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Children with Down syndrome have a 150-fold increased risk of developing myeloid leukaemia (ML-DS). Unusually for a childhood leukaemia, ML-DS arises from a preleukaemic state, termed transient abnormal myelopoiesis (TAM), via a conserved sequence of mutations. Here, we examine the relationship between the genetic and transcriptional evolution of ML-DS from natural variation; a rich collection of primary patient samples and foetal tissues with a range of constitutional karyotypes. We distil transcriptional consequences of each genetic step in ML-DS evolution, utilising single-cell mRNA sequencing, complemented by phylogenetic analyses in progressive disease. We find that transcriptional changes induced by the TAM-defining GATA1 mutations are retained in, and account for most of the ML-DS transcriptome. The GATA1 transcriptome pervades all stages of ML-DS, including progressive disease that had undergone genetic evolution. Our approach delineates the transcriptional evolution of ML-DS and provides an analytical blueprint for distiling consequences of mutations within their pathophysiological context. Development of myeloid leukaemia in children with Down syndrome is a stepwise process. Here, the authors employ scRNA sequencing and phylogenetic analysis on patient samples from various disease stages to define the cellular and molecular features of this stepwise leukaemia development.

Medulloblastomas arise from undifferentiated precursor cells in the cerebellum and account for about 20% of all solid brain tumors during childhood; standard therapies include radiation and chemotherapy, which oftentimes come with severe impairment of the cognitive development of the young patients. Here, we show that the posttranscriptional regulator Y-box binding protein 1 (YBX1), a DNA- and RNA-binding protein, acts as an oncogene in medulloblastomas by regulating cellular survival and apoptosis. We observed different cellular responses upon YBX1 knockdown in several medulloblastoma cell lines, with significantly altered transcription and subsequent apoptosis rates. Mechanistically, PAR-CLIP for YBX1 and integration with RNA-Seq data uncovered direct posttranscriptional control of the heterochromatin-associated gene CBX5; upon YBX1 knockdown and subsequent CBX5 mRNA instability, heterochromatin-regulated genes involved in inflammatory response, apoptosis and death receptor signaling were de-repressed. Thus, YBX1 acts as an oncogene in medulloblastoma through indirect transcriptional regulation of inflammatory genes regulating apoptosis and represents a promising novel therapeutic target in this tumor entity.

A hallmark of acute myeloid leukaemias (AMLs) are chromosomal rearrangements that give rise to novel leukaemia-specific fusion genes. Most of these fusion genes are both initiating and driving events in AML and therefore constitute ideal therapeutic targets but are challenging to target by conventional drug development. siRNAs are frequently used for the specific suppression of fusion gene expression but require special formulations for efficient in vivo delivery. Here we describe the use of siRNA-loaded lipid nanoparticles for the specific therapeutic targeting of the leukaemic fusion gene RUNX1/ETO. Transient knockdown of RUNX1/ETO reduces its binding to its target genes and alters the binding of RUNX1 and its co-factor CBFβ. Transcriptomic changes in vivo were associated with substantially increased median survival of a t(8;21)-AML mouse model. Importantly, transient knockdown in vivo causes long-lasting inhibition of leukaemic proliferation and clonogenicity, induction of myeloid differentiation and a markedly impaired re-engraftment potential in vivo. These data strongly suggest that temporary inhibition of RUNX1/ETO results in long-term restriction of leukaemic self-renewal. Our results provide proof for the feasibility of targeting RUNX1/ETO in a pre-clinical setting and support the further development of siRNA-LNPs for the treatment of fusion gene-driven malignancies.

Acute leukemias are genetic diseases caused by translocations or mutations, which dysregulate hematopoiesis towards malignant transformation. However, the molecular mode of action is highly versatile and ranges from direct transcriptional to post-transcriptional control, which includes RNA-binding proteins (RBPs) as crucial regulators of cell fate. RBPs coordinate RNA dynamics, including subcellular localization, translational efficiency and metabolism, by binding to their target messenger RNAs (mRNAs), thereby controlling the expression of the encoded proteins. In view of the growing interest in these regulators, this review summarizes recent research regarding the most influential RBPs relevant in acute leukemias in particular. The reported RBPs, either dysregulated or as components of fusion proteins, are described with respect to their functional domains, the pathways they affect, and clinical aspects associated with their dysregulation or altered functions.

