Leukaemogenesis requires enhanced self-renewal, which is induced by oncogenes. The underlying molecular mechanisms remain incompletely understood. Here, we identified C/D box snoRNAs and rRNA 2′-O-methylation as critical determinants of leukaemic stem cell activity. Leukaemogenesis by AML1-ETO required expression of the groucho-related amino-terminal enhancer of split (AES). AES functioned by inducing snoRNA/RNP formation via interaction with the RNA helicase DDX21. Similarly, global loss of C/D box snoRNAs with concomitant loss of rRNA 2′-O-methylation resulted in decreased leukaemia self-renewal potential. Genomic deletion of either C/D box snoRNA SNORD14D or SNORD35A suppressed clonogenic potential of leukaemia cells in vitro and delayed leukaemogenesis in vivo. We further showed that AML1-ETO9a, MYC and MLL-AF9 all enhanced snoRNA formation. Expression levels of C/D box snoRNAs in AML patients correlated closely with in vivo frequency of leukaemic stem cells. Collectively, these findings indicate that induction of C/D box snoRNA/RNP function constitutes an important pathway in leukaemogenesis.
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We thank T. Gridley for providing Aes straight knockout (Aes−/−) mice. We are grateful to B. Edemir for help with project organization and manuscript preparation. We thank M. Scheller-Wendorff for support of the mice experiments. This work was supported by Deutsche Forschungsgemeinschaft (DFG, MU1328/15-1 and MU1328/9-2), the Deutsche Jose Carreras Leukämie-Stiftung e. V. (DJCLS R10/35f), by a Wilhelm-Roux-Program grant (28/43) of the Medical Faculty, Martin-Luther-University Halle-Wittenberg to CMT and the grants of the federal state of Saxony-Anhalt (P40). N.B. is supported by Wilhelm Sander Stiftung (2014.054.1), Deutsche Krebshilfe (70112282) and ‘Innovative Medical Research’ of the University of Münster Medical School (IMF) (121314 and 111501). C.P. is supported by a Max-Eder grant of the Deutsche Krebshilfe (70111531). The laboratory of R.S. is supported by Deutsche Forschungsgemeinschaft (DFG SL27/7-2). The laboratory of W.E.B. is supported by DFG grant EXC 1003. The work of 20 AML samples with LSC frequencies was supported by the Government of Canada through Genome Canada and the Ministère de l’enseignement supérieur, de la recherche, de la science et de la technologie du Québec through Génome Québec to G.S. and J.H. and by grants from the Cancer Research Network of the Fonds de recherche du Québec-Santé to J.H.
The authors declare no competing financial interests.
Integrated supplementary information
(a,b) FACS analysis of GFP+ transplanted cells in the peripheral blood of recipients, 4 weeks after transplantation. (a) shows a representative FACS plot. (b) shows quantification of the FACS results. (c) White blood cell counts in peripheral blood of recipients, 4 weeks after transplantation. (d) Survival analysis of primary recipients transplanted with AE9a transduced Aes+/+ (n = 12 mice) or Aes−/− (n = 12 mice) bone marrow cells. P = 0.479, log-rank test. (e) Survival of secondary recipients transplanted with AE9a leukemia blast from Aes+/+ or Aes−/− group. Cells form three donors of each group were used for secondary transplantation. N = 12 mice for Aes+/+ group and 10 mice for Aes−/− group, P = 0.004, log-rank test. (f) Experimental scheme for in vitro serial replating assay with AE9a transduced Aes+/+ or Aesf/f murine fetal liver cells. Excision of the floxed Aes alleles was performed by 4-hydroxytamoxifen (4-OHT) treatment either during the first round of replating or after three round of replating. Colonies were counted at each round of replating. (g) Genotype analysis demonstrated the excision of floxed Aes allele. (h) Colony numbers in each round of replating assay as shown in f. Error bars, mean ± s.d. of n = 3 independent experiments. (i) Number of colonies formed by AE9a transformed Aes+/+or Aesf/f fetal liver cells. Error bars, mean ± s.d. of n = 3 independent experiments; ∗∗∗P < 0.001, Student’s t-test. (j) Representative colonies in methylcellulose. Scale bars, 2.5 mm. (k) Western blot shows knockdown of AES by two independent shRNA in Kasumi-1 cells. (l) Colony numbers from 300 indicated leukemic cells with AES knockdown by shRNA, compared to the control (shCtr). Error bars, mean ± s.d. of n = 3 independent experiments. Unprocessed original scans of blots are shown in Supplementary Figure 7. Statistical source data for Supplementary Fig. 1b, c are provided in Supplementary Table 7.
