Assi, Salam A.

Bonifer, Constanze

Cockerill, Peter N.

Coleman, Daniel J. L.

Imperato, Maria Rosaria

Keane, Peter

Kellaway, Sophie G.

Potluri, Sandeep

English

University of Birmingham Research Portal

Gene regulation in t(6;9) DEK::NUP214 Acute Myeloid Leukemia resembles that of FLT3-ITD/NPM1 Acute Myeloid Leukemia but with an altered HOX/MEIS axis

Kellaway, Sophie G

Coleman, Daniel J L

Assi, Salam A

Cockerill, Peter N

Repository@Nottingham

LETTER OPENACUTE MYELOID LEUKEMIAGene regulation in t(6;9) DEK::NUP214 Acute Myeloid Leukemiaresembles that of FLT3-ITD/NPM1 Acute Myeloid Leukemia butwith an altered HOX/MEIS axisSandeep Potluri 1,4, Sophie G. Kellaway 1,2,4, Daniel J. L. Coleman 1,4, Peter Keane 1,3,4, Maria Rosaria Imperato1, Salam A. Assi1,Peter N. Cockerill 1 and Constanze Bonifer 1✉© The Author(s) 2024Leukemia (2024) 38:403–407; https://doi.org/10.1038/s41375-023-02118-1TO THE EDITOR:Acute Myeloid Leukemia (AML) represents a heterogeneousgroup of hematological malignancies. The t(6;9)(p23;q34) trans-location, generating the DEK::NUP214 fusion protein is found in1% of AML. It causes a highly aggressive disease with poorprognosis in patients with a median age of just 23 [1]. t(6;9) AMLoften harbours a FLT3 internal tandem duplication (ITD) muta-tion as well which contributes to adverse outcomes [1]. Differingmechanisms underlying disrupted differentiation and prolifera-tion in AML challenge treatment improvement, as each AML sub-type forms its own gene regulatory network (GRN) dependent onthe driver mutation, which is distinct from healthy cells [2]. GRNshighlight which transcription factors (TFs) regulate which genesat which level, and inform on specific vulnerabilities [2]. Despiteharbouring different driver oncogenes, gene expression patternsof t(6;9) AML resemble those of NPM1-mutated and NUP98::NSD1AML [3, 4]. However, it remains unclear how DEK::NUP214de-regulates gene expression in AML as the GRN has not beenstudied [5, 6].In healthy hematopoietic cells, DEK is a DNA and RNA bindingprotein with different functions including modulating chromatinaccessibility and histone acetylation [6]. NUP214 is a part ofthe nuclear pore complex, with roles in multiple pathways suchas cell cycle progression and nucleocytoplasmic transport [5].DEK::NUP214 is thought to disrupt various nuclear processesleading to the dysregulation of myeloid differentiation [6]. Thisincludes deregulation of HOX gene clusters, which encode a familyof TFs with crucial roles in normal hematopoietic development andwhich are tightly regulated display spatial-temporal expressionpatterns and are required for normal haematopoietic differentia-tion [7]. Aberrant activation of HOX genes has been associatedwith leukemogenesis in multiple AML sub-types, including t(6;9)[3, 4, 7, 8]. Here, we utilise genome-wide chromatin accessibility toelucidate how the normal haematopoietic progenitor cell GRN isdisrupted by DEK::NUP214. We find that the DEK::NUP214 AMLGRN is related to that of mutant NPM1 AML, but also displays anelevated leukemic stem cell signature, suggesting overlappingand unique therapeutic vulnerabilities.To address the question of how gene expression is de-regulatedin t(6;9) AML, we assessed chromatin accessibility using DNaseI-seq and gene expression by RNA-seq in CD34-purified AML blastsfrom two t(6;9)-positive patients and a t(6;9) cell line, FKH1. Onepatient also carried a FLT3 tyrosine kinase domain mutation. Thepattern of DNaseI hypersensitive sites (DHSs) was similar betweenthe two patients (Fig. 1A, Supplementary Fig. 1A). Unsupervisedclustering of this data together with previously generated DNaseI-seq data from patients with other genotypes [2, 9], showed thatt(6;9) AML is part of the larger FLT3-ITD, NPM1 and FLT3-ITD/NPM1AML cluster (Fig. 1A). Chromatin accessibility data for patients withUBTF-TD, KMT2A-PTD and NUP98-rearranged AML known toderegulate HOX