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. (B) Representative data A from four independent clones are presented. (C) Liquid growth of cultures was AE AE9a Overlay assessed weekly. (D) CD34 progenitor marker expression was determined by flow 250K 250K 100 AE 200K 62.3 200K 75.3 80 x
cytometry. Combination of three independent experiments (cell lines) is shown a A 150K 150K 60 AE9a C-
+ In Vitro S SSC-A S (*P < 0.05). (E) Primary (5,000 cells) colony-forming ability was assessed on CD34 100K 100K %ofM 40 Week 1 SSC UCB cells transduced and sorted as above. Flow cytometry was performed 50K 50K 20 THY1.1-PE 0 0 0 0102 103 104 105 0102 103 104 105 0102 103 104 105 (E, Right). CD33 and CD235a were monitored for myeloid vs. red cell bias. CD34 PE-A PE-A PE-A 100 105 0.224 21.9 105 0.02 41.1 and Thy1.1 expression were also shown. (F) Secondary colony-forming ability was 80 104 104 4 4 x 3 a : :3 60 A A performed with each sample. Representative results from five independent ex- - 3 3 C 10 C- 10 %ofM AP AP 40
– Week 13 CD34 periments are shown for C F.(G) Established long-term AE and AE9a cultures 2 2 10 10 20 0 6.67 71.2 0 0.5 58.4 0 were injected (intrafemorally) into NSGS mice. After 12 wk, BM aspirations were 2 3 4 5 THY1.1-PE 010 10 10 10 0102 103 104 105 0102 103 104 105 performed and analyzed for engraftment. Representative flow plots are shown. PE-A: THY1 PE-A: THY1 PE-A: THY1 B 7 THY1.1 6 AE stem cell factor, granulocyte–macrophage colony-stimulating factor 5 AE9a C 4 (GM-CSF), and interleukin-3 (NSGS) as previously described (32). 3 α-Runx1 AE and AE9a showed comparable engraftment levels at 12 wk, with 2 high variability and no apparent differences among the tested cultures 1 (Fig. 1G). Despite producing a robust AML in a murine model, AE9a 0 was not able to generate leukemia in the human xenotransplant model Relative MFI(Thy1.1) 3813 α--Actin under the conditions tested (Fig. 1G). Weeks D AE AE9a Overlay 5 0.129 57.4 5 0.0186 54.9 100 10 10 AE 80 104 104 AE9a Selects for High Expression in Long-Term Human Cultures. In 4 AE9a :3 60 A 3 3 In Vitro C- 10 10 %ofMax AP weekly monitoring of in vitro cultures by flow cytometry using the APC-A: 34 40 CD34 Week 6 102 102 20 Thy1.1 surface protein as a surrogate marker for AE or mutant ex- 0 1.4 41.1 0 0.567 44.5 0 2 3 4 5 2 3 4 5 010 10 10 10 010 10 10 10 0102 103 104 105 pression,wenoticedareproducibleincreaseinAE9aexpressionover THY1.1 PE-A: THY1 PE-A: THY1 PE-A: THY1 8 weeks in 5 0.975 85.5 5 0.0958 93.4 100 time (Fig. 2 A and B). This intensity shift was evident in both the CD34- vivo 10 10 80 4 4
5 10 10 4
>: 60 A negative and -positive cell fractions. Elevated levels of mutant protein - 103 103
%ofMax 40
9076 | www.pnas.org/cgi/doi/10.1073/pnas.1524225113 Link et al. Downloaded by guest on October 2, 2021 relative to control cells and stopped growing by 6 wk in vitro (Fig. 3B), 600 A AE AE9a B demonstrating that increased protein levels of AE9a are essential for 100 100 1st 80 80 500 this function in human UCB cells. A similar result was obtained in 60 60 2nd 40 40 400 methylcellulose cfu assays, with AE9a-high cells consistently producing 20 20 3rd 0 0 0102 103 104 105 0102 103 104 105 greater colony numbers upon replating than AE9a-low cells, in contrast 300 4th cells to effects seen with full-length AE (Fig. 3C). These data are consistent GFP 200 5th C Week 0 Week 4 Week 10 * 6th with the observations from long-term liquid culture indicating that AE- 80 100 ** 80 3397 1402 1221 Colony # per 10,000 80 * 60 * 7th 60 low and AE9a-high cells have selective advantages for growth and 60 40 0 40 AE 40
20 suggest that high expression of AE9a is necessary for these functions. 20 20 0 0 0 2 3 4 5 2 3 4 5 010 10 10 10 010 10 10 10 0102 103 104 105
