Title: Transcriptional memory of cells of origin overrides β‐catenin requirement of MLL cancer stem cells
Abstract: Article4 October 2017free access Source DataTransparent process Transcriptional memory of cells of origin overrides β-catenin requirement of MLL cancer stem cells Teerapong Siriboonpiputtana Department of Haematological Medicine, Division of Cancer Studies, Leukemia and Stem Cell Biology Team, King's College London, London, UK Search for more papers by this author Bernd B Zeisig Department of Haematological Medicine, Division of Cancer Studies, Leukemia and Stem Cell Biology Team, King's College London, London, UK Search for more papers by this author Magdalena Zarowiecki Department of Haematological Medicine, Division of Cancer Studies, Leukemia and Stem Cell Biology Team, King's College London, London, UK Search for more papers by this author Tsz Kan Fung Department of Haematological Medicine, Division of Cancer Studies, Leukemia and Stem Cell Biology Team, King's College London, London, UK Search for more papers by this author Maria Mallardo Department of Haematological Medicine, Division of Cancer Studies, Leukemia and Stem Cell Biology Team, King's College London, London, UK Search for more papers by this author Chiou-Tsun Tsai Department of Haematological Medicine, Division of Cancer Studies, Leukemia and Stem Cell Biology Team, King's College London, London, UK Search for more papers by this author Priscilla Nga Ieng Lau Department of Haematological Medicine, Division of Cancer Studies, Leukemia and Stem Cell Biology Team, King's College London, London, UK Search for more papers by this author Quoc Chinh Hoang Department of Haematological Medicine, Division of Cancer Studies, Leukemia and Stem Cell Biology Team, King's College London, London, UK Search for more papers by this author Pedro Veiga Department of Haematological Medicine, Division of Cancer Studies, Leukemia and Stem Cell Biology Team, King's College London, London, UK Search for more papers by this author Jo Barnes Department of Haematological Medicine, Division of Cancer Studies, Leukemia and Stem Cell Biology Team, King's College London, London, UK Search for more papers by this author Claire Lynn Department of Haematological Medicine, Division of Cancer Studies, Leukemia and Stem Cell Biology Team, King's College London, London, UK Search for more papers by this author Amanda Wilson Department of Haematological Medicine, Division of Cancer Studies, Leukemia and Stem Cell Biology Team, King's College London, London, UK Search for more papers by this author Boris Lenhard Faculty of Medicine, Institute of Clinical Sciences, Imperial College London, London, UK Computational Regulatory Genomics, MRC London Institute of Medical Sciences, London, UK Sars International Centre for Marine Molecular Biology, University of Bergen, Bergen, Norway Search for more papers by this author Chi Wai Eric So Corresponding Author [email protected] orcid.org/0000-0002-4117-0036 Department of Haematological Medicine, Division of Cancer Studies, Leukemia and Stem Cell Biology Team, King's College London, London, UK Search for more papers by this author Teerapong Siriboonpiputtana Department of Haematological Medicine, Division of Cancer Studies, Leukemia and Stem Cell Biology Team, King's College London, London, UK Search for more papers by this author Bernd B Zeisig Department of Haematological Medicine, Division of Cancer Studies, Leukemia and Stem Cell Biology Team, King's College London, London, UK Search for more papers by this author Magdalena Zarowiecki Department of Haematological Medicine, Division of Cancer Studies, Leukemia and Stem Cell Biology Team, King's College London, London, UK Search for more papers by this author Tsz Kan Fung Department of Haematological Medicine, Division of Cancer Studies, Leukemia and Stem Cell Biology Team, King's College London, London, UK Search for more papers by this author Maria Mallardo Department of Haematological Medicine, Division of Cancer Studies, Leukemia and Stem Cell Biology Team, King's College London, London, UK Search for more papers by this author Chiou-Tsun Tsai Department of Haematological Medicine, Division of Cancer