Role of MNX1-mediated Histone Modifications and PBX Gene Family in MNX1-induced Leukemogenesis

Role of MNX1-mediated Histone Modifications and PBX Gene Family in MNX1-induced Leukemogenesis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Role of MNX1-mediated Histone Modifications and PBX Gene Family in MNX1-induced Leukemogenesis Eric Malmhäll-Bah, Anders Östlund, Tina Nilsson, Dieter Weichenhan, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6480114/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 19 Jan, 2026 Read the published version in Scientific Reports → Version 1 posted 12 You are reading this latest preprint version Abstract The t(7;12)(q36;p13) AML subtype in pediatric patients is associated with the upregulation of homeodomain protein MNX1 as the initiating event for leukemogenesis. In this study, we investigated the downstream targets of MNX1 and their relationship to MNX1-induced histone modifications. Using a comprehensive approach combining TMT mass spectrometry, RNA-sequencing, qPCR, antibody-guided chromatin tagmentation sequencing (ACT-Seq), assay for transposase-accessible chromatin using sequencing (ATAC- seq), and chromatin immunoprecipitation assay (ChIP) followed by qPCR, we identified Pbxip1 along with its associated transcription factor Pbx1, and Pbx4 as downstream targets of MNX1. MNX1 binding to the Pbx1 promoter triggered its transcriptional activation, associated with increased H3K4me3 and decreased H3K27me3 at the Pbx1 promoter. Despite the transient nature of MNX1 ’s promoter interaction, these histone marks persisted, suggesting a “hit-and-run” epigenetic remodeling mechanism. Enrichment of Pbx motifs within MNX1-induced H3K4me1, H3K4me3, and ATAC-seq peaks underscores the role of the Pbx family in MNX1-mediated chromatin dynamics. This was further confirmed by showing that MNX1-induced Pbx1 expression could be downregulated using Sinefungin, a pan-methyltransferase inhibitor, that also prevents MNX1-driven leukemia. Our findings provide insights into how MNX1-driven epigenetic modifications are connected to its downstream targets and may offer new avenues for therapeutic intervention in t(7;12) AML. Biological sciences/Cancer/Haematological cancer Biological sciences/Molecular biology/Epigenetics MNX1 PBX pediatric AML leukemia t(7 12) histone methylation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Acute myeloid leukemia (AML) accounts for approximately 15–20% of childhood leukemias 1 . It is characterized by the clonal expansion of hematopoietic stem and progenitor cells (HSPCs), resulting in ineffective hematopoiesis and bone marrow failure 2 . The overall survival rate for AML is notably lower than that of the more common pediatric acute lymphoblastic leukemia (ALL), largely due to various genetic alterations associated with poor prognosis 3 . One such aberration, the translocation t(7;12)(q36;p13), has been identified in children diagnosed under the age of 24 months 4 – 10 and has been included in the 5th edition of the World Health Organization (WHO) Classification of Hematolymphoid Tumors 11 . In all patients with t(7;12), overexpression of MNX1 has consistently been identified as a common event 7 , 9 , 12 . Using human induced pluripotent stem cells (iPSC), we previously confirmed that this translocation leads to elevated MNX1 expression 4 , through an enhancer-hijacking mechanism that activates the MNX1 promoter via enhancers from the ETV6 locus 6 . MNX1 is a homeobox transcription factor previously recognized for its role in the development of motor neurons and pancreatic beta cells 13 – 15 . However, recent studies have implicated MNX1 in oncogenesis, with its involvement observed in AML and in prostate, colorectal, bladder, and hepatocellular cancers 16 – 21 . In AML, we have shown that MNX1 associates with methyl transferases and components of methionine cycle in AML leading to significant histone modifications, specifically H3K4me3 and H3K27me3, which in turn cause aberrant gene expression, genome-wide chromatin alterations, and DNA damage 5 . In this study, we identify PBXIP1 and PBX4 as MNX1-regulated genes, expanding the potential role of PBX transcription factors in t(7;12) AML. While PBX1 has been extensively studied in leukemia little is known about the contributions of PBXIP1 and PBX4 in this context 22 – 25 . PBXIP1, originally described as a PBX1-interacting protein that modulates PBX-HOX complexes 26 , has been linked to erythrocyte differentiation and is upregulated in AML 27 , suggesting a possible role in hematopoiesis and leukemic transformation. PBX4, though less characterized, has been associated with hematopoietic development and may contribute to transcriptional dysregulation in leukemia 24 . Our findings suggest that MNX1-driven leukemogenesis may involve PBXIP1 and PBX4 through epigenetic modifications that sustain oncogenic gene expression. Results Pbxip1 is a downstream target of MNX1 To investigate the downstream targets of MNX1, we performed an integrative analysis combining protein complexes associated with MNX1 from mass spectrometry with transcriptomic data from RNA-seq. MNX1-associated protein complexes were identified through co-immunoprecipitation of MNX1 followed by TMT mass spectrometry, using BM from mice with MNX1-induced AML, as further detailed in materials and methods. RNA-Seq was performed on FL cells transduced with MNX1 or an empty vector (Ctrl) before transplantation in mice ( Invitro FL; Supplementary Figure 1), or from BM from the leukemic mice 5 (leukemia mice BM) after transplantation with the transduced FL cells. TMT mass spectrometry analysis identified several MNX1-binding partners (Supplementary Table S3), including various methyl transferases and histone variants (Figure 1a), consistent with our previous findings 5 . To identify the group of transcription factors responsible for MNX1-driven differential gene expression, we performed gene set enrichment analysis (GSEA) using transcription factor target gene sets (Supplementary Figure 2). These transcription factors from both in vitro FL cells and leukemic BM cells were mapped against the protein networks of the identified proteins from the mass spectrometry data using the STRING database (Figure 1b). This approach revealed significant enrichment of transcription factors associated with DNA and mitochondrial replication and repair, spliceosome activity, and signaling pathways. The most abundant network of transcription factors identified included mini-chromosome maintenance proteins (Mcm3), RNA Binding Motif Protein 15 (Rbm15), Mitochondrial Transcription Factor 1 (Tfam), Signal Transducer and Activator of Transcription 1 (Stat1), E2F transcription factors and Pbx Homeobox Interacting Protein 1 (Pbxip1). Pbxip1 was of particular interest due to its association with Pbx1, a pioneer transcription factor in the homeobox family, known for its role in development and leukemic transformation 22 . STRING analysis further revealed that Pbx1 interacted with several histone modifiers and methyltransferases identified in our mass spectrometry results, including Kmt2a and Smarca4, as well as with MNX1’s translocation partner Etv6 in t(7;12) AML and several other homeobox proteins (Supplementary Table S7). Using qPCR, we examined Pbxip1 and Pbx1 gene expression in pre-transplant in vitro FL cells and leukemic BM from mice. Pbx1 was upregulated in both settings (Figure 2a), whereas Pbxip1 showed increased expression only in leukemic BM (Figure 2b). Further analysis of other Pbx family members revealed that Pbx4 was also exclusively upregulated in leukemic BM (Figure 2b), similar to Pbxip1 . These findings were consistent with RNA-seq data from paediatric t(7;12) AML samples in the COG-NCI TARGET dataset 5 , where both PBXIP1 and PBX4 were upregulated compared to normal BM (Figure 2c). Together, these results suggest that Pbxip1 and Pbx4 may act as downstream targets of MNX1 that occur during leukemia development, while Pbx1 may present as an early-stage target in preleukemic cells. H3K4 histone methylation as a persistent marker of MNX1-mediated leukemic progression Given Pbx1's potential role as an early target in preleukemic cells, we further examined MNX1’s interaction with the Pbx1 promoter. We found that MNX1 directly binds to this promoter region (Figure 2d). This binding was associated with an increase in H3K4me3 (a histone marker for active promoters) and a decrease in H3K27me3 (a histone marker for inactive transcription) (Figure 2e). These findings align with our previous observations, which demonstrated a global increase in H3K4me3 and a decrease in H3K27me3 following MNX1 ectopic expression. 5 . Interestingly, although Pbx1 expression remained consistently high in both pre-transplantation in vitro FL cells and leukemic BM, MNX1 binding to the Pbx1 promoter was reduced in the leukemic BM (Figure 2d). Despite this reduction in MNX1 binding, H3K4me3 levels slightly increased, whereas H3K27me3 levels showed a slight reduction compared to FL cells (Figure 2e). To further examine these modifications, we employed ACT-seq to assess H3K4me3 and H3K4me1 (a histone marker for active enhancers) binding (Figure 3). Principal component analysis showed no differential binding between control and H3K4me3/H3K4me1 in the in vitro FL cells but revealed significant differences in leukemic BM (Figure 3a-c), similar results also were shown by comparing the differential binding of H3K4me3 and H3K4me1 in in vitro FL cells and leukemic BM (Figure 3b-d). The increase in H3K4me3 levels during leukemia progression was further supported by GSEA analysis, which showed a higher enrichment of differentially expressed genes associated with H3K4 methylation in leukemic BM compared to FL cells (Supplementary Figure 3). Annotation of the differential binding regions of H3K4me3 and H3K4me1 yielded expected results, with H3K4me3 primarily located at promoter regions (~70%), and H3K4me1 mainly found at distal intergenic and intronic regions (~60%; Supplementary Figure 4). The functions of these promoter regions were associated with genes involved in myeloid differentiation, senescence, cell cycle regulation, stem cell differentiation, and mRNA/proteasome catabolic processes (Supplementary Figure 5), consistent with our previous results regarding MNX1 enriched pathways from RNA-seq and mass spectrometry analysis 5 . Pbx motifs show high enrichment in regions marked by MNX1-ATAC and H3K4 peaks To further explore the potential roles of Pbx1 and Pbx4 in MNX1-mediated chromatin and histone modifications, we identified Pbx1 and Pbx4 binding motifs using publicly available ChIP-seq datasets from mouse embryonic trunks (GSE39609) for Pbx1 and mouse testis (GSE224369) for Pbx4, through MEME-suite analysis (Figure 4a; Supplementary Table S4&S5). These motifs were then analyzed for enrichment within MNX1-associated H3K4me3, H3K4me1, and ATAC-seq peaks from leukemic BM samples, using MAST, as described in the materials and methods. The analysis showed significant enrichment for Pbx4 motifs, particularly within ATAC-seq and H3K4me3 peaks, with enrichment levels between 10-15%, while Pbx1 motifs also demonstrated enrichment, albeit to a lesser extent (Figure 4b). To compare the enrichment profiles of Pbx1 and Pbx4 with MNX1, we identified MNX1 motifs using ACT-seq from leukemic BM cells. Although the reproducibility of MNX1 peaks from ACT-seq was low across replicates (Supplementary Figure 6&7), the motifs from each replicate were highly consistent with each other and with other published mouse Mnx1 ChIP-seq datasets (GSE61432) (Figure 4a; Supplementary Figure 8). MNX1 motif enrichment within H3K4me1 was highly significant, reaching approximately 91%. However, MNX1 motif enrichment was lower within H3K4me3 and ATAC-seq peaks (Figure 4b; Supplementary Table S6), supporting our hypothesis that MNX1’s effects might be mediated, in part, through downstream effectors. To examine this in the context of Pbx4 and Pbx1, we compared motif enrichment sites for MNX1, Pbx4, and Pbx1 within ATAC, H3K4me3 and H3K4me1 peaks. As