genes were not available. FKH1 proved to bean unsuitable model for t(6;9) AML as its DHS pattern showedlittle correlation with any primary AML blasts (Fig. 1A), thereforedownstream analysis used only patient cell DNaseI data. AML-specific gene expression, determined by RNA-seq on the t(6;9)primary AML samples compared to healthy peripheral blood stemcells (PBSCs), varied between the patients but showed a significantoverlap of deregulated genes (p= 7.7 × 10−66 upregulated genes,p= 1.3 × 10−99 downregulated genes; Supplementary Fig. 1B).Inspection of known FLT3-ITD and NPM1 de-regulated genesshowed similar expression in t(6;9) AML (Supplementary Fig. 1C).To construct a t(6;9)-specific GRN, we used merged DNaseI andRNA-seq data from both patients filtered against PBSCs, with pan-AML promoter-capture Hi-C to confidently assign enhancers totheir cognate genes, as previously described [10]. This analysisshowed that t(6;9) AML shares key regulatory TF nodes with FLT3-ITD and NPM1 AML such as NFIL3, FOXC1, NFIX, WT1, EGR1 and AP-1 (FOSL2), which are essential for FLT3-ITD/NPM1 AML main-tenance [2, 10] and which were also de-regulated in t(6;9) AML(Fig. 1B, Supplementary Fig. 1C).To evaluate the regulatory phenotype of t(6;9) as comparedto FLT3-ITD or NPM1 AML, we ranked the DNaseI data by theReceived: 21 August 2023 Revised: 7 December 2023 Accepted: 8 December 2023Published online: 4 January 20241Institute of Cancer and Genomic Sciences, University of Birmingham, Birmingham, UK. 2Present address: Blood Cancer and Stem Cells, Centre for Cancer Sciences, School ofMedicine, University of Nottingham, Nottingham, UK. 3Present address: School of Biosciences, University of Birmingham, Birmingham, UK. 4These authors contributed equally:Sandeep Potluri, Sophie G. Kellaway, Daniel J. L. Coleman, Peter Keane. ✉email: c.bonifer@bham.ac.ukwww.nature.com/leuLeukemia1234567890();,:Fig. 1 t(6;9) AML shares chromatin accessibility and gene expression with FLT3-ITD and NPM1-mutated AML. A Heatmap with hierarchicalclustering showing the Pearson correlation of the tag counts at distal DNaseI peaks of t(6;9) patient blasts together with blasts from othermutational backgrounds and a t(6;9) cell line (FKH1). Mutations present in each sample are indicated to the right with common drivers inclusters shown on the heatmap. B t(6;9) AML-specific GRN, based on merged t(6;9)-specific DNase1 and RNA-seq data vs PBSCs. The colour ofthe node indicates the gene expression in FPKM, the edge colour shows how many sites with the source node motif regulate the target genewith the arrow providing directionality between source and target. Nodes outlined in blue and dashed edges are also present in the FLT3-ITD/NPM1 GRN. C Tag counts of the DNaseI distal peaks were ranked by the fold change between the average of two NPM1-only patients and twoFLT3-ITD-only patients. Density plots show the tag counts across a 2 kb window, with the t(6;9) patients plotted on the same axis. Averageprofiles (right) show the average signal of DNaseI in the t(6;9), NPM1 and FLT3-ITD patients across all the 4-fold FLT3-ITD or NPM1 specific sites.S. Potluri et al.404Leukemia (2024) 38:403 – 407fold-change of the DHS tag count between NPM1 and FLT3-ITDpatients, and plotted the t(6;9) DNaseI signal alongside (Fig. 1C, left).These analysis showed that the DHS pattern in t(6;9) AML closelyresembles that of NPM1-mutated AML. Clustering just t(6;9), FLT3-ITD and NPM1 DHS data (Supplementary Fig. 1D) and plotting theaverage signal across NPM1 or FLT3-ITD specific DHSs (Fig. 1C,right), showed that t(6;9) patient chromatin was accessible at theNPM1- but not the FLT3-ITD-AML specific sites. To identify TFsmediating this pattern, we employed digital footprinting followedby de novo motif discovery. TF binding motifs enriched in both setsof t(6;9) specific footprints as compared to those of healthy PBSCs(Supplementary Fig. 1E) included occupied