ABSTRACT
Multiple myeloma (MM) is characterized by recurrent chromosomal translocations. MMSET, identified by its fusion to the IgH locus in t(4;14) MM, is universally overexpressed in t(4;14) MM. In order to identify cell surface biomarkers associated with t(4;14) MM for small molecule or antibody based therapies, we knocked down MMSET expression with shRNA and generated a cell line pair from KMS11, a t(4;14) MM cell line. We used quantitative mass spectrometry to identify plasma membrane proteins associated with MMSET overexpression. Using this approach, 50 cell surface proteins were identified as differentially expressed between KMS11 and KMS11/shMMSET. Western blot and flow cytometry analysis indicated SLAMF7 was over-expressed in t(4;14) MM cell lines and down-regulated by MMSET shRNAs. SLAMF7 expression was also confirmed in primary t(4;14) MM samples by flow cytometry analysis. Quantitative RT-PCR and ChIP analysis indicated MMSET might regulate the transcription level of SLAMF7 and be an important functional element for SLAMF7 promoter activity. Furthermore, SLAMF7 shRNA could induce G1 arrest or apoptosis and reduce clonogenetic capacity in t(4;14) MM cells. Overall, these results illustrated SLAMF7 might be a novel cell surface protein associated with t(4;14) MM. It is potential to develop t(4;14) MM targeted therapy by SLAMF7 antibody mediated drug delivery.
Subject(s)
Biomarkers, Tumor/metabolism , Histone-Lysine N-Methyltransferase/biosynthesis , Multiple Myeloma/metabolism , Repressor Proteins/biosynthesis , Cell Line, Tumor , Cell Membrane/genetics , Cell Membrane/metabolism , Chromosomes, Human, Pair 14 , Chromosomes, Human, Pair 4 , Down-Regulation , Gene Expression Regulation, Neoplastic , HEK293 Cells , Histone-Lysine N-Methyltransferase/genetics , Histone-Lysine N-Methyltransferase/metabolism , Humans , Multiple Myeloma/genetics , Proteomics/methods , Receptors, Immunologic/biosynthesis , Receptors, Immunologic/genetics , Repressor Proteins/genetics , Repressor Proteins/metabolism , Signaling Lymphocytic Activation Molecule Family , Transfection , Translocation, GeneticABSTRACT
The role of enhancer of zeste homolog 2 (EZH2) in cancer is complex and may vary depending on the cellular context. We found that EZH2 is aberrantly overexpressed in the majority of natural killer/T-cell lymphoma (NKTL), an aggressive lymphoid malignancy with very poor prognosis. We show that EZH2 upregulation is mediated by MYC-induced repression of its regulatory micro RNAs and EZH2 exerts oncogenic properties in NKTL. Ectopic expression of EZH2 in both primary NK cells and NKTL cell lines leads to a significant growth advantage. Conversely, knock-down of EZH2 in NKTL cell lines results in cell growth inhibition. Intriguingly, ectopic EZH2 mutant deficient for histone methyltransferase activity is also able to confer growth advantage and rescue growth inhibition on endogenous EZH2 depletion in NKTL cells, indicating an oncogenic role of EZH2 independent of its gene-silencing activity. Mechanistically, we show that EZH2 directly promotes the transcription of cyclin D1 and this effect is independent of its enzymatic activity. Furthermore, depletion of EZH2 using a PRC2 inhibitor 3-deazaneplanocin A significantly inhibits growth of NK tumor cells. Therefore, our study uncovers an oncogenic role of EZH2 independent of its methyltransferase activity in NKTL and suggests that targeting EZH2 may have therapeutic usefulness in this lymphoma.
Subject(s)
Histone-Lysine N-Methyltransferase/metabolism , Killer Cells, Natural/physiology , Lymphoma, T-Cell/physiopathology , Polycomb Repressive Complex 2/genetics , Adenosine/analogs & derivatives , Adenosine/pharmacology , Adolescent , Adult , Aged , Aged, 80 and over , Apoptosis/drug effects , Apoptosis/physiology , Cell Line, Tumor , Cell Proliferation/drug effects , Cyclin D1/genetics , Cyclin D1/metabolism , Enhancer of Zeste Homolog 2 Protein , Female , Gene Expression Regulation, Neoplastic , Histone Methyltransferases , Histone-Lysine N-Methyltransferase/genetics , Humans , Killer Cells, Natural/cytology , Lymphoma, T-Cell/pathology , Male , MicroRNAs/metabolism , Middle Aged , Mutagenesis/physiology , Polycomb Repressive Complex 2/metabolism , Promoter Regions, Genetic/physiology , Up-Regulation/physiology , Young AdultABSTRACT
Recent studies have shown that 3-Deazaneplanocin A (DZNep), a histone methyltransferase inhibitor, disrupts polycomb-repressive complex 2 (PRC2), and preferentially induces apoptosis in cancer cells, including acute myeloid leukemia (AML). However, the underlying molecular mechanisms are not well understood. The present study demonstrates that DZNep induces robust apoptosis in AML cell lines, primary cells, and targets CD34(+)CD38(-) leukemia stem cell (LSC)-enriched subpopulations. Using RNA interference (RNAi), gene expression profiling, and ChIP, we identified that TXNIP, a major redox control molecule, plays a crucial role in DZNep-induced apoptosis. We show that disruption of PRC2, either by DZNep treatment or EZH2 knockdown, reactivates TXNIP, inhibits thioredoxin activity, and increases reactive oxygen species (ROS), leading to apoptosis. Furthermore, we show that TXNIP is down-regulated in AML and is a direct target of PRC2-mediated gene silencing. Consistent with the ROS accumulation on DZNep treatment, we also see a signature of endoplasmic reticulum (ER) stress-regulated genes, commonly associated with cell survival, down-regulated by DZNep. Taken together, we uncover a novel molecular mechanism of DZNep-mediated apoptosis and propose that EZH2 may be a potential new target for epigenetic treatment in AML.