40 200 120 5601 5660 8142 D 100 30 150 Selective Pressure for High Expression of AE9a in a Murine Leukemia 90 20 100 80 AE9a 60 p= 0.02 Model. Murine fetal liver (FL) cells transduced with AE9a and 30 10 50 60 0 0 0 2 3 4 5 2 3 4 5 transplanted into recipient mice develop a robust AML (14). To fur- 010 10 10 10 0102 103 104 105 010 10 10 10 40 GFP 9a Low ther investigate the effects of high versus low AE9a expression in this 20 9a High 250K 400 Survival Percent 0 200K model, E14.5 FL cells were transduced with AE or AE9a [using an E 300 3587 150K 050100150200250 200 AE9a Low FSC A 100K Days
internal ribosome entry site (IRES)-GFP to monitor expression] and 100 50K
0 0 2 3 4 5 sorted for 30% high and 30% low expression based on GFP (Fig. 4A). 010 10 10 10 0102 103 104 105 300 Initial Sort AE9a Leukemia 250K F 200K 4437 200 Sorted cells were plated in methylcellulose, and colony numbers were AE9a High 150K Low High FSC A 100K 100 Low High determined upon weekly replating. AE-low and AE-high cells were 50K 100
0 0 2 3 4 5 AE9a 010 10 10 10 0102 103 104 105 75 equally efficient in serial replatings (Fig. 4B). In contrast, whereas 50 Initial GFP β-Actin AE9a-high cells displayed a sustained replating ability, AE9a-low cells Sort showed significantly diminished replating after week 3. AE- or AE9a- G AE 100 6
Lin- BM Low High leukemia AE Initial transduced FL cells were transplanted into lethally irradiated mice, 100 50 4 AE AE Total Percent
75 Survival and peripheral blood (PB) was analyzed at weeks 0, 4, and 10 for mean 0 2 fluorescence intensity (MFI) of GFP expression. Although initial ex- Actin 0100200 Intensity 123 Days Band Relative pression levels of AE and AE9a were comparable, expression of AE9a 0 was significantly higher at week 10 relative to both AE levels at week Fig. 4. AE9a-high expression facilitates murine replating ability and leukemia 10 and initial AE9a levels (Fig. 4C). These data are consistent with development. (A) Embryonic day (E) 13.5–14.5 fetal liver cells were harvested results obtained in the human UCB model wherein AE9a selects for from C57BL/6 mice and transduced with MIG-AE or MIG-AE9a retrovirus. high expression in vitro and in vivo. Transduced fetal liver cells were sorted for 30% low and 30% high GFP-posi- To evaluate effects of AE9a expression levels on AML development, tive cells to isolate low and high AE and AE9a expression. (B)Low-andhigh- AE9a-transduced FL cells were expanded in vivo for 2–3wk.Sub- sorted cells were plated in methylcellulose colony-forming media. Up to seven sequently, BM was harvested, and c-Kit–positive cells were sorted for serial platings were performed. Combined data from three independent ex- low (30%) and high (30%) GFP expression (Fig. S3A). GFP-positive periments are shown (*P < 0.05). (C) MIG-AE or MIG-AE9a transduced fetal cells (1 × 105) were transplanted into sublethally irradiated mice. liver cells were transplanted into lethally irradiated BoyJ recipient mice. GFP Survival analysis showed a marked latency shift in mice harboring intensity from initial transduction and from peripheral blood at weeks 4 and AE9a-low cells compared with AE9a-high cells, with an overall dif- 10 are shown. The number in each graph is the mean fluorescence intensity (MFI) value for GFP. (D) Fetal liver cells were transduced and transplanted as in ference in penetrance as well (Fig. 4D). Importantly, GFP expression C. AE9a cells were expanded for 2–3 wk in vivo, followed by BM harvest. BM and AE9a protein analysis indicated a selection for high expression – cells were sorted and gated for c-Kit expression, followed by low and high GFP even in AE9a-low expressing tumors (Fig. 4 E and F and Fig. S3B). expression. A limited number of GFP-positive cells (1 × 105) were transplanted Based on Southern blot analysis, the number of viral integrants did not into sublethally irradiated BoyJ recipient mice. Survival analysis is indicated; differ between clones sorted for initial low or high expression of AE9a, P value = 0.02. (E) Representative flow from AE9a low leukemic BM shows with between one and four integrants detected in each clone (Fig. S4). comparable MFI (GFP) to an AE9a-high leukemic BM profile. (F)Western Thus, the high levels of AE9a protein are not directly correlating analysis of a subset of AE9a-low and -high leukemic mice (Top). Anti-ETO and with integrant copy number, indicating that cell clones have other anti–β-actin are shown. Relative intensity is shown (Bottom)(G, Left)Western mechanisms than copy number integration to attain high AE9a levels. analysis of rare AE leukemic mice generated in our studies caused truncation of full-length AE. Anti-runx1 and anti-actin are shown. (Right) Survival curve of AE transplanted mice (48 total mice).