expected, there was minimal overlap between MNX1/Pbx4 and MNX1/Pbx1 binding sites, indicating that Pbx4 and, to a lesser extent, Pbx1, have unique binding sites associated within open chromatin regions in ATAC-seq peaks and promoter regions in H3K4me3 ACT-seq peaks (Figure 4c). Pathway enrichment analysis of promoters associated with Pbx4 and Pbx1 motif enrichment within ATAC-seq and H3K4me3 revealed pathways previously linked to MNX1 activity 5 , including chromosome segregation, erythrocyte differentiation and homeostasis, cell cycle G2/M phase, telomere organization, epigenetic regulation of gene expression, stem cell population maintenance and double-strand break repair (Figure 5a; Supplementary figure 9). These findings suggest that Pbx4 and/or Pbx1 may play roles as MNX1 downstream regulators. To validate this, we treated MNX1-transduced FL cells with the pan-methyltransferase inhibitor Sinefungin, known to inhibit MNX1-mediated histone methylation and leukemia induction 5 . Pre-treatment with Sinefungin significantly reduced the induction of MNX1-induced Pbx1 expression (Figure 5b). Interestingly, Pbx4 and Pbxip1 levels were not significantly affected by Sinefungin (Figure 5c). In conclusion, our findings provide insights into the MNX1 signaling pathway and suggest a potential role for the Pbx family in MNX1-mediated chromatin and histone modifications. Discussion In this study, we aimed to elucidate the downstream targets by which MNX1, a pivotal factor in t(7;12) pediatric AML, drives chromatin and histone modifications. Our findings demonstrate the role of MNX1 in upregulating Ppxip1, Pbx1 , and Pbx4 , highlighting their possible contributions to MNX1-induced epigenetic alterations, and positioning them as potential therapeutic targets for t(7;12) AML. Recently, we have shown that MNX1 activates transcription by interacting with epigenetic modifiers, including histone methyltransferases, which alter the chromatin landscape and regulate gene expression 5 . Consistent with these findings, the current study further reinforces MNX1’s role in transcriptional activation through epigenetic modification. We observed that MNX1 binding to the Pbx1 promoter is associated with increased H3K4me3 and reduced H3K27me3, two key epigenetic marks that regulate gene activation and repression, respectively. The role of MNX1 in inducing methyltransferases was further confirmed by its interactions with various methyltransferases and histone variants identified through TMT mass spectrometry. The significance of H3K4me3 is particularly noteworthy, as it has been implicated in sustaining oncogene expression during leukemogenesis. Studies have shown that H3K4me3 promotes the maintenance of leukemic stem cells 28 and the activation of oncogenes such as HOXA9 and MEIS1 in leukemic stem cells 28 . Additionally H3K4me1, often associated with active enhancers, and H3K4me3 have been associated with MEIS1 binding sites, another member of the homeobox family of transcription factors, during embryological development 29 , and were linked to the regulation of gene networks involved in myeloid differentiation 30 , an important pathway which we have shown to be induced by MNX1 ectopic expression 5 . An intriguing aspect of our data is the transient nature of MNX1 binding to the Pbx1 promoter. Although MNX1 binding diminished over time, the histone modifications, particularly H3K4me3, persist. This suggests a “hit-and-run” mechanism, particularly for Pbx1 , where MNX1 initiates the chromatin modifications but is no longer required to sustain them. This concept is similar to other histone methylation recruiters and writers, such as PRC2 for H3K27me3 methylation and SET1 for H3K4me3 methylation, where histone marks persist long after the dissociation of the methyltransferases 31 , 32 . The sustained histone modifications imply that early MNX1 interactions have long-term consequences in t(7;12) AML, where MNX1 initiates leukemogenesis but may not need to persist for the disease to progress. Moreover, the transient nature of MNX1’s binding could help explain the low reproducibility we observed with MNX1 peaks in ACT-seq, further suggesting that MNX1’s role may primarily be to “mark” key chromatin regions before dissociating. It is also worth noting that MNX1 chromatin profiling was performed using ACT-seq rather than traditional ChIP-seq, which is reported to produce non-specific cleavage of accessible regions 33 . The identification of Pbxip1, Pbx1 , and Pbx4 as downstream targets of MNX1 further builds on existing literature regarding the PBX family’s role in hematopoiesis and leukemic transformation 22 – 24 . PBX1, in particular, has been well-characterized as a partner of HOXA9, collaborating to promote leukemogenesis by activating critical genes involved in cell survival and proliferation 25 . However, PBXIP1 and PBX4 are less extensively studied in the context of leukemia. Beyond PBXIP1's established role as an interacting partner with PBX1, PBX2, and PBX3, blocking the recruitment of PBX-HOX heterodimers and inhibiting the transcriptional activity of PBX 26 , recent study has suggested a potential role for PBXIP1 in erythrocyte differentiation 27 , and elevated PBXIP1 levels have been observed in AML using Oncomine datasets 23 . PBX4 has been implicated in regulating key developmental pathways in hematopoiesis 24 that may drive malignant transformation by altering cell differentiation and proliferation processes. This warrants further investigation into the specific contributions of PBXIP1 and PBX4 to MNX1-driven leukemia and their potential as therapeutic targets. The enrichment of PBX family binding motifs in MNX1-induced H3K4me1, H3K4me3 and ATAC-seq peaks suggests a possible cooperative relationship between MNX1 and the PBX transcriptional network in driving chromatin remodeling as downstream effectors of MNX1. This interaction aligns with findings in MLL-rearranged AML, where PBX1 engages with chromatin-modifying complexes to regulate gene expression 34 . Given that histone methylation plays a crucial role in AML progression, these epigenetic modifications may represent promising therapeutic targets. Methyltransferase inhibitors, such as DOT1L inhibitors, have shown preclinical efficacy in models of MLL-rearranged AML 35 , 36 . In our study, the broad-spectrum methyltransferase inhibitor Sinefungin, previously shown to inhibit the MNX1-drived leukemogenesis 5 , was able to downregulate MNX1-induced PBX1 expression, reinforcing the potential of targeting histone methylation in t(7;12) AML. However, the expression of Pbx4 and Pbxip1 were not significantly affected by Sinefungin treatment, possibly due to their later induction during leukemia development. This temporal regulation suggests that PBX4 and PBXIP1 may play roles in maintaining the leukemic state rather than initiating it, highlighting the complexity of MNX1's downstream effects. In conclusion, our study provides new insights into the MNX1 signaling pathway shedding light on the interplay between MNX1, chromatin regulation, and the PBX family. These findings lay the groundwork for further investigation into the timing and context of PBX1, PBX4 and PBXIP1 involvement in leukemogenesis and their response to methyltransferase inhibition, offering a promising approach to halting or reversing leukemic progression in t(7;12) AML. Methods Plasmids The MNX1 expression vector was constructed using the MSCV Retroviral Expression System (Takara Bio, Cat. No. 634401), which enables stable gene expression under the control of the viral LTR promoter. The MNX1 coding sequence (Supplementary Table S1) was modified to include an N-terminal HA-tag (36 bp), followed by a 24 bp linker sequence, and the first ATG codon was removed to prevent unintended translation initiation. The whole modified MNX1 gene with flanking restriction sites was synthetized at Integrated DNA Technologies and cloned into pMSCV-IRES-GFP and pMSCV-IRES-YFP vectors after digestion with the corresponding restriction enzymes. Animal model and cell cultures Mice were generated and kept at the Gothenburg University Laboratory for Experimental Biomedicine Animal Facility (Gothenburg, Sweden) in an aseptic environment. Bone marrow (BM) cell lines ( In vitro FL) transduced with MNX1 or empty vector control were established as previously detailed in 5 . Briefly, 8–12-week-old C57Bl/6 mice (Charles River Laboratories Inc., Wilmington, MA, USA) were used for mating, and fetal livers (FL) were extracted from embryos at embryonic day E14.5. Whole fetal liver (FL) hematopoietic cells were maintained in culture (DMEM (Merck Millipore, Darmstadt, Germany), supplemented with 18 % fetal calf serum 10 ng/mL human interleukin IL-6 (Peprotech, Cranbury, NJ, USA), 6 ng/mL murine IL-3 (Peprotech), and 50 ng/mL murine stem cell factor (Peprotech)) until transduction with MNX1 or empty vector control. Cells were transduced by cultivation on irradiated E86 producers for 2 days in the presence of 5 μg/mL protamine sulfate (Merck Millipore). FACS-sorted (FACSAria, BD Biosciences, Franklin Lakes, NJ, USA) GFP+ and/or YFP+ cells were maintained in culture 5-7 days post-transduction prior to transplantation into 8–12-week-old NOD.Cg- Kit W-41J Tyr + Prkdc scid Il2rg tm1Wjl /ThomJ (NBSGW) mice (The Jackson Laboratory, Bar Harbor, ME, USA). 0.8x10 6 transduced cells were transplanted via tail vein injections in two experimental arms MNX1 or empty vector control. Engraftment was monitored by FACS-analysis of GFP+ and/or YFP+ expression in peripheral blood every 2 weeks. Each experimental arm consisted of 4–6 mice and was repeated 3 biological times. The number of mice was chosen based on previous studies in similar experimental setups, where this number provides sufficient power to detect significant differences while minimizing animal use. Randomization was used to allocate experimental units to control and MNX1 groups, and the order of measurements was randomized to minimize potential confounders, such as cage location. Exclusion criteria included the removal of animals that suffered injuries due to fighting between mice, which could affect the validity of the results. If any animal exhibited signs of severe injury, it was excluded from the study, and the reason for exclusion was recorded. From each biological replicate, one or two bone marrow (BM) samples were used for PCR, ChIP-qPCR, and ACT-Seq analysis. Blinding was applied during the peripheral blood assessments every 2 weeks, as well as during the collection of BM samples for PCR, ChIP-qPCR, and ACT-Seq analysis, to minimize any potential bias in outcome assessment. Animals were euthanized using the isoflurane (Baxter, Deerfield, IL, USA) drop jar method followed by cervical dislocation. A sealed chamber was prepared by placing a gauze pad soaked with 1.7% isoflurane and the chamber was tightly sealed to prevent the escape of vapors. Upon the onset of deep anesthesia, indicated by the absence of the righting reflex and loss of pedal withdrawal reflex, cervical dislocation was performed manually as per established guidelines. Blood counts were determined on a Sysmex KX-21 Hematology Analyzer (Sysmex, Norderstedt, Germany). Immunoprecipitation and tandem mass spectrometry tag (TMT) Anti HA-magnetic beads from Thermo Fisher Scientific (catalog no. 88836) were used for immunoprecipitation. After blocking with 0.1% BSA in PBS, 1 mg of cell lysates were incubated with HA antibody for 1h at RT. The immune complexes were washed three times with lysis buffer and eluted with 2% SDS in 50mM Tris-HCL with 1mM EDTA (Invitrogen). The samples were then digested with trypsin using modified filter-aided sample preparation (FASP) method 37 . Samples were reduced in 100 mM dithiothreitol at 60°C for 30 min, transferred to Microcon-30kDa Centrifugal Filter Units (Merck), washed with 8 M urea and digestion buffer (50 mM TEAB, 0.5 % sodium deoxycholate (SDC)) prior to alkylation with 10 mM methyl methanethiosulfonate for 30 min at room temperature. Proteins were digested in two steps with trypsin (Pierce MS grade Trypsin, Thermo Fisher Scientific) at 