(a) RT-PCR analysis of AE9a target genes in control (shCtr) and AES (shAES) knockdown Kasumi-1 cells. (b) RT-PCR analysis of snoRNA expression to confirm microarray results. GFP positive (AE9a transduced) bone marrow cells with wildtype (Aes+/+) or knockout (Aesf/f) genotype were sorted from recipient mice. cDNA was reverse transcribed from the same RNA used for microarray analysis. Data shown as average of n = 2 biological replicates each group. (c) Quantitative real-time PCR analysis of Aes mRNA in fetal liver cells retrovirally transduced with Aes or empty vector (Control). GAPDH was used for internal control. (d) Colony numbers from fetal liver cells retrovirally transduced with Aes or empty vector. Error Bar, mean ± s.d. n = 3 independent experiments, P = 0.001, Student’s t-test. (e) Comparison of snoRNA expression in Kasumi-1 cells as determined by snoRNA-seq and by qRT-PCR. The x axis shows the relative expression of indicated snoRNAs in AES knockdown cells compared to control cells. (f) RT-PCR analysis of snoRNA expression in control (shCtr) and AES knockdown (shAES) SKNO-1 cells. (g) Scatterplot comparing pseudouridylation ratios at 72 sites in 18S and 28S rRNA in control (shCtr) and AES knockdown (shAES) Kasumi-1 cells. Pseudouridylation were measured after CMC treatment followed by high throughput sequencing, and the plotted Ψ-ratios were calculated as described in ref. 1. Paired Wilcoxon test. (h) Protein expression of small nucleolar ribonucleoprotein (snoRNPs) components Pontin, NOP56, NOP58 and Fibrillarin in AES knockdown (shAES) and control (shCtr) Kasumi-1 cells (Western blot analysis). (i) Forward Scatter size analysis of control (shCtr) and AES knockdown (shAES) Kasumi-1 cells. One of n = 3 independent experiments is shown. (j) OP-Puro incorporation of control (shCtr) and AES knockdown (shAES) Kasumi-1 cells. One of n = 4 independent experiments is shown. Error bars, mean ± s.d. of n = 3 independent experiments unless otherwise indicated. Unprocessed original scans of blots are shown in Supplementary Figure 7. Statistical source data for Supplementary Fig. 2i, j are provided in Supplementary Table 7.
(a) Schematic diagram of nascent RNA-Seq. Control (shCtr) and AES knockdown (shAES) Kasumi-1 cells (two replicates for each) were cultured in medium containing 4-thiouridine for 1 h. 4-thiouridine was metabolically incorporated into newly transcribed (nascent) RNA during transcription. Following isolation of total cellular RNA and thiol-specific biotinylation, nascent RNA was separated from total RNA using streptavidin-coated magnetic beads. Paired-end libraries prepared from nascent RNA were sequenced on an Illumina Hiseq2000 platform. (b) Levels of Nascent transcripts (FPKM) of snoRNA host genes in control (shCtr) and AES knockdown cells (shAES). 36 host genes for snoRNAs that are downregulated by AES knockdown were shown. (c) Visualization nascent RNA-seq reads on snoRNA host gene RPL3. (d,e) Gene ontology analysis of downregulated nascent transcripts upon AES knockdown. 2180 transcripts were found to be downregulated in AES knockdown Kasumi-1 cells (FPKMshAES/FPKMshCtr < 0.7 and FPKMshCtr > 1). (f) AES and DDX21 interact in vivo. 293T cells were transfected with V5-tagged AES. Immunoprecipitation was performed with anti-V5 antibody. Western blot analysis was performed with anti-DDX21 or anti-V5 antibody. (g) Western blot indicates DDX21 expression in Kasumi-1 leukemic cells with DDX21 knockdown by shRNA, compared to control shRNA (against luciferase gene, shLUC). (h) RT-PCR analysis of snoRNAs in control and DDX21 knockdown Kasumi-1 cells. Data are represented as mean ± s.d. of n = 3 independent experiments. (i) Kasumi-1 cells were infected with lentivirus expressing control shRNA (shLUC), shRNA against AES or DDX21. Percentage of infected cells (GFP+) was analyzed at the indicated time points. Percentage was normalized to the number at day 1. Error Bar, mean ± s.d. n = 3 independent experiments. (j) Cell cycle analysis of control (shLUC) Kasumi-1 cells and Kasumi-1 knockdown of AES (shAES), DDX21 (shDDX21). Error Bar, mean ± s.d. n = 3 independent experiments. Unprocessed original scans of blots are shown in Supplementary Figure 7. Statistical source data for Supplementary Fig. 3j are provided in Supplementary Table 7.