AE AE9a AE Low A W1 W5 W1 W5 B 1.E+12 Because AE9a is attenuated for transcriptional function relative to AE, Low High Low High Low Low High High AE High 100 AE as shown in Fig. 1, we tested the expression level differences of defined 75 AE9a 1.E+10 1234 MIT Low p21 MIT High AE target genes in murine cells sorted for high and low expression of 1.E+08 β-Actin AE Low AE9a. Genes were selected based on previously proposed significance AE High as being dysregulated in, and direct targets for, both mouse (AE9a) and C 120 1st 1.E+06 9a Low
80 2nd cell count Total 9a High human t(8;21) leukemia (35). There was a significant difference in the
Colonies 40 1.E+04 147101316 expression level of multiple AE9a target genes between these two 0 Weeks groups, suggesting that one or more AE9a target genes are insuffi- ciently regulated in the AE9a-low cells (Fig. S5 A–D). Furthermore, we generated an AE gene signature using the sorted murine cells Fig. 3. Sorting for low AE9a expression prevents establishment of long-term + expressing AE in comparison with sorted MIG control cells. Unsu- cultures. Human CD34 cord blood cells were transduced with MIT, AE, or AE9a pervised cluster analysis showed that AE9a-high samples closely re- retrovirus and sorted for 30% low and 30% high Thy1.1-positive cells. sembled the AE samples whereas AE9a-low samples were more MEDICAL SCIENCES (A) Western analysis comparing AE low/high and AE9a low/high samples at time heterogenous and less similar to the AE samples (Fig. S5E). Together, of sort with week 5 samples from liquid culture. Anti-RUNX1, anti-p21, and anti– these data demonstrate that high expression is a prerequisite to facil- β-actin are shown. (B) Sorted cells were grown in liquid culture medium, and weekly cell counts were performed. The Inset highlights the initial slow growth itate AE9a AML formation in a murine transduction/transplantation + of AE-high cultures compared with AE-low. (C)CD34 cells transduced and assay and implicate the transcriptional regulation of AE target genes as sorted as in A were plated in methylcellulose colony-forming media at 5,000 apossibledrivingforcebehindtheincreaseinAE9aexpression. cells per plate (primary) or 20,000 cells per plate (secondary). After 2 wk, total A number of mice that received cells transduced by full-length AE colony number was counted. Representative plating is shown. A combination of also developed AML (4 out of 48 mice developed AML) (Fig. 4G, Right) five independent experiments with three replicates each were performed. that was indistinguishable from AML found in AE9a mice although
Link et al. PNAS | August 9, 2016 | vol. 113 | no. 32 | 9077 Downloaded by guest on October 2, 2021 most mice did not develop any disease, as has been shown by multiple A B laboratories (7, 36). Interestingly, three of these AMLs expressed protein AE AE9a AE + AE9a AE + AE9a culture 105 74.2 0.393 105 0.738 2.66 105 30.2 8.34 100 that was in the same size range expected for AE9a (Fig. 4G, Left). These 104 104 104 103 103 103 75 AE9a GFP 2 2 2 proteins were also highly expressed relative to the initial levels of AE 1 Week 10 10 10 THY1.1 0 25.4 0.0381 0 14.6 82 0 22.4 39 50 AE present in transduced cells. Upon sequence analysis of the proviral 0102 103 104 105 0102 103 104 105 0102 103 104 105 AE+AE9a integrants, all three had insertions/mutations that mimic an AE9a-like 105 0.958 3.72 105 42.6 46.9 25 104 104 Proportion of sequence and would be