37°C at a final enzyme to protein ratio of 1:50. Peptides were collected by centrifugation and labelled using TMT10plex isobaric label reagents (Thermo Fisher Scientific) according to the manufacturer´s instructions. The individually labelled samples were pooled to one TMT-set and purified using HiPPR detergent removal kit (Thermo Fisher Scientific). SDC was removed by acidification with 10 % trifluoroacetic acid. The TMT-set was fractionated to five fractions using Pierce High pH Reversed-Phase Peptide Fractionation columns (Thermo Fisher Scientific) and acetonitrile (ACN) as organic phase. The fractions were evaporated to dryness and reconstituted in 3 % ACN, 0.2 % formic acid (FA) for LC-MS analysis. The fractions were analyzed on an Orbitrap Fusion Lumos Tribrid mass spectrometer equipped with a FAIMS-Pro ion mobility system and interfaced with an Easy-nLC1200 liquid chromatography system (all Thermo Fisher Scientific). Peptides were trapped on an Acclaim Pepmap 100 C18 trap column (100 μm x 2 cm, particle size 5 μm, Thermo Fisher Scientific) and separated on an in-house packed analytical column (38 cm x 75 μm, particle size 3 μm, Reprosil-Pur C18, Dr. Maisch) using a linear gradient from 5 % to 28% ACN in 0.2 % FA over 77 min at a flow of 300 nL/min. FAIMS Pro was alternating between compensation voltages (CVs) of -40, -60 and -80, with the same data-dependent settings for all three CVs. The precursor ion mass spectra were acquired at a resolution of 120 000 and an m/z range of 375-1375. The top eight most abundant precursors with charges 2–7 were isolated with an m/z window of 0.7 and fragmented by collision induced dissociation (CID) at 35 %. Fragment spectra were recorded in the ion trap at Rapid scan rate. The ten most abundant MS2 fragment ions were isolated using multi-notch isolation for further MS3 fractionation. MS3 fractionation was performed using higher-energy collision dissociation (HCD) at 65% and the MS3 spectra were recorded in the Orbitrap at 50 000 resolution and an m/z range of 100–500. Data analysis was performed with Proteome Discoverer (Version 2.4, Thermo Fisher Scientific) using Mascot (Version 2.5.1, Matrix Science) as a search engine. The data was matched against the reviewed swissprot mouse database (May 2020; 17116 entries). Tryptic peptides were accepted with 1 missed cleavage; methionine oxidation was set as variable modification and cysteine methylthiolation, TMT10plex were set as fixed modifications. Precursor mass tolerance was set to 5 ppm and fragment mass tolerance to 0.6 Da. Percolator was used for PSM validation with a strict FDR threshold of 1%. For reporter ion quantification peak integration was set to most confident centroid and a tolerance of 3 mmu. Only unique peptides were used for protein quantification (Supplementary Table S3). Gene expression analysis and next generation sequencing Total RNA was isolated using the miRNeasy Plus micro kit (Qiagen) and cDNA synthesized with SuperScript III First-Strand Synthesis SuperMix for qRT-PCR (Invitrogen/Thermo Fisher Scientific). TaqMan Gene Expression Assays and TaqMan Universal MasterMix II (Applied Biosystems/ Thermo Fisher Scientific) were used for probe-based qPCR assays, listed in Supplementary Table S2. Hprt was used as reference gene for normalization of the expression. RNA-seq was performed as described previously in 5 . Briefly, total RNA was extracted from BM cells from mice with MNX1 induced leukemia and from transduced FL cells after one week of transfection as control. Library preparation and sequencing was carried out by Beijing Genomics Institute. Reads were mapped to the mm10 mouse reference genome using STAR. Differential gene expression was performed using DESeq2 with inclusion criteria of false discovery rate (FDR) less than 0.05 and log2 fold change above |1|. Gene Set Enrichment Analysis 38 (GSEA, Broad Institute) was used with the Gene Transcription Regulation Database (GTRD) transcription factor targets and legacy transcription factor gene set collection. A pathway in GSEA analysis was regarded significant at nominal p-value < 0.05. ChIP-qPCR ChIP-qPCRwas performed as described previously in 5 . In short, iDeal ChIP-qPCR kit (C01010180, Diagenode ) was used to perform the ChIP assay. Crosslinking was achieved incubating cells with formaldehyde for 10-15 min at room temperature (RT). Subsequently, the reactive was quenched by the addition of glycine and further incubation for 5.10 minutes at RT. The cell pellet was then washed with cold PBS and lysed according to manufacturer specifications. DNA was sheared by sonication using a bioruptor for 20 min in 30 second intervals achieving DNA sizes in the 200-600 bp range. Immunoprecipation was then performed using the antibodies against HA (cell signaling), H3K27me3 (Diagenode_C15410195, RRID:AB_2753161), H3K4me3 (Diagenode_C15410003, RRID:AB_2924768), and polyclonal rabbit anti-IgG (Diagenode_C15410206, RRID:AB_2722554) as negative control. DNA was purified and, qPCR was performed using SYBR green chemistry and in-house designed primers shown in Supplementary Table S2. The calculation of enrichment was normalized to the respective inputs and done according to the following formulas: • The % of IP to input formula: % recovery = 2^[(Ct input-log2(5%)) – Ct sample] * 100% • The enrichment was calculated as: %recovery IP / % recovery IgG Negative control was achieved by immunoprecipitation against HA antibody, followed by detection of random region with low CpG islands and promoter binding density corresponding to chr1:168430579+168430704. Designed primers for the random region is shown in Supplementary Table S2 RNA-seq & Mass spec integrative analysis GTRD transcription factor targets gene set from gene set enrichment analysis (GSEA) was used to identify the transcription factor targets from the normalized counts of RNA-seq. Protein network of the identified proteins from tandem mass spectrometry tag (TMT) were obtained from STRING database using medium confidence of 0.4 ATAC and ACT sequencing Assay for Transposase-Accessible Chromatin (ATAC-seq) and Active Chromatin Targeting (ACT-seq) were performed as described previously in 5 . Briefly, bone marrow cells from MNX1-induced leukemic mice, non-transplanted mouse fetal liver (FL) cells containing MNX1-vector as well as FL cells with empty vector were lysed using in resuspension buffer containing 0.1% of NP40 and Tween-20 and 0.01% digitonin. Lysed cells were resuspended in transposition buffer (10 mM Tris-acetate, pH 7.6, 5 mM MgCl2, 10% dimethylformamide, 30% PBS, 0.01% digitonin and 0.1% Tween-20) together with Tn5 transposome and tagementation were allowed to proceed for 30 min at 37°C under agitation. For ACT-seq, targeting antibodies used for the pA-Tn5 transposome-antibody complexes were directed against H3K4me3 (Abcam_ab8580, RRID:AB_306649), H3K4me1 (Abcam_ab8895, RRID:AB_306847), HA-tag (Sigma_H6908, RRID:AB_260070), IgG (Merck Millipore PP64B, RRID:AB_97852) and yeast histone H2B (Hölzel Diagnostika, M30930, Cologne, Germany, RRID:AB_2924769), the latter complexes being used for spike-in to enable sequence read normalization between biological replicates. DNA was purified and checked for quality followed by sequencing on a Illumina next-seq. Reads were trimmed using Trim Galore in conjunction with Cutadapt (v. 1.14) with the options –paired”, “–nextera”, “– length_1 35”, and “–length_2 35. Trimmed reads were mapped against mm10 reference genome using Bowtie2 39 (v. 2.5.2). Conflicting mapped reads with a phred score lower than 20 was removed using SAMTools 40 (v. 1.19). Finally, read ends were shifted to center of the transposition event. HA-tagged MNX1, H3K4me3 and H4K4me1 peaks were retrieved with GoPeaks 41 (v 1.0.0), using a q-value cut-off of 0.05 and the --broad and --mdist 3000 options for the latter. ATAC-seq peaks were called using MACS2 42 with the options --broad, --keep-dup all, --nomodel and a q-value cutoff of 0.05. Differentially binding analysis was determined using Diffbind and DESeq2 43 (v. 3.12) R (v. 4.3.2) package with regions considered differentially accessible or enriched if fulfilling FDR less than 0.05 and a fold change larger than |1|. Peak reproducibility between replicates was determined by Jaccard statistic, using the bedtools jaccard function. Consensus peaks were constructed of replicates using BEDTools 44 (v. 2.30.0) multiinter function only retaining regions/peaks found in all replicates. Subsequently, regions within the 1 kbp range were merged using the BEDTools merge function. To determine where open-chromatin intersected with histone depositions, BEDTools intersect function was used. Peak annotation was performed using ChIPseeker 45 (v. 1.38.0) R package, mapping peaks against TxDb UCSC mm10 known gene 46 reference and defining the promoter region as ± 3 kbp from transcription starting sites. Promoter regions were used to find enriched GO biological pathways using clusterProfiler 47 (v. 4.10.0) R package. Pbx motif analysis ChIP-seq datasets of Pbx1 in mouse embryonic trunks 48 (GSE39609) and Pbx4 in mouse testis 49 (GSE224369) were retrieved from Gene Expression Omnibus (GEO). Sequencing data in FASTQ format was aligned to mm10 reference genome and converted to BAM-file format using Bowtie2 (v. 2.5.2) and SAMTools. Multi-mapped, unmapped and duplicate reads were filtered out using Sambamba (v. 0.7.1). Peaks were retrieved with MACS2 with a q-value of 0.05. When replicates were available, consensus peaks were constructed of replicate peak sets as described in the ATAC and ACT sequencing section. DNA-binding motifs and their enrichment were analyzed using MEME-Suite (v. 5.5.1). Motifs were retrieved from the Pbx ChIP-seq peak regions with the STREME 50 function using default parameters, motif minimum and maximum length of 8 and 15, respectively, and binomial test against a random test set consisting 10 % of the input sequences to evaluate statistical significance. Subsequently, the top scoring motifs, matching >10 % of peaks in each of the Pbx ChIP-seq data sets were used as a motif sets for a MAST 51 scan of the consensus peak sets derived from epigenetic data of the MNX1-induced leukemic mice. A positional p-value of 10 of was used as criteria to consider the sequence significant. The resulting motif-matches of the MAST scan are shown Supplementary Table S4 & S5 for the Pbx1 and Pbx4 motif-sets, respectively. As a control the motif-sets were tested against randomly generated sequences produced with the online tool FaBox 52 , recapitulating number of sequences, sequence size distribution and GC-content of the enriched peak data sets. Regions enriched in Pbx-motifs were submitted to pathway enrichment analysis using Clusterprofiler removing redundancy and filtering for pathways by GO level using the simplify function resulting in the most specific pathways. MNX1 motif analysis MNX1 motifs were identified using the STREME function within MEME-suite on each MNX1 ACT-seq peak set individually, as well as on a publicly available ChIP-seq dataset of Mnx1 in mouse insulinoma cells 53 (GSE61432), applying parameters consistent with those used in the Pbx motif analysis. The top 10 motifs identified were then assessed for correlation using the MAST function's built-in correlation feature within MEME-suite; motifs were considered correlated if their average Pearson correlation exceeded 0.8. Correlated motifs found across all peak sets were subsequently analyzed for enrichment in epigenetic data from MNX1-induced leukemic mice, as outlined in the Pbx motif analysis. The motif matches from the MAST scan for the MNX1 motif set are presented in Supplementary Table S6. Declarations Acknowledgments We thank Tanmoy Mondal for revising the manuscript and Tova Johansson and Hanna Brissman for help with animals. Proteomic analysis was performed at the Proteomics Core Facility, Sahlgrenska academy, Gothenburg University, with financial support from SciLifeLab and BioMS. The computations were enabled by resources in project SNIC 2021/22-754 provided by the Swedish National Infrastructure for