Studies, Leukemia and Stem Cell Biology Team, King's College London, London, UK Search for more papers by this author Priscilla Nga Ieng Lau Department of Haematological Medicine, Division of Cancer Studies, Leukemia and Stem Cell Biology Team, King's College London, London, UK Search for more papers by this author Quoc Chinh Hoang Department of Haematological Medicine, Division of Cancer Studies, Leukemia and Stem Cell Biology Team, King's College London, London, UK Search for more papers by this author Pedro Veiga Department of Haematological Medicine, Division of Cancer Studies, Leukemia and Stem Cell Biology Team, King's College London, London, UK Search for more papers by this author Jo Barnes Department of Haematological Medicine, Division of Cancer Studies, Leukemia and Stem Cell Biology Team, King's College London, London, UK Search for more papers by this author Claire Lynn Department of Haematological Medicine, Division of Cancer Studies, Leukemia and Stem Cell Biology Team, King's College London, London, UK Search for more papers by this author Amanda Wilson Department of Haematological Medicine, Division of Cancer Studies, Leukemia and Stem Cell Biology Team, King's College London, London, UK Search for more papers by this author Boris Lenhard Faculty of Medicine, Institute of Clinical Sciences, Imperial College London, London, UK Computational Regulatory Genomics, MRC London Institute of Medical Sciences, London, UK Sars International Centre for Marine Molecular Biology, University of Bergen, Bergen, Norway Search for more papers by this author Chi Wai Eric So Corresponding Author [email protected] orcid.org/0000-0002-4117-0036 Department of Haematological Medicine, Division of Cancer Studies, Leukemia and Stem Cell Biology Team, King's College London, London, UK Search for more papers by this author Author Information Teerapong Siriboonpiputtana1,†,‡, Bernd B Zeisig1,‡, Magdalena Zarowiecki1,†, Tsz Kan Fung1, Maria Mallardo1, Chiou-Tsun Tsai1, Priscilla Nga Ieng Lau1, Quoc Chinh Hoang1,†, Pedro Veiga1, Jo Barnes1, Claire Lynn1, Amanda Wilson1, Boris Lenhard2,3,4 and Chi Wai Eric So *,1 1Department of Haematological Medicine, Division of Cancer Studies, Leukemia and Stem Cell Biology Team, King's College London, London, UK 2Faculty of Medicine, Institute of Clinical Sciences, Imperial College London, London, UK 3Computational Regulatory Genomics, MRC London Institute of Medical Sciences, London, UK 4Sars International Centre for Marine Molecular Biology, University of Bergen, Bergen, Norway †Present address: Department of Pathology, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand †Present address: Department of Cancer Research, Vinmec Research Institute for Stem Cells and Gene Technology, Hà Nội, Viêtnam †Present address: The Institute of Cancer Research, Sutton, Surrey, UK ‡These authors contributed equally to this work *Corresponding author. Tel: +44 2078485888; E-mail: [email protected] EMBO J (2017)36:3139-3155https://doi.org/10.15252/embj.201797994 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract While β-catenin has been demonstrated as an essential molecule and therapeutic target for various cancer stem cells (CSCs) including those driven by MLL fusions, here we show that transcriptional memory from cells of origin predicts AML patient survival and allows β-catenin-independent transformation in MLL-CSCs derived from hematopoietic stem cell (HSC)-enriched LSK population but not myeloid–granulocyte progenitors. Mechanistically, β-catenin regulates expression of downstream targets of a key transcriptional memory gene, Hoxa9 that is highly enriched in LSK-derived MLL-CSCs and helps sustain leukemic self-renewal. Suppression of Hoxa9 sensitizes LSK-derived MLL-CSCs to β-catenin inhibition resulting in abolishment of CSC transcriptional program and transformation ability. In addition, further molecular and functional analyses identified Prmt1 as a key common downstream mediator for β-catenin/Hoxa9 functions in LSK-derived MLL-CSCs. Together, these findings not only uncover an unexpectedly important role of cells of origin transcriptional memory in regulating CSC self-renewal, but also reveal a novel molecular network