The megakaryocytic transcription factor ARID3A suppresses leukemia pathogenesis

Abstract

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.

RUNX1 isoform disequilibrium in the development of trisomy 21 associated myeloid leukemia

“…Comparative transcriptomic analysis of RNA-sequencing (RNA-seq) data substantiated the synergy between Gata1s mutations and RUNX1A expression. As previously described, gene set enrichment analysis (GSEA) revealed a marked reduction of erythroid genes and concurrent activation of pro-proliferative genes -including MYC and E2F targets -in Gata1s mutated FLCs (Figure 3C-D and Table S3) (Alejo-Valle et al, 2021;Klusmann et al, 2010;Ling et al, 2019). While RUNX1A expression mitigated the reduction of erythroid genes caused by Gata1s, it triggered a similar upregulation of MYC and E2F target genes, suggesting convergence on these oncogenic pathways.…”

“…Importantly, Cre recombinase-mediated excision of the LoxP-flanked RUNX1A cDNA induced rapid depletion of the leukemic blasts in vitro , underlining their dependency on RUNX1A and its importance in ML-DS pathogenesis ( Figure S3H ). Of note, miR-125 further accelerated the leukemic transformation of Gata1s -FLCs by RUNX1A, suggesting synergy between RUNX1A and another well characterized oncogene on chromosome 21 (Alejo-Valle et al ., 2021; Emmrich et al, 2014) ( Figure S3I ).…”

“…We and others have shed light on the additional mutational events that are required for progression to overt ML-DS (Labuhn et al, 2019; Yoshida et al, 2013), however, far less is known about the role of trisomy 21 in creating the TAM/ML-DS disease phenotype. A critical region has been identified on Hsa21 that mediates the expansion of early hematopoietic progenitors observed in DS patients (Banno et al, 2016; Gurbuxani et al, 2004; Roy et al, 2012) and several Hsa21 genes – such as ERG (Birger et al, 2013; Ng et al, 2010), DYRK1A (Malinge et al, 2012), CHAF1B (Volk et al, 2018) and miR-125b (Alejo-Valle et al, 2021; Wagenblast et al, 2021) – have been postulated to play a role in leukemogenesis. On the other hand, Ts65dn mice that are trisomic for 104 orthologs of Hsa21 genes do not fully recapitulate the human phenotype in association with GATA1s (Klusmann et al, 2010), and the postulated factors are either located outside of the critical region, not overexpressed in trisomic fetal progenitor cells (Roy et al ., 2012), or lack full leukemic potential in humans when combined with mutated GATA1s (Chou et al, 2012; Grimm et al, 2021; Malinge et al ., 2012).…”

Deficiencies in the DNA Binding Protein ARID3a Alter Chromatin Structures Important for Early Human Erythropoiesis

“…However, others found discrepancies in protein versus transcript levels during human erythropoiesis, particularly for GATA1 (13), and we found that ARID3a transcripts do not always correlate with protein levels in mature hematopoietic cell subsets (14). Recently, coimmunoprecipitation liquid chromatographymass spectrometry data, using the megakaryoblastic cell line CMK, revealed that both ARID3a and GATA1 acted in concert for proper regulation of megakaryopoiesis (9), but it is unclear if ARID3a is required for early erythropoiesis in human cells. Therefore, it is critical to assess requirements for individual TFs at the protein level during hematopoietic events.…”

“…ENCODE data of K562 cells showed considerable overlap between GATA1, GATA2, and ARID3a binding sites in many genes important for erythropoiesis, suggesting ARID3a may function with those factors, either as a TF or as an epigenetic regulator mediating opening/closing of chromatin in enhancer/promoter regions. Knockdown of ARID3a with shRNA in GATA1-mutated cells revealed a block in both megakaryocytic and erythroid differentiation and revealed 65% of predicted ARID3a binding sites in K562 cells overlap with GATA1 sites (9). Indeed, the globin locus and other differentially regulated genes exhibit close proximity of binding sites for ARID3a, GATA1, GATA2, and TAL1 (Fig.…”