Computing (SNIC) at UPPMAX, partially funded by the Swedish Research Council through grant agreement n. 2018-05973. Author Contributions statement EM-B, DW, CP, LP, and AW designed the research study. EM-B, AW, AÖ, TN, DW, PL, MB, JH, GT, JA and SJ performed the laboratory work and results analysis. LF, CP, LP and AW analyzed the combined data and wrote the paper. Additional information The datasets generated for this study can be found in the Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/) under the following accession IDs:, GSE202137 (RNA-seq) https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE202137 , GSE205697 (ATAC-seq) https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE205697 & GSE269913 (ATAC-seq) https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE269913 , GSE269910 (ACT-seq) https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE269910 and in PRIDE/ProteomeXchange, under accession number PXD056378 https://www.ebi.ac.uk/pride/archive/projects/PXD056378 The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Funding Declaration This work was supported by grants from the Swedish Cancer Society (20 0925 PjF, CAN2017/461), the Swedish Childhood Cancer Foundation (PR2014-0125, PR2019-0013 and TJ2019-0053, TJ2022-0017), Wilhelm och Martina Lundgrens Fond, Assar Gabrielsson Fond and Västra Götalandsregionen (ALFGBG-431881), and the German Funding Agency (DFG) with funding for Collaborative Research Center 1074, Project B11N (to CP). Ethics statement All animal experiments have been accepted by the Swedish Agency for Agriculture (Jordbruksverket) and the animal ethics committee in Gothenburg: Dnr 5.8.18-17008/2021. All procedures were conducted in accordance with applicable ethical guidelines and regulations and in accordance with ARRIVE guidelines. References Tseng, S., Lee, M. E. & Lin, P. C. A Review of Childhood Acute Myeloid Leukemia: Diagnosis and Novel Treatment. Pharmaceuticals (Basel) 16 (2023). https://doi.org:10.3390/ph16111614 Grove, C. S. & Vassiliou, G. S. 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Supplementary Files Supplementaryfiguresandlegends.pdf SupplementaryTableS1.docx SupplementTableS2.docx SupplemetaryTableS3.xlsx SupplementaryTableS4.xlsx SupplementaryTableS5.xlsx SupplementaryTableS6.xlsx SupplementaryTableS7.xlsx Cite Share Download PDF Status: Published Journal Publication published 19 Jan, 2026 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 24 Oct, 2025 Reviews received at journal 23 Oct, 2025 Reviews received at journal 09 Oct, 2025 Reviews received at journal 07 Oct, 2025 Reviewers agreed at journal 07 Oct, 2025 Reviewers agreed at journal 01 Oct, 2025 Reviewers agreed at journal 16 Sep, 2025 Reviewers invited by journal 16 Sep, 2025 Editor invited by journal 22 May, 2025 Editor assigned by journal 22 May, 2025 Submission checks completed at journal 25 Apr, 2025 First submitted to journal 25 Apr, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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1","display":"","copyAsset":false,"role":"figure","size":378337,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIdentification of Pbxip1 as a downstream target of MNX1.\u003c/strong\u003e \u003cstrong\u003e(a)\u003c/strong\u003e Heatmap of TMT-mass spectrometry quantification of MNX1 co-immunoprecipitate comparing mice leukemia BM cells (M) with \u003cem\u003ein vitro\u003c/em\u003eFL cells transduced with empty vector (C)\u003cstrong\u003e. (b)\u003c/strong\u003e Histogram representing the number of proteins in the transcription factor network identified from mass spectrometry data (using the STRING database, medium confidence 0.4) compared to transcription factors identified from gene set enrichment analysis (GSEA) of differentially expressed genes from RNA-seq data.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-6480114/v1/a77012113b0f96dbc66eaa60.png"},{"id":92193635,"identity":"ec45df3e-45b3-4649-b750-e43c06527b66","added_by":"auto","created_at":"2025-09-25 15:36:45","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":304338,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePbx family genes are upregulated in MNX1-driven pathology. \u003c/strong\u003eBox and whisker plots (showing the median, first and third quartiles, minimum and maximum) of: \u003cstrong\u003e(a)\u003c/strong\u003eRelative mRNA expression, shown as 2^(-ΔCt), for \u003cem\u003ePbx1\u003c/em\u003e or (\u003cstrong\u003eb\u003c/strong\u003e) \u003cem\u003ePbx4\u003c/em\u003eand \u003cem\u003ePbxip1\u003c/em\u003e and normalized to \u003cem\u003eHprt\u003c/em\u003e as loading control. Expression levels were measured in MNX1-transduced FL cells relative to \u003cem\u003ein vitro\u003c/em\u003e FL control cells with empty vector (Ctrl), or BM from mice transplanted with MNX1 relative to empty vector (Ctrl). \u003cstrong\u003e(c)\u003c/strong\u003e DESeq2 normalized expression of PBX4 and PBXIP1 in patients samples carrying the t(7;12) translocation compared to normal BM from healthy donors obtained from children’s oncology group (COG)–National Cancer Institute (NCI) TARGET AML initiative data set. \u003cstrong\u003e(d)\u003c/strong\u003e Quantification of of MNX1 or (\u003cstrong\u003eE\u003c/strong\u003e) H3K4me3 and H3K27me3 binding to the promoter regions of \u003cem\u003ePbx1\u003c/em\u003e in FL cells or leukemia BM cells with either MNX1 or empty vector (Ctrl) as determined by ChIP-qPCR. ***indicates p-value \u0026lt;0.05, ****indicates p-value \u0026lt;0.01 by Mann-Whitney U-test. * indicates p-value \u0026lt;0.05, ** indicates p-value \u0026lt;0.01 as determined by Student’s t-test .\u003cstrong\u003e ## \u003c/strong\u003eindicates p-value\u003cstrong\u003e \u003c/strong\u003e\u0026lt;0.001 as determined by Tukey’s post-hoc test following ANOVA test between the groups with p-value \u0026lt;0.001. n represents the number of replicates (n).\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-6480114/v1/e560cd4764a152f68d35992f.png"},{"id":92193641,"identity":"ce2cfc3c-f72a-4722-bd5c-2b5bafbd6dfe","added_by":"auto","created_at":"2025-09-25 15:36:46","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":260213,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAlterations in histone methylation associates with leukemia development. (a \u0026amp; c) \u003c/strong\u003ePrincipal component analysis scatter plot of normalized median of ratios using Diffbind and DESeq2 showing variations between the samples in \u003cstrong\u003e(a)\u003c/strong\u003e H3K4me3 differential binding and \u003cstrong\u003e(c)\u003c/strong\u003e H3K4me1 differential binding. \u003cstrong\u003e(b \u0026amp; d)\u003c/strong\u003eVolcano plot depicting the differential binding of H3K4me3 \u003cstrong\u003e(b)\u003c/strong\u003e, or H3K4me1 \u003cstrong\u003e(d)\u003c/strong\u003e between MNX1-vector transduced FL cells (blue) and empty vector transduced FL cells (Ctrl-grey; left panel) or mice leukemia BM cells (red) and empty vector transduced FL cells (right panel).\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-6480114/v1/5cb8266cd00b0c79abfefb44.png"},{"id":92193642,"identity":"32a972fb-7b30-43d1-a004-d11e24343d9e","added_by":"auto","created_at":"2025-09-25 15:36:46","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":576389,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePbx motifs are enriched in MNX1 peaks corresponding to pathways mediated by MNX1. (a)\u003c/strong\u003eEnrichment analysis using MEME-suite’s STREME-function identifies Pbx1, Pbx4 and MNX1 transcription factor motifs overrepresented at differential accessible promoters and enhancers from ChIP-seq. For a complete list of enriched motifs identified in each condition and genomic region, refer to Supplementary table S4, S5 \u0026amp; S6. \u003cstrong\u003e(b)\u003c/strong\u003e Histogram showing the enrichment of Pbx1 (upper panel), Pbx4 (middle panel) and MNX1(lower panel) motifs identified within MNX1 peaks derived from differentially accessible peaks of ATAC-seq, as well as differential binding peaks for H3K4me3 and H3K4me1. Randomly generated sequences matched for equal size, GC content, and sequence length distribution were used as negative control (Randomized DB). (\u003cstrong\u003ec\u003c/strong\u003e) Venn diagram showing the overlap between MNX1 and Pbx1 motif enrichment (left panel), or MNX1 and Pbx4 motif enrichment (right panel) within ATAC, H3K4me3 and H3K27me3 peaks\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-6480114/v1/b6ae6880398a9684cbc23f96.png"},{"id":92193645,"identity":"3ba36412-7496-46d2-8ef5-4c37df715957","added_by":"auto","created_at":"2025-09-25 15:36:46","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":301247,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePathway enrichment and methyltransferase inhibition effects on Pbx family expression. (a)\u003c/strong\u003eBubble plot showing the pathway enrichment analysis for PBX4 motifs enriched over MNX1 promoters from ATAC-seq (upper panel) and H3K4me3 ACT-seq (lower panel), generated using the ClusterProfiler package. The size of each bubble corresponds to the gene count associated with the respective pathway, while the color gradient represents the adjusted p-value of the enriched pathways. (\u003cstrong\u003eb-c\u003c/strong\u003e) Box and whisker plots (showing the median, first and third quartiles, minimum and maximum) of relative mRNA expression, shown as 2^(-ΔCt), for Pbx1 \u003cstrong\u003e(b)\u003c/strong\u003e, or Pbxip1 and Pbx4 \u003cstrong\u003e(c) \u003c/strong\u003eand normalized to \u003cem\u003eHprt\u003c/em\u003e as loading control. Expression levels were measured in \u003cem\u003ein vitro\u003c/em\u003e FL cells with either MNX1 overexpression or empty vector (Ctrl) after treatment with vehicle or 5μM Sinefungin (Ctrl+S, MNX1+S). Fresh Sinefungin was added daily after viral transduction for one week. ## indicates p-value \u0026lt;0.05 as determined by Tukey’s post-hoc test following ANOVA test between the groups with p-value \u0026lt;0.001. n represents the number of replicates (n).\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-6480114/v1/2a322784a8255627aa19ee3b.png"},{"id":101151897,"identity":"4d9016f4-2e3e-438a-93ab-c9218bad2cfe","added_by":"auto","created_at":"2026-01-26 16:07:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3053817,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6480114/v1/ed85c67d-a2c9-4dd8-afbb-bcf310aef623.pdf"},{"id":92193637,"identity":"c9240bef-96df-4c8a-9349-7c6188db79f1","added_by":"auto","created_at":"2025-09-25 15:36:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1909980,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfiguresandlegends.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6480114/v1/1b40983773c8eb4903d69703.pdf"},{"id":92195724,"identity":"9c05455e-33d7-4951-b268-fb65e0e9a44c","added_by":"auto","created_at":"2025-09-25 15:52:46","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":14322,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-6480114/v1/927f06805b6920db6e54fcef.docx"},{"id":92193639,"identity":"cc4e5f90-ff24-4af7-8ccb-29e1488716d4","added_by":"auto","created_at":"2025-09-25 15:36:46","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":14825,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementTableS2.docx","url":"https://assets-eu.researchsquare.com/files/rs-6480114/v1/2d980c01bac56273f0b9a6ca.docx"},{"id":92193643,"identity":"9f5385ca-6a34-48dd-aea7-7e49396b9210","added_by":"auto","created_at":"2025-09-25 15:36:46","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":237379,"visible":true,"origin":"","legend":"","description":"","filename":"SupplemetaryTableS3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6480114/v1/a89e487d78a4d340c7c4b941.xlsx"},{"id":92193649,"identity":"0738c382-f4c3-40b4-9584-378888c4bc07","added_by":"auto","created_at":"2025-09-25 15:36:46","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":547404,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTableS4.