NUP98::KDM5A-rearranged pediatric acute myeloid leukemia (AML) is a rare, infancy-predominant entity with dismal outcome and no targeted therapeutic options. Given its suspected fetal origin, we hypothesized that leukemic maintenance depends on developmentally restricted vulnerabilities embedded within fetal hematopoietic programs. Using matched fetal and adult hematopoietic stem and progenitor cell models, we integrated transcriptomic and proteomic profiling with CRISPR-Cas9 screenings to define ontogeny-specific dependencies in NUP98::KDM5A leukemia. Fetal-derived NUP98::KDM5A leukemias exhibited greater in vivo aggressiveness, and retained fetal transcriptional signatures compared with adult counterparts. A comparative CRISPR-Cas9 screen using a library targeting fetal gene programs, conducted in both fetal- and adult-derived NUP98::KDM5A leukemias, identified the AAA+; ATPase TRIP13 as a selective and essential dependency in the fetal context. Mechanistically, TRIP13 physically interacted with the TP53 phosphatase PPM1D/WIP1, resulting in suppression of TP53 activation. Genetic ablation of Trip13, or pharmacologic inhibition using DCZ0415, a small-molecule inhibitor targeting TRIP13, reactivated TP53, induced G2/M arrest and mitochondrial apoptosis, and depleted leukemic cells in vitro; these effects were fully rescued by TP53 loss. In competitive transplantation assays, Trip13 ablation significantly impaired leukemic fitness in vivo. Together, these findings define a developmentally restricted TRIP13-PPM1D-TP53 survival axis in NUP98::KDM5A AML and provide mechanistic proof-of-concept that reactivation of the TP53 signaling pathway via TRIP13 inhibition represents a therapeutically targetable vulnerability in this high-risk pediatric leukemia.Competing Interest StatementJ-HK has advisory roles for Boehringer, Roche and Jazz Pharmaceuticals. All other authors declare that the manuscript was written in the absence of any commercial or financial relationships that could be perceived as a potential conflict of interest.Footnotes* The main manuscript text has been revised and improved. The supplementary data have been updated accordingly.Funder Information DeclaredDeutsche José Carreras Leukämie-Stiftung, DJCLS 12 R/2024Frankfurter Stiftung für krebskranke KinderHilfe für krebskranke Kinder Frankfurt, https://ror.org/02ecj1n50European Research Council Horizon 2020 program, #714226German Research Foundation, DFG; FOR 5433 RNA in Focus; KL-2371/7

Children with Down syndrome have a markedly increased risk of developing myeloid leukemia (ML-DS). Although having an excellent prognosis, 10–20% develop relapsed or refractory disease with poor survival, highlighting the need for new targeted approaches. The pathogenesis of ML-DS is tightly linked to fetal hematopoiesis and mutations in GATA1, generating the truncated GATA1short(s) isoform. We identified Delta-like non-canonical Notch ligand 1 (DLK1) as a direct GATA1s target. DLK1, a paternally imprinted transmembrane protein, is highly expressed in fetal liver CD34⁺ cells but absent in adult hematopoiesis, making it an attractive immunotherapeutic target. Chromatin profiling revealed GATA1s occupancy at a distal enhancer within the DLK1–DIO3 locus, driving aberrant DLK1 upregulation in ML-DS. Functional studies demonstrated that DLK1 is a leukemia dependency, as its genetic ablation impaired proliferation and engraftment, induced apoptosis, and altered Notch and β-catenin signaling. Therapeutically, a DLK1-directed antibody-drug conjugate (DLK1-ADC) induced selective cytotoxicity, abrogated colony formation, and significantly prolonged survival in refractory ML-DS PDX models, achieving durable remissions at higher doses. These findings establish DLK1 as a leukemia-specific vulnerability and provide preclinical proof-of-concept for DLK1-targeted therapies in ML-DS and other leukemias with fetal-like expression programs. DLK1 is a GATA1s-driven leukemia dependency in ML-DS, linking fetal hematopoietic programs to leukemic stemness and refractory disease. Therapeutic targeting of DLK1 with an antibody–drug conjugate selectively eradicates leukemia cells and prolongs survival in ML-DS PDX models

