MLL-AF9 Expression in Hematopoietic Stem Cells Drives ... - Cell Press

Download PDF
29 downloads 0 Views 7MB Size Report
Comment
Jun 23, 2016 - chromatin immunoprecipitation (ChIP)-PCR assays on LT- ..... Armstrong, S.A., Staunton, J.E., Silverman, L.B., Pieters, R., den Boer, M.L.,.
Article

MLL-AF9 Expression in Hematopoietic Stem Cells Drives a Highly Invasive AML Expressing EMTRelated Genes Linked to Poor Outcome Highlights d

Generation of an inducible and reversible mouse model for MLL-AF9-driven AML

d

The cell of origin controls aggressiveness and outcome of MLL-AF9-mediated AML

Authors Vaia Stavropoulou, Susanne Kaspar, Laurent Brault, ..., Peter J.M. Valk, Antoine H.F.M. Peters, Juerg Schwaller

Correspondence

d

The EMT transcription factor ZEB1 controls AML cell migration and invasion

[email protected] (A.H.F.M.P.), [email protected] (J.S.)

d

Expression of multiple EMT-related genes is associated with poor outcome in human AML

In Brief Stavropoulou et al. show that MLL-AF9 expression in mouse long-term hematopoietic stem cells causes invasive, chemoresistant acute myeloid leukemia (AML) that expresses genes related to epithelial-mesenchymal transition (EMT). Expression of EMTrelated genes in human AML is associated with poor patient survival.

Accession Numbers GSE65384 E-MTAB-3444

Stavropoulou et al., 2016, Cancer Cell 30, 43–58 July 11, 2016 ª 2016 Elsevier Inc. http://dx.doi.org/10.1016/j.ccell.2016.05.011

Cancer Cell

Article MLL-AF9 Expression in Hematopoietic Stem Cells Drives a Highly Invasive AML Expressing EMT-Related Genes Linked to Poor Outcome Vaia Stavropoulou,1 Susanne Kaspar,2,3 Laurent Brault,1 Mathijs A. Sanders,4 Sabine Juge,1 Stefano Morettini,2 Alexandar Tzankov,5 Michelina Iacovino,6 I-Jun Lau,7 Thomas A. Milne,7 He´le`ne Royo,2 Michael Kyba,8 Peter J.M. Valk,4 Antoine H.F.M. Peters,2,3,9,* and Juerg Schwaller1,9,* 1Department

of Biomedicine, University Children’s Hospital (UKBB), University of Basel, 4031 Basel, Switzerland Miescher Institute for Biomedical Research (FMI), Maulbeerstrasse 66, 4058 Basel, Switzerland 3Faculty of Sciences, University of Basel, 4031 Basel, Switzerland 4Department of Hematology, Erasmus University Medical Center, 3015 CE Rotterdam, the Netherlands 5Institute for Pathology, University Hospital Basel, 4031 Basel, Switzerland 6Department of Pediatrics, LA Biomedical Research Institute, Torrance, CA 90502, USA 7MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, NIHR Oxford Biomedical Research Centre Programme, University of Oxford, Oxford OX3 9DS, UK 8Department of Pediatrics, Lillehei Heart Institute, University of Minnesota, Minneapolis, MN 55455, USA 9Co-senior author *Correspondence: [email protected] (A.H.F.M.P.), [email protected] (J.S.) http://dx.doi.org/10.1016/j.ccell.2016.05.011 2Friedrich

SUMMARY

To address the impact of cellular origin on acute myeloid leukemia (AML), we generated an inducible transgenic mouse model for MLL-AF9-driven leukemia. MLL-AF9 expression in long-term hematopoietic stem cells (LT-HSC) in vitro resulted in dispersed clonogenic growth and expression of genes involved in migration and invasion. In vivo, 20% LT-HSC-derived AML were particularly aggressive with extensive tissue infiltration, chemoresistance, and expressed genes related to epithelial-mesenchymal transition (EMT) in solid cancers. Knockdown of the EMT regulator ZEB1 significantly reduced leukemic blast invasion. By classifying mouse and human leukemias according to Evi1/EVI1 and Erg/ERG expression, reflecting aggressiveness and cell of origin, and performing comparative transcriptomics, we identified several EMT-related genes that were significantly associated with poor overall survival of AML patients. INTRODUCTION Acute myeloid leukemia (AML) is a clinically and genetically heterogeneous disease. In contrast to solid cancers, relatively few cooperating genetic alterations were identified in AML (Miller et al., 2013). Chromosomal translocations leading to fusions of the mixed lineage leukemia gene (MLL1 or KMT2A) are recurrent alterations associated with aggressive pediatric and adult de novo and therapy-related acute leukemia (Muntean and Hess, 2012). The t(9; 11) (p22; q23) reciprocal translocation results in the expression of MLL-AF9 fusion gene and myelo-monoblastic