mediated by β-catenin/Hoxa9/Prmt1 in governing leukemic self-renewal. Synopsis Genetic and functional in vivo analyses reveal a novel molecular network mediated by Hoxa9/Prmt1 that can bypass β-catenin dependent leukemic self-renewal. Deletion of β-catenin abolishes leukemic self-renewal and maintenance of granulocyte/ macrophage progenitor (GMP)-derived but not hematopoietic stem/progenitor cell (LSK)-derived cancer stem cells. Transcriptional memory signatures of cells of origin are retained after transformation and predict AML patient survival. Hoxa9 self-renewal factor resembles β-catenin's function in the cells of origin-specific development of MLL leukemia. Prmt1 histone methyltransferase is a common target of β-catenin and Hoxa9 in LSK-derived MLL-ENL cells. Suppression of Prmt1 sensitizes LSK-MLL cells to β-catenin- and Hoxa9- inhibition. Introduction Self-renewal is a critical feature of stem cells, but is diminished upon differentiation into their progenitors. During the differentiation process, gene expression programs responsible for self-renewal are down-regulated and frequently replaced by lineage-specific transcriptional programs. Increasing evidence suggests that genes involved in promoting normal stem cell self-renewal are commonly hijacked in cancer stem cells (CSCs), which are believed to sustain the disease and be responsible for relapse of various cancers including acute myeloid leukemia (AML; Zeisig et al, 2012; Fung et al, 2013). Among them, one of the most striking molecules is β-catenin, which is required for leukemic stem cells (LSCs) driven by MLL-fusion proteins or their downstream targets, Meis1/Hoxa9 (Wang et al, 2010; Yeung et al, 2010). Suppression of β-catenin reversed MLL-LSC to pre-LSC stage (Yeung et al, 2010), and its complete inactivation prevented development of leukemia driven by MLL fusions or Meis1/Hoxa9 (Wang et al, 2010; Yeung et al, 2010). While β-catenin is critical for embryonic and a number of somatic stem cells including fetal hematopoietic stem cells (HSCs; Zhao et al, 2007; Malhotra & Kincade, 2009), it is largely dispensable for adult HSCs, which can function normally when β-catenin alone or even together with γ-catenin is deleted (Cobas et al, 2004; Jeannet et al, 2008; Koch et al, 2008), highlighting the therapeutic potentials of targeting β-catenin for eradication of LSCs. In spite of the fundamental difference between stem cells and their progenitors in self-renewal ability, we and others have shown that phenotypically and genetically indistinguishable cancers, including MLL-rearranged leukemia, can arise from not only stem cells, but also their immediate downstream short-lived progenitors with distinctive transcriptional programs (Cozzio et al, 2003; So et al, 2003; Huntly et al, 2004; Krivtsov et al, 2006; Visvader, 2011; Blanpain, 2013). Consistently, gene expression signatures associated with stem cells and progenitors correlate with different clinical outcomes in AML (Eppert et al, 2011; Krivtsov et al, 2013). MLL leukemia derived from mouse HSC-enriched Lin−Sca-1+c-kit+ (LSK) populations can be more aggressive and less responsive to standard chemotherapy than those derived from granulocyte–myeloid progenitors (GMPs; Krivtsov et al, 2013). In line with this, a more recent study also reveals that HSC-derived leukemia drives an invasive EMT-related gene expression program, which may partly account for the aggressive nature of the disease (Stavropoulou et al, 2016). In spite of these recent evidences indicating the importance of cancer cells of origin in disease pathogenesis, we still do not know whether and how they may govern the utilization of molecular pathways for self-renewal, which is a defining feature of CSC and has been a major focus for development of effective cancer therapeutics in the past decade. Given the important function of β-catenin in CSC biology, we carried out detailed functional biology and molecular studies examining β-catenin requirement in MLL-CSCs originated from different cells of origin. Here, we report that transcriptional memory from cells of origin that robustly predicts AML patient survival can govern and help to override the β-catenin requirement