EGR, NF1 and AP-1S. Potluri et al.405Leukemia (2024) 38:403 – 407motifs, confirming that these core nodes of the GRN are involved inregulating t(6;9)-specific AML gene expression. Footprints were alsoenriched with RUNX, ETS and C/EBP motifs which are boundby these global haematopoietic regulators. Together these datashow that the GRN driven by DEK::NUP214 is similar to FLT3-ITD andclosest to that of NPM1-driven AML, but does not completelyoverlap.NPM1-mutated AML is associated with a more favourableprognosis than t(6;9) AML. We therefore evaluated the differencesin gene regulation underpinning this phenotype. Whilst t(6;9) andNPM1 AML gene expression patterns (Fig. 2A, left) and theenrichment of footprinted TF motifs such as for AP-1 and HOX(Fig. 2A, right) were overall similar, differences were evident. Toinvestigate these, we this time ranked the DHS tag counts by thefold change between NPM1 and t(6;9) (Fig. 2B) demonstrating thatNPM1-specific DHSs were enriched with HOX and MEIS motifs. Incontrast, HIF1A and STAT motifs were enriched in t(6;9) specificsites (Fig. 2B) which suggests increased signalling activity in t(6;9)patients and could drive leukemic stem cell (LSC) growth [11]. Thisnotion was supported by a higher LSC17 score, a gene signatureindicating the stemness and associated prognosis of an AML(Fig. 2B, right) in t(6;9) patients. Although NPM1 patients typicallylack CD34 cell surface marker expression, the same trend was seenwhen excluding CD34 from the calculation (SupplementaryFig. 2A). The poorer prognosis of t(6;9) AML patients maytherefore be due to higher LSC numbers or growth but furtherinvestigation would be needed to test this hypothesis.The HOX binding motifs in the NPM1-specific DHSs could bebound by any of several HOX factors. The HOXA and HOXBclusters, together with MEIS1 are upregulated in NPM1 AML whichcontributes to the differentiation block [8]. HOX genes have alsobeen reported as upregulated in t(6;9) AML as compared to othersubtypes such as t(8;21) [4]. MLL-Menin modulates upregulation ofHOX genes in NPM1-mutated and SET::NUP214 AML [5, 12], whilstother NUP fusion proteins cause upregulation of HOX genes [3]through co-operation of NUP proteins with XPO1 (CRM1) [13]enriched at HOX promoters [14]. In line with these findings, theHOX loci of t(6;9) patients display accessible chromatin (Fig. 2C).However, accessibility at the HOX loci of t(6;9) patients differedwhen compared to that of patients carrying the FLT3-ITD withoutthe NPM1 mutation (with additional mutations in BCOR, DNMT3A,TET2, TP53 and Tri(13)), and patients carrying NPM1 mutation butnot the FLT3-ITD. Patients with t(6;9) AML displayed less accessiblechromatin at several HOX DHSs, including at the HOXA7, HOXA9and HOXB2 promoters and at distal cis-regulatory elements(Fig. 2C). Moreover, mRNA expression of HOXA7, HOXA9, HOXA11(in both patient samples but not FKH1 cells) and HOXB2 geneswere significantly lower in t(6;9) AML compared to FLT3-ITD andNPM1 AML (Fig. 2C, Supplementary Table 1), although stillupregulated and more accessible than in, for example, t(8;21)AML. Of the AMLs studied and with the exception of HOXB9, HOXgene expression patterns in t(6;9) AML most closely resembledNUP98::NSD1 [2, 9, 15].HOX genes contribute to maintaining an immature phenotypeof NPM1 AML [8], but compared to DHS patterns from healthystage-specific datasets, NPM1, FLT3-ITD and t(6;9) AML showedsimilar maturity (Supplementary Fig. 2B). Therefore, specific HOXexpression patterns are due to the driver mutation, rather than thecell stage of the differentiation block. Genes with HOX and/orMEIS sites at associated DHSs included CD34, which displayshigher gene and cell surface expression in t(6;9) AML compared toNPM1 AML (Supplementary Fig. 2C, D). However, the majority ofdifferentially expressed genes with HOX/MEIS motifs, includinglymphoid genes, MEIS1 and AP-1 family member genes, wereexpressed at least 2-fold higher in NPM1 patients (SupplementaryFig. 