NK-like homeodomain proteins activate NOTCH3-signaling in leukemic T-cells.
BACKGROUND
Homeodomain proteins control fundamental cellular processes in development and in cancer if deregulated. Three members of the NK-like subfamily of homeobox genes (NKLs), TLX1, TLX3 and NKX2-5, are implicated in T-cell acute lymphoblastic leukemia (T-ALL). They are activated by particular chromosomal aberrations. However, their precise function in leukemogenesis is still unclear. Here we screened further NKLs in 24 T-ALL cell lines and identified the common expression of MSX2. The subsequent aim of this study was to analyze the role of MSX2 in T-cell differentiation which may be disturbed by oncogenic NKLs.


METHODS
Specific gene activity was examined by quantitative real-time PCR, and globally by expression profiling. Proteins were analyzed by western blot, immuno-cytology and immuno-precipitation. For overexpression studies cell lines were transduced by lentiviruses.


RESULTS
Quantification of MSX2 mRNA in primary hematopoietic cells demonstrated higher levels in CD34+ stem cells as compared to peripheral blood cells and mature CD3+ T-cells. Furthermore, analysis of MSX2 expression levels in T-cell lines after treatment with core thymic factors confirmed their involvement in regulation. These results indicated that MSX2 represents an hematopoietic NKL family member which is downregulated during T-cell development and may functionally substituted by oncogenic NKLs. For functional analysis JURKAT cells were lentivirally transduced, overexpressing either MSX2 or oncogenic TLX1 and NKX2-5, respectively. These cells displayed transcriptional activation of NOTCH3-signaling, including NOTCH3 and HEY1 as analyzed by gene expression profiling and quantitative RT-PCR, and consistently attenuated sensitivity to gamma-secretase inhibitor as analyzed by MTT-assays. Furthermore, in addition to MSX2, both TLX1 and NKX2-5 proteins interacted with NOTCH-pathway repressors, SPEN/MINT/SHARP and TLE1/GRG1, representing a potential mechanism for (de)regulation. Finally, elevated expression of NOTCH3 and HEY1 was detected in primary TLX1/3 positive T-ALL cells corresponding to the cell line data.


CONCLUSIONS
Identification and analysis of MSX2 in hematopoietic cells implicates a modulatory role via NOTCH3-signaling in early T-cell differentiation. Our data suggest that reduction of NOTCH3-signaling by physiological downregulation of MSX2 expression during T-cell development is abrogated by ectopic expression of oncogenic NKLs, substituting MSX2 function.
Nagel
Stefan
S
Dept. of Human and Animal Cell Lines, DSMZ - German Collection of Microorganisms and Cell Cultures, Inhoffenstr, 7B, 38124 Braunschweig, Germany. sna@dsmz.de
Venturini
Letizia
L
Przybylski
Grzegorz K
GK
Grabarczyk
Piotr
P
Meyer
Corinna
C
Kaufmann
Maren
M
Battmer
Karin
K
Schmidt
Christian A
CA
Drexler
Hans G
HG
Scherr
Michaela
M
Macleod
Roderick Af
RA
D002454:Cell Differentiation; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D015458:Leukemia, T-Cell / Q000235:genetics / Q000378:metabolism* / Q000503:physiopathology; D000071656:Receptor, Notch3; D051880:Receptors, Notch / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D013601:T-Lymphocytes / Q000166:cytology / Q000378:metabolism
D018398:Homeodomain Proteins; C084119:MSX2 protein; C499374:NOTCH3 protein, human; D000071656:Receptor, Notch3; D051880:Receptors, Notch