Epigenetic silencing of the tetraspanin CD9 during disease progression in multiple myeloma cells and correlation with survival.
OBJECTIVE
The purpose of this study was to investigate expression and epigenetic regulation of CD9 in multiple myeloma (MM) cells during disease progression.


METHODS
CD9 expression was retrospectively analyzed on bone marrow myeloma samples from 81 patients by immunophenotyping. CD9 expression by murine 5TMM cells was detected by flow cytometric staining and quantitative PCR. The methylation status of the CD9 promoter was determined by bisulfite PCR sequencing.


RESULTS
Primary plasma cells in the majority of MM patients with nonactive disease (n = 28) showed CD9 expression, whereas most cases with active disease (n = 53) were CD9 negative. CD9 expression in diagnostic bone marrow samples (n = 74) correlated with survival. Moreover, CD9 expression on murine 5T33 and 5T2MM cells was significantly down-regulated during disease development. Treatment of CD9-nonexpressing 5T33MMvt cells with the clinically relevant histone deacetylase inhibitor LBH589 resulted in a significant increase in CD9 expression. In contrast, cells treated with the demethylation agent 5-aza-2'deoxycytidine barely showed any increase. A combination study with both compounds resulted in a strong synergistic reactivation of CD9. CD9-expressing 5T33MMvv cells and 5T33MMvt cells stably transduced with a mCD9 lentiviral transferplasmid were shown to be more susceptible to natural killer cell-mediated cytolysis than CD9-negative 5T33MMvt cells.


CONCLUSIONS
CD9 expression correlates with disease status and survival of MM patients. In the murine 5T33MM model, we show that histone modifications, and to a lesser extent CpG methylation, are key epigenetic events in CD9 down-regulation. Furthermore, as CD9 expression becomes down-regulated, 5T33MM cells become less susceptible to natural killer cell-mediated cytolysis.
De Bruyne
Elke
E
Department of Hematology and Immunology, Vrije Universiteit Brussel, Brussels, Belgium.
Bos
Tomas Jan
TJ
Asosingh
Kewal
K
Vande Broek
Isabelle
I
Menu
Eline
E
Van Valckenborgh
Els
E
Atadja
Peter
P
Coiteux
Valérie
V
Leleu
Xavier
X
Thielemans
Kris
K
Van Camp
Ben
B
Vanderkerken
Karin
K
Van Riet
Ivan
I
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000818:Animals; D015703:Antigens, CD / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D003602:Cytotoxicity, Immunologic; D019175:DNA Methylation; D018450:Disease Progression; D004791:Enzyme Inhibitors / Q000494:pharmacology; D044127:Epigenesis, Genetic; D005260:Female; D005434:Flow Cytometry; D006655:Histone Deacetylases / Q000187:drug effects; D006657:Histones / Q000378:metabolism; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology; D016130:Immunophenotyping; D007211:Indoles; D053208:Kaplan-Meier Estimate; D007694:Killer Cells, Natural / Q000276:immunology; D008297:Male; D008562:Membrane Glycoproteins / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008875:Middle Aged; D009101:Multiple Myeloma / Q000235:genetics* / Q000276:immunology / Q000401:mortality*; D000077767:Panobinostat; D012189:Retrospective Studies; D020133:Reverse Transcriptase Polymerase Chain Reaction; D060245:Tetraspanin 29
D015703:Antigens, CD; C554869:CD9 protein, human; C554870:Cd9 protein, mouse; D004791:Enzyme Inhibitors; D006657:Histones; D006877:Hydroxamic Acids; D007211:Indoles; D008562:Membrane Glycoproteins; D060245:Tetraspanin 29; D000077767:Panobinostat; D006655:Histone Deacetylases