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 the 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 behind this synergy. We demonstrate that, during normal hematopoiesis, this transcription factor promotes megakaryocytic differentiation in concert with GATA1 and mediates TGFβ-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 can interplay to block multiple routes of hematopoietic differentiation and cause leukemia. In AMKL, surmounting this differentiation blockade through restoration of the tumor suppressor ARID3A represents a promising strategy for treating this lethal pediatric disease.

Gain of chromosome 21 (Hsa21) is among the most frequent aneuploidies in leukemia. However, it remains unclear how partial or complete amplifications of Hsa21 promote leukemogenesis and why children with Down syndrome (i.e. trisomy 21) are particularly at risk of leukemia development. Here, we propose that RUNX1 isoform disequilibrium with RUNX1A bias is key to Down syndrome-associated myeloid leukemia (ML-DS). Starting with Hsa21-focused CRISPR-Cas9 screens, we uncovered a strong and specific RUNX1 dependency in ML-DS cells. Expression of the RUNX1A isoform is elevated in ML-DS patients, and mechanistic studies using murine ML-DS models and patient-derived xenografts (PDXs) revealed that excess RUNX1A synergizes with the pathognomonic Gata1s mutation during leukemogenesis by displacing RUNX1C from its endogenous binding sites and inducing oncogenic programs in complex with the MYC cofactor MAX. These effects were reversed by restoring the RUNX1A:RUNX1C equilibrium in PDXs in vitro and in vivo. Moreover, pharmacological interference with MYC:MAX dimerization using MYCi361 exerted strong anti-leukemic effects. Thus, our study highlights the importance of alternative splicing in leukemogenesis, even on a background of aneuploidy, and paves the way for the development of specific and targeted therapies for ML-DS, as well as for other leukemias with Hsa21 aneuploidy or RUNX1 isoform disequilibrium.

Aneuploidy is a hallmark of cancer, but its complex nature limits our understanding of how it drives oncogenesis. Gain of chromosome 21 (Hsa21) is among the most frequent aneuploidies in leukemia and is associated with markedly increased leukemia risk. Here, we propose that disequilibrium of the RUNX1 isoforms is key to the pathogenesis of trisomy 21 (i.e. Down syndrome)-associated myeloid leukemia (ML-DS). Hsa21-focused CRISPR-Cas9 screens uncovered a strong and specific RUNX1 dependency in ML-DS. Mechanistic studies revealed that excess of RUNX1A isoform - as seen in ML-DS patients - synergized with the pathognomonic Gata1s mutation in leukemogenesis by displacing RUNX1C from its endogenous binding sites and inducing oncogenic programs in complex with the MYC cofactor MAX. These effects were reversed by restoring the RUNX1A:RUNX1C equilibrium or pharmacological interference with MYC:MAX dimerization. Our study highlights the importance of alternative splicing in leukemogenesis, even on a background of aneuploidies, and opens new avenues for developing specific and targeted therapies.