of the paternally expressed chromosome 15q11-q13 region to chromosome 4. The resulting iPSCs match all standard criteria of bona fide reprogramming and could be readily differentiated into tissues derived from the three germ layers, including neurons. Moreover, these iPSCs retain a high level of DNA methylation in the imprinting center of the maternal allele and show concomitant reduced expression of the disease-associated small nucleolar RNA HBII-85/SNORD116. These results indicate that iPSCs may be a useful tool to study PWS and perhaps other genetic imprinting diseases as well.

Mutations of the TET2 and CBL genes: novel molecular markers in myeloid malignancies.

Despite recent progress in molecular research in myeloid malignancies, in subsets of patients with myelodysplastic syndrome (MDS) so far no underlying mutation was identified. In the myeloproliferative neoplasms (MPNs), the JAK2V617F alone cannot explain the phenotypic heterogeneity. In acute myeloid leukemia (AML), clinical variability exists within distinct subgroups. Thus, the search for novel molecular markers continues. Recently, mutations of the tet oncogene family member 2 (TET2) and Casitas B-cell lymphoma (CBL) genes became the focus of interest. With diverse genetic methods, TET2 on chromosome 4q24 was identified as candidate tumor suppressor gene. Sequencing studies revealed heterogeneous mutations in 10-25% of patients with acute myeloid leukemia (AML), MDS, and MPNs, while the frequency might be higher in chronic myelomonocytic leukemia (CMML). The prognostic impact is being explored. The CBL gene is involved in the degradation of tyrosine kinases. In rare cases of human AML (<2%), CBL mutants were identified, with a higher frequency in core binding factor leukemias. Presence of these mutations was suggested to be involved in aberrant FLT3 expression. In the MPNs, a 2-8% frequency of CBL mutations was reported. These novel mutations deepened insights in the mechanisms of leukemogenesis, might contribute to the identification of new therapeutic targets, and improve diagnostics in the myeloid malignancies.

Two independent gene signatures in pediatric t(4;11) acute lymphoblastic leukemia patients.

OBJECTIVE: Gene expression profiles become increasingly more important for diagnostic procedures, allowing clinical predictions including treatment response and outcome. However, the establishment of specific and robust gene signatures from microarray data sets requires the analysis of large numbers of patients and the application of complex biostatistical algorithms. Especially in case of rare diseases and due to these constrains, diagnostic centers with limited access to patients or bioinformatic resources are excluded from implementing these new technologies. METHOD: In our study we sought to overcome these limitations and for proof of principle, we analyzed the rare t(4;11) leukemia disease entity. First, gene expression data of each t(4;11) leukemia patient were normalized by pairwise subtraction against normal bone marrow (n = 3) to identify significantly deregulated gene sets for each patient. RESULT: A 'core signature' of 186 commonly deregulated genes present in each investigated t(4;11) leukemia patient was defined. Linking the obtained gene sets to four biological discriminators (HOXA gene expression, age at diagnosis, fusion gene transcripts and chromosomal breakpoints) divided patients into two distinct subgroups: the first one comprised infant patients with low HOXA genes expression and the MLL breakpoints within introns 11/12. The second one comprised non-infant patients with high HOXA expression and MLL breakpoints within introns 9/10. CONCLUSION: A yet homogeneous leukemia entity was further subdivided, based on distinct genetic properties. This approach provided a simplified way to obtain robust and disease-specific gene signatures even in smaller cohorts.

Exon organization and novel alternative splicing of the human ANK2 gene: implications for cardiac function and human cardiac disease.

