P190^BCR-ABL1 Signaling Modulates The Function Of Tumor Suppressor Protein IKZF1

The chromosomal translocation BCR-ABL1 is frequently present in adult B cell precursor acute lymphoblastic leukemia (BCP-ALL) in about 30% of the patients, while in pediatric BCP-ALL it occurs only in 3% of the patients. However, in both cases the disease is characterized by the almost obligatory presence of IKZF1 gene deletions and mutations, arguing that loss of IKZF1 function is required for oncogenic transformation by p190^BCR-ABL1. The IKZF1 gene encodes a transcription factor that belongs to the Ikaros family of zinc-finger proteins, which mainly acts as a transcriptional repressor protein through the recruitment of both HDAC-dependent and HDAC-independent co-repressor molecules. However, in some cases IKZF1 has also been shown to transcriptional activate specific target genes through association with the SWI/SNF chromatin remodeling complexes. We hypothesized that IKZF1-mediated transcription in a direct or indirect manner is modulated by BCR-ABL1 signaling. Therefore, we performed cell biological assays and proteomic studies to investigate the effect of p190^BCR-ABL1 expression on IKZF1 protein function.

Using a luciferase reporter assay employing the human BAX- promoter, we established that IKZF1-induced transcriptional repression was alleviated by p190^BCR-ABL1 expression. This effect could be reversed by Imatinib treatment, suggesting that BCR-ABL1 signaling interferes with the normal function of IKZF1. Next, we assessed the effect of p190^BCR-ABL1 on doxycycline-induced expression of IKZF1 using the murine lymphoid Tet-on Ba/F3 (TonB) cell line. Gene expression analysis showed that several target genes that are repressed by IKZF1 in TonB cells, such as p16Ink4a, Cnot6, Dscc1 and Tspan5, are transcriptionally induced by co-expression of p190^BCR-ABL1. In order to understand how p190^BCR-ABL1 signaling affects IKZF1 protein function, mass spectrometry was performed on FLAG-affinity purified IKZF1 from transiently transfected HEK293 cells in the absence or presence of p190^BCR-ABL1. These analyses revealed that p190^BCR-ABL1 expression induces phosphorylation of IKZF1 on specific serine, threonine and tyrosine residues as well lysine acetylation. Transient transfection of lysine acetyltransferase PCAF (KAT2B) confirmed that IKZF1 is modified by lysine acetylation. Western blot analysis using phospho-specific antibodies showed that IKZF1 is subject to tyrosine phosphorylation by p190^BCR-ABL1, both in HEK293 cells and TonB cells. Using an in vitro kinase assay, we demonstrated that IKZF1 can be directly phosphorylated by active recombinant ABL kinase.

Our studies show that p190^BCR-ABL1 signaling induces a multitude of different post-translational modifications on IKZF1, which could modify its properties as transcriptional regulator. We propose that modulation of IKZF1 tumor suppressor function by p190^BCR-ABL1 signaling is the driving force for IKZF1 gene deletions in BCP-ALL patients harboring a BCR-ABL1 translocation.

No relevant conflicts of interest to declare.