AML (Meyer et al., 2013) associated with extramedullary tumor infiltration, frequent relapses, and poor survival (Tamai and Inokuchi, 2010). Next to the age and health status of patients, coexisting mutations in FLT3 or in the RAS pathway may contribute to the aggressiveness of MLL-fusion related AML (Lavallee et al., 2015). Whether the cellular origin of the leukemic transformation event contributes to the wide heterogeneity in AML patients is under investigation. Previous work suggested that certain stages of the hematopoietic hierarchy are particularly permissive for distinct leukemia-inducing alterations. For example,

Significance The cellular origin is a critical determinant of the biology of AML. Expression of MLL-AF9 in mouse LT-HSC but not in more committed progenitor cells resulted in an invasive and chemoresistant AML expressing EMT-related genes akin to solid tumors. Cross-species comparative expression profiling focusing on origin and aggressiveness identified many genes with important functions in migration and invasion with significant association to poor overall survival of AML patients. Our data indicate that aggressive AML can result from activation of a driver fusion oncogene in mouse HSC and possibly in humans as well. In addition, our work led to the identification of origin-related genes as potential biomarkers or targets for future personalized therapeutic strategies. Cancer Cell 30, 43–58, July 11, 2016 ª 2016 Elsevier Inc. 43

the BCR-ABL1 fusion, associated with chronic myeloid leukemia, initiated the disease only in hematopoietic stem cells (HSC), whereas the AML-associated MOZ-TIF2 or MLL-AF9 fusions drove leukemia in HSC and committed progenitor cells (Huntly et al., 2004). The strong association of MLL-AF9 with myelo-monoblastic AML suggested granulocyte-macrophage progenitors (GMP) as the origin of the disease. However, MLL-AF9 was also found in a few patients with acute lymphoblastic leukemia or acute megakaryoblastic leukemia, implying that stem cells or early progenitor cells may also function as cells of origin. Transplantation of irradiated mice with lineage-marker-negative, Sca1+, c-Kit+ (LSK) early hematopoietic progenitor cells retrovirally expressing MLL-AF9 (rMLLAF9) resulted in a more rapid disease than reconstitution with GMP expressing the same fusion (Krivtsov et al., 2013). In contrast, transplantation experiments with bone marrow (BM) of Mll-AF9 knockin mice suggested that expression of the fusion in common lymphoid progenitors (CLP) rather than in LSK or GMP results in a more aggressive disease associated with distinct origin-related gene-expression profiles (Chen et al., 2008). Together, these studies suggested that cellular origin might determine the biology of AML. We carried out this study to address the impact of cellular origin on AML. RESULTS A Reversible Transgenic Mouse Model of MLL-AF9-Induced AML The transgenic mouse model for inducible expression of the human MLL-AF9 (iMLL-AF9) is based on the reversed tetracyclinecontrolled transactivator (rtTA) system (Iacovino et al., 2011) (Figure 1A). Increasing doxycycline (DOX) amounts induced a dose-dependent expression of iMLL-AF9 in in vitro cultured BM cells, reaching maximal levels at 2 mg/ml (Figure 1B). iMLLAF9 expression was 10- to 20-fold lower than rMLL-AF9 in vitro and in vivo (Figures 1B and S1A). iMLL-AF9 expression supported the expansion of BM cells in liquid cultures in a DOX dose-dependent manner (Figures S1B and S1C) and provided self-renewal capacity by efficient replating in methylcellulose (MC) (Figure 1C). With DOX, most colonies had an immature, round, and dense morphology (known as type I colonies) and only few were more differentiated (types II and III) (Somervaille and Cleary, 2006) (Figure 1D). The cells expressed high levels of Gr-1, Mac-1, and c-Kit surface markers, similar to rMLL-AF9 cells (Figure 1D and data not shown). Upon DOX removal, iMLL-AF9 expression rapidly decreased to background levels (Figure 1B) and the cells differentiated into monocytes and macrophages, reflected by >30% increased Mac-1 and Gr-1 expression (Figure 1D and data not shown). Next, we exposed transgenic mice to different DOX doses and performed primary and secondary BM transplantations (BMT) (Figure 1E). Mice receiving 1.2 mg, but not 0.6 mg, of DOX per day developed AML with elevated white blood cell count (WBC), presence of mono- and myeloblasts in the peripheral blood, and hepatosplenomegaly (median latency: 118 ± 22.0 days) (Figures 1F–1H and Table S1). A higher dose of 3.2 mg/day of DOX resulted in earlier symptoms, yet lower tumor burden and WBC, suggesting potential toxicity (Figures 1G and 1H; Table S1). Histologic analysis revealed infiltration of 44 Cancer Cell 30, 43–58, July 11, 2016

