Supraphysiologic levels of the AML1-ETO isoform AE9a are essential for transformation

Abstract

Chromosomal translocation 8;21 is found in 40% of the FAB M2 subtype of acute myeloid leukemia (AML). The resultant in-frame fusion protein AML1-ETO (AE) acts as an initiating oncogene for leukemia development. AE immortalizes human CD34 + cord blood cells in longterm culture. We assessed the transforming properties of the alternatively spliced AE isoform AE9a (or alternative splicing at exon 9), which is fully transforming in a murine retroviral model, in human cord blood cells. Full activity was realized only upon increased fusion protein expression. This effect was recapitulated in the AE9a murine AML model. Cotransduction of AE and AE9a resulted in a strong selective pressure for AE-expressing cells. In the context of AE, AE9a did not show selection for increased expression, affirming observations of human t(8;21) patient samples where full-length AE is the dominant protein detected. Mechanistically, AE9a showed defective transcriptional regulation of AE target genes that was partially corrected at high expression. Together, these results bring an additional perspective to our understanding of AE function and highlight the contribution of oncogene expression level in t(8;21) experimental models.T he t(8;21)(q22;q22) chromosomal translocation comprises the N-terminal DNA binding domain of AML1 (RUNX1) and nearly the entire ETO (RUNX1T1) gene, forming the fusion protein AML1-ETO (AE) (1, 2). Conditional expression and transduction-transplantation approaches have demonstrated that expression of AE provides self-renewal signaling to hematopoietic stem/progenitor cells (HSPCs) but does not induce transformation in the absence of additional cooperating events (3-7). Several such cooperating events have been identified, including overexpression of WT1, mutant c-KIT, TEL-PDGFRB, FLT3-ITD, loss of p21, and treatment with the DNA-damaging agent ENU (6,(8)(9)(10)(11)(12)(13). Conversely, C-terminal truncation of AE through frameshift mutation (AML1-ETOtr) or alternative splicing at exon 9 (AE9a) leads to acute myeloid leukemia (AML) transformation of murine HSPCs (14, 15). The AE9a transcript is expressed in ∼70% of t(8;21) + AML patients, along with full-length AE (14, 16). AE9a lacks the conserved ETO domains NHR3/4, whose interaction with corepressor proteins such as N-CoR/SMRT and HDACs is important for transcriptional repression (17)(18)(19)(20). Despite maintaining some corepressor interaction, AE9a has greatly diminished N-CoR and SMRT interaction and is a much less potent transcriptional repressor than full-length AE (17,(19)(20)(21)(22). The AML1-ETOtr mutant showed altered regulation of cell cycle proteins, thereby providing a proliferative advantage in K562 cells relative to AE (15). Recently, DeKelver et al. showed that the interaction between N-CoR and the NHR4 domain plays an inhibitory role in leukemia development in the context of full-length AE (23). However, analysis of a small cohort of t(8;21) patient samples did not detect any mutations in the NHR4 domain, indicating that such mutations are probabl...

A FOXO1-induced oncogenic network defines the AML1-ETO preleukemic program

“…MSCV-IRES-GFP/THY.1 (MIG/MIT) and MSCV-AE-IRES-GFP/THY.1 retroviral vectors were as described. 10,20 pSIN-TREtight-DsRED-IRES and MSCV-GFP-IRES-tTA retroviral vectors were generously provided by Johannes Zuber. Insertion of AE was subcloned into pSIN-TREtight-DsRED-IRES to build an AE-tet-off system.…”

“…We and others have shown that expression of AE in human CD34 1 hematopoietic stem and progenitor cells (HSPCs) causes dysregulated differentiation and increased self-renewal of cells but without inducing AML, 9,10 serving as an ideal model to study the preleukemic stage of t(8;21) AML (AE cells). Until now, the mechanism by which AE programs progenitor cells into preleukemia with aberrant self-renewal has not been completely understood.…”

Definition of a Small Core Transcriptional Circuit Regulated by AML1-ETO

“…Mouse models of AML1-ETO-induced leukemogenesis require a truncated protein that is dramatically diminished in its ability to mediate repression (see ∆NH4 and W692A in Fig. 4), and therefore, may not be reflective of the transcriptional networks established by AML1-ETO in human disease 54 . Thus, we turned to long-term cultures of CD34 + cells from human cord blood to establish a pre-leukemic system to study AML1-ETO transcription functions.…”

Epigenetic Modifications in Acute Myeloid Leukemia: Prognosis, Treatment, and Heterogeneity

“…Wang et al. found median survival in leukemic mouse models was increased via inhibition of the lysine acetyltransferase p300 (Wang et al., 2011), which reduced Lys 43 acetylation levels in AML1-ETO9a, a splice isoform of AML1-ETO (Zhang et al., 2007; Link et al., 2016). Though p300 knockdown leads to decreased acetylation, the therapeutic effects of p300 knockdown could be due to effects largely unrelated to AML1-ETO9a acetylation, indicating p300 may be a broader therapeutic target (Wang et al., 2011).…”