We have identified expression of NKL MSX2 in T-ALL cell lines as well as in primary HSCs and T-cells. MSX2 expression in cell lines was regulated by core thymic factors, suggesting that MSX2 is a physiological NKL in hematopoietic cells, notably in T-cells/thymocytes. Analysis of transduced JURKAT cells overexpressing either MSX2 or oncogenic TLX1 and NKX2-5 identified activation of NOTCH3-signaling, including bHLH target gene HEY1. Expression levels of NOTCH3 and HEY1 were elevated in primary TLX1/3 positive T-ALL cells, underpinning the cell line data.
MSX2 expression has been described in stem cells, progenitor cells and derivates of neural crest cells, indicating a role in cell differentiation [47
]. Core stem cell transcription factors directly regulate expression of MSX2, demonstrating a prominent function of this homeobox gene in the regulatory network of undifferentiated cells [48
]. MSX2 knockout mice exhibit several abnormalities in tissues derived from neural crest cells but none in the hematopoietic system [49
]. However, homeostatic compensations among MSX genes as described for some tissues may explain the absence of any hematopoietic phenotype hitherto [51
]. Of note, expression of oncogenic NKLs have also been reported in neural crest derivates, including TLX1 in teeth, TLX3 in dorsal root ganglia and NKX2-5 in heart and teeth which may suggest correlations in the regulation of differentiation processes [28
In T-cell lines we analyzed several core thymic factors which differently influenced MSX2 expression. The activity of these factors correlates with particular stages of thymocyte development, showing IL7 and TGFbeta activity in early stages, and BMP4, LEF1 and TCR-signaling in later stages of thymocyte differentiation [2
]. Combined data, concerning both higher MSX2 expression levels in primary HSCs as compared to CD3+ T-cells and particular MSX2 regulation, activating by early and suppressing by late core thymic factors, indicate physiological downregulation of MSX2 during T-cell development. Accordingly, PER-117 which expresses high MSX2 levels represents a very immature T-cell line corresponding to stage I thymocytes [54
]. Additionally, our data indicated an impact of DNA methylation on MSX2 expression. However, whether this mechanism represents the physiological situation is unclear.
Interestingly, we detected chromosomal deletions of MSX2 loci at 5q35 in three T-ALL cell lines which contain t(5;14)(q35;q32) rearrangements, targeting TLX3 or NKX2-5 (also located at 5q35 centromeric of MSX2), respectively [22
]. Cooperation of genetic defects t(5;14)(q35;q32) and del(5)(q35) has been reported in primary T-ALL samples [55
], suggesting that MSX2 may be a target of del(5)(q35) in this entity. But the poor correlation with expression data indicated that MSX2 is not a plausible target of this chromosomal aberration. We suggest that TLX3 and NKX2-5 are main targets and deletion of MSX2 is incidental because of its physiological downregulation and functional substitution by those oncogenic NKLs.
NOTCH3 has a major impact on T-cell development, by regulating differentiation and survival of thymocytes [56
]. Here we identified MSX2 mediated activation of the NOTCH3-pathway in T-cells reminiscent of MSX1 in neuroblastoma [44
]. We failed in detecting direct binding of MSX2 protein to a far upstream located potential binding site [45
]. However, other studies demonstrate indirect impacts of MSX2 in target gene activities [37
]. Furthermore, MSX2 colocalized with SPEN in JURKAT cells but not with PML and interacted with SPEN and TLE1, both components of the CBF1 associated repressor complex. This complex has been localized to subnuclear aggregates which differ from those containing PML protein and which comprise CBF1, SPEN, TLE1, SKIP and activated NOTCH [7
]. Therefore, our results reveal the impact of MSX2 on CBF1 target gene regulation. Since bHLH genes HES1 and HEY1 are regulated by CBF1 and NOTCH3 [13
], we conclude that MSX2 regulates HES1 and HEY1 via both CBF1 repressor complex modulation and activation of NOTCH3 expression. The subnuclear distribution of these aggregates seems to be important for their activity. Noteworthy in this context, MSX2 has been described to interact with SUMO-ligase PIAS2 [62
]. PIAS proteins may regulate distribution and activity of NK-like homeodomain proteins via SUMOylation as recently described for MSX1 and NKX2-5 [63
Additional downstream effects of NOTCH1/3-signaling comprise activation of NFkB and of PI3K-pathway, enhancing survival of thymocytes [13
]. Accordingly, both activities were elevated in JURKAT cells overexpressing MSX2 or TLX1, as demonstrated by reduced sensitivities to inhibitors of gamma-secretase, NFkB and PI3K-signaling.
Oncogenic NKLs TLX1 and NKX2-5 resemble MSX2 in interacting with SPEN and TLE1. Accordingly, a direct interaction between TLX1 and TLE1 has been shown recently [68
]. In addition, both TLX1 and NKX2-5 activate the NOTCH3 target gene HEY1. These results suggest that both proteins substitute MSX2 in modulating the CBF1 associated repressor complex. In JURKAT cells overexpressing oncogenic NKLs we observed a shift in target gene activity from HES1 to HEY1, suggesting differences in their mode of interaction with SPEN. However, both HES1 and HEY1 are bHLH proteins and involved in inhibition of differentiation via interaction with E2A proteins E12 and E47 [12
]. Therefore, our results imply that NK-like homeodomain proteins activate CBF1 target genes, thereby inhibiting T-cell differentiation.
Furthermore, we have identified S100A genes as targets of NKLs. S100A9/11 are calcium-binding proteins which inhibit PKC-mediated phosphorylation of bHLH proteins and activate apoptosis in ALL cells [69
]. Our data suggest a correlation between S100A expression and sensitivity for Calphostin C. Mechanistically, Calphostin C may enhance apoptosis either via elevation of intracellular calcium levels or via inhibition of PKC, modulating activities of bHLH proteins [46
]. However, this promising apoptotic effect merits further examination.
Recently, we have identified MEF2C as specific oncogenic target of NKX2-5 in T-ALL cells, reflecting the physiological function of NKX2-5 in the heart [29
]. Here, we identified NOTCH3-signaling activated by both physiological MSX2 and oncogenic TLX/NKX2-5 in T-ALL cells. Thus, both modes of leukemic action may be displayed by NKLs in T-ALL, ectopic activations related to their physiological origin and dysregulations due to structural similarities to physiological members of this homeobox gene family.