leukemic cells in many organs with occasional formation of extramedullary tumors (Figure S1D). The blasts expressed c-Kit, Gr-1, and Mac-1 (Figure S1E). The disease was also induced by BM reconstitution. Transplantation of 106 BM cells from non-induced naive iMLL-AF9 mice into lethally irradiated wildtype (WT) mice kept on DOX resulted in AML after a median latency of 73 ± 6.6 days. BMT of 105 leukemic blasts from diseased mice induced AML in secondary recipients after a shorter latency (32 ± 5.7 days) (Figure 1I). Culturing blasts from diseased mice without DOX resulted in rapid differentiation (Figure S1F). Continuous expression of iMLL-AF9 was required for AML maintenance in vivo, as DOX removal when 50% of the transplanted mice developed leukemia resulted in long-term survival of the remaining animals (Figure 1J). Two months after DOX removal, organs of surviving mice only had occasional small infiltrations of cells with pyknotic nuclei (Figure S1G). Thus, iMLL-AF9 expression induces AML and is required for disease maintenance in vivo. The Cellular Origin Determines MLL-AF9 Transformation In Vitro Next, we characterized the in vitro transformation of naive HSC and progenitor cells by culturing iMLL-AF9 LT-HSC and GMP in MC with DOX and growth factors (murine interleukin-3 [mIL3], human interleukin-6 [hIL6], and murine stem cell factor [mSCF]) (Figure 2A). Induction of iMLL-AF9 in GMP resulted in expansion of mostly compact type I colonies (Figure 2B), as reported for rMLL-AF9 (Somervaille and Cleary, 2006). iMLL-AF9 expression in LT-HSC also resulted in type I colony formation. However, in the first three passages, about 15% of LT-HSC-derived colonies showed a ‘‘grape-like’’ morphology not resembling any of the classical colony types (Figure 2B), here referred to as ‘‘type IV’’ (Figure 2C). Their dispersed morphology suggests a higher migratory capacity. Thus, beside committed progenitor cells iMLL-AF9 can also transform HSC and/or multipotent progenitors (MPP). Surface marker analysis showed that a subset of cells in single type IV colonies express lower levels of FcgRII/III than cells generally do in type I and type III colonies (Figures 2C and 2D). Dispersed type IV colonies grown at low density maintained their type IV growth characteristic but also converted to a type I phenotype. In contrast, type I colony-forming cells propagated only as type I colonies (Figures 2E and S2). Immunophenotypically, both LT-HSC- and GMP-derived cells expressed c-Kit, Mac-1, and Gr-1 (Figure 2F and data not shown). Upon DOX removal in medium favoring myelo- and erythropoiesis (with mIL3, hIL6, mSCF, human erythropoietin [hEPO]), LT-HSC-derived cells displayed a broad differentiation potential into several c-Kit/Mac-1 subpopulations, whereas GMP-derived cells rapidly differentiated into c-KitLow and Mac1High-expressing cells (Figure 2F). Only LT-HSC-derived cells grew in medium favoring stem cell expansion (StemSpan) (Figure 2G) and were more resistant to the cytotoxic drug cytosine arabinoside (Ara-C) than GMP-derived cells (Figure 2H). Thus, upon in vitro iMLL-AF9 transformation LT-HSC-derived cells retained some characteristics of HSC and/or MPP multipotent progenitors, whereas iMLL-AF9 GMP-derived cells resembled more rMLL-AF9 transduced BM cells.

