The Ikaros (IKZF1) gene encodes a DNA-binding zinc finger protein that functions as a tumor suppressor in leukemia. Defects in the Ikaros gene that lead to its decreased activity are associated with the development of B-cell precursor acute lymphoblastic leukemia (pre-B-ALL). However, the mechanisms by which Ikaros exerts its tumor suppressor activity, as well as the mechanisms that control the tumor suppressor function of Ikaros are poorly understood. Here, we report the identification of two novel Ikaros target genes, as well as a signal transduction pathway that regulates the Ikaros-mediated transcriptional control of these genes in pre-B ALL. We also present evidence that targeting the signal transduction pathway which regulates Ikaros transcriptional control is a potent therapeutic tool for treating pre-B ALL.
Analysis of the promoter sequences of the CDC7 and the CDK6 genes revealed multiple evolutionarily-conserved Ikaros consensus binding sites. Using qChIP (quantitative chromatin immunoprecipitation assay) we found that Ikaros binds in vivo to the promoters of the CDC7 and CDK6 genes in the human Nalm6 pre-B ALL cell line, and in primary human pre-B ALL cells. This led to the hypothesis that Ikaros regulates transcription of CDC7 and CDK6 - genes whose expression is essential for cell cycle progression and cellular proliferation. The effect of Ikaros on CDC7 and CDK6 expression was studied using a transient co-transfection assay. Luciferase reporter plasmids containing the CDC7 or CDK6 promoter regions were co-transfected with or without Ikaros into 293T cells. Co-transfection of Ikaros led to decreased luciferase activity, suggesting that Ikaros acts as a repressor of the CDC7 and CDK6 upstream regulatory elements. Increased expression of Ikaros via retroviral transduction in Nalm6 cells resulted in decreased transcription of CDC7 and CDK6. Decreased transcription of these genes was associated with increased binding of Ikaros to their promoter regions as measured by qChIP. These results suggest that Ikaros negatively regulates transcription of CDC7 and CDK6, and thus negatively regulates cell cycle progression in pre-B cell leukemia.
We have shown previously that phosphorylation of Ikaros by casein kinase II (CK2) inhibits Ikaros' ability to bind DNA and to regulate transcription of its target genes. CK2 activity is elevated in human leukemia. We tested whether the inhibition of CK2 affects the transcription of CDC7 and CDK6 in pre-B ALL in vitro and in vivo. In vitro treatment of Nalm6 pre-B ALL with TBB, a specific CK2 inhibitor, decreased transcription of CDC7 and CDK6 and was associated with increased binding of Ikaros to the promoters of these genes. Using an in vivo preclinical model of pre-B ALL we tested whether targeting of CK2 would affect transcription of CDC7 and CDK6. In vivo treatment of a human-mouse xenograft model of pre-B ALL with a CK2 inhibitor resulted in decreased transcription of CDC7 and CDK6 and strongly increased Ikaros binding to the promoters of these genes. The in vivo targeting of CK2 provided a strong anti-leukemia effect that resulted in the prolonged survival of treated mice as compared to controls.
In summary, our results suggest that Ikaros exerts its tumor suppressor activity in pre-B ALL by repressing transcription of cell cycle-promoting genes. Targeting CK2 in vivo enhances Ikaros-mediated repression of cell cycle progression resulting in an anti-leukemia effect. These data demonstrate the efficacy of CK2 targeting as a treatment for pre-B ALL in a preclinical model. These results provide a mechanistic rationale and support for the use of CK2 inhibitors as a targeted treatment of pre-B ALL in early-stage clinical trials. Supported by National Institutes of Health R01 HL095120 and a St. Baldrick's Foundation Career Development Award (to S.D.).
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.