2C, Supplementary Table 1) suggesting reduced reliance onHOX factors in t(6;9) AML. Taken together, these data indicatedifferential regulation of the HOX/MEIS genes in t(6;9) AML ascompared to NPM1 AML, with downstream HOX/MEIS bindingsites also differentially regulated.In conclusion, our study sheds light on the unique molecularcharacteristics of t(6;9) AML. It reveals that t(6;9) AML exhibits aGRN comparable to that of the NPM1 and FLT3-ITD AML subtypesbut shows altered HOX expression and diminished downstreamregulation by HOX-related pathways. However, t(6;9) AML exhibitsa stronger LSC signature. These findings emphasize the need fortailored therapeutic approaches based on the broader generegulatory program to target this highly aggressive AML sub-type.We have recently shown that targeting essential components inFLT3-ITD/NPM1 AML GRN, such as AP-1, FOXC1 or NFIX interfereswith AML maintenance [2, 10]. We also have shown that LSCgrowth can be specifically inhibited [11]. Our data here indicatethat similar strategies could be considered for t(6;9) AML.DATA AVAILABILITYRNA-seq and DNaseI-seq produced in this study have been deposited in the GeneExpression Omnibus (GEO) under accession code GSE240272.REFERENCES1. Slovak ML, Gundacker H, Bloomfield CD, Dewald G, Appelbaum FR, Larson RA,et al. A retrospective study of 69 patients with t(6;9)(p23;q34) AML emphasizesthe need for a prospective, multicenter initiative for rare ‘poor prognosis’myeloidmalignancies. Leukemia. 2006;20:1295–7. https://doi.org/10.1038/sj.leu.2404233.2. Assi SA, Imperato MR, Coleman DJL, Pickin A, Potluri S, Ptasinska A, et al. Subtype-specific regulatory network rewiring in acute myeloid leukemia. Nat Genet.2019;51:151–62. https://doi.org/10.1038/s41588-018-0270-1.3. Hollink IH, van den Heuvel-Eibrink MM, Arentsen-Peters ST, Pratcorona M, AbbasS, Kuipers JE, et al. NUP98/NSD1 characterizes a novel poor prognostic group inacute myeloid leukemia with a distinct HOX gene expression pattern. Blood.2011;118:3645–56. https://doi.org/10.1182/blood-2011-04-346643.4. Sandahl JD, Coenen EA, Forestier E, Harbott J, Johansson B, Kerndrup G, et al.t(6;9)(p22;q34)/DEK-NUP214-rearranged pediatric myeloid leukemia: an interna-tional study of 62 patients. Haematologica. 2014;99:865–72. https://doi.org/10.3324/haematol.2013.098517.5. Mendes A, Fahrenkrog B. NUP214 in Leukemia: It’s More than Transport. Cells.2019;8:76. https://doi.org/10.3390/cells8010076.Fig. 2 t(6;9) AML differs from NPM1-mutated AML in the HOX/MEIS axis. A. Scatterplot of gene expression as log2 FPKM in t(6;9) vs PBSCs.Genes shown in red are at least 4-fold upregulated in blue at least 4-fold downregulated in NPM1 AML compared to PBSCs. Dashed lines de-lineate 4-fold up and downregulated in t(6;9) vs PBSCs. Heatmap with hierarchical clustering showing the log2 motif score for footprintedmotifs identified in t(6;9) and NPM1 DNase1 datasets and not found in PBSCs. B Tag counts of the DNaseI distal peaks were ranked by the foldchange between the average of two NPM1-only patients and two t(6;9)-only patients. Density plots show the tag counts across a 2 kb window,with enriched motifs plotted on the same axis. LSC17 scores were calculated from the normalised FPKM values of the RNA-seq for thesepatients, the horizontal bar indicates the mean of the two patients which are shown by individual points. C UCSC Genome Browser screenshotof the HOXA and HOXB loci showing normalised DNaseI read coverage, boxes indicate sites which display less accessible chromatin in botht(6;9) patients as compared to all FLT3-ITD and NPM1 patients, average 1.94-fold less accessible across all peaks in each patient compared toeach FLT3-ITD/NPM1 patient (range 1.17–5.49-fold), top. Normalised counts of key HOX genes and MEIS1 shown for cells from t(6;9) patientsand a t(6;9) cell line, healthy PBSCs