in MLL-CSCs. Mechanistically, we identify a novel transcriptional network mediated by Hoxa9/Prmt1 in sustaining leukemic self-renewal in the absence of β-catenin in HSC-derived MLL-CSCs. These findings reveal previously unrecognized functions and molecular networks from cancer cells of origin that allow override of β-catenin-dependent leukemic self-renewal, adding a new dimension to the ongoing research efforts in developing effective therapeutics for eradication of CSCs. Results LSK- but not GMP-derived MLL-CSCs can override β-catenin requirements for leukemic self-renewal To determine the functional requirement of β-catenin in MLL-CSCs derived from different cells of origin, we employed the previously described retroviral transduction/transformation assays (RTTA; Yeung & So, 2009; Zeisig & So, 2009) using HSC-enriched Lin−Sca-1+c-Kit+ population (LSK), granulocyte/macrophage progenitors (GMPs), and control c-Kit+ cells (mixed population consisting of mostly progenitors) from Ctnnb1fl/fl CreER (Brault et al, 2001) conditional knockout mice (Fig 1A, Appendix Fig S1A and B). Consistent with previous findings (Yeung et al, 2010), β-catenin was not required for MLL-ENL in vitro transformation of c-Kit+ cells (Appendix Fig S1C–E), but essential for in vivo development of CSCs (Appendix Fig S1F). Similarly, MLL-ENL could transform LSK and GMPs independently of β-catenin in vitro and formed compact colonies with early myeloid phenotypes (Fig 1B–D, Appendix Fig S1G and H). However, while β-catenin deletion in GMP-MLL-ENL abolished its leukemogenic potentials in vivo (Fig 1E), β-catenin deletion had little impact on LSK-MLL-ENL, which could still induce leukemia with indistinguishable phenotypes and largely similar latencies as compared with the wild-type controls (Fig 1F–H). More importantly, LSK-MLL-ENL β-catenin-deficient cells could competently induce AML upon secondary transplant (Fig 1F–H, Appendix Fig S1I and J), which readout the self-renewal property of CSCs and indicate the largely uncompromised CSC property in the absence of β-catenin in LSK-derived but not GMP-derived MLL-CSCs. The results could also be reproduced using a different MLL-ENL construct carrying the minimal transformation domain (Slany et al, 1998) and MLL-AF9 (Smith et al, 2011), and were not due to different expression levels of the MLL fusions in these populations (Appendix Fig S1K–M). Figure 1. MLL-ENL leukemic stem cells derived from LSK or GMP populations have contrasting functional requirements of β-catenin for their initiation and maintenance of disease A. Schematic overview of the experimental procedures. Keys and color codes in the legend box indicate the cells of origin and the β-catenin status of MLL-ENL-transduced cells in the following experiments (B–K). B. Colony numbers in serial replating assay of the different MLL-ENL-transduced cells. Data are represented as mean ± SD (n = 4). C. PCR validation of Ctnnb1 deletion on genomic DNA isolated from the indicated MLL-ENL-transduced GMP and LSK cells. L, 100-bp ladder; W, wild-type control; F, Ctnnb1-floxed allele; D, Ctnnb1-deleted allele; N, negative control. D. Cell lysates from indicated MLL-ENL-transduced GMP and LSK cells after the fourth round of plating were blotted with anti-β-catenin (top) and anti-actin (bottom) antibodies. E, F. Kaplan–Meier survival curves of indicated MLL-ENL-transduced cells transplanted into primary recipient (solid lines) and secondary recipient mice (dotted lines). N = 10 mice per group were used in primary transplants (solid lines) in (E). N = 15 mice were used for Ctnnb1fl/fl (blue solid line), and n = 10 mice were used for Ctnnb1del/del (green solid line) in (F). For all secondary transplants (dotted lines), n = 5 mice were used per group. G. PCR validation of Ctnnb1 deletion on genomic DNA isolated from leukemic cells. L, 100-bp ladder; W, wild-type control; F, Ctnnb1-floxed allele; D, Ctnnb1-deleted allele; N, negative control. H. Cell lysates from indicated leukemic mice were blotted with anti-β-catenin (top) and anti-actin (bottom) antibodies. I. Percentage of CD45.2+ donor cells in the bone marrow of recipient mice at the indicated time points post-transplantation of the