BCOR Mutations Define a Therapeutic Vulnerability to DHODH Inhibition in Acute Myeloid Leukemia

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In this study, we reveal a synthetic lethal interaction between BCOR and dihydroorotate dehydrogenase (DHODH). We demonstrate that BCOR -deficient cells have a heightened sensitivity to DHODH inhibitors such as brequinar and leflunomide, that are already in clinical use. We confirm that DHODH inhibition selectively induces cell death in BCOR-mutant cells in multiple cellular models, in malignant and non-malignant cells, through chemical and genetic manipulation. Interestingly, we find that the dependency on DHODH does not stem from its role in de novo pyrimidine biosynthesis disruption. Rather, DHODH’s role in the electron transport chain, essential for mitigating reactive oxygen species, may be the physiological vulnerability that pushes BCOR-mutant cells toward cell death when DHODH is inhibited. DHODH inhibitors could be repurposed as targeted therapies for BCOR-mutant tumors, offering a promising strategy for precision medicine in AML and other cancers. Figures Figure 1 Figure 2 Figure 3 INTRODUCTION Acute Myeloid Leukemia (AML) is a heterogeneous hematologic malignancy marked by uncontrolled proliferation of immature myeloid progenitors [ 1 ]. Despite molecular advances, prognosis remains poor, particularly in relapsed or therapy-resistant disease [ 2 ]. BCL-6 co-repressor (BCOR) mutations occur in ~ 3–6% of AML [ 3 ], enriched in RUNX1-mutant or secondary AML following myelodysplastic syndromes [ 4 ]. BCOR, a transcriptional co-repressor within the noncanonical PRC1.1 complex [ 5 ], regulates gene expression via epigenetic interactions. Most BCOR mutations are loss-of-function, impairing hematopoietic differentiation and driving leukemogenesis [ 6 ]. Clinically, BCOR-mutant AML has inferior outcomes (Figure S.1.A), especially when co-mutated with cohesin, RUNX1, or DNMT3A [ 6 , 7 ], with increased chemoresistance at relapse [ 7 ]. Thus, new therapeutic approaches are urgently needed. Previously, we observed that human induced pluripotent stem cells (hiPSCs) acquired BCOR mutations during culture, despite absence in patient-derived starting material [ 8 ]. While probing mutagenesis from dNTP-pool imbalance, we found BCOR-mutant hiPSCs were selectively sensitive to brequinar, a potent inhibitor of dihydroorotate dehydrogenase (DHODH). DHODH catalyzes the mitochondrial conversion of dihydroorotate to orotate in de novo pyrimidine synthesis [ 9 ], essential for uridine, cytidine, and thymidine nucleotide pools. A screen of > 330,000 compounds in a murine AML model also identified DHODH inhibitors as predominant hits [ 10 ]. Subsequent studies of brequinar, leflunomide, and teriflunomide support DHODH inhibition as effective and tolerable in AML, T-ALL, and breast cancer [ 10 ]. Calls to repurpose brequinar for AML have followed, though a predictive biomarker remains lacking. Given the aggressiveness of AML, limited therapeutic options, and our serendipitous finding of BCOR-mutant sensitivity to DHODH inhibition, we tested whether BCOR mutations establish a synthetic lethal interaction with DHODH blockade. If validated, BCOR could represent a biomarker to guide selective DHODH inhibitor use in AML. METHODS Cell culture OCI-AML2/3 were maintained in α-MEM with 20% FCS; MOLM13, HL60, and SKM-1 in RPMI with 10–20% FCS; RPE1 in DMEM/F12 with 10% FCS; and hiPSCs on Vitronectin in Essential E8 medium (all Gibco). siRNA transfection and compound treatment OCI-AML2/3 and RPE1 cells were transfected with siRNAs targeting BCOR, DHODH, or controls (Thermo Fisher) using Lipofectamine 2000. Cells were harvested after 72 h for RNA isolation, viability, or proliferation assays. For pharmacologic studies, cells were treated with brequinar (Cayman Chemical), TP-021, leflunomide, teriflunomide, AG-636, farudodstat, sparfosic acid (MedChemExpress), or 6-azauridine (Thermo Fisher). Viability was assessed by CellTiter-Glo (Promega). CRISPR–Cas9 editing CRISPR–Cas9 editing BCOR knockout in OCI-AML3 was achieved using Cas9 RNP nucleofection with sgRNAs (Synthego) and HDR templates (IDT) via a Lonza 4D-Nucleofector. RNA sequencing RNA libraries (PureLink RNA Mini Kit, Thermo Fisher) were sequenced on Illumina NovaSeq (150 bp paired-end). Reads were aligned with STAR, quantified by featureCounts, and analyzed using DESeq2; pathway enrichment was performed with ShinyGO. Whole-genome sequencing Single-cell clones of RPE1 cells from control and brequinar-treated conditions were subjected to whole-genome sequencing (Illumina HiSeq X-Ten, 30×). Variants were called with CaVEMan, and mutational signatures analyzed using the Mutational Signatures framework and signature.tools.lib. ROS, ATP, and Fe²⁺ assays ROS, ATP, and cytoplasmic Fe²⁺ were quantified using commercial detection kits (OZ Biosciences, Promega, Dojindo) and measured on a Pherastar plate reader. Imaging was performed with an EVOS FL Auto 2 microscope. Proteomics Protein extracts were digested, TMT-labeled, fractionated by high-pH reversed-phase chromatography, and analyzed by Orbitrap Lumos mass spectrometry. Spectra were searched against UniProt with Proteome Discoverer, and reporter ion quantification normalized across TMT batches for differential analysis in R. Statistical analysis Statistical tests were performed in GraphPad Prism 10 using two-tailed t tests or one-/two-way ANOVA with post-hoc corrections. P < 0.05 was considered significant. RESULTS BCOR INHIBITION SENSITISES CELLS TO BREQUINAR We tested two human AML cell lines, OCI-AML2 and OCI-AML3, derived from male patients aged 65 and 57 years, both BCOR wildtype (cellmodelpassports.sanger.ac.uk). Cells were treated with the BCOR inhibitor TP-021 and increasing doses of brequinar (Fig. 1 .A). Clonogenic assays showed that 10 µM TP-021 sensitized both lines to brequinar, reducing the SF50 from ~ 25 µM in wildtype cells to ~ 0.1 µM in BCOR-inhibited cells, a 250-fold increase in sensitivity. To confirm BCOR suppression, we examined downstream targets. The Beat AML study [ 11 ] reported elevated FOXO1, PDGFA, and JAG1 expression in BCOR-mutant AML. Consistently, TP-021 treatment (6–24h) upregulated these genes in both OCI-AML2/3 (Figure S.1.D). We next used a non-malignant hTERT-immortalised RPE1 model and observed a similar phenotype, with a 166-fold increase in brequinar sensitivity upon BCOR inhibition (Fig. 1 .A). Collectively, chemical BCOR inhibition upregulates canonical target genes and confers strong brequinar sensitivity, independent of tissue type or malignant state. BCOR INHIBITION SENSITISES CELLS TO DHODH INHIBITORS To test whether BCOR inhibition broadly sensitizes cells to DHODH blockade, we evaluated four additional inhibitors: leflunomide and teriflunomide, used clinically in multiple sclerosis (MS) and rheumatoid arthritis (RA) [ 12 ], and the AML investigational agents AG-636 and farudodstat [ 13 , 14 ]. OCI-AML2 and OCI-AML3 cells were treated with these compounds ± TP-021 (10 µM) (Figure S.1.C). In all cases, BCOR-inhibited cells showed selective sensitivity, with brequinar and leflunomide most potent at low concentrations (0.01–0.1 µM). We then tested SKM-1, an AML line harboring a BCOR loss-of-function mutation (c.4977-1G > A; cellmodelpassports.sanger.ac.uk) (Fig. 1 .E). Consistent with chemical inhibition, genetically BCOR-deficient SKM-1 cells displayed strong sensitivity to all DHODH inhibitors, with brequinar and leflunomide again most effective. These results support DHODH inhibition, including repurposed leflunomide, as a targeted strategy for BCOR-dysfunctional AML. SYNTHETIC