Figure 1. iMLL-AF9: Modeling MLL-AF9-Dependent AML (A) iMLL-AF9 mice carry an rtTA cDNA in the Rosa26 locus and a human MLL-AF9 cDNA with a Tet-responsive element (TRE) in the Hprt locus. (B) iMLL-AF9 mRNA expression in BM cells grown in vitro with DOX, normalized to Gapdh mRNA level. Mean ± SD of triplicates. (C) Colony formation of iMLL-AF9 BM cells in MC without ( ), with (+), or after DOX removal (+/ ). Mean ± SD of duplicates. (D) Colony morphology (a, d), cellular morphology (Giemsa-Wright stains, b, e), and % Mac-1 and Gr-1 cell-surface expression (c, f) of iMLL-AF9 BM cells. (E) Schematic illustrating iMLL-AF9 induction in transgenic mice or after primary (1 ) and secondary (2 ) BMT. (F) Myelo-monocytic leukemic blasts on blood smears of sick mice (Giemsa-Wright stain). (G) WBC of iMLL-AF9 mice exposed to different DOX concentrations. Lines indicate the median values. (H) Kaplan-Meier survival (KM) curves of DOX-exposed iMLL-AF9 mice (n = 5 per group), median latencies ± SD. (I) KM curves of syngeneic DOX-exposed recipients after transfer of uninduced iMLL-AF9 BM (primary, n = 10) and of blasts obtained from primary BMT (secondary, n = 5). Median latencies ± SD. Control mice received naive iMLL-AF9 BM cells but no DOX (n = 5 mice). (J) KM curve after DOX removal at median latency (n = 20). See also Figure S1 and Table S1.

LT-HSC- and GMP-Derived Cells Express an iMLL-AF9-Dependent Oncogenic Program We next compared the transcriptomes of in vitro immortalized LT-HSC- and GMP-derived iMLL-AF9 cells (Figure 3A). We established multiple LT-HSC- and GMP-derived cell lines by

plating cells in MC with growth factors (mIL3, hIL6, mSCF) and DOX for three passages, expanded them for 48 or 72 hr in liquid medium with or without DOX, and profiled their transcriptomes. Principal component analysis (PCA) clearly separated LTHSC- and GMP-derived cells based on iMLL-AF9 expression Cancer Cell 30, 43–58, July 11, 2016 45

Figure 2. Cell-of-Origin-Dependent Growth of In Vitro Transformed iMLL-AF9 BM Cells (A) Generation of iMLL-AF9 LT-HSC- and GMP-derived cell cultures. (B) Representative images (left panels) and quantification (right panels) of colonies with different morphologies formed by GMP- and LT-HSC-derived iMLL-AF9 cells, respectively. Mean ± SD of three experiments. Output cell numbers were 2.4–4 3 105 cells/replating. (C) Representative images (upper panels) and FcgRII/III surface levels upon dispersal (lower panels) of types I, III, and IV colonies formed by LT-HSC-derived cells. The cutoff value defining FcgRII/IIILow and FcgRII/IIIHigh cell populations was set according to the distribution plot for type I colony cells. (D) Box plot showing the distribution of median FcgRII/III levels for the FcgRII/IIILow cell populations for 15 dispersed single colonies per type (central bar, median; lower and upper box limits, 25th and 75th percentiles, respectively; whiskers, maximum and minimum values). (E) Types of colonies obtained by replating single type I and type IV colonies (n = 10). Mean ± SD. (legend continued on next page)

