NOTCH SIGNALING INHIBITED BY IKAROS1 IN HUMAN T-CELL ACUTE LYMPHOBLASTIC LEUKEMIA

The highly conserved Notch signaling pathway regulates cell growth, differentiation, survival and apoptosis. In hematopoiesis, Notch signaling drives commitment to the T-cell fate and promotes differentiation, T-cell receptor signaling and immune function. In T-cells, Notch signaling is oncogenic when constitutively active, promoting proliferation and survival while inhibiting differentiation. The majority of patients with T-cell acute lymphoblastic leukemia (TALL) have activating Notch mutations. Similarly, Notch activation is present in more than 75% of murine T-ALL models. In murine T-ALL, loss of Ikaros, a zinc finger transcriptional regulator, leads to disrupted differentiation and also cooperates with activated Notch signaling to promote T-ALL. Loss of Ikaros function most frequently results from alternative RNA splicing, which leads to expression of non-DNA-binding isoforms. In murine T-ALL, Notch and Ikaros are reciprocally regulated. Specifically, constitutive Notch3 activation promotes expression of non-DNA-binding Ikaros isoforms in vivo, leading to loss of Ikaros function. Additionally, Ikaros regulates Notch by competing for binding sites in the promoter regions of Notch target genes, Hes1 and pTα. To determine whether this reciprocal regulation of Notch and Ikaros occurs in human T-ALL, we expressed active intracellular Notch receptors in human T-ALL cell lines and showed that Notch activation does not significantly increase the expression of non-DNAbinding Ikaros isoforms. While Notch activation has little effect on the pattern of Ikaros isoform expression in human T-ALL cell lines, Ikaros exerts a repressive effect on Notch signaling, similar to the Notch repression described in murine T-ALLs. Specifically, we demonstrated that Ikaros overexpression inhibits growth of human T-ALLs and that Ikaros downregulates expression of Notch target genes Hes1 and Hes5 at the transcriptional level. Interestingly, Ikaros inhibits growth more effectively in the cell lines with stronger Notch activation. In addition, Ikaros was shown to inhibit the Notch target gene Hes1 in both Notch dependent and independent manners. These data support the hypothesis that disruption of Ikaros contributes to proliferation of human T-ALL through de-repression of Notch signaling. In summary, we have demonstrated that Ikaros represses Notch signaling in human T-ALL