Overexpression of transcripts originating from the MMSET locus characterizes all t(4;14)(p16;q32)-positive multiple myeloma patients

Abstract

Multiple myeloma (MM) is a B-lineage malignancy characterized by diverse genetic subtypes and clinical outcomes. The recurrent immunoglobulin heavy chain (IgH) switch translocation, t(4;14)(p16;q32), is associated with poor outcome, though the mechanism is unclear. Quantitative reverse-transcription-polymerase chain reaction (RT-PCR) for proposed target genes on a panel of myeloma cell lines and purified plasma cells showed that only transcripts originating from the WHSC1/MMSET/NSD2 gene are uniformly dysregulated in all t(4;14)POS patients. The different transcripts detected, multiple myeloma SET domain containing protein (MMSET I), MMSET II, Exon 4a/MMSET III, and response element II binding protein (RE-IIBP), are produced by alternative splicing and alternative transcription initiation events. Translation of the various transcripts, including those from major breakpoint region 4-2 (MB4-2) and MB4-3 breakpoint variants, was confirmed by transient transfection and immunoblotting. Green fluorescent protein (GFP)-tagged MMSET I and II, corresponding to proteins expressed in MB4-1 patients, localized to the nucleus but not nucleoli, whereas the MB4-2 and MB4-3 proteins concentrate in nucleoli. Cloning and localization of the Exon 4a/MMSET III splice variant, which contains the protein segment lost in the MB4-2 variant, identified a novel protein domain that prevents nucleolar localization. Kinetic studies using photobleaching suggest that the breakpoint variants are functionally distinct from wild-type proteins. In contrast, RE-IIBP is universally dysregulated and also potentially functional in all t(4;14)POS patients irrespective of fibroblast growth factor receptor 3 (FGFR3) expression or breakpoint type.

Figure 1.

Descriptive diagram of 4p16, MMSET locus, and MMSET proteins. (A) Representation of the 0.5-Mb region of 4p16.3 from the human genome project Build 34.3 flanking known t(4;14) breakpoints. Arrows indicate the direction of transcription. Empty boxes are predicted genes based on mapped image consortium clones. (B) Exon-intron structure diagram of the MMSET gene. The breakpoint cluster regions for MB4-1, MB4-2, and MB4-3 are shown. Solid lines indicate the proper splicing pattern that leads to the production of the MMSET II mRNA species. Alternative splicing events that produce MMSET III and MMSET I are indicated by dotted gray lines. In-frame stop codons are indicated by asterisks. The proper translation initiation site of MMSET is indicated by a solid black arrow, while the alternative translation initiation sites in exon 4 and 6 identified by MC and PLB are indicated by gray arrows. The point of transcription initiation of RE-IIBP is indicated by gray square boxes and the translation initiation site is indicated by a dotted black arrow. The approximate locations of the individual qRT-PCR reactions are indicated by dotted black lines. The MMSET total (T) reaction spans exons 8 and 9, while MMSET II spans exons 16 and 17. (C) Conserved domains present in MMSET variants as predicted by SMART protein prediction program., Shaded boxes indicate the position of the identified protein domains.

Figure 2.

Kaplan-Meier survival plots. (A) Survival comparison of the 43 t(4;14)^POS patients expressing or not expressing FGFR3, 31 and 12 patients, respectively. (B) Survival comparison of MB4-1 versus MB4-2/MB4-3 combined t(4;14)^POS patients, 30 and 13 patients, respectively, subgrouped based on their ability to encode a full-length MMSET protein as a result of their respective breakpoint types.

Figure 3.

Quantitative RT-PCR for RE-IIBP on BMMCs. The relative expression level of RE-IIBP from a panel of unfractionated BMMCs from t(4;14)^POS and t(4;14)^NEG patients was determined by the ΔΔCt analysis method using the Raji cell line as a reference expression level of 1. Patients positive for the translocation are denoted by a black line along the x-axis and they are grouped into their respective breakpoint types as indicated. represents t(4;14)^POS FGFR3 nonexpressing patients. Patients, t(4;14)^POS, were selected on the basis of a BM plasmacytosis more than 35%, and then best match t(4;14)^NEG samples were selected based on sex, M-protein isotype, bone marrow plasmacytosis, and age. Error bars represent the standard deviation.

Figure 4.

Localization of wild-type MMSET proteins. (A) Live cell localization of MMSET I, MMSET II, and RE-IIBP tagged with GFP at the N-terminus in transiently transfected HeLa cells. The location of the nucleus and nucleoli is identified by the live cell permeable DNA dye, Hoechst. (B) Live cell localization of MMSET I, MMSET II, and RE-IIBP tagged with GFP at the C-terminus. (C) Colocalization of MMSET II–GFP with DNA/chromatin. The fluorescence profile is generated from the area covered by the red arrow. The blue plot represents the intensity of the Hoechst stain; the green plot, the intensity of MMSET II–GFP.

Figure 5.

Alternative translation sites produce mislocalized MMSET variants. (A) Immunoblot of transiently transfected HeLa cells with various MMSET constructs. GFP-protein indicates N-terminally tagged constructs and protein-GFP indicates C-terminally tagged constructs. The detection of the predicted protein products in the C-terminally tagged MB4-2, MB4-3, and RE-IIBP constructs confirms that the alternative translation initiation sites in exon 4, 6, and 15 are functional. Interestingly, the MB4-2 construct resulted in 2 protein products, indicating that neither alternative translation site is dominant. (B) Live cell localization of type I and II constructs of MB4-2 and MB4-3 MMSET variants C-terminally tagged with GFP in transiently transfected HeLa cells. The live cell permeable DNA stain, Hoechst, is used to identify the nucleus and nucleoli. All 4 novel MMSET constructs, which are unique to the MB4-2 and MB4-3 breakpoint variants, result in proteins that enrich in nucleoli unlike the wild-type variants that are excluded from nucleoli.

Figure 6.

The N-terminus of MMSET regulates its localization pattern. Live cell localization of N- and C-terminally GFP-tagged Exon 4a/MMSET III, and N-terminally GFP-tagged B23 and MMSET III–B23 hybrid constructs, in transiently transfected HeLa cells. The live cell permeable DNA stain, Hoechst, is used to identify the nucleus and nucleoli. The Exon 4a/MMSET III construct shows a nuclear localization with nucleolar exclusion pattern. The B23 construct shows the characteristic nucleolar localization pattern, while the MMSET III–B23 hybrid construct shows the typical MMSET phenotype (nuclear and excluded from nucleoli). Immunoblotting experiments of the Exon 4a/B23 constructs confirmed that only the hybrid GFP–Exon 4a–B23 protein product is produced by this expression vector (not shown).

Figure 7.

FRAP kinetics of wild-type and breakpoint MMSET variants. FRAP recovery curves from nucleoplasmic bleaching experiments of C-terminally tagged MMSET II/MB4-1 II (□), MB4-2 II (⋄), and MB4-3 II (○). Error bars represent the standard deviation. Only the initial recovery of MMSET II is represented as the x-axis has been shortened to allow the differences in the t_1/2 times to be more evident.