A hallmark of acute myeloid leukaemias (AMLs) are chromosomal rearrangements that give rise to novel leukaemia-specific fusion genes. Most of these fusion genes are both initiating and driving events in AML and therefore constitute ideal therapeutic targets but are challenging to target by conventional drug development. siRNAs are frequently used for the specific suppression of fusion gene expression but require special formulations for efficient in vivo delivery. Here we describe the use of siRNA-loaded lipid nanoparticles for the specific therapeutic targeting of the leukaemic fusion gene RUNX1/ETO. Transient knockdown of RUNX1/ETO reduces its binding to its target genes and alters the binding of RUNX1 and its co-factor CBFβ. Transcriptomic changes in vivo were associated with substantially increased median survival of a t(8;21)-AML mouse model. Importantly, transient knockdown in vivo causes long-lasting inhibition of leukaemic proliferation and clonogenicity, induction of myeloid differentiation and a markedly impaired re-engraftment potential in vivo. These data strongly suggest that temporary inhibition of RUNX1/ETO results in long-term restriction of leukaemic self-renewal. Our results provide proof for the feasibility of targeting RUNX1/ETO in a pre-clinical setting and support the further development of siRNA-LNPs for the treatment of fusion gene-driven malignancies.

Children with Down syndrome have a 150-fold increased risk of developing myeloid leukaemia (ML-DS). Unusually for a childhood leukaemia, ML-DS arises from a preleukaemic state, termed transient abnormal myelopoiesis (TAM), via a conserved sequence of mutations. Here, we examined the relationship between the genetic and transcriptional evolution of ML-DS from natural variation; a rich collection of primary patient samples and fetal tissues with a range of constitutional karyotypes. We distilled transcriptional consequences of each genetic step in ML-DS evolution, utilising single cell mRNA sequencing, complemented by phylogenetic analyses in progressive disease. We found that transcriptional changes induced by the TAM-defining GATA1 mutations are retained in, and account for most of the ML-DS transcriptome. The GATA1 transcriptome pervaded all stages of ML-DS, including progressive disease that had undergone genetic evolution. Our approach delineates the transcriptional evolution of ML-DS and provides an analytical blueprint for distilling consequences of mutations within their pathophysiological context.

Given the plasticity of hematopoietic stem/progenitor cells, multiple routes of differentiation must be blocked during acute myeloid leukemia pathogenesis - the molecular basis of which is incompletely understood. Here we report that post-transcriptional repression of transcription factor ARID3A by miR-125b is a key event in megakaryoblastic leukemia (AMKL) pathogenesis. AMKL is frequently associated with trisomy 21 and GATA1 mutations (GATA1s), and children with Down syndrome are at a high risk of developing this disease. We show that chromosome 21-encoded miR-125b synergizes with Gata1s to drive leukemogenesis in this context. Leveraging forward and reverse genetics, we uncover Arid3a as the main miR-125b target underlying this synergy. We demonstrate that during normal hematopoiesis this transcription factor promotes megakaryocytic differentiation in concert with GATA1 and mediates TGFbeta-induced apoptosis and cell cycle arrest in complex with SMAD2/3. While Gata1s mutations perturb erythroid differentiation and induce hyperproliferation of megakaryocytic progenitors, intact ARID3A expression assures their megakaryocytic differentiation and growth restriction. Upon knockdown, these tumor suppressive functions are revoked, causing a dual megakaryocytic/erythroid differentiation blockade and subsequently AMKL. Inversely, restoring ARID3A expression relieves the megakaryocytic differentiation arrest in AMKL patient-derived xenografts. This work illustrates how mutations in lineage-determining transcription factors and perturbation of post-transcriptional gene regulation interplay to block multiple routes of hematopoietic differentiation and cause leukemia. Surmounting this differentiation blockade in megakaryoblastic leukemia by restoring the tumor suppressor ARID3A represents a promising strategy for treating this lethal pediatric disease. Competing Interest Statement D.R. has advisory roles for Celgene Corporation, Novartis, Bluebird Bio, Janssen, and receives research funding from CLS Behring and Roche. J.H.K. has advisory roles for Bluebird Bio, Novartis, Roche and Jazz Pharmaceuticals.