Contributions
Abstract: EP398
Type: E-Poster Presentation
Session title: Acute myeloid leukemia - Biology & Translational Research
Background
Down syndrome-associated myeloid leukemia (ML-DS) is characterized by the triad of trisomy 21, fetal origin and mutations in GATA1 (GATA1s mutations). We previously described that miR-125b synergized with mutated GATA1s via downregulation of the transcription factor ARID3A. However, the mechanisms of this synergy remained to be understood.
Aims
We aimed to understand the function of ARID3A in normal hematopoiesis and unravel its role in the pathogenesis of Down syndrome (trisomy21)-associated leukemia in cooperation with oncogenic GATA1s.
Methods
We combined CRISPR-Cas9 genome editing with lentiviral gain- and loss-of-function studies in hematopoietic stem/progenitor cells (HSPCs) and primary patient blasts. We integrated gene expression profiling after Arid3a modulation in GATA1s fetal cells with CUT&RUN and mass spectrometry analyses to identify key ARID3A-regulated pathways involved in leukemic transformation.
Results
RNA-Seq data of sorted blood cells indicated increased ARID3A expression in megakaryocytes compared to erythroid cells. Accordingly, Arid3a overexpression in fetal and adult HSPCs promoted megakaryocytic differentiation (1.73x and 2.89x fold increased CD41a+CD42d+ megakaryocytes, p=0.0.17 and p=0.018, respectively), while shRNA-mediated Arid3a knockdown in fetal HSPCs caused a 40% reduction (p=0.04). Interestingly, while Gata1s perturbs erythroid differentiation and induces hyperproliferation of fetal HSPCs, Arid3a knockdown blocks their otherwise unperturbed megakaryocytic differentiation, leading to the expansion (3.6x fold) of CD117+CD41+ megakaryocytic progenitors in vitro and development of ML-DS-like leukemia (CD41+CD117+) upon transplantation into syngeneic C57BL/6J recipients in vivo (71 days median disease-free survival and 100% penetrance).
To understand the ARID3A molecular network and its role in leukemia progression, we leveraged gene expression data (RNA-Seq), chromatin occupancy (CUT&RUN) and mass spectrometry (LC-MS/MS). Integrated analysis uncovered that ARID3A promotes terminal differentiation by occupying and inducing megakaryocytic genes in concert with GATA1. In parallel, ARID3A binds SMAD2/3, forming a complex that mediates TGFβ-induced cell cycle arrest and apoptosis. In line with a role as tumor suppressor, low expression in pediatric AML patients (NCI-TARGET) associates with poorer overall survival (53.9% vs. 67.7%; P-log-rank = 0.025). Importantly, restoring expression of ARID3A in ML-DS patient-derived xenografts suppressed leukemic growth and promoted megakaryocytic differentiation in vitro and in vivo.
Conclusion
We uncovered a novel function of ARID3A in promoting megakaryocytic differentiation in concert with GATA1 and mediating TGFβ-induced cell cycle arrest and apoptosis in complex with SMAD2/3. Inversely, ARID3A repression is a key event in the pathogenesis of ML-DS as Arid3a knockdown blocks the otherwise unperturbed megakaryocytic differentiation of hyperproliferative Gata1s megakaryocytic progenitors arresting them in an undetermined differentiation status and causing leukemia. Our work provides a basis to develop refined treatment approaches by restoring the ARID3A-induced differentiation program.
Keyword(s): Acute myeloid leukemia, Down Syndrome, Megakaryopoiesis, Tumor suppressor
Abstract: EP398
Type: E-Poster Presentation
Session title: Acute myeloid leukemia - Biology & Translational Research
Background
Down syndrome-associated myeloid leukemia (ML-DS) is characterized by the triad of trisomy 21, fetal origin and mutations in GATA1 (GATA1s mutations). We previously described that miR-125b synergized with mutated GATA1s via downregulation of the transcription factor ARID3A. However, the mechanisms of this synergy remained to be understood.
Aims
We aimed to understand the function of ARID3A in normal hematopoiesis and unravel its role in the pathogenesis of Down syndrome (trisomy21)-associated leukemia in cooperation with oncogenic GATA1s.
Methods
We combined CRISPR-Cas9 genome editing with lentiviral gain- and loss-of-function studies in hematopoietic stem/progenitor cells (HSPCs) and primary patient blasts. We integrated gene expression profiling after Arid3a modulation in GATA1s fetal cells with CUT&RUN and mass spectrometry analyses to identify key ARID3A-regulated pathways involved in leukemic transformation.
Results
RNA-Seq data of sorted blood cells indicated increased ARID3A expression in megakaryocytes compared to erythroid cells. Accordingly, Arid3a overexpression in fetal and adult HSPCs promoted megakaryocytic differentiation (1.73x and 2.89x fold increased CD41a+CD42d+ megakaryocytes, p=0.0.17 and p=0.018, respectively), while shRNA-mediated Arid3a knockdown in fetal HSPCs caused a 40% reduction (p=0.04). Interestingly, while Gata1s perturbs erythroid differentiation and induces hyperproliferation of fetal HSPCs, Arid3a knockdown blocks their otherwise unperturbed megakaryocytic differentiation, leading to the expansion (3.6x fold) of CD117+CD41+ megakaryocytic progenitors in vitro and development of ML-DS-like leukemia (CD41+CD117+) upon transplantation into syngeneic C57BL/6J recipients in vivo (71 days median disease-free survival and 100% penetrance).
To understand the ARID3A molecular network and its role in leukemia progression, we leveraged gene expression data (RNA-Seq), chromatin occupancy (CUT&RUN) and mass spectrometry (LC-MS/MS). Integrated analysis uncovered that ARID3A promotes terminal differentiation by occupying and inducing megakaryocytic genes in concert with GATA1. In parallel, ARID3A binds SMAD2/3, forming a complex that mediates TGFβ-induced cell cycle arrest and apoptosis. In line with a role as tumor suppressor, low expression in pediatric AML patients (NCI-TARGET) associates with poorer overall survival (53.9% vs. 67.7%; P-log-rank = 0.025). Importantly, restoring expression of ARID3A in ML-DS patient-derived xenografts suppressed leukemic growth and promoted megakaryocytic differentiation in vitro and in vivo.
Conclusion
We uncovered a novel function of ARID3A in promoting megakaryocytic differentiation in concert with GATA1 and mediating TGFβ-induced cell cycle arrest and apoptosis in complex with SMAD2/3. Inversely, ARID3A repression is a key event in the pathogenesis of ML-DS as Arid3a knockdown blocks the otherwise unperturbed megakaryocytic differentiation of hyperproliferative Gata1s megakaryocytic progenitors arresting them in an undetermined differentiation status and causing leukemia. Our work provides a basis to develop refined treatment approaches by restoring the ARID3A-induced differentiation program.
Keyword(s): Acute myeloid leukemia, Down Syndrome, Megakaryopoiesis, Tumor suppressor