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6480114/v1/89408dbe22b728312966f82a.xlsx"},{"id":92193659,"identity":"3d023455-1427-4a58-a51f-9f15c0ce6483","added_by":"auto","created_at":"2025-09-25 15:36:46","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":2036140,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTableS5.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6480114/v1/c754ef057655859be8fc4f46.xlsx"},{"id":92193676,"identity":"f6f673f3-b1b0-4edc-8c6a-f069e3cf9f77","added_by":"auto","created_at":"2025-09-25 15:36:47","extension":"xlsx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":37102052,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTableS6.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6480114/v1/15d35cc9cf2072c3ce0632a6.xlsx"},{"id":92193667,"identity":"da5ada0f-823d-439d-894b-30023bdd022f","added_by":"auto","created_at":"2025-09-25 15:36:46","extension":"xlsx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":35432,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTableS7.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6480114/v1/c15a4a6137804116882cb006.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Role of MNX1-mediated Histone Modifications and PBX Gene Family in MNX1-induced Leukemogenesis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAcute myeloid leukemia (AML) accounts for approximately 15\u0026ndash;20% of childhood leukemias \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. It is characterized by the clonal expansion of hematopoietic stem and progenitor cells (HSPCs), resulting in ineffective hematopoiesis and bone marrow failure \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. The overall survival rate for AML is notably lower than that of the more common pediatric acute lymphoblastic leukemia (ALL), largely due to various genetic alterations associated with poor prognosis \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. One such aberration, the translocation t(7;12)(q36;p13), has been identified in children diagnosed under the age of 24 months \u003csup\u003e\u003cspan additionalcitationids=\"CR5 CR6 CR7 CR8 CR9\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e and has been included in the 5th edition of the World Health Organization (WHO) Classification of Hematolymphoid Tumors \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. In all patients with t(7;12), overexpression of \u003cem\u003eMNX1\u003c/em\u003e has consistently been identified as a common event \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Using human induced pluripotent stem cells (iPSC), we previously confirmed that this translocation leads to elevated \u003cem\u003eMNX1\u003c/em\u003e expression \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e, through an enhancer-hijacking mechanism that activates the \u003cem\u003eMNX1\u003c/em\u003e promoter via enhancers from the \u003cem\u003eETV6\u003c/em\u003e locus \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eMNX1 is a homeobox transcription factor previously recognized for its role in the development of motor neurons and pancreatic beta cells \u003csup\u003e\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. However, recent studies have implicated MNX1 in oncogenesis, with its involvement observed in AML and in prostate, colorectal, bladder, and hepatocellular cancers \u003csup\u003e\u003cspan additionalcitationids=\"CR17 CR18 CR19 CR20\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. In AML, we have shown that MNX1 associates with methyl transferases and components of methionine cycle in AML leading to significant histone modifications, specifically H3K4me3 and H3K27me3, which in turn cause aberrant gene expression, genome-wide chromatin alterations, and DNA damage \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn this study, we identify PBXIP1 and PBX4 as MNX1-regulated genes, expanding the potential role of PBX transcription factors in t(7;12) AML. While PBX1 has been extensively studied in leukemia little is known about the contributions of PBXIP1 and PBX4 in this context \u003csup\u003e\u003cspan additionalcitationids=\"CR23 CR24\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. PBXIP1, originally described as a PBX1-interacting protein that modulates PBX-HOX complexes \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, has been linked to erythrocyte differentiation and is upregulated in AML \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e, suggesting a possible role in hematopoiesis and leukemic transformation. PBX4, though less characterized, has been associated with hematopoietic development and may contribute to transcriptional dysregulation in leukemia \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Our findings suggest that MNX1-driven leukemogenesis may involve PBXIP1 and PBX4 through epigenetic modifications that sustain oncogenic gene expression.\u003c/p\u003e"},{"header":"Results","content":"\u003ch2\u003e\u003cem\u003ePbxip1\u003c/em\u003e is a downstream target of \u003cem\u003eMNX1\u003c/em\u003e\u003c/h2\u003e\n\u003cp\u003eTo investigate the downstream targets of MNX1, we performed an integrative analysis combining protein complexes associated with MNX1 from mass spectrometry with transcriptomic data from RNA-seq. MNX1-associated protein complexes were identified through co-immunoprecipitation of MNX1 followed by TMT mass spectrometry, using BM from mice with MNX1-induced AML, as further detailed in materials and methods. RNA-Seq was performed on FL cells transduced with \u003cem\u003eMNX1\u003c/em\u003e or an empty vector (Ctrl) before transplantation in mice (\u003cem\u003eInvitro\u003c/em\u003e FL; Supplementary Figure 1), or from BM from the leukemic mice \u003csup\u003e5\u003c/sup\u003e (leukemia mice BM) after transplantation with the transduced FL cells. TMT mass spectrometry analysis identified several MNX1-binding partners (Supplementary Table S3), including various methyl transferases and histone variants (Figure 1a), consistent with our previous findings \u003csup\u003e5\u003c/sup\u003e. To identify the group of transcription factors responsible for MNX1-driven differential gene expression, we performed gene set enrichment analysis (GSEA) using transcription factor target gene sets (Supplementary Figure 2). These transcription factors from both \u003cem\u003ein vitro\u003c/em\u003e FL cells and leukemic BM cells were mapped against the protein networks of the identified proteins from the mass spectrometry data using the STRING database (Figure 1b). This approach revealed significant enrichment of transcription factors associated with DNA and mitochondrial replication and repair, spliceosome activity, and signaling pathways. The most abundant network of transcription factors identified included mini-chromosome maintenance proteins (Mcm3), RNA Binding Motif Protein 15 (Rbm15), Mitochondrial Transcription Factor 1 (Tfam), Signal Transducer and Activator of Transcription 1 (Stat1), E2F transcription factors and Pbx Homeobox Interacting Protein 1 (Pbxip1). Pbxip1 was of particular interest due to its association with Pbx1, a pioneer transcription factor in the homeobox family, known for its role in development and leukemic transformation \u003csup\u003e22\u003c/sup\u003e. STRING analysis further revealed that Pbx1 interacted with several histone modifiers and methyltransferases identified in our mass spectrometry results, including Kmt2a and Smarca4, as well as with MNX1’s translocation partner Etv6 in t(7;12) AML and several other homeobox proteins (Supplementary Table S7).\u003c/p\u003e\n\u003cp\u003eUsing qPCR, we examined \u003cem\u003ePbxip1\u003c/em\u003e and \u003cem\u003ePbx1\u003c/em\u003e gene expression in pre-transplant \u003cem\u003ein vitro\u003c/em\u003e FL cells and leukemic BM from mice. \u003cem\u003ePbx1\u003c/em\u003e was upregulated in both settings (Figure 2a), whereas \u003cem\u003ePbxip1\u003c/em\u003e showed increased expression only in leukemic BM (Figure 2b). Further analysis of other Pbx family members revealed that \u003cem\u003ePbx4\u003c/em\u003e was also exclusively upregulated in leukemic BM (Figure 2b), similar to \u003cem\u003ePbxip1\u003c/em\u003e. These findings were consistent with RNA-seq data from paediatric t(7;12) AML samples in the COG-NCI TARGET dataset \u003csup\u003e5\u003c/sup\u003e, where both \u003cem\u003ePBXIP1\u003c/em\u003e and \u003cem\u003ePBX4\u003c/em\u003e were upregulated compared to normal BM (Figure 2c). Together, these results suggest that Pbxip1 and Pbx4 may act as downstream targets of MNX1 that occur during leukemia development, while Pbx1 may present as an early-stage target in preleukemic cells.\u003c/p\u003e\n\u003ch2\u003eH3K4 histone methylation as a persistent marker of MNX1-mediated leukemic progression\u003c/h2\u003e\n\u003cp\u003eGiven Pbx1's potential role as an early target in preleukemic cells, we further examined MNX1’s interaction with the \u003cem\u003ePbx1\u003c/em\u003e promoter. We found that MNX1 directly binds to this promoter region (Figure 2d). This binding was associated with an increase in H3K4me3 (a histone marker for active promoters) and a decrease in H3K27me3 (a histone marker for inactive transcription) (Figure 2e). These findings align with our previous observations, which demonstrated a global increase in H3K4me3 and a decrease in H3K27me3 following MNX1 ectopic expression. \u003csup\u003e5\u003c/sup\u003e. Interestingly, although \u003cem\u003ePbx1\u003c/em\u003e expression remained consistently high in both pre-transplantation \u003cem\u003ein vitro\u003c/em\u003e FL cells and leukemic BM,\u0026nbsp;MNX1 binding to the \u003cem\u003ePbx1\u003c/em\u003e promoter was reduced in the leukemic BM (Figure 2d). Despite this reduction in MNX1 binding, H3K4me3 levels slightly increased, whereas H3K27me3 levels showed a slight reduction compared to FL cells (Figure 2e).\u003c/p\u003e\n\u003cp\u003eTo further examine these modifications, we employed ACT-seq to assess H3K4me3 and H3K4me1 (a histone marker for active enhancers) binding (Figure 3). Principal component analysis showed no differential binding between control and H3K4me3/H3K4me1 in the \u003cem\u003ein vitro\u003c/em\u003e FL cells but revealed significant differences in leukemic BM (Figure 3a-c), similar results also were shown by comparing the differential binding of H3K4me3 and H3K4me1 in \u003cem\u003ein vitro\u003c/em\u003e FL cells and leukemic BM (Figure 3b-d). The increase in H3K4me3 levels during leukemia progression was further supported by GSEA analysis, which showed a higher enrichment of differentially expressed genes associated with H3K4 methylation in leukemic BM compared to FL cells (Supplementary Figure 3).\u003c/p\u003e\n\u003cp\u003eAnnotation of the differential binding regions of H3K4me3 and H3K4me1 yielded expected results, with H3K4me3 primarily located at promoter regions (~70%), and H3K4me1 mainly found at distal intergenic and intronic regions (~60%; Supplementary Figure 4). The functions of these promoter regions were associated with genes involved in myeloid differentiation, senescence, cell cycle regulation, stem cell differentiation, and mRNA/proteasome catabolic processes (Supplementary Figure 5), consistent with our previous results regarding MNX1 enriched pathways from RNA-seq and mass spectrometry analysis \u003csup\u003e5\u003c/sup\u003e.