expected to give rise to a truncated AE protein 103 103 GFP 0 Transduced Cells Transduced Week 5 102 102 THY1.1 0 0 0123456 (Fig. S6). These data indicate that there is a strong selective pressure for 5.69 89.6 5 5.47 2 3 4 5 2 3 4 5 Weeks high expression of a truncated AE protein in the murine transduction/ 010 10 10 10 010 10 10 10 transplantation assay. Fig. 5. Coexpression of AE and AE9a in murine BM cells necessitates the presence of AE fusion in vitro. Murine BM cells were transduced with MIT-AE, Full-Length AE Dominates Under Conditions of AE Plus AE9a Coexpression MIG-AE9a, or MIT-AE plus MIG-AE9a. Posttransduction, cells were plated in in Murine BM Cells in Vitro. To identify potential selective pressures methylcellulose. Cultures were monitored weekly by flow cytometry to mea- upon coexpression of both AE and AE9a, murine BM cells were sure the percentage of culture of each population. Cells were replated each isolated and transduced with AE, AE9a, or AE plus AE9a. Cells week for up to 6 wk. (A) Representative flow from week 1 and week 5 cultures were plated in methylcellulose and monitored weekly by flow demonstrating a push toward AE containing cells at week 5. (B) Percentages of cytometry for the percentage of transduced cells (Fig. 5). Despite cultures from AE plus AE9a transduced cells are shown. Representative of two initial robust transduction efficiency, cells expressing AE9a were independent experiments. gradually outcompeted by cells expressing full-length AE or AE plus AE9a, suggesting functional dominance of full-length AE under these conditions. In vivo analysis of coexpression was also understood regarding the functional differences with full-length AE. examined. Despite several attempts at recapitulating AE9a leu- Interestingly, AE9a showed a partial deficiency of function in the hu- kemia in the context of full-length AE, AE9a alone prevailed as man preleukemia model, including aberrant activation and repression the dominant expressing protein (Fig. S7), implying that the pres- of select target genes. When an AE-like effect was observed, as in the ence of AE under these conditions may be deleterious to leukemia case of long-term liquid cultures, increased oncogene expression was formation. necessary for this effect. These data bring important insight into the pressures driving transformation in these model systems and raise Coexpression of AE and AE9a in Human Cells Results in AE Dominance questions as to the proper interpretation of the data relative to mech- and Relieves Selective Pressure for Increased AE9a Expression. The anisms of transformation operative in human AML patient samples. differential selective pressures observed in the murine system prompted Oncogene expression level has been previously identified as a crit- investigation of AE and AE9a coexpression studies in the human ical parameter in modulating leukemia development. The MLL-AF9 + model. Human CD34 UCB cells were transduced with AE, AE9a, or fusion gene produces very distinct results depending on the expression AE plus AE9a. Transduction frequencies at week 2 of culture ranged level of the fusion protein. Both low expression (under the control from 20% to 50% for each sample (Fig. 6 A and B). AE and AE9a of the endogenous promoter) and high expression (retroviral trans- single-transduced cultures showed a gradual selection for the transduced duction) of MLL-AF9 led to leukemia development when targeting cells, with nearly 100% purity being reached at weeks 6–8(Fig.6B). the stem cell population, but only high levels of oncogene expression Similar to the results obtained in the murine studies, coexpression in the drove leukemia development upon targeting the granulocyte-macro- human system led to an accumulation of AE single-transduced and AE phage committed progenitor (33). In addition, a recent study showed plus AE9a double-transduced cells, with loss of AE9a single-transduced the frequency of leukemia-initiating cells in acute lymphocytic