Abstract
Translocations involving the IgH locus are common genetic events in multiple myeloma (MM). A number of recurrent IgH translocations exist with t(11;14)(q13;q32) and t(4;14)(p16;q32) being the most common. These translocations predict for differential clinical outcomes, good versus poor, respectively. We have shown that ~70% of t(4;14) POS patients express the initially proposed target gene FGFR3. However, the t(4;14)POS/FGFR3NEG group of patients still fare poorly (P=0.01). Therefore, either the transformation event associated with t(4;14) is multifactorial or independent of FGFR3. The loss of FGFR3 expression is associated with the loss of der(14). However, der(4) is detectable in all t(4;14)POS patients at diagnosis and relapse, suggesting that it is biologically and clinically relevant. The der(4) chromosome is thought to result in the overexpression of MMSET. The genomic breakpoints associated with t(4;14) occur in the 5′ end of the MMSET locus. In 70% of patients (MB4-1), the breakpoints maintain the full length open reading frame of MMSET. In the remaining 30% (MB4-2 & MB4-3), the breakpoints occur downstream of the proper translation initiation site. Two principle transcripts originate from the MMSET gene. The first transcript initiates in the beginning of the MMSET locus and, as a result of alternative splicing of exon 12, produces either MMSET I or MMSET II. The second transcript initiates upstream of exon 10 and uses an alternative translation initiation site to produces RE-IIBP. MMSET I and II transcripts, produced by each breakpoint variant, and the RE-IIBP transcript, produced in all patients irrespective of breakpoint type, were cloned. Transcripts were C-terminally tagged with GFP and transiently transfected into HeLa cells. Anti-GFP immunoblots showed that all transcripts produced a protein product, even the MB4-2 and MB4-3 variants that utilize alternative translation initiation sites in exon 4 and 6, respectively. The wildtype/MB4-1 MMSET I and II constructs localized to the nucleus and were excluded from nucleoli. MMSET II is almost exclusively associated with chromatin while MMSET I localized diffusely. RE-IIBP localized primarily in cytoplasmic foci and to nucleoli. Unlike the full length MMSET proteins, the MB4-2/MB4-3 constructs localized to the nucleus but also localized in nucleoli. To determine if the N-terminus regulates the nuclear localization pattern, we cloned the N-terminal portion of MMSET, which is lost in the MB4-2 transcripts. As this construct localized to the nucleus and was excluded from nucleoli, therefore a domain required for the proper localization of MMSET is lost in the MB4-2/MB4-3 variants. Kinetic analysis of MMSET variants localized to the nucleoplasm shows that the association of MMSET II with chromatin is very stable, t1/2 130 sec, while the type II MB4-2 and MB4-3 breakpoint variants have reduced kinetics, t1/2 19 and 12 sec, respectively, suggesting a decreased stability of association. The reduction in kinetics is also seen in the type I variants. We verified the overexpression of RE-IIBP by quantitative RT-PCR on a panel of purified plasma cells and unpurified BMMC. RE-IIBP was overexpressed in t(4;14)POS patients, P=0.0009 and P=0.00006, respectively, making it the only overexpressed protein without altered function in all t(4;14)POS patients irrespective of FGFR3 expression or breakpoint type. Therefore, RE-IIBP may be of central importance to the poor outcome of t(4;14)POS MM patients.
Migraine-Associated TRESK Mutations Increase Neuronal Excitability through Alternative Translation Initiation and Inhibition of TREK
