Paper Details

ABSTRACT

Myeloproliferative neoplasms (MPNs) are clonal disorders initiated by activating mutations in JAK2, CALR, or MPL in hematopoietic stem/progenitor cells. Although JAK inhibitors improve symptoms, up to 20% of patients progress to secondary acute myeloid leukemia (sAML), an aggressive malignancy with limited therapeutic options and poor outcomes. Notably, several high-molecular-risk genes, including ASXL1, EZH2, and IDH1/2, encode epigenetic regulators, suggesting that epigenetic dysregulation contributes to leukemic transformation; however, studies leveraging multiomic datasets to define the transformation mechanisms remain limited.

To identify epigenetic drivers of leukemic transformation, we integrated bulk (11 NBM, 45 MPN, 34 sAML) and single-cell (6 NBM, 5 MPN, 4 sAML) transcriptomic datasets from the WashU cohort with adverse prognostic genes identified by genome-wide Cox proportional hazards analysis (P < 0.05, HR > 1.0) in BeatAML sAML cohort (n=31), together with a Gene-Ontology (GO)-derived list of epigenetic regulators. We prioritized the hematopoietic transcription factor Helios (IKZF2) as a candidate driver of leukemic transformation. Immunofluorescence confirmed progressive IKZF2 protein upregulation from NBM to MPN to sAML bone marrow specimens. Single-cell RNA sequencing localized IKZF2 upregulation predominantly to sAML HSC–MPP cells, while pseudotime and gene regulatory network analyses revealed progressive activation of the IKZF2 regulon.

We next examined the functional role of IKZF2 in MPN/sAML models. IKZF2 expression was highest in JAK2-mutant SET-2 and UKE-1 cells, which were also the most sensitive to the IKZF2-targeting PROTAC DEG-35. Pharmacologic degradation or CRISPR-mediated knockout of IKZF2 impaired proliferation and mitochondrial respiration, induced apoptosis and G1/S arrest in SET-2 cells, while reducing clonogenicity of primary sAML CD34? cells. Conversely, IKZF2 overexpression promoted proliferation and ruxolitinib resistance in SET-2 cells. Additionally, Jak2^V617F c-Kit? progenitors subjected to enforced overexpression (OE) of IKZF2 displayed enhanced serial replating ex vivo. Following transplantation, IKZF2-OE accelerated disease progression, producing leukocytosis, monocytosis, anemia, thrombocytopenia, splenomegaly, expansion of immature bone marrow cells, and shortened survival.

We then interrogated the epigenetic mechanism of IKZF2 activation. ATAC-seq and H3K27ac CUT&Tag across serial patient samples identified a candidate enhancer at the IKZF2 locus that was selectively activated after transformation to sAML and largely inactive in normal CD34? cells. Hi-C and H3K27ac HiChIP demonstrated enhancer-promoter interactions. CRISPR interference (CRISPRi) with dCas9-KRAB supported its functional role in governing IKZF2 expression in SET-2 cells. Motif analysis and ChIP-seq revealed occupancy of the transcriptional repressor GFI1B and activator SPI1 at this enhancer. Mechanistically, pharmacologic LSD1 inhibition disrupted the GFI1B–LSD1 repressive complex, leading to reduced GFI1B occupancy, increased SPI1 binding, enriched H3K27ac marks at the enhancer, and consequent de-repression of IKZF2 expression. These effects were abolished in LSD1 inhibitor-resistant cells. Consistently, GFI1B knockout increased IKZF2 expression and SPI1 knockout reduced IKZF2 expression, demonstrating that competitive GFI1B-SPI1 occupancy regulates enhancer-dependent IKZF2 activation.

To define downstream programs regulated by IKZF2, we performed multiomic profiling of SET-2 cells. IKZF2 loss suppressed MYC pathways and leukemia-associated genes, including ITGB3, TERT, and SPP1. In contrast, IKZF2 depletion induced differentiation-associated regulators, including CEBPA and the MYC antagonist and tumor suppressor PRDM1. Consistently, IKZF2 degradation reduced phosphorylation of RB, S6, and 4EBP1, indicating attenuation of cell-cycle progression and proliferative signaling. We further identified a putative IKZF2-associated enhancer at the PRDM1 locus, supporting a model in which IKZF2 rewires downstream enhancer programs to sustain proliferation while restraining differentiation.

Collectively, our study identifies IKZF2 as a previously unrecognized regulator of post-MPN leukemic transformation through integrative multiomic profiling and mechanistic interrogation. These findings link enhancer dysregulation to leukemic transcriptional reprogramming, highlighting IKZF2 as a potential therapeutic target in sAML.