indicated GMP-MLL-ENL- and LSK-MLL-ENL-transduced cells. Data are represented as mean ± SD. N = 4, two-tailed t-test was performed. J, K. Kaplan–Meier survival curves of secondary transplanted GMP-MLL-ENL (J) and LSK-MLL-ENL (K) primary leukemia bone marrow cells treated with DMSO as floxed controls or tamoxifen for β-catenin deletion prior to secondary transplantation (n = 3 per group). Data information: See also Appendix Fig S1. Source data are available online for this figure. Source Data for Figure 1D [embj201797994-sup-0007-SDataFig1.png] Download figure Download PowerPoint To gain further insights into the role of β-catenin in disease development, we followed the in vivo kinetics of the MLL-transformed cells derived from different cellular origins with or without β-catenin. The results showed a similar percentage of engraftment across all samples of different cellular origins and genotypes at 16 and 96 h post-transplant (Fig 1I), suggesting that β-catenin deletion did not significantly affect homing and early in vivo proliferation abilities. In contrast to LSK-derived MLL-CSCs that continued to expand and induced leukemia in the absence of β-catenin, the expansion of GMP-MLL-ENL Ctnnb1del/del cells slowed down at 15 days and were gradually lost in vivo over a 4-month period (Fig 1I), consistent with an impaired self-renewal. β-Catenin is also not required for leukemia maintenance by LSK-derived MLL-CSCs To explore the function of β-catenin in the maintenance of leukemia derived from different origin-specific CSCs, full-blown leukemic cells harvested from primary leukemic mice carrying Ctnnb1-floxed alleles were then treated with either EtOH or tamoxifen prior to transplantation into secondary recipients (Fig 1A). As expected, both EtOH-treated LSK- and GMP-derived MLL leukemic cells could competently induce leukemia. Inactivation of β-catenin in GMP-MLL-ENL totally abolished their leukemogenic potential (Fig 1J), while β-catenin-deficient LSK-MLL-ENL still efficiently induced leukemia and even with a slightly shorter latency in secondary recipients (Fig 1K). In contrast to the absolute requirement of β-catenin for development of various LSCs (Zhao et al, 2007; Wang et al, 2010; Yeung et al, 2010), the finding of largely dispensable function of β-catenin in LSK-derived MLL-CSCs reveals an unexpected and previously unrecognized role of cells of origin in governing leukemic self-renewal for both cancer initiation and maintenance. Genomic variations do not account for contrasting β-catenin dependence in MLL-CSCs from different cells of origin To assess whether the observed cell type-specific differences could be explained by random genetic changes associated with particular cell types, nucleotide variations were called from all actively transcribed genes using RNA-Seq. Among 23,766,084 high-quality base pairs (depth ≥ 10 and quality ≥ 30), the vast majority of sites (23,747,763) were invariant in all our primary cell lines, and identical to the reference genome GRCm38, while a very small proportion, 4,663 SNPs (single nucleotide polymorphisms), were invariant and different from GRCm38. In-depth variance analysis revealed that the difference between the LSK-MLL-ENL and GMP-MLL-ENL cells was not larger than between normal cells (Fig 2A, Dataset EV1A–C), and there were no fixed differences between the samples that could have caused the observed phenotypic differences. To further profile the non-coding genomes, whole-genome sequence analysis on LSK-MLL-ENL and GMP-MLL-ENL cells covering 918,583,518 high-quality base pairs revealed 917,764,811 invariant sites, and a very small number of variants in the samples; 39,846 variants were different between the two biological replicates (mice), and only 17,225 were found different between the two cell types (Fig 2B, Dataset EV1C). Interestingly, the distribution of SNPs differing between cell types or mice was comparable for both comparisons in non-coding and coding regions (Appendix Fig S2A). Consistently, SNPs in coding regions occurred in similar proportions in exon, intron, and UTR regions in both comparisons (Appendix Fig S2B). Moreover, the number of derived SNPs in GMP-MLL-ENL (9,309) was higher