(a) AES knockdown did not affect transcription from DDX21 bound promoters. Nascent transcripts levels (FPKM) in control and AES knockdown Kasumi-1 cells were used from Nascent RNA-seq as described in Supplementary Fig. 3. DDX21 bound genomic regions were obtained from publically available ChIP-Seq data (GSE56802). Box plots represent fold change (shAES/shCtr, log2 scale) of transcripts originating from promoter regions either bound or not bound by DDX21. As indicated, DDX21-bound gene transcripts were not generally suppressed or enhanced upon loss of AES. The central mark in box plot is mean, with 5/95 percentiles at the whiskers and 25/75 percentiles at the box. (b) DDX21 interaction was enriched for ribosome complex. Mass Spectrometry analysis of SILAC labelled proteins was performed for DDX21 interaction partners at the presence or absence of AES. IP with anti-DDX21 antibody (or IgG control) was performed in protein lysates from Kasumi-1 cells transduced with either shAES or shControl. The ratio of precipitated interaction partners (anti-DDX21/IgG) was analyzed. Circles in red indicate proteins found to interact with DDX21. Proteins labelled in gray are known ribosome complex members that were not identified in our Mass Spectrometry analysis. Gray lines designate protein interactions.
(a) Sanger sequencing of Kasumi-1 CRISPR snoRNA knockout cells. Arrow shows the mutations induced by CRISPR/CAS9 in snoRNA loci. (b) to (c) SnoRNA knockout did not affect host gene expression. SnoRNA host gene RPL13A was analyzed both on mRNA level by RT-PCR (b) and on protein level by Western blot (c) in control and SNORD34 or 35A knockouts. Two single clones for each knockout were checked. (d) SnoRNA knockout did not affect total RNA content. Total RNA from 1 × 105 indicated Kasumi-1cells were eluted in 25 μl RNase-free H2O. Error Bar, mean ± s.d. of n = 5 measurement of 2 independent replicates. (e) Colony numbers formed by MV4-11 CRISPR snoRNA knockout cells, compared to control. (f) Kaplan–Meier survival analysis of NSG mice injected with snoRNA knockout or control MV4-11 cells. N = 12 (Control_gRNA), 7 (SNORD14D_gRNA) and 6 mice (SNORD35_gRNA), log-rank test. (g) Kaplan–Meier survival curve of NSG mice receiving 1 × 104 control (Ctr_gRNA, n = 12 mice) or SNORD43 CRISPR knockout (SNORD43_gRNA, n = 6 mice) MV4-11 cells. Error Bar, means ± s.d. of n = 3 independent experiments unless otherwise indicated. Unprocessed original scans of blots are shown in Supplementary Figure 7.
(a) RT-PCR analysis shows expression of snoRNAs in AE9a transduced lin− bone marrow cells, compared to control. (b) and (c) Scatterplot shows snoRNA expression in myc- (b), MLL/AF9-transduced (c) and control (empty vector) lin− bone marrow cells determined by snoRNA-seq. Paired Wilcoxon test. (d) RT-PCR analysis of Aes mRNA expression in lin− bone marrow cells transduce with AE9a, MLL-AF9 or c-Myc, compared to control. (e) and (f) Scatterplot shows snoRNA expression in MLL-AF9 (e) and c-Myc (f) transduced AES wildtype (AES WT) and knockout (AES KO) lin− bone marrow cells determined by snoRNA-seq. Lin− bone marrow cells isolated from ROSA26-Cre/ERT; Aes+/+ or ROSA26-Cre/ERT; Aesf/f were retrovirally transduced with oncogene. Transduced cells were treated with 4-hydroxytamoxifen for three days for Aes deletion. (g) Kaplan–Meier survival analysis of mice injected with MLL-AF9 transduced lin− bone marrow cells isolated from Cre/ERT; Aes+/+ or ROSA26-Cre/ERT; Aesf/f mice. Tamoxifen induction was performed at two weeks after transplantation. N = 7 and 8 mice, log-rank test. (h) Kaplan–Meier survival analysis of mice injected with c-Myc transduced lin− bone marrow cells isolated from AES wildtype (Aes+/+) or straight knockout (Aes−/−) mice. N = 12 mice each group, log-rank test. (i) Cell cycle analysis of U937 cells expressing empty vector (MIG) or AML1-ETO. One of n = 3 independent experiment is shown. (j) Cell cycle distribution of control (MIG) or AML1-ETO-expressing (AE) U937 cells. (k) Scatterplot shows snoRNA expression in control (MIG) or AML-ETO-expressing U937 cells determined by snoRNA-seq. (l) RT-PCR analysis shows expression of snoRNAs in control (MIG) or AML-ETO-expressing (AE) U937 cells. (m) Pseudouridylation ratios were analyzed at 72 sites in 18S and 28S rRNA in human primary AML1-ETO (n = 5) and CD34+ cell samples (n = 5). (n) Heatmap shows significant snoRNAs associated with chemotherapy response. Error bars, mean ± s.d. of n = 3 independent experiments unless otherwise indicated. Statistical source data for Supplementary Fig. 6j are provided in Supplementary Table 7.
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Zhou, F., Liu, Y., Rohde, C. et al. AML1-ETO requires enhanced C/D box snoRNA/RNP formation to induce self-renewal and leukaemia. Nat Cell Biol 19, 844–855 (2017). https://doi.org/10.1038/ncb3563
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