LETHALITY BETWEEN BCOR AND DHODH To validate synthetic lethality between BCOR dysfunction and DHODH, we first performed siRNA knockdown of BCOR, DHODH, or both in OCI-AML2, OCI-AML3, and RPE1 cells. Single knockdowns had little effect, but combined siBCOR + siDHODH significantly reduced viability across all lines (Fig. 1 .B), further enhanced when siBCOR was paired with brequinar. This likely reflects full DHODH inhibition by brequinar versus partial knockdown by siRNA (Figure S.1.E). Live-cell imaging confirmed increased cell death with dual suppression, whether by knockdown or siBCOR plus brequinar (Fig. 1 .C–D). Next, we used CRISPR editing to introduce BCOR loss-of-function mutations in exon 3 of OCI-AML3. Sanger sequencing and Western blot verified edits in three independent clones (BCOR_KO.03, -17 and − 24) (Figure S.1.F–G). All three clones showed synthetic lethality with brequinar, leflunomide, and AG-636 (Fig. 1 .F). Attempts to restore BCOR expression caused profound cell death (data not shown), suggesting BCOR-KO cells had adapted to loss of BCOR. Together, these findings confirm that genetic or pharmacologic DHODH inhibition selectively impairs viability of BCOR-deficient cells, establishing a synthetic lethal relationship. DEPENDENCY OF BCOR-DEFICIENT CELLS ON DHODH IS DUE TO ITS ROLE IN ROS HOMEOSTASIS As shown in the supplementary data (Figure S.2), the observed lethality was not attributable to disruptions in the de novo pyrimidine synthesis pathway, prompting us to investigate the mitochondrial role of DHODH. By catalyzing the conversion of dihydroorotate to orotate, DHODH reduces ubiquinone (coenzyme Q, CoQ) to ubiquinol, which transfers electrons to complex III of the Electron Transport Chain (ETC). This supports mitochondrial membrane potential and ATP production by sustaining electron flow [ 15 , 16 ]. Impaired DHODH activity disrupts bioenergetics, elevates reactive oxygen species (ROS), and triggers ferroptosis [ 10 , 18 ]. In cancer, DHODH is thought to mitigate ROS accumulation, enabling evasion of apoptosis [ 17 ]. Moreover, the BCOR P483L mutation in SKM-1 cells has been linked to impaired mitochondrial function and increased ROS [ 18 ]. We therefore hypothesized that BCOR-mutant cells exist under ROS stress, and that DHODH depletion may elevate ROS to a lethal threshold. Supporting this, Gene Ontology analysis of published BCOR-mutant hiPSC proteomic and transcriptomic data [ 8 ] revealed upregulation of oxidative phosphorylation and ROS production genes (Fig. 2 .A – S.3.A), with increased expression of components of all five ETC complexes (Fig. 2 .B – S.3.B). Metallothioneins, ROS scavengers induced under oxidative stress [ 19 ], were also elevated (Figure S.3.C). These findings suggest BCOR-mutant cells experience basal oxidative stress. We next examined whether DHODH inhibition elevates ROS. RPE1 cells treated with brequinar at its EC50 (0.55 µM) for 24h showed increased expression of ROS-generating genes and decreased expression of Epidermal Growth Factor (EGF) and Hepatocyte Growth Factor (HGF) pathway genes (Fig. 2 .C – S.3.E), consistent with impaired proliferation under oxidative stress [ 20 ]. Upregulated transcripts included PPIF and NDUFB2–10 (ROS production), as well as ROS scavengers COX6B2, GSTO2, GSTT2/2B, GSTM1, and GSTM3 (Fig. 2 .C – S.3.F–G). Chronic brequinar treatment (42 days) of RPE1 cells induced a ten-fold increase in mutations (Fig. 2 .E) with mutational signature SBS18, linked to oxidative lesions and 8-oxo-dG accumulation [ 21 ] (Fig. 2 .F–G). Together, these findings show that DHODH inhibition elevates ROS independently of BCOR. We next evaluated whether synthetic lethality arises from DHODH inhibition exacerbating ROS in oxidatively stressed BCOR-mutant cells. DHODH INHIBITION INDUCES POTENTIALLY LETHAL OXIDATIVE STRESS IN BCOR-MUTANT CELLS We compared ROS levels in OCI-AML3 BCOR-wildtype and three BCOR-mutant clones following 24h brequinar treatment. TBHP, a glutathione inhibitor and ROS inducer, served as a positive control. At baseline, BCOR-mutant clones showed significantly higher ROS than wildtype (Fig. 2 .H). Even low-dose brequinar (0.1 µM) markedly increased ROS, approaching TBHP-induced levels (3h of treatment) (Fig. 2 .I), indicating DHODH inhibition exacerbates ROS stress in BCOR-mutant cells. Notably, TBHP treatment for six days did not induce cell death, unlike DHODH inhibition, suggesting DHODH’s role extends beyond ROS scavenging to sustaining mitochondrial metabolism (Figure S.3.J). Supporting this, BCOR-mutant clones displayed elevated baseline ATP compared to wildtype (Fig. 2 .J). Brequinar or TBHP increased ATP in wildtype but not mutant clones (Fig. 2 .K), consistent with BCOR-deficient cells already operating at maximal ATP output and crossing a survival threshold under DHODH inhibition. Because ferroptosis has been linked to DHODH inhibition [ 17 ], we examined Fe²⁺ accumulation. BCOR-mutant cells showed higher baseline Fe²⁺ than wildtype (Fig. 2 .L). Brequinar (0.1 or 10 µM, 24h) further elevated Fe²⁺ in both groups, supporting ferroptosis induction. Transcriptomics from brequinar-treated RPE1 cells confirmed increased expression of pro-ferroptosis genes (Figure S.3.H) and decreased expression of protective genes (Figure S.3.I). Altogether, these findings highlight elevated ROS, ATP metabolic stress, and ferroptosis susceptibility as features of BCOR-mutant cells, supporting DHODH inhibition as a biomarker-driven therapeutic strategy in AML. DISCUSSION In this study, we identify a synthetic lethal interaction between BCOR and DHODH , uncovering a therapeutic vulnerability in BCOR -mutant cells across multiple cancer and primary cell models. We establish a class effect for DHODH inhibitors, confirmed by both chemical inhibition and genetic manipulation, with efficacy varying among compounds. Mechanistically, lethality stems from DHODH ’s role in controlling ROS rather than de novo pyrimidine biosynthesis (Fig. 3 ). Although in vivo validation is lacking, DHODH inhibitors already have strong clinical and preclinical support, including AML trials where they show activity in resistant patients. A screen of > 330,000 compounds in a murine AML model also identified DHODH inhibitors as predominant hits [ 10 ]. Our work introduces BCOR loss as a biomarker for DHODH therapy, relevant since BCOR mutations are poor prognostic markers in AML and other cancers [ 22 ]. Several DHODH inhibitors are clinically advanced: AG-636 shows antitumor activity in lymphoma models (10–100 mg/kg BID, 14 days) and has entered Phase 1 testing (NCT03834584) [ 23 ]. Leflunomide, approved for autoimmune disease, is dosed at 20 mg/day (~ 74 µM). Brequinar is orally bioavailable and under evaluation in AML (NCT03760666) and COVID-19 (NCT04425252) at ~ 0.3 mM daily. Notably, these doses are much higher than those effective in our BCOR -mutant cell models. We further show that BCOR -deficient cells exhibit elevated ROS and ATP compared to wildtype. DHODH inhibition increases ROS, but in BCOR -mutant cells this fails to trigger compensatory ATP production, suggesting they already operate at their metabolic ceiling. This metabolic stress likely underlies synthetic lethality. Clinically, BCOR mutations may also impact responses to other therapies: they cooperate with FLT3 mutations to drive FLT3 inhibitor resistance [ 24 ]. Supporting this, brequinar sensitized FLT3-mutant MOLM13 cells (Supplementary Figure S.4.A). BCOR mutations are also enriched in salivary gland and endometrial cancers (Supplementary Figure S.4.B), extending DHODH inhibitors’ potential beyond AML. Preclinical studies combining brequinar with venetoclax in high-grade B-cell lymphoma further highlight therapeutic promise [ 25 ]. Altogether, our results position BCOR mutations as a genomic biomarker for repurposing DHODH inhibitors, advancing precision medicine strategies for AML and other cancers. Declarations ACKNOLEDGMENTS The discovery described in this manuscript has been filed as a patent application through Cambridge Enterprise. Conflict -of-interest disclosure : The authors declare no competing financial interests. Ethics and Consent to Participate declarations Not applicable. BCOR Mutations Define a Therapeutic Vulnerability to DHODH Inhibition in Acute Myeloid Leukemia AUTHORS Florian Robert 1,2 , Cherif Badja 1,2 , Soraya Boushaki 1,2 , Andrea Degasperi 1,2 , Yasin Memari 1,2 , Sophie Momen 1,2 , Theodoros I. Roumeliotis 4 , Zuza Kozik 4 , Malgorzata Gozdecka 3 , Jyoti Choudhary 4 , George Vassiliou 3 , Gene CC Koh 1,2 , Serena Nik-Zainal 1,2 FUNDINGS The work performed by S.N.