Characterizing a Molecular Pathway Through Which Ikaros Functions as a Tumor Suppressor in BCR-ABL1+ pre-B Acute Lymphoblastic Leukemia (pre-B ALL)

Abstract

Although tyrosine kinase inhibitors have greatly improved patient outcome in pre-B acute lymphoblastic leukemia (pre-B ALL), this disease is still the deadliest childhood cancer. Ikaros is a tumor suppressor in pre-B ALL. More than 75% of BCR-ABL1+ pre-B ALL cases were found to harbor an intragenic deletion in at least 1 allele of IKZF1 resulting in the expression of a dominant negative (DN) Ikaros isoform, Ik6. Ik6 expression abrogates Ikaros functions in pre-B ALL. The prognosis of BCR-ABL1+ pre-B ALL is very poor, and loss of Ikaros functions has been linked to poor prognosis and therapy resistance in other types of pre-B ALL. Therefore, Ikaros is an interesting target for the treatment of BCR-ABL1+ pre-B ALL. Not much is known about the mechanism of Ikaros tumor suppression in pre-B ALL. The aim of this project was to characterize a hypothesized Ikaros tumor suppression pathway in pre-B ALL. Our group and others have shown that the gene CTNND1 is a conserved Ikaros target, and it has been reported in other cell types that CTNND1 regulates gene expression in the nucleus by removing repression of gene expression by the transcriptional regulator Kaiso. Our group also found that Ctnnd1 and Ccnd1 were positively regulated due to a loss of Ikaros tumor suppressor function in mouse BCR-ABL1+ pre-B ALL cells. We hypothesized that one of the ways through which Ikaros suppresses leukemic growth in human BCR-ABL1+ pre-B ALL is by suppressing CTNND1 expression, allowing Kaiso to repress cyclin D2 expression. To test our hypothesis in human pre-B ALL, we first profiled Ikaros in patient-derived pre-B ALL samples and analyzed the correlation between Ikaros status and CTNND1 and CCND2 expression. We observed the expression of a dominant negative isoform of Ikaros, Ik6, and point mutations in the DNA binding domain of Ikaros in pre-B ALL. These genetic changes in Ikaros correlated with a positive regulation in CTNND1 and CCND2 expression. We next restored wild-type Ikaros in a BCR-ABL1+ pre-B ALL subtype expressing DN-Ik6, and analyzed for resulting phenotypes. We found that the restoration of wild-type Ikaros induced a slow growing BCR-ABL1+ pre-B ALL phenotype. We also found that cell cycle progression in these cells was inhibited. Analysis of protein expression from these cells revealed a negative regulation of CTNND1 and CCND2 expression. A genetic deletion of Ctnnd1 in a mouse model of BCR-ABL1+ pre-B ALL cells showed that the increased CTNND1 expression in Ikaros mutant pre-B ALL gave a growth advantage to these cells. Ongoing work in this study suggests that blocking Ikaros functions in pre-B ALL that express wild-type Ikaros resulted in a positive deregulation of CTNND1 and CCND2 expression, supporting our above findings. Preliminary results also suggested that a combination of blocking cKit and Ctnnd1 severely inhibited Ikaros mutant pre-B ALL cell growth. An experiment to test Kaiso gain of function in pre-B ALL is yet to be completed. In conclusion, our study provided evidence to suggest that the negative regulation of CTNND1 by Ikaros, with a corresponding downregulation of cyclin D2 expression represents a molecular pathway through which Ikaros functions as a tumor suppressor in pre-B ALL