leuke- cells over time. Western blot analysis of cells from week 14 cultures mia correlated with increased expression of the oncogene c-myc (34). showed increased AE9a expression in the single-transduced population Our study supports the hypothesis that AE9a preferentially requires in comparison with single-transduced AE cells, similar to our previous selection for high-expressing clones to promote long-term growth and findings (Fig. 6C). However, in the context of transduction with full- leukemia development whereas low expression is insufficient to elicit a length AE, AE9a levels were substantially reduced relative to full-length transforming effect. These data are in sharp contrast to results observed AE, with no sign of selective pressure for high expression. Expression with full-length AE, wherein high expression in human cells is inhibitory analysis was also performed on double-positive AEplusAE9a sorted to cell growth and low expression is preferred. These differences may cells. Again, lower levels of AE9a were present in the double-positive relate to the differential regulation of target genes, in particular the population compared with AE9a single-transduced cultures (Fig. S8A). repression of CEBPA and SPI1 and up-regulation of CDKN1A, the p21 Furthermore, although AE9a single-transduced cultures selected for cell cycle inhibitor. A study from Alcalay and coworkers showed sig- high percentages of CD34-expressing cells over time (Figs. 2B and 6D), nificantly reduced recruitment of AE9a to target promoters relative to AEplusAE9a cells displayed a phenotype resembling that of AE, with AE in U937 cells engineered to express one or the other oncogene, much lower percentages of CD34 surface expression (Fig. 6D). To de- supporting our data showing a need for increased expression to elicit an termine the relevance of these findings to t(8;21) AML patient samples, AE-like transcriptional regulation (37). These data could indicate that we screened a subset of t(8;21)-positive samples for AE/AE9a protein many of the targets and signaling pathways are in common between AE expression by Western blot (23 patient samples in all). Analysis revealed and AE9a and that increased expression of AE9a is needed to mirror a dominant band within the size range of full-length AE, with no clear thesametranscriptionalchangesasAE.Ourgeneexpressiondata evidence of an AE9a band, similar to results observed with the Kasumi-1 comparing AE9a-high and AE9a-low cells would support this idea. t(8;21) cell line (Fig. 6E and Fig. S8 B and C). Our result that none of Despite the absence of strong selective pressure for high AE expres- the 23 randomly selected t(8;21) patient samples has detectable 9a sion in our mouse and human models, clinical evidence supports the protein predicts that between 0% and 14.3% of t(8;21) patients express importance for high AE levels in t(8;21) leukemia. Disease recurrence 9a protein (Wilson method with 95% confidence), which is significantly and therapeutic response both correlate with transcript levels of the lower than the reported percentage (∼70%) of patients harboring the t(8;21) oncogene. At diagnosis, low levels of transcript (on a per-cell 9a transcript (binomial exact test P value < 0.05) (14, 16). Collectively, basis) trend toward better overall and event-free survival (38). Fur- these data underscore the significance of oncogene expression to dis- thermore, disease relapse is significantly correlated with higher tran- ease phenotype and suggest that, whereas AE9a is a potent oncogene script levels (39–42). Similarly, high transcript levels of AE9a also at high expression levels in murine transduction/transplantation assays, correlated with t(8;21) disease relapse (16, 43). Jiao et al. (16) showed its contribution to t(8;21) disease requires careful verification. a strong positive correlation between c-Kit expression and high levels of AE9a transcript. Although these studies are strictly based on RNA Discussion transcript levels by quantitative RT-PCR, our data showing the im- Although an increasing number of studies have used the AE9a onco- portance of high AE9a protein expression in human and mouse model gene as representative of t(8;21) human leukemia, much remains to be systems support the findings of these clinical studies. It is possible that