-Z.’s laboratory was funded by the Cancer Research UK (CRUK) Advanced Clinician Scientist Award (C60100/A23916), Dr. Josef Steiner Cancer Research Award 2019, Basser Gray Prime Award 2020, CRUK Pioneer Award (C60100/A23433), CRUK Grand Challenge Awards (C60100/A25274 and CGCATF-2021/100013), CRUK Early Detection Project Award (C60100/A27815), and the National Institute of Health Research (NIHR) Research Professorship (NIHR301627). This work was also supported by the NIHR Cambridge Biomedical Research Centre (BRC-1215-20014). The views expressed are those of the author(s) and not necessarily those of the NIHR or the Department of Health and Social Care. Author Contribution F.R designed and conducted experiments, analyzed the data, and wrote the manuscript; C.B assisted in designing and conducting experiments and helped analyzing the data; S.B assisted in conducting experiments; A.G conducted WGS analysis, Y.M conducted WGS and RNAseq alignments; S.M assisted in performing proteomic and transcriptomic data on hiPSC; T.R, Z.K and J.C conducted proteomic experiments and analysis, and provided correction on the manuscript; M.G and G.V provided technical and scientific expertise, AML cell lines and Manuscript Reviewing; G.CC.K supervised the study; S.N.Z supervised the study and wrote the manuscript. References Döhner H, Weisdorf DJ, Bloomfield CD (2015) Acute Myeloid Leukemia. N Engl J Med 373(12):1136–1152 Estey E (2014) Acute myeloid leukemia: 2014 update on risk-stratification and management. 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Supplementary Files BCORmutantcancersDHODHinhibitionMethods.docx WesternBlotSupplData.png BCORmutantcancersDHODHinhibitionSupplData.docx Cite Share Download PDF Status: Published Journal Publication published 19 Jan, 2026 Read the published version in Annals of Hematology → Version 1 posted Editorial decision: Revision requested 10 Sep, 2025 Reviews received at journal 10 Sep, 2025 Reviewers agreed at journal 08 Sep, 2025 Reviewers invited by journal 07 Sep, 2025 Editor assigned by journal 02 Sep, 2025 Submission checks completed at journal 02 Sep, 2025 First submitted to journal 27 Aug, 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7470212","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":513338090,"identity":"6b7c35c5-b8e9-4e97-85b6-7f4567a8437b","order_by":0,"name":"Florian Robert","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6ElEQVRIie3PvWoCQRDA8VkG5ppBWw/jOywcCDbeq5wsWAkpUgWFIAexs/ZJtpeBs7QVtNAErLVI0EbcExubu7MLZP+wHyz7g10An+8PFkzclLhRx2C+dSvXygjLnYQTNjo/oEokTy+h3cg35QQxO2xHm1cQ6L//DLovBLj7WhUSMrMk23fGqcrWLWvcwyiKBgUkRo4gIdGI0F+HFh1hahYRvpGLaEJov4X2oyLpfYpmR9TRShVCBnpT0Q1k01R2wYQlf+EgFXX6FR0vF/Pj2Q7jepDuvovIQ8i3uer1PHV65rbP5/P9m64WrTzEPvfkpwAAAABJRU5ErkJggg==","orcid":"","institution":"University of Cambridge","correspondingAuthor":true,"prefix":"","firstName":"Florian","middleName":"","lastName":"Robert","suffix":""},{"id":513338091,"identity":"43b4ceb0-6b30-45ca-b9a5-e26c2552a943","order_by":1,"name":"Cherif Badja","email":"","orcid":"","institution":"University of Cambridge","correspondingAuthor":false,"prefix":"","firstName":"Cherif","middleName":"","lastName":"Badja","suffix":""},{"id":513338092,"identity":"2b7a26d7-04f0-44e2-990d-8c4c1f154d0f","order_by":2,"name":"Soraya Boushaki","email":"","orcid":"","institution":"University of Cambridge","correspondingAuthor":false,"prefix":"","firstName":"Soraya","middleName":"","lastName":"Boushaki","suffix":""},{"id":513338093,"identity":"10896fb7-443f-4f36-87f0-9770dc984271","order_by":3,"name":"Andrea Degasperi","email":"","orcid":"","institution":"University of Cambridge","correspondingAuthor":false,"prefix":"","firstName":"Andrea","middleName":"","lastName":"Degasperi","suffix":""},{"id":513338094,"identity":"29871394-a960-4d9a-88a1-e1a94f3ceb19","order_by":4,"name":"Yasin Memari","email":"","orcid":"","institution":"University of Cambridge","correspondingAuthor":false,"prefix":"","firstName":"Yasin","middleName":"","lastName":"Memari","suffix":""},{"id":513338095,"identity":"f2eb0a2c-cd0c-4929-a23d-75f477a21f0a","order_by":5,"name":"Sophie Momen","email":"","orcid":"","institution":"University of Cambridge","correspondingAuthor":false,"prefix":"","firstName":"Sophie","middleName":"","lastName":"Momen","suffix":""},{"id":513338096,"identity":"92f1cba9-efb1-41a3-a084-f49fd57bac97","order_by":6,"name":"Theodoros I. Roumeliotis","email":"","orcid":"","institution":"Chester Betty Labs","correspondingAuthor":false,"prefix":"","firstName":"Theodoros","middleName":"I.","lastName":"Roumeliotis","suffix":""},{"id":513338097,"identity":"ce89a3b6-c8c1-4611-860f-0e85b19bed1f","order_by":7,"name":"Zuza Kozik","email":"","orcid":"","institution":"Chester Betty Labs","correspondingAuthor":false,"prefix":"","firstName":"Zuza","middleName":"","lastName":"Kozik","suffix":""},{"id":513338098,"identity":"222931b3-924a-4ace-8bf2-76681fe4da44","order_by":8,"name":"Malgorzata Gozdecka","email":"","orcid":"","institution":"Wellcome/MRC Cambridge Stem Cell Institute","correspondingAuthor":false,"prefix":"","firstName":"Malgorzata","middleName":"","lastName":"Gozdecka","suffix":""},{"id":513338099,"identity":"5269d7e6-c438-4f35-995b-c32142eb026e","order_by":9,"name":"Jyoti Choudhary","email":"","orcid":"","institution":"Chester Betty Labs","correspondingAuthor":false,"prefix":"","firstName":"Jyoti","middleName":"","lastName":"Choudhary","suffix":""},{"id":513338100,"identity":"37a51832-b50e-4b66-a545-78b62a4f7b4a","order_by":10,"name":"George Vassiliou","email":"","orcid":"","institution":"Wellcome/MRC Cambridge Stem Cell Institute","correspondingAuthor":false,"prefix":"","firstName":"George","middleName":"","lastName":"Vassiliou","suffix":""},{"id":513338101,"identity":"ca4ea228-a024-4ee5-a2c9-29882a60e98a","order_by":11,"name":"Gene CC Koh","email":"","orcid":"","institution":"University of Cambridge","correspondingAuthor":false,"prefix":"","firstName":"Gene","middleName":"CC","lastName":"Koh","suffix":""},{"id":513338102,"identity":"02d4d8ee-241c-4e13-9add-2a790e2e89d8","order_by":12,"name":"Serena Nik-Zainal","email":"","orcid":"","institution":"University of Cambridge","correspondingAuthor":false,"prefix":"","firstName":"Serena","middleName":"","lastName":"Nik-Zainal","suffix":""}],"badges":[],"createdAt":"2025-08-27 09:38:29","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7470212/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7470212/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00277-026-06773-z","type":"published","date":"2026-01-19T15:59:05+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":91305284,"identity":"0ce8843c-81a2-42fd-a278-39882801489b","added_by":"auto","created_at":"2025-09-15 