ALBERT

Description: Background Glucocorticoids (GCs) such as prednisolone and dexamethasone are critical components of multi-agent chemotherapy regimens used in the treatment of acute lymphoblastic leukemia (ALL). Children with ALL are stratified into risk groups based on diagnostic features (i.e. age and cytogenetics) and therapy response. It has been established that the initial response to prednisolone is a major prognostic factor. Moreover, at relapse, de novo or acquired resistance to GCs is common and represents an important determinant in treatment failure. Recent studies performed by us and others have identified IKZF1 gene deletions and mutations as an independent prognostic factor that predicts prognosis and treatment outcome of children with B cell precursor ALL (BCP-ALL). These monoallelic IKZF1 gene deletions either affect the whole gene or may result in expression of dominant-negative IKZF1 isoforms due to intragenic deletions. However, it has not been established whether loss of IKZF1 function directly impacts the response to glucocorticoids. Results We examined whether haplodeficiency for Ikzf1 gene expression in mouse lymphocytes affects glucocorticoid-induced apoptosis. Splenocytes from Ikzf1+/- knockout mice were activated with lipopolysaccharide (LPS) and treated with increasing concentrations of either prednisolone or dexamethasone for 48 hours. B-lymphocytes haplodeficient for IKZF1 showed a significantly enhanced survival after treatment with GCs compared to wild type cells, as measured in an MTS assay and by AnnexinV staining. In case of prednisolone, the inhibitory concentration (IC50) was about ∼200-fold higher in the Ikzf1+/- splenocytes as compared to the wild-type cells. Gene expression analysis revealed that Ikzf1+/- splenocytes displayed lower overall expression levels as well as diminished transcriptional activation of several glucocorticoid receptor (GR)-induced target genes (i.e. Sgk1, Irs2, Zfp36L2). Furthermore, in luciferase reporter assays we established that IKZF1 overexpression enhances GR-mediated transcriptional activation in response to prednisolone. Finally, lentivirus-mediated IKZF1-shRNA expression in Nalm6 cell line, which reduces endogenous IKZF1 protein levels to around 50%, inhibits prednisolone and dexamethasone-induced apoptosis, demonstrating that also in human leukemia cells reduced IKZF1 expression levels protect against GC-induced cell death. In conclusion, our data provide evidence that loss of IKZF1 function mediates resistance to glucocorticoid-induced apoptosis, which may contribute to the poor outcome of IKZF1-deleted BCP-ALL. Disclosures: No relevant conflicts of interest to declare.

Description: Background 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 p190BCR-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 p190BCR-ABL1 expression on IKZF1 protein function. Results Using a luciferase reporter assay employing the human BAX- promoter, we established that IKZF1-induced transcriptional repression was alleviated by p190BCR-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 p190BCR-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 p190BCR-ABL1. In order to understand how p190BCR-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 p190BCR-ABL1. These analyses revealed that p190BCR-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 p190BCR-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. Conclusion Our studies show that p190BCR-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 p190BCR-ABL1 signaling is the driving force for IKZF1 gene deletions in BCP-ALL patients harboring a BCR-ABL1 translocation. Disclosures: No relevant conflicts of interest to declare.