9078 | www.pnas.org/cgi/doi/10.1073/pnas.1524225113 Link et al. Downloaded by guest on October 2, 2021 A B
C D
E
+ Fig. 6. Coexpression of AE and AE9a relieves AE9a selective pressure in human cells. CD34 cells were transduced with MIT vector, MIG-AE, MIT-AE9a, or MIG-AE plus MIT-AE9a. (A) Representative flow cytometry at weeks 2, 4, and 14 showing expression of AE (GFP) and/or AE9a (Thy1.1). (B) Cultures were monitored on a weekly basis for GFP and Thy1.1 expression. Percentages of culture are shown for MIT, AE, and AE9a single transductions (Top) and AE plus AE9a (Bottom). Representative of two independent experiments. (C) Week 14 AE, AE9a, and AE plus AE9a bulk cultures were subjected to Western blot analysis for anti-runx1 and anti-actin. (D) CD34 expression was monitored on AE, AE9a, and AE plus AE9a transduced cultures. (E) AML patient samples were analyzed for AE and AE9a expression using an anti-runx1 antibody (Left). The t(8;21) samples are designated. (Right) AE/AE9a ratio of band intensity is shown.
signals from cooperating events play a role in how AE or AE9a affect From these data, we conclude that the transformation elicited by AE9a downstream signals. A number of cooperating oncogenes have been in murine myeloid cells in vivo is particular to this approach and may + shown to promote leukemic transformation with full-length AE (6, 8–13). not reflect conditions found in t(8;21) human cells. This conclusion A recent study demonstrated that various c-Kit–activating mutations co- mayreflecttherelativeeasewithwhich murine cells are transformed operate with AEtr (the truncated form of AE, similar to AE9a) to initiate compared with human cells or may be due to differential activation of + an oncogenic-like phenotype in human mobilized PB CD34 cells (44). signals between the two species in response to AE9a expression. Future Interestingly, expression of AEtr alone had minimal effects in their study, studies are needed to explore these hypotheses. indicating possible experimental differences compared with the present What, then, is the contribution of AE9a to t(8;21) AML? It is still study. Along with cell source differences, the use of GM-CSF and serum possible that there exist a group of patients whose blast cells express in the study by Wichmann et al. (44) could have contributed to the ob- high levels of AE9a protein. Alternatively, the rapid turnover of the + served decline in CD34 cells. Furthermore, it is important to note the AE9a protein could contribute to the apparent absence in patient limited positive selection elicited by AEtr in their experiments, which may samples, leaving open the possibility that AE9a is functionally con- be attributable to low initial transduction frequency and potentially low tributing to t(8;21) disease but at much lower expression levels and levels of AEtr expression. Future studies should include examination of in ways that cannot be evaluated in model systems. Upon reaching cooperating events in the presence of both AE and AE9a. supraphysiologic levels of expression as in the murine and human ex- It is apparent that selective pressure for increased AE9a expression is perimental systems, AE9a seems to resemble full-length AE signaling occurring as a result of necessity. Exactly how this high oncogene ex- to a significant extent, with an additional gain-of-function in murine pression is achieved in these model systems is unclear. Although we cells in vivo that has not yet been fully characterized. Thus, independent have ruled out increased protein stability and integration as driving of its role in t(8;21) human disease, AE9a may serve as a valuable factors, it