06:25:11","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":871424,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eBCOR deficiency sensitizes cells to DHODH Inhibitors. \u003c/strong\u003e\u003c/em\u003e\u003cem\u003e(A) Heatmaps representing survival fractions of OCI-AML2, OCI-AML3 and RPE1 treated with increasing doses of TP-021 (BCOR inhibitor) and brequinar, for 6 days (n=3 per line). Survival fractions of OCI-AML2, OCI-AML3 and RPE1 treated with working dose of TP-021 (10 µM) compared to DMSO, with increasing doses of brequinar, for each cell line, for 6 days (two-way ANOVA, n=3; p\u0026lt;0.0001****; p\u0026lt;0.001***). (B) Survival fractions of OCI-AML2, OCI-AML3 and RPE1; transfected with siRNA and ± treated with brequinar 1 µM (two-way ANOVA, n=3; p\u0026lt;0.0001****). (C) Cell proliferation assay of RPE1 transfected with siRNA, ± treated with brequinar 1 µM (two-way ANOVA, n=2; p\u0026lt;0.0001****) with (D) associated pictures taken at 48hrs (basic analyzer – Incucyte, 10x magnification). (E) SKM-1 (BCOR-mutated AML) was treated with increasing doses of DHODH inhibitors for 6 days (two-way ANOVA, n=3; p\u0026lt;0.0001****). (F) OCI-AML3\u003c/em\u003e\u003csup\u003e\u003cem\u003eBCOR_WT\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e, OCI-AML3\u003c/em\u003e\u003csup\u003e\u003cem\u003eBCOR_KO.03\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e, OCI-AML3\u003c/em\u003e\u003csup\u003e\u003cem\u003eBCOR_KO.17\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e and OCI-AML3\u003c/em\u003e\u003csup\u003e\u003cem\u003eBCOR_KO.24\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e were treated for 6 days with increasing doses of 5 different DHODH inhibitors (two-way ANOVA, n=3; p\u0026lt;0.0001****, p\u0026lt;0.001***, p\u0026lt;0.01**, p\u0026lt;0.05*, n.s = non-significant).\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7470212/v1/eeb045a89ce8df4f22793d99.png"},{"id":91305288,"identity":"36deb769-5af9-4190-84c9-2119aebfd51e","added_by":"auto","created_at":"2025-09-15 06:25:11","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":969618,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eBCOR-mutant cells exhibit higher ROS levels compared to wildtype, with brequinar further elevating this level to potential lethal thresholds. \u003c/strong\u003e\u003c/em\u003e\u003cem\u003e(A)\u003c/em\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eGene Ontology of upregulated Differentially Expressed Proteins (DEPs) in BCOR-mutant contrasted to BCOR-wildtype hiPSCs, showing significant upregulation of Oxidative Phosphorylation, Reactive Oxygen Species and Metabolic Pathways indicating BCOR-mutant cells live in higher oxidative stress environment. (B) Heatmap of DEPs involved in mitochondrial electron transport chain (ETC) in BCOR-mutant and Wildtype hiPSCs. This significant increase of ETC-involved gene expression in BCOR-mutant cells indicate higher mitochondrial metabolism and potentially higher oxidative stress. (C) Volcano plot of significantly differentially expressed genes (DEGs) (-Log\u003c/em\u003e\u003csub\u003e\u003cem\u003e10\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e[P\u003c/em\u003e\u003csub\u003e\u003cem\u003eadj\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e] \u0026gt; 1,30) RPE1 cells treated with brequinar (0.55µM, 24Hrs) versus untreated, highlighting upregulation of ROS-pathway genes and downregulation of EGF/HGF-pathway genes. (D) Experimental schematic of Whole Genome Sequencing experiment. RPE1 cells were chronically treated with brequinar 0.55µM or untreated for 42 days, then subcloned and amplified for WGS.\u0026nbsp;\u0026nbsp; (E) Single base substitution counts comparing 3 untreated RPE1 clones to 3 brequinar-treated RPE1 clones. (SBS count plot, t-test, n=3; p\u0026lt;0.01**). (F) Exposure proportions of brequinar and control signatures, showing specific enrichment of brequinar signature in brequinar-treated RPE1 cells. (G) Single base substitution plots showing a high similarity of brequinar signature to SBS18 (Cosine similarity = 0.92) (Degasperi et al, 2022), which is induced by an excess of 8-oxo-dG indicating oxidative damages in DNA. (H) Basal ROS levels amongst OCI-AML3\u003c/em\u003e\u003csup\u003e\u003cem\u003eBCOR_WT\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e, OCI-AML3\u003c/em\u003e\u003csup\u003e\u003cem\u003eBCOR_KO.03\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e, OCI-AML3\u003c/em\u003e\u003csup\u003e\u003cem\u003eBCOR_KO.17\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e and OCI-AML3\u003c/em\u003e\u003csup\u003e\u003cem\u003eBCOR_KO.24\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e. BCOR-mutant cells show significantly higher basal ROS level compared to wildtype (Relative fluorescent units per 100,000 cells - two-way ANOVA, n=3; p\u0026lt;0.0001****). (I) DHODH-inhibition through brequinar treatment further exacerbate ROS levels in OCI-AML3\u003c/em\u003e\u003csup\u003e\u003cem\u003eBCOR_WT\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e, OCI-AML3\u003c/em\u003e\u003csup\u003e\u003cem\u003eBCOR_KO.03\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e, OCI-AML3\u003c/em\u003e\u003csup\u003e\u003cem\u003eBCOR_KO.17\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e and OCI-AML3\u003c/em\u003e\u003csup\u003e\u003cem\u003eBCOR_KO.24\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e (two-way ANOVA, n=3; p\u0026lt;0.0001****, p\u0026lt;0.001***, p\u0026lt;0.01**). (J) Basal ATP levels amongst OCI-AML3\u003c/em\u003e\u003csup\u003e\u003cem\u003eBCOR_WT\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e, OCI-AML3\u003c/em\u003e\u003csup\u003e\u003cem\u003eBCOR_KO.03\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e, OCI-AML3\u003c/em\u003e\u003csup\u003e\u003cem\u003eBCOR_KO.17\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e and OCI-AML3\u003c/em\u003e\u003csup\u003e\u003cem\u003eBCOR_KO.24\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e. BCOR-mutant cells show significantly higher basal ATP level compared to wildtype, possibly a sign of enhanced oxidative phosphorylation (Relative luminescent units per 100,000 cells - two-way ANOVA, n=3; p\u0026lt;0.0001****, p\u0026lt;0.001***). (K) DHODH-inhibition through brequinar treatment further exacerbate ATP levels in OCI-AML3\u003c/em\u003e\u003csup\u003e\u003cem\u003eBCOR_WT\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e, but not BCOR-mutant cell lines, implying that BCOR-mutant cells may be operating at their saturating limit of metabolic ATP production (two-way ANOVA, n=3; p\u0026lt;0.001***, p\u0026lt;0.01**, p\u0026lt;0.05*, n.s=non-significant). (L) Basal cytoplasmic Fe\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e levels amongst OCI-AML3\u003c/em\u003e\u003csup\u003e\u003cem\u003eBCOR_WT\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e, OCI-AML3\u003c/em\u003e\u003csup\u003e\u003cem\u003eBCOR_KO.03\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e, OCI-AML3\u003c/em\u003e\u003csup\u003e\u003cem\u003eBCOR_KO.17\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e and OCI-AML3\u003c/em\u003e\u003csup\u003e\u003cem\u003eBCOR_KO.24\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e. BCOR-mutant cells show significantly higher basal cytoplasmic Fe\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e level compared to wildtype (Relative fluorescent units per 100,000 cells - two-way ANOVA, n=3; p\u0026lt;0.0001****). (M) DHODH-inhibition through brequinar treatment further exacerbate cytoplasmic Fe\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e levels in OCI-AML3\u003c/em\u003e\u003csup\u003e\u003cem\u003eBCOR_WT\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e, OCI-AML3\u003c/em\u003e\u003csup\u003e\u003cem\u003eBCOR_KO.03\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e, OCI-AML3\u003c/em\u003e\u003csup\u003e\u003cem\u003eBCOR_KO.17\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e and OCI-AML3\u003c/em\u003e\u003csup\u003e\u003cem\u003eBCOR_KO.24\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e, where accumulation of Fe\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e is directly linked to Ferroptosis (two-way ANOVA, n=3; p\u0026lt;0.0001****, p\u0026lt;0.001***, p\u0026lt;0.01**). (N) Representative images of cells captured under a fluorescence microscope using GFP excitation at 10× magnification, highlighting reactive oxygen species (ROS) as GFP fluorescence. Scale bar = 500 µm.