Supraphysiologic levels of the AML1-ETO isoform AE9a are essential for transformation Kevin A. Linka, Shan Lina, Mahesh Shresthaa, Melissa Bowmana, Mark Wunderlicha, Clara D. Bloomfieldb, Gang Huanga,c, and James C. Mulloya,1 aDivision of Experimental Hematology and Cancer Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH 45229; bThe Ohio State University Comprehensive Cancer Center, Columbus, OH 43210; and cDivision of Pathology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH 45229 Edited by Dennis A. Carson, University of California, San Diego, La Jolla, CA, and approved June 17, 2016 (received for review December 8, 2015) Chromosomal translocation 8;21 is found in 40% of the FAB M2 sub- of t(8;21) disease (25–27). We have also established human-based type of acute myeloid leukemia (AML). The resultant in-frame fusion models using the CBFB-MYH11 and MLL-AF9 fusion proteins (28– protein AML1-ETO (AE) acts as an initiating oncogene for leukemia 30). These models seem to be faithful representations of human dis- development. AE immortalizes human CD34+ cord blood cells in long- ease at multiple levels, demonstrating a high degree of relevance to term culture. We assessed the transforming properties of the alterna- primary patient samples in a tractable model system. In the present tively spliced AE isoform AE9a (or alternative splicing at exon 9), study, we examined the functional consequences of AE9a expression in which is fully transforming in a murine retroviral model, in human comparison with full-length AE in human HSPCs. Our data identify cord blood cells. Full activity was realized only upon increased fusion oncogene expression level as a critical parameter for AE9a function. protein expression. This effect was recapitulated in the AE9a murine Results AML model. Cotransduction of AE and AE9a resulted in a strong se- + lective pressure for AE-expressing cells. In the context of AE, AE9a did AE9a Promotes Self-Renewal of Human CD34 Hematopoietic Cells. not show selection for increased expression, affirming observations of We examined the function of the AML1-ETO isoform AE9a (and the Δ + human t(8;21) patient samples where full-length AE is the dominant AE9a 2 mutant as a negative control) in human CD34 umbilical cord protein detected. Mechanistically, AE9a showed defective transcrip- blood (UCB) cells (Fig. 1A). Cells were transduced with MSCV-HA-AE- IRES-Thy1.1-,AE9a-,orAE9aΔ2-expressing retrovirus and sorted for tional regulation of AE target genes that was partially corrected at Thy1.1 expression. AE9a partially recapitulated the effects of AE tran- high expression. Together, these results bring an additional perspec- scriptional activation and repression although most target genes were tive to our understanding of AE function and highlight the contribu- regulatedtoalesserextentcomparedwithAE,includingCEBPA,SPI1, tion of oncogene expression level in t(8;21) experimental models. OGG1, CDKN1A, and MPL, indicating some loss of function for AE9a + (Fig. 1B). AE9a extended cell growth and maintained a CD34 pop- oncogene dosage | AML | isoform | transformation ulation throughout the lifespan of the cultures, similar to the effects of AE (Fig. 1 C and D). AE9a cultures consistently trended toward a higher he t(8;21)(q22;q22) chromosomal translocation comprises the percentage of CD34-expressing cells over time, unlike AE cultures, TN-terminal DNA binding domain of AML1 (RUNX1) and which showed relatively constant levels of CD34-expressing cells. How- nearly the entire ETO (RUNX1T1) gene, forming the fusion ever, this selection was not advantageous, given the similar growth ki- protein AML1-ETO (AE) (1, 2). Conditional expression and netics of the cells. As expected, deletion of NHR2 in the context of AE9a transduction–transplantation approaches have demonstrated that resulted in a fully nonfunctional mutant, in line with previous findings expression of AE provides self-renewal signaling to hematopoietic (31). AE and AE9a dramatically repressed erythroid colony formation stem/progenitor cells (HSPCs) but does not induce transformation and showed a myeloid colony bias, with significantly fewer colonies in the absence of additional cooperating events (3–7). Several such formed overall compared with controls (Fig. 1E). CD34-expressing cells cooperating events have been identified, including overexpression were maintained in colony-forming unit (cfu) assays, consistent with colony-replating potential for both AE and AE9a (Fig. 1 E and F). of WT1, mutant c-KIT, TEL-PDGFRB, FLT3-ITD, loss of p21, – The ability of the AE9a mutant to transform murine HSPC to AML and treatment with the DNA-damaging agent ENU (6, 8 13). prompted examination of the engraftment and transformation potential Conversely, C-terminal truncation of AE through frameshift muta- of human cells expressing AE9a in comparison with full-length AE. tion (AML1-ETOtr) or alternative splicing at exon 