\u003c/p\u003e\n\u003ch2\u003ePbx motifs show high enrichment in regions marked by MNX1-ATAC and H3K4 peaks\u003c/h2\u003e\n\u003cp\u003eTo further explore the potential roles of Pbx1 and Pbx4 in MNX1-mediated chromatin and histone modifications, we identified Pbx1 and Pbx4 binding motifs using publicly available ChIP-seq datasets from mouse embryonic trunks (GSE39609) for Pbx1 and mouse testis (GSE224369) for Pbx4, through MEME-suite analysis (Figure 4a; Supplementary Table S4\u0026amp;S5). These motifs were then analyzed for enrichment within MNX1-associated H3K4me3, H3K4me1, and ATAC-seq peaks from leukemic BM samples, using MAST, as described in the materials and methods. The analysis showed significant enrichment for Pbx4 motifs, particularly within ATAC-seq and H3K4me3 peaks, with enrichment levels between 10-15%, while Pbx1 motifs also demonstrated enrichment, albeit to a lesser extent (Figure 4b). To compare the enrichment profiles of Pbx1 and Pbx4 with MNX1, we identified MNX1 motifs using ACT-seq from leukemic BM cells. Although the reproducibility of MNX1 peaks from ACT-seq was low across replicates (Supplementary Figure 6\u0026amp;7), the motifs from each replicate were highly consistent with each other and with other published mouse Mnx1 ChIP-seq datasets (GSE61432) (Figure 4a; Supplementary Figure 8). MNX1 motif enrichment within H3K4me1 was highly significant, reaching approximately 91%. However, MNX1 motif enrichment was lower within H3K4me3 and ATAC-seq peaks (Figure 4b; Supplementary Table S6), supporting our hypothesis that MNX1’s effects might be mediated, in part, through downstream effectors.\u003c/p\u003e\n\u003cp\u003eTo examine this in the context of Pbx4 and Pbx1, we compared motif enrichment sites for MNX1, Pbx4, and Pbx1 within ATAC, H3K4me3 and H3K4me1 peaks. As expected, there was minimal overlap between MNX1/Pbx4 and MNX1/Pbx1 binding sites, indicating that Pbx4 and, to a lesser extent, Pbx1, have unique binding sites associated within open chromatin regions in ATAC-seq peaks and promoter regions in H3K4me3 ACT-seq peaks (Figure 4c).\u003c/p\u003e\n\u003cp\u003ePathway enrichment analysis of promoters associated with Pbx4 and Pbx1 motif enrichment within ATAC-seq and H3K4me3 revealed pathways previously linked to MNX1 activity \u003csup\u003e5\u003c/sup\u003e, including chromosome segregation, erythrocyte differentiation and homeostasis, cell cycle G2/M phase, telomere organization, epigenetic regulation of gene expression, stem cell population maintenance and double-strand break repair (Figure 5a; Supplementary figure 9). These findings suggest that Pbx4 and/or Pbx1 may play roles as MNX1 downstream regulators.\u003c/p\u003e\n\u003cp\u003eTo validate this, we treated MNX1-transduced FL cells with the pan-methyltransferase inhibitor Sinefungin, known to inhibit MNX1-mediated histone methylation and leukemia induction \u003csup\u003e5\u003c/sup\u003e. Pre-treatment with Sinefungin significantly reduced the induction of MNX1-induced \u003cem\u003ePbx1\u003c/em\u003e expression (Figure 5b). Interestingly, \u003cem\u003ePbx4\u003c/em\u003e and \u003cem\u003ePbxip1\u003c/em\u003e levels were not significantly affected by Sinefungin (Figure 5c).\u003c/p\u003e\n\u003cp\u003eIn conclusion, our findings provide insights into the MNX1 signaling pathway and suggest a potential role for the Pbx family in MNX1-mediated chromatin and histone modifications.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we aimed to elucidate the downstream targets by which MNX1, a pivotal factor in t(7;12) pediatric AML, drives chromatin and histone modifications. Our findings demonstrate the role of MNX1 in upregulating \u003cem\u003ePpxip1, Pbx1\u003c/em\u003e, and \u003cem\u003ePbx4\u003c/em\u003e, highlighting their possible contributions to MNX1-induced epigenetic alterations, and positioning them as potential therapeutic targets for t(7;12) AML.\u003c/p\u003e\u003cp\u003eRecently, we have shown that MNX1 activates transcription by interacting with epigenetic modifiers, including histone methyltransferases, which alter the chromatin landscape and regulate gene expression \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Consistent with these findings, the current study further reinforces MNX1\u0026rsquo;s role in transcriptional activation through epigenetic modification. We observed that MNX1 binding to the \u003cem\u003ePbx1\u003c/em\u003e promoter is associated with increased H3K4me3 and reduced H3K27me3, two key epigenetic marks that regulate gene activation and repression, respectively. The role of MNX1 in inducing methyltransferases was further confirmed by its interactions with various methyltransferases and histone variants identified through TMT mass spectrometry. The significance of H3K4me3 is particularly noteworthy, as it has been implicated in sustaining oncogene expression during leukemogenesis. Studies have shown that H3K4me3 promotes the maintenance of leukemic stem cells \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e and the activation of oncogenes such as \u003cem\u003eHOXA9\u003c/em\u003e and \u003cem\u003eMEIS1\u003c/em\u003e in leukemic stem cells \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Additionally H3K4me1, often associated with active enhancers, and H3K4me3 have been associated with MEIS1 binding sites, another member of the homeobox family of transcription factors, during embryological development \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e, and were linked to the regulation of gene networks involved in myeloid differentiation \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e, an important pathway which we have shown to be induced by \u003cem\u003eMNX1\u003c/em\u003e ectopic expression \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eAn intriguing aspect of our data is the transient nature of MNX1 binding to the \u003cem\u003ePbx1\u003c/em\u003e promoter. Although MNX1 binding diminished over time, the histone modifications, particularly H3K4me3, persist. This suggests a \u0026ldquo;hit-and-run\u0026rdquo; mechanism, particularly for \u003cem\u003ePbx1\u003c/em\u003e, where MNX1 initiates the chromatin modifications but is no longer required to sustain them. This concept is similar to other histone methylation recruiters and writers, such as PRC2 for H3K27me3 methylation and SET1 for H3K4me3 methylation, where histone marks persist long after the dissociation of the methyltransferases \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. The sustained histone modifications imply that early MNX1 interactions have long-term consequences in t(7;12) AML, where MNX1 initiates leukemogenesis but may not need to persist for the disease to progress. Moreover, the transient nature of MNX1\u0026rsquo;s binding could help explain the low reproducibility we observed with MNX1 peaks in ACT-seq, further suggesting that MNX1\u0026rsquo;s role may primarily be to \u0026ldquo;mark\u0026rdquo; key chromatin regions before dissociating. It is also worth noting that MNX1 chromatin profiling was performed using ACT-seq rather than traditional ChIP-seq, which is reported to produce non-specific cleavage of accessible regions \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe identification of \u003cem\u003ePbxip1, Pbx1\u003c/em\u003e, and \u003cem\u003ePbx4\u003c/em\u003e as downstream targets of MNX1 further builds on existing literature regarding the PBX family\u0026rsquo;s role in hematopoiesis and leukemic transformation \u003csup\u003e\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. PBX1, in particular, has been well-characterized as a partner of HOXA9, collaborating to promote leukemogenesis by activating critical genes involved in cell survival and proliferation \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. However, PBXIP1 and PBX4 are less extensively studied in the context of leukemia. Beyond PBXIP1's established role as an interacting partner with PBX1, PBX2, and PBX3, blocking the recruitment of PBX-HOX heterodimers and inhibiting the transcriptional activity of PBX \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, recent study has suggested a potential role for PBXIP1 in erythrocyte differentiation \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e, and elevated PBXIP1 levels have been observed in AML using Oncomine datasets \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. PBX4 has been implicated in regulating key developmental pathways in hematopoiesis \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e that may drive malignant transformation by altering cell differentiation and proliferation processes. This warrants further investigation into the specific contributions of PBXIP1 and PBX4 to MNX1-driven leukemia and their potential as therapeutic targets.\u003c/p\u003e\u003cp\u003eThe enrichment of PBX family binding motifs in MNX1-induced H3K4me1, H3K4me3 and ATAC-seq peaks suggests a possible cooperative relationship between MNX1 and the PBX transcriptional network in driving chromatin remodeling as downstream effectors of MNX1. This interaction aligns with findings in MLL-rearranged AML, where PBX1 engages with chromatin-modifying complexes to regulate gene expression \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Given that histone methylation plays a crucial role in AML progression, these epigenetic modifications may represent promising therapeutic targets. Methyltransferase inhibitors, such as DOT1L inhibitors, have shown preclinical efficacy in models of MLL-rearranged AML \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. In our study, the broad-spectrum methyltransferase inhibitor Sinefungin, previously shown to inhibit the MNX1-drived leukemogenesis \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e, was able to downregulate MNX1-induced \u003cem\u003ePBX1\u003c/em\u003e expression, reinforcing the potential of targeting histone methylation in t(7;12) AML. However, the expression of \u003cem\u003ePbx4\u003c/em\u003e and \u003cem\u003ePbxip1\u003c/em\u003e were not significantly affected by Sinefungin treatment, possibly due to their later induction during leukemia development. This temporal regulation suggests that PBX4 and PBXIP1 may play roles in maintaining the leukemic state rather than initiating it, highlighting the complexity of MNX1's downstream effects.\u003c/p\u003e\u003cp\u003eIn conclusion, our study provides new insights into the MNX1 signaling pathway shedding light on the interplay between MNX1, chromatin regulation, and the PBX family. These findings lay the groundwork for further investigation into the timing and context of PBX1, PBX4 and PBXIP1 involvement in leukemogenesis and their response to methyltransferase inhibition, offering a promising approach to halting or reversing leukemic progression in t(7;12) AML.\u003c/p\u003e"},{"header":"Methods","content":"\u003ch2\u003ePlasmids\u003c/h2\u003e\n\u003cp\u003eThe MNX1 expression vector was constructed using the MSCV Retroviral Expression System (Takara Bio, Cat. No. 634401), which enables stable gene expression under the control of the viral LTR promoter. The MNX1 coding sequence (Supplementary Table S1) was modified to include an N-terminal HA-tag (36 bp), followed by a 24 bp linker sequence, and the first ATG codon was removed to prevent unintended translation initiation. The whole modified MNX1 gene with flanking restriction sites was synthetized at Integrated DNA Technologies and cloned into pMSCV-IRES-GFP and pMSCV-IRES-YFP vectors after digestion with the corresponding restriction enzymes.\u003c/p\u003e\n\u003ch2\u003eAnimal model and cell cultures\u003c/h2\u003e\n\u003cp\u003eMice were generated and kept at the Gothenburg University Laboratory for Experimental Biomedicine Animal Facility (Gothenburg, Sweden) in an aseptic environment. Bone marrow (BM) cell lines (\u003cem\u003eIn vitro\u0026nbsp;\u003c/em\u003eFL) transduced with \u003cem\u003eMNX1\u003c/em\u003e or empty vector control were established as previously detailed in \u003csup\u003e5\u003c/sup\u003e. Briefly, 8–12-week-old C57Bl/6 mice (Charles River Laboratories Inc., Wilmington, MA, USA) were used for mating, and fetal livers (FL) were extracted from embryos at embryonic day E14.5. Whole fetal liver (FL) hematopoietic cells were maintained in culture (DMEM (Merck Millipore, Darmstadt, Germany), supplemented with 18 % fetal calf serum 10 ng/mL human interleukin IL-6 (Peprotech, Cranbury, NJ, USA), 6 ng/mL murine IL-3 (Peprotech), and 50 ng/mL murine stem cell factor (Peprotech)) until transduction with \u003cem\u003eMNX1\u003c/em\u003e or empty vector control. Cells were transduced by cultivation on irradiated E86 producers for 2 days in the presence of 5 μg/mL protamine sulfate (Merck Millipore). FACS-sorted (FACSAria, BD Biosciences, Franklin Lakes, NJ, USA) GFP+ and/or YFP+ cells were maintained in culture 5-7 days post-transduction prior to transplantation into 8–12-week-old NOD.Cg-\u003cem\u003eKit\u003csup\u003eW-41J\u003c/sup\u003e Tyr\u003csup\u003e+\u0026nbsp;\u003c/sup\u003ePrkdc\u003csup\u003escid\u0026nbsp;\u003c/sup\u003eIl2rg\u003csup\u003etm1Wjl\u003c/sup\u003e\u003c/em\u003e/ThomJ (NBSGW) mice (The Jackson Laboratory, Bar Harbor, ME, USA). 