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7470212/v1/b23193a8067d8898a2082d35.png"},{"id":91305266,"identity":"77258c30-8e57-44ca-9eea-289dd22a508e","added_by":"auto","created_at":"2025-09-15 06:25:09","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":530914,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eGraphical summary of DHODH-inhibitors in BCOR-mutant cells. \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eHere, we demonstrate that BCOR-deficient cells exhibit heightened sensitivity to DHODH inhibitors, such as brequinar. DHODH is a key enzyme in the de novo pyrimidine synthesis pathway, responsible for converting dihydroorotate to orotate, ultimately leading to the production of dUTP, dCTP, and dTTP necessary for DNA replication. When BCOR-deficient cells were treated with inhibitors targeting other components of the pathway, such as CAD-inhibitor (sparfosic acid) or UMPS-inhibitor (6-azauridine), the same sensitivity seen with DHODH inhibitors was not replicated. This led us to hypothesize that the DHODH dependency in BCOR-deficient cells is linked to its role within the mitochondrial Electron Transport Chain (ETC). DHODH reduces ubiquinone (CoQ) to ubiquinol (CoQH2), which subsequently facilitates electron transfer from complexes I and II to complex III. BCOR-mutant cells show increased ROS levels compared to wild-type cells, which are further amplified by brequinar treatment and associated with enhanced ATP production and elevated Fe2+ levels. Thus, brequinar and other DHODH inhibitors are effective tools for selectively increasing ROS in BCOR-deficient cells, thereby inducing targeted cell death.\u003c/em\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eCAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, dihydroorotase); DHO (dihydroorotate); DHODH (dihydroorotate dehydrogenase); UMPS (uridine monophospate synthetase); UMP (uridine monophosphate); CoQ (coenzyme Q - ubiquinone); CoQH2 (ubiquinol); Cyt-c (cytochrome c); ROS (reactive oxygen species) (adapted from Boukalova et al., 2020).\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage310.png","url":"https://assets-eu.researchsquare.com/files/rs-7470212/v1/8dfe14386d2cfca92a79e603.png"},{"id":101151823,"identity":"f9685acf-6927-40cc-a718-06ed175ce76f","added_by":"auto","created_at":"2026-01-26 16:06:15","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3022739,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7470212/v1/22284a27-61c9-4680-91f0-c2ef364e23ca.pdf"},{"id":91305272,"identity":"cdf078fe-ffb6-4633-8a01-06aea37393be","added_by":"auto","created_at":"2025-09-15 06:25:10","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":27586,"visible":true,"origin":"","legend":"","description":"","filename":"BCORmutantcancersDHODHinhibitionMethods.docx","url":"https://assets-eu.researchsquare.com/files/rs-7470212/v1/19f0b4abaf296ccee8d63056.docx"},{"id":91305267,"identity":"5cad46e0-79e8-4d9d-a892-4b16e588c3cb","added_by":"auto","created_at":"2025-09-15 06:25:09","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":991154,"visible":true,"origin":"","legend":"","description":"","filename":"WesternBlotSupplData.png","url":"https://assets-eu.researchsquare.com/files/rs-7470212/v1/5c619390b090d9797304ea70.png"},{"id":91305240,"identity":"3a2e37f4-d4e0-4d8c-86bb-02710ed3fba8","added_by":"auto","created_at":"2025-09-15 06:25:06","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1814311,"visible":true,"origin":"","legend":"","description":"","filename":"BCORmutantcancersDHODHinhibitionSupplData.docx","url":"https://assets-eu.researchsquare.com/files/rs-7470212/v1/2b61e09627e7fe3a33cf6b60.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"BCOR Mutations Define a Therapeutic Vulnerability to DHODH Inhibition in Acute Myeloid Leukemia","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eAcute Myeloid Leukemia (AML) is a heterogeneous hematologic malignancy marked by uncontrolled proliferation of immature myeloid progenitors [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Despite molecular advances, prognosis remains poor, particularly in relapsed or therapy-resistant disease [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. BCL-6 co-repressor (BCOR) mutations occur in ~\u0026thinsp;3\u0026ndash;6% of AML [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], enriched in RUNX1-mutant or secondary AML following myelodysplastic syndromes [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. BCOR, a transcriptional co-repressor within the noncanonical PRC1.1 complex [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], regulates gene expression via epigenetic interactions. Most BCOR mutations are loss-of-function, impairing hematopoietic differentiation and driving leukemogenesis [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Clinically, BCOR-mutant AML has inferior outcomes (Figure S.1.A), especially when co-mutated with cohesin, RUNX1, or DNMT3A [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], with increased chemoresistance at relapse [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Thus, new therapeutic approaches are urgently needed.\u003c/p\u003e\u003cp\u003ePreviously, we observed that human induced pluripotent stem cells (hiPSCs) acquired BCOR mutations during culture, despite absence in patient-derived starting material [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. While probing mutagenesis from dNTP-pool imbalance, we found BCOR-mutant hiPSCs were selectively sensitive to brequinar, a potent inhibitor of dihydroorotate dehydrogenase (DHODH).\u003c/p\u003e\u003cp\u003eDHODH catalyzes the mitochondrial conversion of dihydroorotate to orotate in de novo pyrimidine synthesis [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], essential for uridine, cytidine, and thymidine nucleotide pools. A screen of \u0026gt;\u0026thinsp;330,000 compounds in a murine AML model also identified DHODH inhibitors as predominant hits [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Subsequent studies of brequinar, leflunomide, and teriflunomide support DHODH inhibition as effective and tolerable in AML, T-ALL, and breast cancer [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Calls to repurpose brequinar for AML have followed, though a predictive biomarker remains lacking.\u003c/p\u003e\u003cp\u003eGiven the aggressiveness of AML, limited therapeutic options, and our serendipitous finding of BCOR-mutant sensitivity to DHODH inhibition, we tested whether BCOR mutations establish a synthetic lethal interaction with DHODH blockade. If validated, BCOR could represent a biomarker to guide selective DHODH inhibitor use in AML.