Recent findings illustrate a critical role for ankyrin-B function in normal cardiovascular physiology. Specifically, decreased expression of ankyrin-B in mice or human mutations in the ankyrin-B gene (ANK2) results in potentially fatal cardiac arrhythmias. Despite the clear role of ankyrin-B in heart, the mechanisms underlying transcriptional regulation of ANK2 are unknown. In fact, to date there is no description of ANK2 genomic organization. The aims of this study were to provide a comprehensive description of the ANK2 gene and to evaluate the relative expression of alternative splicing events associated with ANK2 transcription in heart. Using reverse-transcriptase PCR on mRNA isolated from human hearts, we identify seven new exons associated with the ANK2 gene including an alternative first exon located approximately 145 kb upstream of the previously-identified first exon. In addition, we identify over thirty alternative splicing events associated with ANK2 mRNA transcripts. Using real-time PCR and exon boundary-spanning primers to selectively amplify these splice variants, we demonstrate that these variants are expressed at varying levels in human heart. Finally, ankyrin-B immunoblot analysis demonstrates the expression of a heterogeneous population of ankyrin-B polypeptides in heart. ANK2 consists of 53 exons that span approximately 560 kb on human chromosome 4. Additionally, our data demonstrates that ANK2 is subject to complex transcriptional regulation that likely results in differential ankyrin-B polypeptide function.

Prospective tracing of MLL-FRYL clone with low MEIS1 expression from emergence during neuroblastoma treatment to diagnosis of myelodysplastic syndrome.

We prospectively observed a child exposed to intensive multimodality therapy for metastatic neuroblastoma from emergence of a MLL translocation to disease diagnosis. The t(4;11)(p12;q23) was detected in the marrow 17 months after starting treatment following topoisomerase II poisons, alkylating agents, local radiation, hematopoietic stem cell transplantation, anti-GD2 monoclonal antibody with granulocyte macrophage-colony-stimulating factor, and a high cumulative dose of oral etoposide. Reciprocal genomic breakpoint junctions and fusion transcripts joined MLL with FRYL, the Drosophila melanogaster protein homologue of which regulates cell fate. Etoposide metabolites induced topoisomerase II cleavage complexes that could form both breakpoint junctions. Cells harboring the translocation replaced the marrow without clinical evidence of leukemia and differentiation appeared unaffected for 37 months. Subsequent bilineage dysplasia and increased blasts in addition to the translocation fulfilled criteria for MDS. The MEIS1 target gene of typical MLL fusion oncoproteins was underexpressed before and at MDS diagnosis. These results are consistent with repair of topoisomerase II cleavage from etoposide metabolites as the translocation mechanism, whereas other agents in the regimen may have contributed to progression of the clone with the translocation to MDS. MLL-FRYL did not increase MEIS1 expression, conferred a proliferative advantage without altering differentiation, and had protracted latency to disease.

The multiple myeloma associated MMSET gene contributes to cellular adhesion, clonogenic growth, and tumorigenicity.

Multiple myeloma (MM) is an incurable hematologic malignancy characterized by recurrent chromosomal translocations. Patients with t(4;14)(p16;q32) are the worst prognostic subgroup in MM, although the basis for this poor prognosis is unknown. The t(4;14) is unusual in that it involves 2 potential target genes: fibroblast growth factor receptor 3 (FGFR3) and multiple myeloma SET domain (MMSET). MMSET is universally overexpressed in t(4;14) MM, whereas FGFR3 expression is lost in one-third of cases. Nonetheless, the role of MMSET in t(4;14) MM has remained unclear. Here we demonstrate a role for MMSET in t(4;14) MM cells. Down-regulation of MMSET expression in MM cell lines by RNA interference and by selective disruption of the translocated MMSET allele using gene targeting dramatically reduced colony formation in methylcellulose but had only modest effects in liquid culture. In addition, MMSET knockdown led to cell-cycle arrest of adherent MM cells and reduced the ability of MM cells to adhere to extracellular matrix. Finally, MMSET knockdown and knockout reduced tumor formation by MM xenografts. These results provide the first direct evidence that MMSET plays a significant role in t(4;14) MM and suggest that therapies targeting this gene could impact this particular subset of poor-prognosis patients.