DEK::NUP214 acts as an XPO1-dependent transcriptional activator of essential leukemia genes

Abstract

The t(6;9)(p22.3;q34.1) translocation/DEK::NUP214 fusion protein defines a distinct subgroup of younger AML patients classified as a separate disease entity by the World Health Organization. DEK is a nuclear factor with multifunctional roles, including gene regulation, while its fusion partner, NUP214, plays a pivotal role in nuclear export by interacting with transport receptors such as XPO1. However, the precise mechanism by which DEK::NUP214 drives leukemia remains unclear. A comprehensive multi-omics comparison of 57 AML primary samples (including whole genome sequencing, targeted sequencing, transcriptomics, and drug screening with >500 compounds) revealed that t(6;9) cases display a selective response to XPO1 inhibitors (Selinexor & Eltanexor) and a distinct transcriptomic signature characterized by the overexpression of FOXC1 and HOX genes that are key leukemia mediators. CUT&RUN experiments demonstrated the direct binding of DEK::NUP214 to the promoters of FOXC1 and HOXA/B clusters. Strikingly, the expression of these genes and the binding of DEK::NUP214 to their regulatory regions were selectively reduced upon XPO1 inhibition in t(6;9) cells. Altogether, these results identified a novel function of DEK::NUP214 as an XPO1-dependent transcriptional activator of key leukemia drivers and provide a rationale to explore the use of XPO1 inhibitors in this patient population.

Fig. 1. AML primary samples with t(6;9)/DEK::NUP214 display a unique transcriptional signature.

A RNA-seq unsupervised clustering of 57 cytogenetically poor-risk AML samples [10], including four t(6;9) cases. Different colors represent distinct cytogenetic AML subtypes. B Volcano plot representing differentially expressed genes in t(6;9) primary AML samples vs other cytogenetic AML subtypes (RNA-seq analysis integrating our cohort of 57 poor-risk cases and 691 cases from the Leucegene cohort (Leucegene project, https://leucegene.ca/) (significance determined by Kernel MCC > 0.105 and log₂FoldChange (l2fc) > 0.58). Kernel MCC is a shorthand for Matthews Correlation Coefficient of a kernel density estimation (KDE)-based classifier. C mRNA expression levels HOXA and HOXB genes in t(6;9) samples compared to KMT2A-rearranged and other poor-risk AMLs (n = 57). D mRNA expression level of FOXC1 in t(6;9) patients compared to other poor-risk AMLs (n = 57). E FOXC1 protein expression across the AML cell line panel (OCI-AML3, KASUMI-1, THP-1, FKH-1, P31-FUJ, MV4;11 and K562). GAPDH was used as an endogenous control. F Bar chart showing FOXC1 silencing efficiency by RT-qPCR after shRNA/scramble transduction of FKH-1 cells. Relative expression levels were calculated using the ΔCT method, normalized to the average of GAPDH and 18S rRNA. G Representative flow cytometry plot for the apoptosis assay, with FOXC1 knockdown (KD)/scramble (scr) FKH-1 cells stained with Annexin V (X-axis) and DAPI (Y-axis). The bar graph illustrates the percentage of live cells (DAPI^– Annexin V^–), early apoptosis (DAPI^– Annexin V⁺), late apoptosis (DAPI⁺ Annexin V⁺), and dead cells (DAPI⁺ Annexin V^–). H Bar graph representing cell cycle analysis by flow cytometry based on DNA content stained with DAPI of FOXC1 KD/scr FKH-1 cells. Each group of bars indicates the percentage distribution of cells in G0/G1, S, and G2/M phases. I Bar graph showing the number of colonies of FOXC1 KD/scr FKH-1 cells after the colony forming unit (CFU) assay. J Representative images of colonies corresponding to the CFU assay. Asterisks indicate statistical significance (*: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001 and ‘ns’ denotes non-significant differences).

Fig. 2. DEK::NUP214 specifically binds to the regulatory regions of key leukemia genes in an XPO1-dependent manner.

A In vitro drug screening using a 527-drug panel in 4 t(6;9) patients and 145 primary AML samples from other cytogenetic groups [10, 14]. The table shows the top-ranked compounds most specific and efficient for t(6;9) patients, with the XPO1 inhibitors (Selinexor and Eltanexor) highlighted in gray. B Volcano plot representing differentially expressed genes in two t(6;9) primary AML samples treated with Selinexor vs DMSO treatment control (RNA-seq results). Genes highlighted in the plot were significantly downregulated (p < 0.05) after XPO1 inhibition. C IGV genome browser plots representing the binding peaks on the FOXC1, HOXA and HOXB promoters corresponding to the CUT&RUN experiments performed in DN-HA-293T and EV-293T cells using anti-HA antibody (and anti-IgG as a control). D CUT&RUN-qPCR results show fold enrichment at the promoter regions of the FOXC1, HOXA and HOXB genes clusters in the t(6;9) AML FKH-1 and non-t(6;9) AML KASUMI-1 cells, compared to their respective IgG controls. FKH-1_NUP214 and KASUMI-1_NUP214 refer to CUT&RUN performed with the NUP214 antibody, while FKH-1_IgG and KASUMI-1_IgG indicate the corresponding controls performed with the IgG antibody. Fold enrichment was calculated relative to the FKH-1_IgG sample. Statistical significance is indicated as ***: p < 0.001, ***: p < 0.0001, and ns non-significant differences. (n = 3). E IGV genome browser plots representing the binding peaks on the FOXC1, HOXA and HOXB promoters corresponding to the CUT&RUN experiments performed in FKH-1 and KASUMI-1 cells after treatment with 200 nM Selinexor for 48 h and using anti-NUP214 antibody (and anti-IgG as a control) (Selix: treated with Selinexor; DMSO: treated with DMSO).