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eCell culture\u003c/h2\u003e\u003cp\u003eOCI-AML2/3 were maintained in α-MEM with 20% FCS; MOLM13, HL60, and SKM-1 in RPMI with 10\u0026ndash;20% FCS; RPE1 in DMEM/F12 with 10% FCS; and hiPSCs on Vitronectin in Essential E8 medium (all Gibco).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003esiRNA transfection and compound treatment\u003c/h3\u003e\n\u003cp\u003eOCI-AML2/3 and RPE1 cells were transfected with siRNAs targeting BCOR, DHODH, or controls (Thermo Fisher) using Lipofectamine 2000. Cells were harvested after 72 h for RNA isolation, viability, or proliferation assays. For pharmacologic studies, cells were treated with brequinar (Cayman Chemical), TP-021, leflunomide, teriflunomide, AG-636, farudodstat, sparfosic acid (MedChemExpress), or 6-azauridine (Thermo Fisher). Viability was assessed by CellTiter-Glo (Promega).\u003c/p\u003e\n\u003ch3\u003eCRISPR–Cas9 editing\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003eCRISPR\u0026ndash;Cas9 editing\u003c/div\u003e\u003cp\u003eBCOR knockout in OCI-AML3 was achieved using Cas9 RNP nucleofection with sgRNAs (Synthego) and HDR templates (IDT) via a Lonza 4D-Nucleofector.\u003c/p\u003e\n\u003ch3\u003eRNA sequencing\u003c/h3\u003e\n\u003cp\u003eRNA libraries (PureLink RNA Mini Kit, Thermo Fisher) were sequenced on Illumina NovaSeq (150 bp paired-end). Reads were aligned with STAR, quantified by featureCounts, and analyzed using DESeq2; pathway enrichment was performed with ShinyGO.\u003c/p\u003e\n\u003ch3\u003eWhole-genome sequencing\u003c/h3\u003e\n\u003cp\u003eSingle-cell clones of RPE1 cells from control and brequinar-treated conditions were subjected to whole-genome sequencing (Illumina HiSeq X-Ten, 30\u0026times;). Variants were called with CaVEMan, and mutational signatures analyzed using the Mutational Signatures framework and signature.tools.lib.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eROS, ATP, and Fe\u0026sup2;⁺ assays\u003c/h2\u003e\u003cp\u003eROS, ATP, and cytoplasmic Fe\u0026sup2;⁺ were quantified using commercial detection kits (OZ Biosciences, Promega, Dojindo) and measured on a Pherastar plate reader. Imaging was performed with an EVOS FL Auto 2 microscope.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eProteomics\u003c/h3\u003e\n\u003cp\u003eProtein extracts were digested, TMT-labeled, fractionated by high-pH reversed-phase chromatography, and analyzed by Orbitrap Lumos mass spectrometry. Spectra were searched against UniProt with Proteome Discoverer, and reporter ion quantification normalized across TMT batches for differential analysis in R.\u003c/p\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eStatistical tests were performed in GraphPad Prism 10 using two-tailed t tests or one-/two-way ANOVA with post-hoc corrections. P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eBCOR INHIBITION SENSITISES CELLS TO BREQUINAR\u003c/h2\u003e\u003cp\u003eWe tested two human AML cell lines, OCI-AML2 and OCI-AML3, derived from male patients aged 65 and 57 years, both BCOR wildtype (cellmodelpassports.sanger.ac.uk). Cells were treated with the BCOR inhibitor TP-021 and increasing doses of brequinar (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.A). Clonogenic assays showed that 10 \u0026micro;M TP-021 sensitized both lines to brequinar, reducing the SF50 from ~\u0026thinsp;25 \u0026micro;M in wildtype cells to ~\u0026thinsp;0.1 \u0026micro;M in BCOR-inhibited cells, a 250-fold increase in sensitivity. To confirm BCOR suppression, we examined downstream targets. The Beat AML study [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] reported elevated FOXO1, PDGFA, and JAG1 expression in BCOR-mutant AML. Consistently, TP-021 treatment (6\u0026ndash;24h) upregulated these genes in both OCI-AML2/3 (Figure S.1.D).\u003c/p\u003e\u003cp\u003eWe next used a non-malignant hTERT-immortalised RPE1 model and observed a similar phenotype, with a 166-fold increase in brequinar sensitivity upon BCOR inhibition (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.A). Collectively, chemical BCOR inhibition upregulates canonical target genes and confers strong brequinar sensitivity, independent of tissue type or malignant state.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eBCOR INHIBITION SENSITISES CELLS TO DHODH INHIBITORS\u003c/h2\u003e\u003cp\u003eTo test whether BCOR inhibition broadly sensitizes cells to DHODH blockade, we evaluated four additional inhibitors: leflunomide and teriflunomide, used clinically in multiple sclerosis (MS) and rheumatoid arthritis (RA) [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], and the AML investigational agents AG-636 and farudodstat [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. OCI-AML2 and OCI-AML3 cells were treated with these compounds\u0026thinsp;\u0026plusmn;\u0026thinsp;TP-021 (10 \u0026micro;M) (Figure S.1.C). In all cases, BCOR-inhibited cells showed selective sensitivity, with brequinar and leflunomide most potent at low concentrations (0.01\u0026ndash;0.1 \u0026micro;M).\u003c/p\u003e\u003cp\u003eWe then tested SKM-1, an AML line harboring a BCOR loss-of-function mutation (c.4977-1G\u0026thinsp;\u0026gt;\u0026thinsp;A; cellmodelpassports.sanger.ac.uk) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.E). Consistent with chemical inhibition, genetically BCOR-deficient SKM-1 cells displayed strong sensitivity to all DHODH inhibitors, with brequinar and leflunomide again most effective. These results support DHODH inhibition, including repurposed leflunomide, as a targeted strategy for BCOR-dysfunctional AML.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eSYNTHETIC LETHALITY BETWEEN BCOR AND DHODH\u003c/h2\u003e\u003cp\u003eTo validate synthetic lethality between BCOR dysfunction and DHODH, we first performed siRNA knockdown of BCOR, DHODH, or both in OCI-AML2, OCI-AML3, and RPE1 cells. Single knockdowns had little effect, but combined siBCOR\u0026thinsp;+\u0026thinsp;siDHODH significantly reduced viability across all lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.B), further enhanced when siBCOR was paired with brequinar. This likely reflects full DHODH inhibition by brequinar versus partial knockdown by siRNA (Figure S.1.E). Live-cell imaging confirmed increased cell death with dual suppression, whether by knockdown or siBCOR plus brequinar (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.C\u0026ndash;D).\u003c/p\u003e\u003cp\u003eNext, we used CRISPR editing to introduce BCOR loss-of-function mutations in exon 3 of OCI-AML3. Sanger sequencing and Western blot verified edits in three independent clones (BCOR_KO.03, -17 and \u0026minus;\u0026thinsp;24) (Figure S.1.F\u0026ndash;G). All three clones showed synthetic lethality with brequinar, leflunomide, and AG-636 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.F). Attempts to restore BCOR expression caused profound cell death (data not shown), suggesting BCOR-KO cells had adapted to loss of BCOR.\u003c/p\u003e\u003cp\u003eTogether, these findings confirm that genetic or pharmacologic DHODH inhibition selectively impairs viability of BCOR-deficient cells, establishing a synthetic lethal relationship.