than those in LSK-MLL-ENL cells (7,916) that exhibited β-catenin-independent phenotypes (Dataset EV1C). Additionally, we examined and compared the copy number variances (CNVs) between the LSK-MLL-ENL and GMP-MLL-ENL genomes (Fig 2C and D). As a result, we observed very little CNVs in both genomes. There is only a very small genomic region of about 1 kb showing CNV between same cell types (i.e., LSK-MLL-ENL vs. GMP-MLL-ENL), whereas multiple chromosomal regions of about 50 kb exhibiting CNV were detected between samples (i.e., mouse Exp60 vs. mouse Exp69 in Dataset EV1D). Importantly, there is also no known coding gene in the 1-kb CNV region shared between cell types (Fig 2D), consistently indicating insignificant genomic difference between LSK-MLL-ENL vs. GMP-MLL-ENL cells, which could account for their contrasting β-catenin dependence. Together, these data reveal relatively few genomic variation in LSK-MLL-ENL compared with GMP-MLL-ENL and the controls, suggesting that non-genomic influence from the cells of origin can be a key factor in governing the self-renewal property of genetically and phenotypically indistinguishable cancers. Figure 2. Cells of origin transcriptional memory predicts survival in AML patients A, B. Number of identified genomic variants in indicated MLL-ENL transformed cells using RNA-Seq (A) and genomic sequencing (B). A two-tailed t-test was performed in (A). LSK, wild-type LSK cells; GMP, wild-type GMP cells. C, D. Manhattan plots indicating estimated length of CNVs (C) or number of genes in CNV areas (D) on the y-axis in the respective chromosomal positions shown in the x-axis. E. MA-plots showing the log2-fold gene expression changes in the normal (left panel) and MLL-ENL transformed (right panel) cells as indicated. F. Transcriptional memory signature; the overlap of differentially expressed genes in GMP-LSK in normal vs. MLL-ENL transformed cells is significantly enriched using a hypergeometric test. G. Survival differences between patients clustered using transcriptional memory signatures with a log-rank test. Data information: See also Appendix Fig S2. Download figure Download PowerPoint Transcriptional memory from cells of origin governs self-renewal pathways and predicts AML patient survival As self-renewal in normal stem cells is maintained by specific transcriptional programs, we hypothesized that the transcriptional memories from LSK and GMPs would be partially preserved even after transformation, resulting in transcriptional and functional differences observed in the respective CSCs (Zeisig et al, 2012). Thus, RNA-seq analyses of normal LSK, GMPs, and their MLL-ENL-transformed counterparts were carried out. There were, as expected, large transcriptional differences between normal LSK and GMPs with 4,768 significantly differentially expressed genes, including Hox genes, Meis1 and Evi1 (Fig 2E, Appendix Fig S2C, Dataset EV2A and B), while overall gene expression differences between cells of different origin decreased after MLL-ENL transformation (Fig 2E, Appendix Fig S2D). Nevertheless, a significantly larger than expected by chance number of genes remained differentially expressed between LSK and GMP even after transformation (Fig 2F, Appendix Fig S2C, Dataset EV2C), indicating the presence of “transcriptional memory” retained from the cells of origin. Toppgene functional annotation revealed genes associated with AML are consistently present in both signatures (Appendix Fig S2F–I, Dataset EV2D). To further investigate the relevance of this cells of origin transcriptional memory gene signature in human leukemia, we employed it to stratify 1,290 human AML patients from multiple independent centers (Valk et al, 2004, Raponi et al, 2007, 2008; Metzeler et al, 2008; Wouters et al, 2009; Cancer Genome Atlas Research N, 2013; Dataset EV2E). AML patients with LSK-transcriptional memory signature had much worse prognosis with a median survival 14.5 months as compared to patients with GMP-transcriptional memory signature with median survival 22.7 months (Fig 2G), even though the two groups had similar WBC count (means = 40.3, 45.3, t-test P = 0.30), age distributions (means = 48.0, 50,0, t-test P = 0.07), and