and patient cells from other AML backgrounds. Horizontal line indicates the mean, individual patients/cellline are shown by circles. *indicates p < 0.05, **p < 0.01 with green asterisks showing the significance between t(6;9) and FLT3-ITD onlyexpression and orange asterisks showing significance between t(6;9) and NPM1 only expression by Student’s T-test.S. Potluri et al.406Leukemia (2024) 38:403 – 4076. Wilcher KE, Page ERH, Privette Vinnedge LM. The impact of the chromatinbinding DEK protein in hematopoiesis and acute myeloid leukemia. Exp Hematol.2023;S0301-472X:00225–4. https://doi.org/10.1016/j.exphem.2023.05.002.7. Alharbi RA, Pettengell R, Pandha HS, Morgan R. The role of HOX genes in normalhematopoiesis and acute leukemia. Leukemia. 2013;27:1000–8. https://doi.org/10.1038/leu.2012.356.8. 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Targetingchromatin regulators inhibits leukemogenic gene expression in NPM1 mutantleukemia. Cancer Discov. 2016;6:1166–81. https://doi.org/10.1158/2159-8290.CD-16-0237.13. Saito S, Cigdem S, Okuwaki M, Nagata K. Leukemia-associated Nup214 fusionproteins disturb the XPO1-mediated nuclear-cytoplasmic transport pathwayand thereby the NF-κB signaling pathway. Mol Cell Biol. 2016;36:1820–35.https://doi.org/10.1128/MCB.00158-16.14. Oka M, Mura S, Yamada K, Sangel P, Hirata S, Maehara K, et al. Chromatin-prebound Crm1 recruits Nup98-HoxA9 fusion to induce aberrant expression ofHox cluster genes. Elife. 2016;5:e09540. https://doi.org/10.7554/eLife.09540.15. Rasouli M, Blair H, Troester S, Szoltysek K, Cameron R, Ashtiani M, et al. The MLL-menin interaction is a therapeutic vulnerability in NUP98-rearranged AML.Hemasphere. 2023;7:e935.ACKNOWLEDGEMENTSThis study was supported by grants from Blood Cancer UK (20006) and the MedicalResearch Council (MR/S021469/1), awarded to CB and PNC. The authors would like tothank the West Midlands Regional Genetics Laboratory for supplying mutation datalinked to AML patient samples. We also thank Genomics Birmingham for expertsequencing services.AUTHOR CONTRIBUTIONSSP and CB conceived the study, PNC and CB directed the work, SP, DJLC and MRIperformed experiments, SGK, DJLC, PK and SAA performed analysis, SGK, PNC and CBwrote the manuscript, all authors provided feedback on the data and manuscript.COMPETING INTERESTSThe authors declare no competing interests.ADDITIONAL INFORMATIONSupplementary information The online version contains supplementary materialavailable at https://doi.org/10.1038/s41375-023-02118-1.Correspondence and requests for materials should be addressed to ConstanzeBonifer.Reprints and permission information is available at http://www.nature.com/reprintsPublisher’s note Springer Nature remains neutral with regard to jurisdictional claimsin published maps and institutional affiliations.Open Access This article is licensed under a Creative CommonsAttribution 4.0 International License, which permits use, sharing,adaptation, distribution and reproduction in anymedium or format, as long as you giveappropriate credit to the original author(s) and the source, provide a link to the CreativeCommons licence, and indicate if changes were made. The images or other third partymaterial in this article are included in the article’s Creative Commons licence, unlessindicated otherwise in a credit line to the material. If material is not included inthe article’s Creative Commons licence and your intended use is not permitted bystatutory regulation or exceeds the permitted use, you will need to obtain permissiondirectly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.© The Author(s) 2024S. Potluri et al.407Leukemia (2024) 38:403 – 407

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Gene regulation in t(6;9) DEK::NUP214 Acute Myeloid Leukemia resembles that of FLT3-ITD/NPM1 Acute Myeloid Leukemia but with an altered HOX/MEIS axis

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