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eDEPENDENCY OF BCOR-DEFICIENT CELLS ON DHODH IS DUE TO ITS ROLE IN ROS HOMEOSTASIS\u003c/h2\u003e\u003cp\u003eAs shown in the supplementary data (Figure S.2), the observed lethality was not attributable to disruptions in the de novo pyrimidine synthesis pathway, prompting us to investigate the mitochondrial role of DHODH. By catalyzing the conversion of dihydroorotate to orotate, DHODH reduces ubiquinone (coenzyme Q, CoQ) to ubiquinol, which transfers electrons to complex III of the Electron Transport Chain (ETC). This supports mitochondrial membrane potential and ATP production by sustaining electron flow [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Impaired DHODH activity disrupts bioenergetics, elevates reactive oxygen species (ROS), and triggers ferroptosis [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In cancer, DHODH is thought to mitigate ROS accumulation, enabling evasion of apoptosis [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Moreover, the BCOR\u003csup\u003eP483L\u003c/sup\u003e mutation in SKM-1 cells has been linked to impaired mitochondrial function and increased ROS [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. We therefore hypothesized that BCOR-mutant cells exist under ROS stress, and that DHODH depletion may elevate ROS to a lethal threshold.\u003c/p\u003e\u003cp\u003eSupporting this, Gene Ontology analysis of published BCOR-mutant hiPSC proteomic and transcriptomic data [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] revealed upregulation of oxidative phosphorylation and ROS production genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.A \u0026ndash; S.3.A), with increased expression of components of all five ETC complexes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.B \u0026ndash; S.3.B). Metallothioneins, ROS scavengers induced under oxidative stress [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], were also elevated (Figure S.3.C). These findings suggest BCOR-mutant cells experience basal oxidative stress.\u003c/p\u003e\u003cp\u003eWe next examined whether DHODH inhibition elevates ROS. RPE1 cells treated with brequinar at its EC50 (0.55 \u0026micro;M) for 24h showed increased expression of ROS-generating genes and decreased expression of Epidermal Growth Factor (EGF) and Hepatocyte Growth Factor (HGF) pathway genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.C \u0026ndash; S.3.E), consistent with impaired proliferation under oxidative stress [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Upregulated transcripts included PPIF and NDUFB2\u0026ndash;10 (ROS production), as well as ROS scavengers COX6B2, GSTO2, GSTT2/2B, GSTM1, and GSTM3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.C \u0026ndash; S.3.F\u0026ndash;G).\u003c/p\u003e\u003cp\u003eChronic brequinar treatment (42 days) of RPE1 cells induced a ten-fold increase in mutations (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.E) with mutational signature SBS18, linked to oxidative lesions and 8-oxo-dG accumulation [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.F\u0026ndash;G). Together, these findings show that DHODH inhibition elevates ROS independently of BCOR.\u003c/p\u003e\u003cp\u003eWe next evaluated whether synthetic lethality arises from DHODH inhibition exacerbating ROS in oxidatively stressed BCOR-mutant cells.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eDHODH INHIBITION INDUCES POTENTIALLY LETHAL OXIDATIVE STRESS IN BCOR-MUTANT CELLS\u003c/h2\u003e\u003cp\u003eWe compared ROS levels in OCI-AML3 BCOR-wildtype and three BCOR-mutant clones following 24h brequinar treatment. TBHP, a glutathione inhibitor and ROS inducer, served as a positive control. At baseline, BCOR-mutant clones showed significantly higher ROS than wildtype (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.H). Even low-dose brequinar (0.1 \u0026micro;M) markedly increased ROS, approaching TBHP-induced levels (3h of treatment) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.I), indicating DHODH inhibition exacerbates ROS stress in BCOR-mutant cells.\u003c/p\u003e\u003cp\u003eNotably, TBHP treatment for six days did not induce cell death, unlike DHODH inhibition, suggesting DHODH\u0026rsquo;s role extends beyond ROS scavenging to sustaining mitochondrial metabolism (Figure S.3.J). Supporting this, BCOR-mutant clones displayed elevated baseline ATP compared to wildtype (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.J). Brequinar or TBHP increased ATP in wildtype but not mutant clones (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.K), consistent with BCOR-deficient cells already operating at maximal ATP output and crossing a survival threshold under DHODH inhibition.\u003c/p\u003e\u003cp\u003eBecause ferroptosis has been linked to DHODH inhibition [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], we examined Fe\u0026sup2;⁺ accumulation. BCOR-mutant cells showed higher baseline Fe\u0026sup2;⁺ than wildtype (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.L). Brequinar (0.1 or 10 \u0026micro;M, 24h) further elevated Fe\u0026sup2;⁺ in both groups, supporting ferroptosis induction. Transcriptomics from brequinar-treated RPE1 cells confirmed increased expression of pro-ferroptosis genes (Figure S.3.H) and decreased expression of protective genes (Figure S.3.I).\u003c/p\u003e\u003cp\u003eAltogether, these findings highlight elevated ROS, ATP metabolic stress, and ferroptosis susceptibility as features of BCOR-mutant cells, supporting DHODH inhibition as a biomarker-driven therapeutic strategy in AML.\u003c/p\u003e\u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eIn this study, we identify a synthetic lethal interaction between \u003cem\u003eBCOR\u003c/em\u003e and \u003cem\u003eDHODH\u003c/em\u003e, uncovering a therapeutic vulnerability in \u003cem\u003eBCOR\u003c/em\u003e-mutant cells across multiple cancer and primary cell models. We establish a class effect for DHODH inhibitors, confirmed by both chemical inhibition and genetic manipulation, with efficacy varying among compounds. Mechanistically, lethality stems from \u003cem\u003eDHODH\u003c/em\u003e\u0026rsquo;s role in controlling ROS rather than \u003cem\u003ede novo\u003c/em\u003e pyrimidine biosynthesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAlthough in vivo validation is lacking, DHODH inhibitors already have strong clinical and preclinical support, including AML trials where they show activity in resistant patients. A screen of \u0026gt;\u0026thinsp;330,000 compounds in a murine AML model also identified DHODH inhibitors as predominant hits [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Our work introduces \u003cem\u003eBCOR\u003c/em\u003e loss as a biomarker for DHODH therapy, relevant since \u003cem\u003eBCOR\u003c/em\u003e mutations are poor prognostic markers in AML and other cancers [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eSeveral DHODH inhibitors are clinically advanced: AG-636 shows antitumor activity in lymphoma models (10\u0026ndash;100 mg/kg BID, 14 days) and has entered Phase 1 testing (NCT03834584) [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Leflunomide, approved for autoimmune disease, is dosed at 20 mg/day (~\u0026thinsp;74 \u0026micro;M). Brequinar is orally bioavailable and under evaluation in AML (NCT03760666) and COVID-19 (NCT04425252) at ~\u0026thinsp;0.3 mM daily. Notably, these doses are much higher than those effective in our \u003cem\u003eBCOR\u003c/em\u003e-mutant cell models.\u003c/p\u003e\u003cp\u003eWe further show that \u003cem\u003eBCOR\u003c/em\u003e-deficient cells exhibit elevated ROS and ATP compared to wildtype. DHODH inhibition increases ROS, but in \u003cem\u003eBCOR\u003c/em\u003e-mutant cells this fails to trigger compensatory ATP production, suggesting they already operate at their metabolic ceiling. This metabolic stress likely underlies synthetic lethality.