Neomorphic chromatin engagement and epigenetic reprogramming by the IKZF1 N159Y tandem duplication mutant in b-acute lymphoblastic leukemia Free

Introduction IKZF1 regulates hematopoietic cell development and gene expression through DNA-binding. IKZF1 mutants, such as the IK6 deletion mutant, occur in 15% of B-cell precursor acute lymphoblastic leukemia (B-ALL) including in BCR::ABL1 and DUX4-rearranged subtypes. IKZF1 N159Y defines a subtype of B-ALL, in which the N159Y variant in the DNA-binding domain is in a tandem duplication (TD) of exon 5. The mechanistic role of IKZF1 N159Y in leukemogenesis is unknown. We hypothesized that IKZF1 N159Y harbors altered DNA binding properties, distinct from wild-type IKZF1 (WT), which drive leukemogenic gene expression.

Methods We generated novel patient-derived cell line (PD CL) models with heterozygous (het) or hemizygous (hemi) IKZF1 N159Y-TD expression, NALM6 (DUX4-r) CLs expressing WT or IKZF1 N159Y in an IKZF1-null background, and BCR::ABL1 PD CLs expressing WT or the IK6 mutant as comparators. Chromatin occupancy was profiled using ChIP-seq for IKZF1, H3K27ac, H3K4me1, H3K4me3, H3K36me3, H3K9me3, H3K27me3, EP300 and CTCF. Chromatin states were inferred using ChromHMM. Transcriptomes were assessed by RNA-seq. Motif enrichment was analyzed using HOMER. A luciferase reporter driven by the MCL1 promoter and AlphaFold 3.0 modeling were used to study functional and structural consequences of WT and TD binding. Cytotoxicity assays were used to assess sensitivity to 3 epigenetic modulators including inhibitors of histone acetylation (A-485 and JQ1) or deacetylation (vorinostat).

Results ChromHMM analysis of the IKZF1 N159Y heterozygous patient-derived CL revealed enrichment of IKZF1 proteins (WT/TD) in active chromatin and transcription start sites, and exclusion from polycomb-repressed domains. H3K27ac enrichment and H3K27me3 depletion were observed at sites bound by the TD mutant in the hemi CL. Compared to a BCR::ABL1 cell line, both the het and hemi IKZF1 N159Y cell lines showed dysregulation of 30% of the IKZF1 N159Y leukemia gene expression signature (GES), characterized by positive enrichment of IKZF1 and H3K27ac and depletion of H3K27me3 at upregulated genes, with the opposite pattern at downregulated genes. These findings suggest that the TD mutant binds chromatin and drives leukemogenic transcription and epigenetic changes.

We identified sites bound by both WT and TD or sites specific to either WT or TD in IKZF1 N159Y CLs. A higher proportion of promoters of IKZF1 N159Y signature genes were bound by both WT and TD (65%) compared to WT alone (6%) or TD alone (6%). In the hemi CL, the TD mutant bound 56% of IKZF1 N159Y GES promoters, albeit with reduced IKZF1 peak intensity at shared WT/TD targets and gene expression overlapped with the het CL and with 17 patients with IKZF1 N159Y leukemia.

In the NALM6 IKZF1-null CL, a subset of sites bound by the TD mutant were target sites shared with WT (42% of TD-bound sites). Compared to WT, at putative target promoters, the TD mutant bound more sites (4942 vs 2383), a higher proportion of IKZF1 N159Y GES genes (24% vs 9%) and dysregulated gene expression at a higher proportion of sites (13% vs 1%). TD mutant binding was associated with dysregulation of 45 IKZF1 N159Y GES genes and pathways like TGF-beta signaling which are enriched in IKZF1 N159Y leukemia.

Sites bound by WT or TD alone or both were enriched for ETS motifs. Motif discovery identified a novel bHLH motif enriched among sites bound by the TD mutant in the presence or absence of WT. Both WT and TD bound the MCL1promoter, which contains ETS motifs. Luciferase assays showed repression by WT (1.6-fold), TD (1.9-fold), and both (2.4-fold). AlphaFold modeling predicted increased interactions between exon 5 ZnF2 amino acids and an ETS motif in the MCL1 promoter in a model with both WT and the TD mutant, compared to models with WT or TD alone.

Functionally, the IKZF1 N159Y het CLs were more sensitive to A-485, JQ1, and vorinostat than BCR::ABL1 CLs, suggesting a dependency on acetylation-coupled chromatin regulation.

Conclusion We demonstrate that the IKZF1 N159Y TD mutant exhibits altered chromatin-binding specificity and transcription activity compared to WT IKAROS. Cooperative binding with WT IKZF1 and binding at de novo sites, not bound by WT, suggest partial dominant and neomorphic functions of the TD mutant. Our findings define a distinct chromatin regulatory program in IKZF1 N159Y leukemia and suggest therapeutic vulnerability to epigenetic modulators targeting histone acetylation.