Characterization of Ikaros zinc finger protein 1 mutations in acute myeloid leukemia

By Tarun Sai LomteFeb 2 2023Reviewed by Aimee Molineux

In a recent study posted to Preprints with The Lancet*, researchers evaluated the clinical and genetic characteristics of acute myeloid leukemia (AML) patients with a mutant Ikaros zinc finger protein 1 (IKZF1).

*Important notice: Preprints with The Lancet publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Background

AML is a group of hematopoietic malignancies in adults. Chromosomal abnormalities and sequence variants define AML subtypes. IKZF1 encodes Ikaros, an important zinc-finger transcriptional factor for immune hematopoiesis and lymphopoiesis. It involves germline and somatic mutations of acute leukemia (AL) and potentially functions as a tumor suppressor.

Previously, the authors identified several small sequence variants of IKZF1 with distinct expression profiles in AML. One of them is a recurrent hotspot mutation (N159S) clustered in some patients with myelodysplasia-related mutations and intermediate expression of homeobox A/B (HOXA/B) family genes. Immune-modulating drugs inducing Ikaros degradation exhibit potential therapeutic efficacy in AML, suggesting an emergent synergistic role of Ikaros in high-risk AML.

The study and findings

In the present study, researchers explored the mutation landscape of IKZF1 in a large Chinese AML cohort. Primary blasts were collected from the bone marrow of 475 patients with AML having at least 20% abnormal blasts. RNA sequencing and targeted screening of common leukemia-associated genes were performed. FLAG-tagged wild-type or mutant IKZF1 was expressed in the K562 cell line.  

Twenty-three small sequence variants of IKZF1 were identified in 4.84% of the AML patients. Of note, the hotspot mutation, N159S, was observed in nine AML cases, which accounted for 39.13% of all IKZF1 mutations in the cohort. All IKZF1 N159S cases clustered with myelodysplasia-associated AML, consistent with previous findings. In contrast, other IKZF1 mutant loci had similar expression profiles as biallelic CCAAT enhancer binding protein alpha (biCEBPA).

Hemoglobin and bone marrow blasts were significantly elevated in biCEBPA­-like IKZF1 cases, whereas platelets and variant allele frequency were higher in N159S-positive patients and those with other IKZF1 mutations. Unsupervised clustering confirmed the genomic classification in IKZF1-positive AML. Patients with biCEBPA­­-like IKZF1 and N159S were mutually exclusive and belonged to two hierarchical branches – low and high expression of HOXA/B family genes.

Gene set enrichment analysis (GSEA) revealed that B-cell receptor, tumor-growth factor (TGF)-beta, MYC, NOTCH, MYC-associated factor X (MAX), vascular endothelial growth factor (VEGF), and mitogen-activated protein kinase (MAPK) were upregulated in N159S-positive AML patients. At the same time, nucleotide excision and DNA mismatch repair pathways were downregulated.

Multivariable cox regression indicated that mutant IKZF1 might be a critical predictor of overall survival among AML patients. moreover, T regulatory (T_reg) cells, native B lymphocytes, eosinophils, and neutrophils were significantly elevated in N159S-positive AML cases. Next, the team tested the impact of different variants on Ikaros activity.

The most frequent mutations, G158S and N159S, were selected for further in vitro analysis, with N159Y acting as the positive control that affects the normal function of Ikaros. Ectopic expression of wild-type IKZF1 resulted in profound apoptosis. In contrast, N159S/Y expression caused much less apoptosis. G158S expression-induced apoptosis was similar to that induced by wild-type Ikaros.

More wild-type and G158S-positive cells were arrested in the G1 phase of the cell cycle than N159S/Y-positive cells. Further, cell growth and differentiation were significantly inhibited in G158S and wild-type cells relative to N159S/Y cells, suggesting that N159S might disturb Ikaros’ normal function, induce apoptosis, and inhibit cell cycle and proliferation of leukemic cells.

Next, the team performed CUT&Tag sequencing in the IKZF1 N159S mutant cell line to examine the genomic binding profiles in AML. This revealed that N159S reshaped the genome binding profile of IKZF1. Functional enrichment analysis revealed that interacting proteins were enriched in pathways related to cancer development and cell cycle regulation.

MYC, copine 7 (CPNE7), and CPNE8 were significantly upregulated in N159S-positive cells relative to wild-type cells. Finally, the team treated N159S-positive cells with 10058-F4 (MYC inhibitor) and observed a dose-dependent MYC inhibition after 24 days. Moreover, the proportion of cells in the G1 phase increased in a dose-dependent manner.

Conclusions

In summary, the researchers analyzed AML cases and illustrated the genomic landscape of IKZF1 mutations in AML. They identified three classes of mutations – N159S, biCEBPA-like IKZF1, and others. Many of these lead to abnormal gene expression regulation and genomic binding patterns. Integrating IKZF1 into prognostic AML classification may improve diagnosis and facilitate the development of tailored AML therapies.

*Important notice: Preprints with The Lancet publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.