9 (AE9a) leads Cells were transplanted by intrafemoral injection into immunodeficient to acute myeloid leukemia (AML) transformation of murine HSPCs −/− + NOD/SCID IL2RG (NSG) mice transgenic for the human cytokine (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 con- Significance served ETO domains NHR3/4, whose interaction with corepressor proteins such as N-CoR/SMRT and HDACs is important for tran- The AE9a protein (alternative splicing at exon 9) is often used to scriptional repression (17–20). Despite maintaining some corepres- model t(8;21) leukemia. Our study demonstrates that increased sor interaction, AE9a has greatly diminished N-CoR and SMRT oncogene dosage is a critical parameter of AE9a transformation, interaction and is a much less potent transcriptional repressor likely as a result of impaired transcriptional regulation of AML1-ETO than full-length AE (17, 19–22). The AML1-ETOtr mutant showed target genes. This insight could assist in identifying those down- altered regulation of cell cycle proteins, thereby providing a stream genes most critical for t(8;21)-associated transformation. proliferative advantage in K562 cells relative to AE (15). Re- cently, DeKelver et al. showed that the interaction between N-CoR Author contributions: K.A.L., S.L., and J.C.M. designed research; K.A.L., S.L., M.S., M.B., and and the NHR4 domain plays an inhibitory role in leukemia de- M.W. performed research; C.D.B. and G.H. contributed new reagents/analytic tools; K.A.L., MEDICAL SCIENCES velopment in the context of full-length AE (23). However, analysis S.L., M.S., M.W., and J.C.M. analyzed data; and K.A.L., S.L., and J.C.M. wrote the paper. of a small cohort of t(8;21) patient samples did not detect any The authors declare no conflict of interest. mutations in the NHR4 domain, indicating that such mutations This article is a PNAS Direct Submission. are probably very infrequent (24). The role of AE9a in the genesis Data deposition: The data reported in this paper have been deposited in the Gene Ex- of t(8;21)-associated AML is currently unclear. pression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE84513). + Previously, we showed that expression of AE in human CD34 1To whom correspondence should be addressed. Email: [email protected]. HSPCs promotes enhanced self-renewal and long-term expansion of This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. + CD34 progenitors, making this model a valuable preleukemia model 1073/pnas.1524225113/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1524225113 PNAS | August 9, 2016 | vol. 113 | no. 32 | 9075–9080 Downloaded by guest on October 2, 2021 AB protein synthesis inhibitor cycloheximide (Fig. S1A). Protein turnover for AE9a was actually more rapid than for full-length AE, suggesting that increased protein stability was not contributing to high AE9a ex- pression. Given the decreased ability of AE9a to efficiently repress AE target genes, it was possible that AE9a does not repress expression from the murine stem cell virus (MSCV) promoter [which contains a core binding factor (CBF) recognition site] whereas AE does. However, AE C D and AE9a showed similar activity on an MSCV luciferase reporter, suggesting that hyperactivation of the MSCV promoter by AE9a is unlikely to be driving the increased AE9a expression (Fig. S1B). High AE9a Expression Is Essential for Establishment of Long-Term Human E Cultures. To determine whether high expression is necessary for AE9a function, human UCB cells transduced with vector, AE, or AE9a were sorted for expression of the vector-encoded Thy1.1 surface marker immediately after transduction. Fractions with the lowest (30% posi- tive) and highest (30% positive) expression were sorted, and cell growth was monitored on a weekly basis (Fig. 3 A and B). AE-low cells showed robust growth, and expression analysis at week 5 showed that F G AE-low cultures reached a median level of AE expression, based on both AE protein and Thy1.1 expression (Fig. 3A and Fig. S2). AE-high cells initially showed a lag in proliferation (Fig. 3B, Inset), possibly due to an up-regulation of the p21 cell cycle inhibitor (Fig. 3A), but re- covered growth potential after 2–3 wk, presumably due to outgrowth of clones expressing lower levels of AE (Fig. 3 A and B). In contrast, AE9a-high cells had excellent proliferative ability but, interestingly, Fig. 1. AE9a extends CD34+ UCB culture life, retains CD34 progenitor marker maintained or even increased AE9a levels over time (Fig. 3 A and B and expression, and engrafts immunodeficient mice. (A) Schematic of HA-tagged Fig. S2). Strikingly, AE9a-low cells showed no enhanced proliferation + AE, AE9a, or AE9aΔ2. CD34 umbilical cord blood (UCB) cells were transduced with MIT-HA-AE, -AE9a, or -AE9aΔ2, or empty vector and sorted for Thy1.1 surface marker expression. (B–G) Quantitative PCR (QPCR) analysis was per- formed on week 1 transduced AE and mutant cultures.