MMSET I Acts As an Oncoprotein and Regulates GLO1 Expression in T(4;14) Multiple Myeloma Cells

Recurrent chromosomal translocations are central to the pathogenesis of multiple myeloma (MM). The translocation t(4;14)(p16;q32) is one of the most common translocation and associated with poor prognosis. T(4;14) MM universally overexpresses MMSET that exists in three major isoforms, full length MMSET II, and short isoforms REIIBP and MMSET I. MMSET II has been suggested to play an important tumorigenic role in t(4;14) MM, but little is yet known about whether and how the MMSET short isoforms contribute to MM tumorigenesis. Survival analysis indicated there was no significant difference in outcomes between t(4;14) positive patients expressing MMSET II (breakpoint MB4-1) or not expressing MMSET II (breakpoint MB4-2/MB4-3). Therefore overexpression of MMSET II is not necessary for the poor outcome of t(4;14) MM. The truncated or short isoforms of MMSET might also be important for t(4;14) MM. The aim of this study is to characterize the role of short isoform MMSET I and determine its downstream targets in t(4;14) MM.

MMSET I was knocked down by shRNAs in four t(4;14) MM cell lines (KMS11, KMS18, KMS28BM and KMS34). MMSET I knockdown (KD) reduced cell cycle S phase and induced cell apoptosis. MMSET I KD significantly inhibited the capacity to produce colonies in t(4;14) MM cells on methylcellulose colony formation assay. MMSET I KD inhibited KMS11 tumor formation or reduced tumor size (ShLuc vs ShMMSET I, 8/8 vs 2/8) in xenograft studies. Gene array analysis indicated 46 genes down-regulated, and 22 genes up-regulated upon MMSET I KD in t(4;14) MM cell lines. Glyoxalase I (GLO1) was one of the top 5 down-regulated genes and was down-regulated in all 4 cell lines upon MMSET I KD. qPCR and western blot (WB) analysis confirmed GLO1 expression was down-regulated upon MMSET I KD. When MMSET I was transfected into U266 cells, a non-t(4;14) MM cell line, the ectopic MMSET I decreased cell apoptosis, promoted U266 cell colony formation and tumorigenicity in vivo (VCon vs MMSET I, 1/7 vs 4/7). qPCR and WB analysis indicated ectopic MMSET I also increased GLO1 expression. ChIP analysis showed binding of MMSET I at upstream of the GLO1 transcription start site (-350bp). When U266 cells were transfected with MMSET I mutant vectors, WB analysis showed that both the MMSET I N and C-terminus participated in regulating GLO1 expression, but only the C-terminus HMG-b domain was necessary to regulate GLO1 expression. These observations confirm that MMSET I is a bona fide oncogene and promotes GLO1 expression.

GLO1 belongs to the glyoxalase (GLO) system, which catalyzes the conversion of deleterious methylglyoxal (MG) to non-toxic D-lactate in cytoplasm. GLO1 KD induced apoptosis and reduced colony formation greatly in t(4;14) MM cells. WB analysis showed MMSET I or GLO1 KD increased MG-modified protein significantly, and reduced the levels of anti-apoptosis factors such as MCL1 and BCL2. In cell growth media, ascorbate is oxidized to dehydroascorbate (DHA). DHA is transported across cellular membrane and reduced to ascorbate in the cytoplasm by depleting glutathione (GSH), which is required for GLO detoxification pathway. Therefore we hypothesize that ascorbate will inhibit GLO pathway through decreasing GSH level, result in MG accumulation and inhibition of cell growth. As expected, ascorbate reduced GSH level and increased MG-modified protein level in t(4;14) MM cells. CTG assays indicated ascorbate less than 0.2 mM inhibited t(4;14) MM cell growth significantly in 48 h. Apoptosis analysis showed that ascorbate was cytotoxic instead of cytostatic to t(4;14) MM cells. The effect of ascorbate was then investigated in a NOD/scid KMS11 xenograft model. Compared with vehicle control, ascorbate extended survival of the mice significantly (on day 54 after cell injection, 0/8 vs 6/8, P < 0.01). Flow cytometric analysis indicated that IV injection of KMS11 led to spleen and bone marrow infiltration, which was inhibited by ascorbate treatment. Femur weight showed ascorbate treatment also reduced bone lesion greatly.

Taken together, our data indicated that MMSET I acts as an oncoprotein and regulates GLO1 expression in t(4;14) multiple myeloma cells. We found that ascorbate inhibits GLO pathway through depleting GSH and is cytotoxic to t(4;14) MM cells. Furthermore ascorbate alone inhibits tumor formation and extends mouse survival in MM xenograft model. Our study suggested that ascorbate might be exploited therapeutically for t(4;14) MM.