Transformation Mechanisms of the Nfia-ETO2 Fusion Gene Associated with Pediatric Pure Acute Erythroleukemia

Abstract

Pure erythroleukemia (PEL) is a very aggressive, but poorly understood form of acute myeloid leukemia characterized by malignant accumulation of erythroid progenitor cells. A novel t(1;16)(p31;q24) chromosomal translocation leading to expression of a fusion between the nuclear factor I A (NFIA) and the ETO2 transcriptional co-regulator (a.k.a. CBFA2T3 or MTG16) has been identified in pediatric patients with PEL. Based on the function of the fusion partners, we hypothesized that NFIA-ETO2 (N-E) might initiate PEL by interfering with erythroid differentiation. To investigate its function, we cloned a full-length ORF and retrovirally expressed N-E in primary mouse bone marrow (BM)- and fetal liver (FL)-derived erythroblasts (EB). N-E expression significantly increased proliferation and blocked differentiation of EB. N-E expressing BM-derived hematopoietic stem and progenitor cells (HSPC) could be plated in erythropoietin (EPO)-containing methylcellulose (MC) for up to 3 rounds. Expression of N-E deletion mutants lacking the NFIA DNA-binding, the ETO2 NHR2 or NHR4 (ΔNHR4) transcriptional repression domains were unable to block erythroid differentiation. Notably, interfering with the ETO2-NHR2 oligomerization domain by overexpressing a competing peptide overcame the N-E-mediated differentiation block. Transplantation of N-E-expressing BM-derived HSPC into irradiated syngenic mice did not induce any disease suggesting the need of genetic cooperation. As TP53 gain-of-function (GOF) mutations are molecular hallmarks of PEL, we explored functional cooperation by using a conditional TP53R248Q allele. Interestingly, the TP53 status did not affect EB in vitro proliferation or differentiation. However, N-E expression increased proliferation of TP53R248Q+ EB and resulted in the formation of abnormal round and dense colonies in MC that could be serially propagated. In addition, transplantation of N-E-expressing TP53R248Q+ EB into irradiated recipients induced a transplantable PEL-like disease after a median latency of 4 months. Symptomatic mice presented with anemia, thrombocytopenia, multi-organ tumor cell infiltration and increased white blood cell counts. To better understand the molecular mechanism, we compared the gene expression signatures before and 24 hours after induced differentiation of FL-derived EB expressing WT or the inactive ΔNHR4 N-E mutant, in presence or absence of TP53R248Q. Principal component analysis (PCA) revealed a clear separation between the transcriptomes of WT EB expressing either the active or the inactive ΔNHR4 N-E (PC1:54.7%) and by their erythroid differentiation stage (PC2:9.07%). Overall, we observed 3753 (FDR<0.05, logFC>1.5) differentially expressed genes. Many of the significantly higher expressed genes (2313/3753) were related to hematopoietic stemness (GSEAs, p<0.001). Almost 10% of the significantly lower expressed genes (92/1440) were linked to the erythroid lineage development and to erythropoietic targets of NFIA or the erythroid master regulator GATA1. Interestingly, we also found reduced expression of genes encoding for ETO2-interacting transcription factors including TAL1 and KLF1. Despite a critical role on disease progression, PCA showed only minimal changes in the N-E expression signature in presence or absence of TP53R248Q with only 12 genes differently expressed (FDR<0.05, logFC>1). These genes were previously shown to be oncogenic mediators of TP53-GOF mutations, related to metabolism and transcriptional regulation. Interestingly, the signature of differentially expressed genes in N-E transformed FL-derived EB were significantly differentially expressed in tumor cells from pediatric but not adult PEL patients (p=0.00045), indicating the pediatric origin of the fusion. Collectively, we found that the PEL-associated N-E fusion blocks erythroid differentiation, and cooperates with a TP53-GOF mutation to induce a PEL-like disease in mice that phenocopies the human disease. Mechanistically, its activity seems to correlate with repression of erythroid regulatory genes controlled by the fusion partners NFIA, ETO2, and the erythroid master regulator GATA1. Disclosures No relevant conflicts of interest to declare.