0.8x10\u003csup\u003e6\u0026nbsp;\u003c/sup\u003etransduced cells were transplanted via tail vein injections in two experimental arms \u003cem\u003eMNX1\u003c/em\u003e or empty vector control. Engraftment was monitored by FACS-analysis of GFP+ and/or YFP+ expression in peripheral blood every 2 weeks. Each experimental arm consisted of 4–6 mice and was repeated 3 biological times. The number of mice was chosen based on previous studies in similar experimental setups, where this number provides sufficient power to detect significant differences while minimizing animal use. Randomization was used to allocate experimental units to control and \u003cem\u003eMNX1\u003c/em\u003e groups, and the order of measurements was randomized to minimize potential confounders, such as cage location.\u003c/p\u003e\n\u003cp\u003eExclusion criteria included the removal of animals that suffered injuries due to fighting between mice, which could affect the validity of the results. If any animal exhibited signs of severe injury, it was excluded from the study, and the reason for exclusion was recorded.\u003c/p\u003e\n\u003cp\u003eFrom each biological replicate, one or two bone marrow (BM) samples were used for PCR, ChIP-qPCR, and ACT-Seq analysis. Blinding was applied during the peripheral blood assessments every 2 weeks, as well as during the collection of BM samples for PCR, ChIP-qPCR, and ACT-Seq analysis, to minimize any potential bias in outcome assessment.\u003c/p\u003e\n\u003cp\u003eAnimals were euthanized using the isoflurane (Baxter, Deerfield, IL, USA) drop jar method followed by cervical dislocation. A sealed chamber was prepared by placing a gauze pad soaked with 1.7% isoflurane and the chamber was tightly sealed to prevent the escape of vapors. Upon the onset of deep anesthesia, indicated by the absence of the righting reflex and loss of pedal withdrawal reflex, cervical dislocation was performed manually as per established guidelines. Blood counts were determined on a Sysmex KX-21 Hematology Analyzer (Sysmex, Norderstedt, Germany).\u003c/p\u003e\n\u003ch2\u003eImmunoprecipitation and tandem mass spectrometry tag (TMT)\u003c/h2\u003e\n\u003cp\u003eAnti HA-magnetic beads from Thermo Fisher Scientific (catalog no. 88836) were used for immunoprecipitation. After blocking with 0.1% BSA in PBS, 1 mg of cell lysates were incubated with HA antibody for 1h at RT. The immune complexes were washed three times with lysis buffer and eluted with 2% SDS in 50mM Tris-HCL with 1mM EDTA (Invitrogen).\u003c/p\u003e\n\u003cp\u003eThe samples were then digested with trypsin using modified filter-aided sample preparation (FASP) method \u003csup\u003e37\u003c/sup\u003e. Samples were reduced in 100 mM dithiothreitol at 60°C for 30 min, transferred to Microcon-30kDa Centrifugal Filter Units (Merck), washed with 8 M urea and digestion buffer (50 mM TEAB, 0.5 % sodium deoxycholate (SDC)) prior to alkylation with 10 mM methyl methanethiosulfonate for 30 min at room temperature. Proteins were digested in two steps with trypsin (Pierce MS grade Trypsin, Thermo Fisher Scientific) at 37°C at a final enzyme to protein ratio of 1:50. Peptides were collected by centrifugation and labelled using TMT10plex isobaric label reagents (Thermo Fisher Scientific) according to the manufacturer´s instructions. The individually labelled samples were pooled to one TMT-set and purified using HiPPR detergent removal kit (Thermo Fisher Scientific). SDC was removed by acidification with 10 % trifluoroacetic acid. The TMT-set was fractionated to five fractions using Pierce High pH Reversed-Phase Peptide Fractionation columns (Thermo Fisher Scientific) and acetonitrile (ACN) as organic phase. The fractions were evaporated to dryness and reconstituted in 3 % ACN, 0.2 % formic acid (FA) for LC-MS analysis.\u003c/p\u003e\n\u003cp\u003eThe fractions were analyzed on an Orbitrap Fusion Lumos Tribrid mass spectrometer equipped with a FAIMS-Pro ion mobility system and interfaced with an Easy-nLC1200 liquid chromatography system (all Thermo Fisher Scientific). Peptides were trapped on an Acclaim Pepmap 100 C18 trap column (100 μm x 2 cm, particle size 5 μm, Thermo Fisher Scientific) and separated on an in-house packed analytical column (38 cm x 75 μm, particle size 3 μm, Reprosil-Pur C18, Dr. Maisch) using a linear gradient from 5 % to 28% ACN in 0.2 % FA over 77 min at a flow of 300 nL/min. FAIMS Pro was alternating between compensation voltages (CVs) of -40, -60 and -80, with the same data-dependent settings for all three CVs. The precursor ion mass spectra were acquired at a resolution of 120 000 and an m/z range of 375-1375. The top eight most abundant precursors with charges 2–7 were isolated with an m/z window of 0.7 and fragmented by collision induced dissociation (CID) at 35 %. Fragment spectra were recorded in the ion trap at Rapid scan rate. The ten most abundant MS2 fragment ions were isolated using multi-notch isolation for further MS3 fractionation. MS3 fractionation was performed using higher-energy collision dissociation (HCD) at 65% and the MS3 spectra were recorded in the Orbitrap at 50 000 resolution and an m/z range of 100–500.\u003c/p\u003e\n\u003cp\u003eData analysis was performed with Proteome Discoverer (Version 2.4, Thermo Fisher Scientific) using Mascot (Version 2.5.1, Matrix Science) as a search engine. The data was matched against the reviewed swissprot mouse database (May 2020; 17116 entries). Tryptic peptides were accepted with 1 missed cleavage; methionine oxidation was set as variable modification and cysteine methylthiolation, TMT10plex were set as fixed modifications. Precursor mass tolerance was set to 5 ppm and fragment mass tolerance to 0.6 Da. Percolator was used for PSM validation with a strict FDR threshold of 1%. For reporter ion quantification peak integration was set to most confident centroid and a tolerance of 3 mmu. Only unique peptides were used for protein quantification (Supplementary Table S3).\u003c/p\u003e\n\u003ch2\u003eGene expression analysis and next generation sequencing\u003c/h2\u003e\n\u003cp\u003eTotal RNA was isolated using the miRNeasy Plus micro kit (Qiagen) and cDNA synthesized with SuperScript III First-Strand Synthesis SuperMix for qRT-PCR (Invitrogen/Thermo Fisher Scientific). TaqMan Gene Expression Assays and TaqMan Universal MasterMix II (Applied Biosystems/ Thermo Fisher Scientific) were used for probe-based qPCR assays, listed in Supplementary Table S2. Hprt was used as reference gene for normalization of the expression.\u003c/p\u003e\n\u003cp\u003eRNA-seq was performed as described previously in \u003csup\u003e5\u003c/sup\u003e. Briefly, total RNA was extracted from BM cells from mice with MNX1 induced leukemia and from transduced FL cells after one week of transfection as control. Library preparation and sequencing was carried out by Beijing Genomics Institute. Reads were mapped to the mm10 mouse reference genome using STAR. Differential gene expression was performed using DESeq2 with inclusion criteria of false discovery rate (FDR) less than 0.05 and log2 fold change above |1|. Gene Set Enrichment Analysis \u003csup\u003e38\u003c/sup\u003e (GSEA, Broad Institute) was used with the Gene Transcription Regulation Database (GTRD) transcription factor targets and legacy transcription factor gene set collection. A pathway in GSEA analysis was regarded significant at nominal p-value \u0026lt; 0.05.\u003c/p\u003e\n\u003ch2\u003eChIP-qPCR\u003c/h2\u003e\n\u003cp\u003eChIP-qPCRwas performed as described previously in \u003csup\u003e5\u003c/sup\u003e. In short, iDeal ChIP-qPCR kit (C01010180, Diagenode\u003cem\u003e)\u0026nbsp;\u003c/em\u003ewas used to perform the ChIP assay. Crosslinking was achieved incubating cells with formaldehyde for 10-15 min at room temperature (RT). Subsequently, the reactive was quenched by the addition of glycine and further incubation for 5.10 minutes at RT. The cell pellet was then washed with cold PBS and lysed according to manufacturer specifications. DNA was sheared by sonication using a bioruptor for 20 min in 30 second intervals achieving DNA sizes in the 200-600 bp range. Immunoprecipation was then performed using the antibodies against HA (cell signaling), H3K27me3 (Diagenode_C15410195, RRID:AB_2753161), H3K4me3 (Diagenode_C15410003, RRID:AB_2924768), and polyclonal rabbit anti-IgG (Diagenode_C15410206, RRID:AB_2722554) as negative control. DNA was purified and, qPCR was performed using SYBR green chemistry and in-house designed primers shown in Supplementary Table S2. \u0026nbsp; The calculation of enrichment was normalized to the respective inputs and done according to the following formulas:\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;• The % of IP to input formula: % recovery = 2^[(Ct input-log2(5%)) – Ct sample] * 100%\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;• The enrichment was calculated as: %recovery IP / % recovery IgG\u003c/p\u003e\n\u003cp\u003eNegative control was achieved by immunoprecipitation against HA antibody, followed by detection of random region with low CpG islands and promoter binding density corresponding to chr1:168430579+168430704. Designed primers for the random region is shown in Supplementary Table S2\u003c/p\u003e\n\u003ch2\u003eRNA-seq \u0026amp; Mass spec integrative analysis\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eGTRD transcription factor targets gene set from gene set enrichment analysis (GSEA) was used to identify the transcription factor targets from the normalized counts of RNA-seq. Protein network of the identified proteins from tandem mass spectrometry tag (TMT) were obtained from STRING database using medium confidence of 0.4\u003c/p\u003e\n\u003ch2\u003eATAC and ACT sequencing\u003c/h2\u003e\n\u003cp\u003eAssay for Transposase-Accessible Chromatin (ATAC-seq) and Active Chromatin Targeting (ACT-seq) were performed as described previously in \u003csup\u003e5\u003c/sup\u003e. Briefly, bone marrow cells from MNX1-induced leukemic mice, non-transplanted mouse fetal liver (FL) cells containing MNX1-vector as well as FL cells with empty vector were lysed using in resuspension buffer containing 0.1% of NP40 and Tween-20 and 0.01% digitonin. Lysed cells were resuspended in transposition buffer (10 mM Tris-acetate, pH 7.6, 5 mM MgCl2, 10% dimethylformamide, 30% PBS, 0.01% digitonin and 0.1% Tween-20) together with Tn5 transposome and tagementation were allowed to proceed for 30 min at 37°C under agitation. For ACT-seq, targeting antibodies used for the pA-Tn5 transposome-antibody complexes were directed against H3K4me3 (Abcam_ab8580, RRID:AB_306649), H3K4me1 (Abcam_ab8895, \u0026nbsp;RRID:AB_306847), HA-tag (Sigma_H6908, RRID:AB_260070), \u0026nbsp;IgG (Merck Millipore PP64B, RRID:AB_97852) and yeast histone H2B (Hölzel Diagnostika, M30930, Cologne, Germany, RRID:AB_2924769), the latter complexes being used for spike-in to enable sequence read normalization between biological replicates. DNA was purified and checked for quality followed by sequencing on a Illumina next-seq. Reads were trimmed using Trim Galore in conjunction with Cutadapt (v. 1.14) with the options –paired”, “–nextera”, “– length_1 35”, and “–length_2 35. Trimmed reads were mapped against mm10 reference genome using Bowtie2\u003csup\u003e39\u003c/sup\u003e (v. 2.5.2). Conflicting mapped reads with a phred score lower than 20 was removed using SAMTools\u003csup\u003e40\u003c/sup\u003e (v. 1.19). Finally, read ends were shifted to center of the transposition event.