is possible that the specific chromatin geography at integration model that could potentially provide insight into the molecular mech- MEDICAL SCIENCES sites could allow for increased expression. In addition, although we show anisms of AE leukemia. However, care must be taken in interpreting that AE9a does not positively regulate the MSCV promoter in transient and extrapolating results from this model to t(8;21) human leukemia. transfection assays, this finding does not rule out that, in the context of Our studies identified expression level as a critical factor for AE9a intact chromatin, AE is a repressor of the MSCV promoter whereas leukemia development. Full-length AE may also require such selection AE9a has an attenuated function. Interestingly, in the presence of AE for leukemia development but only rarely reached high levels to pro- expression in both human and murine cells in vitro, high AE9a ex- mote leukemia in our model systems. It will be of interest to examine pression is not positively selected, and, in patient samples, we are unable AE protein levels in murine leukemia samples generated in the context to readily detect AE9a protein even in cells that express transcript. of cooperating events. Further studies will require focusing on factors
Link et al. PNAS | August 9, 2016 | vol. 113 | no. 32 | 9079 Downloaded by guest on October 2, 2021 that control AE expression level and examining the possibility of tar- occasional limb paralysis, and the necessity to sacrifice the animal due to ill- geting the signaling cascades that respond to increased expression. ness, as well as a blast population of greater than 20% in the bone marrow. All antibodies used were from BD Biosciences. Materials and Methods Retroviral Plasmids and Transduction. pMSCV-HA-AE-IRES-Thy1.1 (MIT-AE) and Fetal Liver Experiments. Transduced fetal liver cells were injected i.v. into BoyJ pMSCV-Flag-AE-IRES-GFP were described (45, 46). pMSCV-Flag-AE9a-IRES-GFP recipient mice. Cells were expanded 2–3 wk in vivo, mice were killed, and bone was from Dong Er Zhang, University of California, San Diego, La Jolla, CA. marrow (BM) was harvested. c-Kit+ cells were sorted (BD FACS Aria) for 30% + + AE9a cDNA was cloned into the BamHI and EcoRI sites of MIT-AE. UCB prep- low and 30% high GFP-expressing populations. Then, 1 × 105 sorted c-Kit /GFP + aration, CD34 isolation, and virus production were as described (28). cells were injected into sublethally irradiated recipient mice.
Colony-Forming Unit Assays. Transduced cells were sorted for total or 30% low AML Patient Samples. Patient samples were used under Cincinnati Child- and 30% high Thy1.1 or GFP expression. Thy1.1-positive cells were plated in ren’s Hospital Medical Center (CCHMC) institutional review board (IRB)- MethoCult 4100 (Stem Cell Technologies). Colony assays were as described with approved protocol “Tissue Repository (Tumors & Vascular Anomalies).” the addition of THPO (20 ng/mL) (28). For fetal liver cells, 1,000 GFP+ cells were Informed consent was obtained from all subjects whose samples were plated in MethoCult GF 3434 (Stem Cell Technologies). Colonies were counted included here. 7 d later. Mouse colony-forming assays were performed in triplicate with 2–3 repeats. ACKNOWLEDGMENTS. We thank Adam Lane for statistical analyses and the
−/− CCHMC core facilities [Comprehensive Mouse and Cancer Core (irradiations/ Xenotransplant Assays. NOD/SCID/IL2rg (NSG) mice transgenic for the hu- injection); the Research Flow Core (sorting); and the Translational Core Lab man cytokines stem cell factor (SCF), GM-CSF, and IL-3 (NSGS) (32) were in- (cord blood)]. This work was supported by Hope Street Kids, CancerFreeKids, jected intrafemorally with AE or AE9a cultures. Mice were killed and analyzed NIH Training Grant 5 T32 CA117846 (to K.A.L.), and the Leukemia & Lymphoma by flow cytometry. Leukemia was characterized by lethargy, scruffy coat, Society (J.C.M.).
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