Central gene transcriptional regulatory networks shaping monocyte development in bone marrow

“…Forkhead box proteins are a group of transcriptional factors implicated in different cellular functions such as differentiation, proliferation and senescence ( 72 ) to affect hematopoietic tumor, while EP300 suppresses leukemia development in myelodysplastic syndromes through inhibiting Myb ( 73 ). It has also been reported that mutations in the human Ank1 ( 74 , 75 ), Runx2 ( 76 – 79 ), Nfia ( 80 ), Hmga2 ( 81 ), and Klf1 ( 82 , 83 ) genes caused diseases of the hematopoietic system, such as hereditary spherocytosis, fetal anemia, myelodysplastic syndrome, and acute erythroleukemia. The diseases caused by mutations in these genes strongly support our speculation that the transcription factors Ank1, Runx2, Nfia, Hmga2, Klf1, and Bmyc may play important roles in monocyte development.…”

Molecular Landscapes and Models of Acute Erythroleukemia

“…In contrast, NFIA-ETO2 -expressing erythroblasts harboring one of the most frequent cancer and AEL-associated TP53 mutation, TP53 R248Q , could be serially plated in MC and when transplanted into irradiated recipients, induced a fully penetrant transplantable lethal erythroleukemia-like disease characterized by hepatosplenomegaly, anemia, thrombocytopenia, and the presence of erythroid progenitor cells on peripheral blood smear. 102 Molecular studies suggested that NFIA-ETO2 primarily blocks erythroid differentiation by repressing NFIA as well as GATA1 target genes, and that the TP53 R248Q mutation endowed cells with aberrant stemness and aberrant activity of the polycomb complex 2 (PRC2). In addition, similar to other ETO-protein containing fusions, NFIA-ETO2 immortalized cells may also be sensitive to small peptides that disrupt ETO-NHR-domain-mediated protein/protein interactions, suggesting a potential therapeutic vulnerability.…”

“…In addition, similar to other ETO-protein containing fusions, NFIA-ETO2 immortalized cells may also be sensitive to small peptides that disrupt ETO-NHR-domain-mediated protein/protein interactions, suggesting a potential therapeutic vulnerability. 102 , 112 …”

“…19,21 Similarly, as outlined before, mouse models have shown that genetic TP53 inactivation cooperates with several mutations, such as loss of function alterations of CEBPA or BCOR or constitutively active mutated tyrosine kinases such as JAK2 V617F or NTRK1 H498R to induce erythroleukemia in mice. 19,23,[100][101][102][103]114 Hence, treatment of xenografted AEL patient cells carrying either JAK2 or EPOR amplification with a JAK2 inhibitor significantly suppressed cell growth and prolonged overall survival. 130 Together, these observations strongly suggest that TP53 mutations are essential players not only in AEL development but also crucial for the maintenance of the disease.…”

“…However, NFIA‐ETO2 ‐expressing cells could not be serially propagated in growth‐factor containing methylcellulose and transplantation of NFIA‐ETO2 ‐expressing BM or fetal liver erythroid progenitor cells into irradiated mice did not result in any disease. In contrast, NFIA‐ETO2 ‐expressing erythroblasts harboring one of the most frequent cancer and AEL‐associated TP53 mutation, TP53 R248Q , could be serially plated in MC and when transplanted into irradiated recipients, induced a fully penetrant transplantable lethal erythroleukemia‐like disease characterized by hepatosplenomegaly, anemia, thrombocytopenia, and the presence of erythroid progenitor cells on peripheral blood smear 102 . Molecular studies suggested that NFIA‐ETO2 primarily blocks erythroid differentiation by repressing NFIA as well as GATA1 target genes, and that the TP53 R248Q mutation endowed cells with aberrant stemness and aberrant activity of the polycomb complex 2 (PRC2).…”