\u003c/p\u003e\u003cp\u003eClinically, \u003cem\u003eBCOR\u003c/em\u003e mutations may also impact responses to other therapies: they cooperate with \u003cem\u003eFLT3\u003c/em\u003e mutations to drive FLT3 inhibitor resistance [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Supporting this, brequinar sensitized FLT3-mutant MOLM13 cells (Supplementary Figure S.4.A). \u003cem\u003eBCOR\u003c/em\u003e mutations are also enriched in salivary gland and endometrial cancers (Supplementary Figure S.4.B), extending DHODH inhibitors\u0026rsquo; potential beyond AML. Preclinical studies combining brequinar with venetoclax in high-grade B-cell lymphoma further highlight therapeutic promise [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAltogether, our results position \u003cem\u003eBCOR\u003c/em\u003e mutations as a genomic biomarker for repurposing DHODH inhibitors, advancing precision medicine strategies for AML and other cancers.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eACKNOLEDGMENTS\u003c/h2\u003e\n\u003cp\u003eThe discovery described in this manuscript has been filed as a patent application through Cambridge Enterprise.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict\u003c/strong\u003e\u003cstrong\u003e-of-interest disclosure\u003c/strong\u003e: The authors declare no competing financial interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics and Consent to Participate declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003eBCOR Mutations Define a Therapeutic Vulnerability to DHODH Inhibition in Acute Myeloid Leukemia\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHORS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFlorian Robert\u003csup\u003e1,2\u003c/sup\u003e, Cherif Badja\u003csup\u003e1,2\u003c/sup\u003e, Soraya Boushaki\u003csup\u003e1,2\u003c/sup\u003e, Andrea Degasperi\u003csup\u003e1,2\u003c/sup\u003e, Yasin Memari\u003csup\u003e1,2\u003c/sup\u003e, Sophie Momen\u003csup\u003e1,2\u003c/sup\u003e, Theodoros I. Roumeliotis\u003csup\u003e4\u003c/sup\u003e, Zuza Kozik\u003csup\u003e4\u003c/sup\u003e, Malgorzata Gozdecka \u003csup\u003e3\u003c/sup\u003e, Jyoti Choudhary\u003csup\u003e4\u003c/sup\u003e, George Vassiliou\u003csup\u003e3\u003c/sup\u003e, Gene CC Koh\u003csup\u003e1,2\u003c/sup\u003e, Serena Nik-Zainal\u003csup\u003e1,2\u003c/sup\u003e\u003c/p\u003e\n\u003ch2\u003eFUNDINGS\u003c/h2\u003e\n\u003cp\u003eThe work performed by S.N.-Z.\u0026rsquo;s laboratory was funded by the Cancer Research UK (CRUK) Advanced Clinician Scientist Award (C60100/A23916), Dr. Josef Steiner Cancer Research Award 2019, Basser Gray Prime Award 2020, CRUK Pioneer Award (C60100/A23433), CRUK Grand Challenge Awards (C60100/A25274 and CGCATF-2021/100013), CRUK Early Detection Project Award (C60100/A27815), and the National Institute of Health Research (NIHR) Research Professorship (NIHR301627). This work was also supported by the NIHR Cambridge Biomedical Research Centre (BRC-1215-20014). The views expressed are those of the author(s) and not necessarily those of the NIHR or the Department of Health and Social Care.\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eF.R designed and conducted experiments, analyzed the data, and wrote the manuscript; C.B assisted in designing and conducting experiments and helped analyzing the data; S.B assisted in conducting experiments; A.G conducted WGS analysis, Y.M conducted WGS and RNAseq alignments; S.M assisted in performing proteomic and transcriptomic data on hiPSC; T.R, Z.K and J.C conducted proteomic experiments and analysis, and provided correction on the manuscript; M.G and G.V provided technical and scientific expertise, AML cell lines and Manuscript Reviewing; G.CC.K supervised the study; S.N.Z supervised the study and wrote the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eD\u0026ouml;hner H, Weisdorf DJ, Bloomfield CD (2015) Acute Myeloid Leukemia. 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BMC Cancer 24(1):761. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s12885-024-12534-w\u003c/span\u003e\u003cspan address=\"10.1186/s12885-024-12534-w\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003ePMID: 38918775; PMCID: PMC11197201\u003c/span\u003e\u003c/li\u003e\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":"annals-of-hematology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"aohe","sideBox":"Learn more about [Annals of Hematology](http://link.springer.com/journal/277)","snPcode":"277","submissionUrl":"https://submission.nature.com/new-submission/277/3","title":"Annals of Hematology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7470212/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7470212/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAcute Myeloid Leukemia (AML) remains challenging to treat, especially in cases with mutations in the BCL-6 co-repressor (BCOR), which are associated with poor prognosis and chemo-resistance. In this study, we reveal a synthetic lethal interaction between \u003cem\u003eBCOR\u003c/em\u003e and dihydroorotate dehydrogenase (DHODH). We demonstrate that \u003cem\u003eBCOR\u003c/em\u003e-deficient cells have a heightened sensitivity to DHODH inhibitors such as brequinar and leflunomide, that are already in clinical use. We confirm that DHODH inhibition selectively induces cell death in BCOR-mutant cells in multiple cellular models, in malignant and non-malignant cells, through chemical and genetic manipulation. Interestingly, we find that the dependency on DHODH does not stem from its role in \u003cem\u003ede novo\u003c/em\u003e pyrimidine biosynthesis disruption. Rather, DHODH\u0026rsquo;s role in the electron transport chain, essential for mitigating reactive oxygen species, may be the physiological vulnerability that pushes BCOR-mutant cells toward cell death when DHODH is inhibited. DHODH inhibitors could be repurposed as targeted therapies for BCOR-mutant tumors, offering a promising strategy for precision medicine in AML and other cancers.\u003c/p\u003e","manuscriptTitle":"BCOR Mutations Define a Therapeutic Vulnerability to DHODH Inhibition in Acute Myeloid Leukemia","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-15 06:24:33","doi":"10.21203/rs.3.rs-7470212/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-11T00:30:04+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-10T07:26:39+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"296072633326076901528130333525363480266","date":"2025-09-08T05:19:21+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-07T13:01:30+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-02T08:22:58+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-02T08:22:15+00:00","index":"","fulltext":""},{"type":"submitted","content":"Annals of Hematology","date":"2025-08-27T09:27:30+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"annals-of-hematology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"aohe","sideBox":"Learn more about [Annals of Hematology](http://link.springer.com/journal/277)","snPcode":"277","submissionUrl":"https://submission.nature.com/new-submission/277/3","title":"Annals of Hematology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"9c060a86-742c-476f-b525-6bfc3e29ab22","owner":[],"postedDate":"September 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-01-26T16:02:30+00:00","versionOfRecord":{"articleIdentity":"rs-7470212","link":"https://doi.org/10.1007/s00277-026-06773-z","journal":{"identity":"annals-of-hematology","isVorOnly":false,"title":"Annals of Hematology"},"publishedOn":"2026-01-19 15:59:05","publishedOnDateReadable":"January 19th, 2026"},"versionCreatedAt":"2025-09-15 06:24:33","video":"","vorDoi":"10.1007/s00277-026-06773-z","vorDoiUrl":"https://doi.org/10.1007/s00277-026-06773-z","workflowStages":[]},"version":"v1","identity":"rs-7470212","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7470212","identity":"rs-7470212","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}