A previously unidentified alternatively spliced isoform of t(8;21) transcript promotes leukemogenesis

Abstract

The t(8;21)(q22;q22) translocation is one of the most common genetic abnormalities in acute myeloid leukemia (AML), identified in 15% of all cases of AML, including 40-50% of FAB M2 subtype and rare cases of M0, M1 and M4 subtypes. The most commonly known AML1-ETO fusion protein (full-length AML1-ETO) from this translocation has 752 amino acids and contains the N-terminal portion of RUNX1 (also known as AML1, CBFalpha2 or PEBP2alphaB), including its DNA binding domain, and almost the entire RUNX1T1 (also known as MTG8 or ETO) protein. Although alterations of gene expression and hematopoietic cell proliferation have been reported in the presence of AML1-ETO, its expression does not lead to the development of leukemia. Here, we report the identification of a previously unknown alternatively spliced isoform of the AML1-ETO transcript, AML1-ETO9a, that includes an extra exon, exon 9a, of the ETO gene. AML1-ETO9a encodes a C-terminally truncated AML1-ETO protein of 575 amino acids. Expression of AML1-ETO9a leads to rapid development of leukemia in a mouse retroviral transduction-transplantation model. More importantly, coexpression of AML1-ETO and AML1-ETO9a results in the substantially earlier onset of AML and blocks myeloid cell differentiation at a more immature stage. These results indicate that fusion proteins from alternatively spliced isoforms of a chromosomal translocation may work together to induce cancer development.

Mtg16/Eto2 Contributes to Murine T-Cell Development

“…NHR4 mediates the weaker of the two contacts between Mtg16 and NCoR/SMRT but is not required for association with NCoR or for transcriptional repression (1,31), whereas the NHR3 domain contributes to binding the regulatory subunit of protein kinase A (PKA RII) (12,16). Rather than contributing to the formation of acute leukemia, deletion of these domains has been associated with increasing the activity of the t(8;21) fusion protein (46), suggesting that these domains play a negative regulatory role.…”

Combined gene expression and DNA occupancy profiling identifies potential therapeutic targets of t(8;21) AML

“…To enrich for target genes bound to the HA-tagged AE9a, we immunoprecipitated the resulting chromatin fragments (2 mg) with 10 g of rabbit-anti-HA (Santa Cruz) or rabbit-anti-AML1. 20 A control experiment was performed with rabbit-immunoglobulin (Ig)G (Sigma-Aldrich). After reversal of crosslinks and purification, the enriched DNA was amplified by ligation-mediated polymerase chain reaction (PCR) and subsequently labeled with the Cy5 fluorophore by random priming.…”

“…20 AE9a/CD45 coexpression for serial replating assays and bone marrow transplantation Fetal liver cells (embryonic day 14.5) or bone marrow lineage negative cells from MF-1 or C57BL/6 mice were transduced with retroviruses expressing MigR1-AE9a or coexpressing MigR1-AE9a and MSCV-CD45.1. EGFP ϩ and EGFP ϩ /CD45.1 ϩ cells were sorted by fluorescence-activated cell sorter (FACS), respectively, and cultured in methylcellulose media for serial replating assays (MethoCult GF M3434; StemCell Technologies) at 7-day intervals.…”

“…19 Subsequently, we identified a C-terminally truncated variant of AML1-ETO named AML1-ETO9a (AE9a), resulting from alternative splicing and found to coexist with full-length AML1-ETO in most analyzed t(8;21) AML patients. 20 Similar to AEtr, AE9a causes a rapid onset of leukemia in mice, 20 which provides a useful mouse model to study the molecular biology of t(8;21) leukemia.…”

“…19 Subsequently, we identified a C-terminally truncated variant of AML1-ETO named AML1-ETO9a (AE9a), resulting from alternative splicing and found to coexist with full-length AML1-ETO in most analyzed t(8;21) AML patients. 20 Similar to AEtr, AE9a causes a rapid onset of leukemia in mice, 20 which provides a useful mouse model to study the molecular biology of t(8;21) leukemia.To understand the molecular mechanism of AML1-ETOrelated leukemia development and to explore novel therapeutic targets to treat this type of leukemia, in this study we combined gene expression microarray and promoter occupancy (ChIP-chip) analyses to identify genes directly modulated by AE9a in primary murine leukemia cells. Among the common targets of microarray and ChIP-chip assays, approximately 30% show human t(8;21)-specific up or down-regulation compared with AML that have other chromosomal abnormalities.…”