cytogenetic risk (cytogenetic risk (1/2/3) = 71/199/85, 82/165/66, chi-square test P = 0.16). When compared with the previously identified human HSC signature (Eppert et al, 2011) and MLL leukemic-GMP (LGMP) signatures from different cells of origin (Krivtsov et al, 2013), the current transcriptional memory signature represents a stronger predictor to stratify patients into different prognostic subgroups based on both resultant median survivals and P-values (Appendix Fig S2J). Moreover, multivariate analyses consistently resulted in significant Cox proportional hazards ratios > 1; z-score < 0.1 with both human HSC signature and transcriptional memory signature (Dataset EV2F). Together, these data indicate functional and pathological relevance of the newfound cells of origin transcriptional memory in governing human cancer biology beyond known cytogenetic/genetic risk factors. Hoxa9 as a key transcriptional memory gene phenocopies β-catenin function in development of origin-specific MLL leukemia Given the largely dispensable function of β-catenin in LSK-derived MLL-CSCs, we hypothesize that some self-renewal programs from normal stem cells may persist after transformation, and can sustain self-renewal in the absence of β-catenin. In the transcriptional memory signature, there were a small number of self-renewal genes such as Hoxa9, Hoxa10, and Meis1 (Fig 2E and F), which are known downstream targets of MLL fusions (Milne et al, 2002; Zeisig et al, 2004; Huang et al, 2012), indicating that their degrees of activation are in part also determined by the cellular origins. Strikingly, RNA-sequencing analysis on MLL-ENL transformed cells upon β-catenin inactivation revealed a specific up-regulation of targets genes suppressed by Hoxa9/Meis1, suggesting a critical function of β-catenin in regulation Hox/Meis1 axis for leukemic self-renewal (Fig 3A, Dataset EV3A–C). Moreover, various stem cell-related gene sets were positively enriched in β-catenin-deleted LSK-MLL-ENL cells as compared with β-catenin-deleted GMP-MLL-ENL (Fig 3B, Appendix Fig S3A, Dataset EV3C). β-catenin-deleted LSK-MLL-ENL not only expressed higher levels of Hoxa9 (Fig 3C and D) but also showed a negative enrichment for genes repressed by Hoxa9 (Fig 3E, Dataset EV3C). Together, the data consistently suggest a potential Hoxa9 complementation function in replacing β-catenin in LSK-derived MLL-CSCs. Figure 3. Key transcriptional memory gene Hoxa9 may help to overcome β-catenin-dependent transformation in LSK-derived MLL-CSCs A. Gene set enrichment analysis (GSEA) shows “Targets of Hoxa9/Meis1, down” (Hess et al, 2006) for the indicated comparison. B. Log10-fold FDR q-values of the indicated gene sets positively enriched in β-catenin-deleted LSK-MLL-ENL compared to β-catenin-deleted GMP-MLL-ENL. C–E. RNA-seq log2-fold change of key self-renewal genes (C), RT–qPCR validation of Hox/Meis1 expression represented as mean ± SD of three independent experiments. Two-tailed t-test was performed (D), and GSEA showing “Hoxa9_dn.v1_up” (Faber et al, 2009) for the β-catenin-deleted LSK-MLL-ENL to β-catenin-deleted GMP-MLL-ENL comparison (E). F. Colony numbers in serial replating assay of indicated MLL-ENL-transduced cells. Data are represented as mean ± SD (n = 3). G. Kaplan–Meier survival curve of mice transplanted with Hoxa9−/− (n = 18) or WT LSK-MLL-ENL (n = 13) transformed cells (solid lines) and secondary recipient mice (n = 5 for Hoxa9−/− and n = 4 for wt, dotted lines) as indicated. Comparisons between Hoxa9 WT and Hoxa9−/− were not significantly different (ns). Data information: See also Appendix Fig S3. Download figure Download PowerPoint Similar to β-catenin, activation of Hoxa9 enhances self-renewal, while its deletion does not have significant impact on HSCs (So et al, 2004; Lawrence et al, 2005; Smith et al, 2011), consistent with the existence of multiple complementary self-renewal pathways in HSCs. We hypothesize whether there is indeed a functional complementation between Hoxa9 and β-catenin, Hoxa9 requirement for MLL transformation may also be influenced by cells of origin. To address this issue, we used purified hematopoietic populations from Hoxa9-knockout mice for RTTA. While MLL-E