\u003c/p\u003e\n\u003cp\u003eHA-tagged MNX1, H3K4me3 and H4K4me1 peaks were retrieved with GoPeaks \u003csup\u003e41\u003c/sup\u003e (v 1.0.0), using a q-value cut-off of 0.05 and the --broad and \u0026nbsp; --mdist 3000 options for the latter. ATAC-seq peaks were called using MACS2\u003csup\u003e42\u003c/sup\u003e with the options --broad, --keep-dup all, --nomodel and a q-value cutoff of 0.05. Differentially binding analysis was determined using Diffbind and DESeq2 \u003csup\u003e43\u003c/sup\u003e (v. 3.12) R (v. 4.3.2) package with regions considered differentially accessible or enriched if fulfilling FDR less than 0.05 and a fold change larger than |1|. Peak reproducibility between replicates was determined by Jaccard statistic, using the bedtools jaccard function. Consensus peaks were constructed of replicates using BEDTools \u003csup\u003e44\u003c/sup\u003e (v. 2.30.0) multiinter function only retaining regions/peaks found in all replicates. Subsequently, regions within the 1 kbp range were merged using the BEDTools merge function. To determine where open-chromatin intersected with histone depositions, BEDTools intersect function was used.\u003c/p\u003e\n\u003cp\u003ePeak annotation was performed using ChIPseeker \u003csup\u003e45\u003c/sup\u003e (v. 1.38.0) R package, mapping peaks against TxDb UCSC mm10 known gene\u003csup\u003e46\u003c/sup\u003e reference and defining the promoter region as ± 3 kbp from transcription starting sites. Promoter regions were used to find enriched GO biological pathways using clusterProfiler \u003csup\u003e47\u003c/sup\u003e (v. 4.10.0) R package.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003ePbx motif analysis\u003c/h2\u003e\n\u003cp\u003eChIP-seq datasets of Pbx1 in mouse embryonic trunks \u003csup\u003e48\u003c/sup\u003e (GSE39609) and Pbx4 in mouse testis \u003csup\u003e49\u003c/sup\u003e (GSE224369) were retrieved from Gene Expression Omnibus (GEO). Sequencing data in FASTQ format was aligned to mm10 reference genome and converted to BAM-file format using Bowtie2 (v. 2.5.2) and SAMTools. Multi-mapped, unmapped and duplicate reads were filtered out using Sambamba (v. 0.7.1). Peaks were retrieved with MACS2 with a q-value of 0.05. When replicates were available, consensus peaks were constructed of replicate peak sets as described in the ATAC and ACT sequencing section. DNA-binding motifs and their enrichment were analyzed using MEME-Suite (v. 5.5.1). Motifs were retrieved from the Pbx ChIP-seq peak regions with the STREME \u003csup\u003e50\u003c/sup\u003e function using default parameters, motif minimum and maximum length of 8 and 15, respectively, and binomial test against a random test set consisting 10 % of the input sequences to evaluate statistical significance. Subsequently, the top scoring motifs, matching \u0026gt;10 % of peaks in each of the Pbx ChIP-seq data sets were used as a motif sets for a MAST \u003csup\u003e51\u003c/sup\u003e scan of the consensus peak sets derived from epigenetic data of the MNX1-induced leukemic mice. A positional p-value of \u0026lt;0.0001 for a motif was deemed as a match and a E-value (combined positionally p-value for all matches within a sequence times the number of sequences in the database) of \u0026gt;10 of was used as criteria to consider the sequence significant. The resulting motif-matches of the MAST scan are shown Supplementary Table S4 \u0026amp; S5 for the Pbx1 and Pbx4 motif-sets, respectively. As a control the motif-sets were tested against randomly generated sequences produced with the online tool FaBox \u003csup\u003e52\u003c/sup\u003e, recapitulating number of sequences, sequence size distribution and GC-content of the enriched peak data sets. Regions enriched in Pbx-motifs were submitted to pathway enrichment analysis using Clusterprofiler removing redundancy and filtering for pathways by GO level using the simplify function resulting in the most specific pathways.\u003c/p\u003e\n\u003ch2\u003eMNX1 motif analysis\u003c/h2\u003e\n\u003cp\u003eMNX1 motifs were identified using the STREME function within MEME-suite on each MNX1 ACT-seq peak set individually, as well as on a publicly available ChIP-seq dataset of Mnx1 in mouse insulinoma cells \u003csup\u003e53\u003c/sup\u003e (GSE61432), applying parameters consistent with those used in the Pbx motif analysis. The top 10 motifs identified were then assessed for correlation using the MAST function's built-in correlation feature within MEME-suite; motifs were considered correlated if their average Pearson correlation exceeded 0.8. Correlated motifs found across all peak sets were subsequently analyzed for enrichment in epigenetic data from MNX1-induced leukemic mice, as outlined in the Pbx motif analysis. The motif matches from the MAST scan for the MNX1 motif set are presented in Supplementary Table S6.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAcknowledgments\u003c/p\u003e\n\u003cp\u003eWe thank Tanmoy Mondal for revising the manuscript and Tova Johansson and Hanna Brissman for help with animals.\u003c/p\u003e\n\u003cp\u003eProteomic analysis was performed at the Proteomics Core Facility, Sahlgrenska academy, Gothenburg University, with financial support from SciLifeLab and BioMS. The computations were enabled by resources in project SNIC 2021/22-754 provided by the Swedish National Infrastructure for Computing (SNIC) at UPPMAX, partially funded by the Swedish Research Council through grant agreement n. 2018-05973.\u003c/p\u003e\n\u003cp\u003eAuthor Contributions statement\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eEM-B, DW, CP, LP, and AW designed the research study. EM-B, AW, A\u0026Ouml;, TN, DW, PL, MB, JH, GT, JA and SJ performed the laboratory work and results analysis. LF, CP, LP and AW analyzed the combined data and wrote the paper.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAdditional information\u003c/p\u003e\n\u003cp\u003eThe datasets generated for this study can be found in the Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/) under the following accession IDs:, GSE202137 (RNA-seq) https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE202137 , GSE205697 (ATAC-seq) https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE205697 \u0026amp; GSE269913 (ATAC-seq) https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE269913 , GSE269910 (ACT-seq) https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE269910 and in PRIDE/ProteomeXchange, under accession number PXD056378 https://www.ebi.ac.uk/pride/archive/projects/PXD056378\u003c/p\u003e\n\u003cp\u003eThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by grants from the Swedish Cancer Society (20 0925 PjF, CAN2017/461), the Swedish Childhood Cancer Foundation (PR2014-0125, PR2019-0013 and TJ2019-0053, TJ2022-0017), Wilhelm och Martina Lundgrens Fond, Assar Gabrielsson Fond and V\u0026auml;stra G\u0026ouml;talandsregionen (ALFGBG-431881), and the German Funding Agency (DFG) with funding for Collaborative Research Center 1074, Project B11N (to CP).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments have been accepted by the Swedish Agency for Agriculture (Jordbruksverket) and the animal ethics committee in Gothenburg: Dnr 5.8.18-17008/2021. All procedures were conducted in accordance with applicable ethical guidelines and regulations and in accordance with ARRIVE guidelines.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eTseng, S., Lee, M. E. \u0026amp; Lin, P. C. A Review of Childhood Acute Myeloid Leukemia: Diagnosis and Novel Treatment. \u003cem\u003ePharmaceuticals (Basel)\u003c/em\u003e\u003cstrong\u003e16\u003c/strong\u003e (2023). https://doi.org:10.3390/ph16111614\u003c/li\u003e\n \u003cli\u003eGrove, C. S. \u0026amp; Vassiliou, G. S. Acute myeloid leukaemia: a paradigm for the clonal evolution of cancer? \u003cem\u003eDis Model Mech\u003c/em\u003e\u003cstrong\u003e7\u003c/strong\u003e, 941-951 (2014). https://doi.org:10.1242/dmm.015974\u003c/li\u003e\n \u003cli\u003eTomizawa, D. \u0026amp; Tsujimoto, S. I. 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Pro-oncogenic Roles of HLXB9 Protein in Insulinoma Cells through Interaction with Nono Protein and Down-regulation of the c-Met Inhibitor Cblb (Casitas B-lineage Lymphoma b). \u003cem\u003eJ Biol Chem\u003c/em\u003e\u003cstrong\u003e290\u003c/strong\u003e, 25595-25608 (2015). https://doi.org:10.1074/jbc.M115.661413\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"MNX1, PBX, pediatric AML, leukemia, t(7;12), histone methylation","lastPublishedDoi":"10.21203/rs.3.rs-6480114/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6480114/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe t(7;12)(q36;p13) AML subtype in pediatric patients is associated with the upregulation of homeodomain protein MNX1 as the initiating event for leukemogenesis. In this study, we investigated the downstream targets of MNX1 and their relationship to MNX1-induced histone modifications. Using a comprehensive approach combining TMT mass spectrometry, RNA-sequencing, qPCR, antibody-guided chromatin tagmentation sequencing (ACT-Seq), assay for transposase-accessible chromatin using sequencing (ATAC- seq), and chromatin immunoprecipitation assay (ChIP) followed by qPCR, we identified Pbxip1 along with its associated transcription factor Pbx1, and Pbx4 as downstream targets of MNX1. MNX1 binding to the \u003cem\u003ePbx1\u003c/em\u003e promoter triggered its transcriptional activation, associated with increased H3K4me3 and decreased H3K27me3 at the \u003cem\u003ePbx1\u003c/em\u003epromoter. Despite the transient nature of \u003cem\u003eMNX1\u003c/em\u003e’s promoter interaction, these histone marks persisted, suggesting a “hit-and-run” epigenetic remodeling mechanism. Enrichment of Pbx motifs within MNX1-induced H3K4me1, H3K4me3, and ATAC-seq peaks underscores the role of the Pbx family in MNX1-mediated chromatin dynamics. This was further confirmed by showing that MNX1-induced \u003cem\u003ePbx1\u003c/em\u003eexpression could be downregulated using Sinefungin, a pan-methyltransferase inhibitor, that also prevents MNX1-driven leukemia. Our findings provide insights into how MNX1-driven epigenetic modifications are connected to its downstream targets and may offer new avenues for therapeutic intervention in t(7;12) AML.\u003c/p\u003e","manuscriptTitle":"Role of MNX1-mediated Histone Modifications and PBX Gene Family in MNX1-induced Leukemogenesis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-25 15:36:41","doi":"10.21203/rs.3.rs-6480114/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-24T14:56:39+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-23T05:22:21+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-09T09:17:51+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-08T00:28:00+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"123690395008056038523064856186649693519","date":"2025-10-07T15:27:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"146050963183576004738235228765126254593","date":"2025-10-01T23:20:18+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"267980601987746955085916095188243436795","date":"2025-09-16T14:48:50+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-16T12:25:16+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-05-22T16:05:10+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-22T16:04:21+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-25T10:07:20+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-04-25T10:06:11+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"46d39744-11a9-46b4-a28c-34d06a4b7c86","owner":[],"postedDate":"September 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":55080734,"name":"Biological sciences/Cancer/Haematological cancer"},{"id":55080735,"name":"Biological sciences/Molecular biology/Epigenetics"}],"tags":[],"updatedAt":"2026-01-26T16:03:41+00:00","versionOfRecord":{"articleIdentity":"rs-6480114","link":"https://doi.org/10.1038/s41598-026-36367-8","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2026-01-19 15:58:48","publishedOnDateReadable":"January 19th, 2026"},"versionCreatedAt":"2025-09-25 15:36:41","video":"","vorDoi":"10.1038/s41598-026-36367-8","vorDoiUrl":"https://doi.org/10.1038/s41598-026-36367-8","workflowStages":[]},"version":"v1","identity":"rs-6480114","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6480114","identity":"rs-6480114","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}