46 Cancer Cell 30, 43–58, July 11, 2016

(PC1) and cellular origin (PC2) (Figure 3B). To investigate the iMLL-AF9 dependency, we determined genes that were significantly differentially expressed upon DOX removal. We found 52 genes more highly expressed with DOX, irrespective of cellular origin (Figure 3C). Among them, many were known MLL-fusion target genes such as Hoxa9, Hoxa10, Meis1, Eya1, Mef2c, Myb, and Six1 (Wang et al., 2011) (Figure 3D and Table S2). 156 genes showed increased expression upon DOX removal, including genes previously associated with myelomonocytic differentiation (Brown et al., 2006) (Figures 3C and 3E; Table S2). Ingenuity Pathway Analysis (IPA) provided terms such as ‘‘leukemogenesis’’ and ‘‘proliferation of hematopoietic progenitor cells’’ that were significantly enriched for differentially expressed genes (fold change >1.5, adjusted p value 60 days; ‘‘LTHSC-late-AML’’) (Figure 4B). The reduced latency was related to the number of transplanted cells. In two additional BMT with 1,000 LT-HSC, however, we observed a broad range of disease latencies, with about 50 days between the first and last mouse developing AML (Figure S4A), suggesting that only a subpopulation of LT-HSC-derived cells can induce the more aggressive phenotype. iMLL-AF9 induced AML in short-term HSC and common myeloid progenitors with similar kinetics as in GMP, while CLP were refractory (Figure S4B and data not shown). Histopathology showed extensive organ infiltration of leukemic blasts in all sick mice. Notably, LT-HSC-derived disease, and in particular LT-HSC-early-AML, presented with massive infiltrations with almost complete loss of organ architecture. LT-HSC-derived AML showed extensive diffuse lung infiltration, whereas in GMP-derived disease the tumor cells were mostly localized around large airways (Figure 4D). LT-HSC-early-derived AML cells formed significantly more colonies in vitro in the first round of plating, suggesting that they were enriched for leukemia-initiating cells (Figure 4E). In line with this, BMT of LT-HSC- and GMP-AML blasts in limited dilutions showed that 1,000 LT-HSC-early-AML blasts induced leukemia in all recipients but only 25% of mice transplanted with 1,000 GMP-derived blasts developed AML (Figure 4F). Since leukemia-initiating cells were proposed to be less sensitive to cytotoxic chemotherapy, we compared the effects of Ara-C. Similarly to ex vivo immortalized cells (Figure 2H), Ara-C treatment significantly prolonged the disease latency induced by BMT of GMP-AML-derived blasts, but had no significant effect on disease induced by LT-HSC-early-AML-derived blasts (Figure S4C).

(F) c-Kit and Mac-1 levels of iMLL-AF9 LT-HSC- and GMP-derived cells, cultured in MC with myelo- and erythropoiesis promoting factors (mIL3, hIL6, mSCF, hEPO), and with (+) or after removing (+/ ) DOX. (G) Growth of cells cultured in StemSpan medium with growth factors (mSCF, mFlt-3L, TPO) and + or +/ DOX. Mean ± SD of two experiments. (H) Sensitivity of LT-HSC- versus GMP-derived iMLL-AF9 cells to Ara-C (IC50 ± SE = 0.236 ± 0.04 and 0.112 ± 0.03, respectively). See also Figure S2.

Cancer Cell 30, 43–58, July 11, 2016 47

Figure 3. Origin-Dependent Gene-Expression Signatures of In Vitro Transformed iMLL-AF9 Cells (A) Schematic representation of the transcriptome analysis of iMLL-AF9 LT-HSC- and GMP-derived cells cultured in liquid medium + or +/ DOX for the indicated times. (B) PCA of iMLL-AF9 LT-HSC- and GMP-derived cells. (C) Correlation plot showing log-fold changes (lfc) between GMP + DOX versus GMP DOX 48 hr and LT-HSC + DOX versus LT-HSC DOX 72 hr. Probe sets of significantly more highly expressed in +DOX condition are in red (‘‘upregulated’’) and those of more highly expressed in DOX in blue (‘‘downregulated’’) (lfc > 0.585; adjusted p < 0.05). (D and E) Heatmaps showing expression values of upregulated (D) and downregulated (E) probe sets of in vitro cultured LT-HSC and GMP-derived cells grown with or without DOX for indicated time points with examples of differentially expressed genes. (F) Correlation plot showing mean expression values in LT-HSC- versus GMP-derived + DOX treated cells. Probe sets of more highly expressed LT-HSC-derived cells (purple) and GMP-derived cells (orange) (lfc > 0.585; adjusted p < 0.05). Probe sets showing DOX-dependent expression are in black. (G and H) Heatmaps comparing gene-expression values among different samples originating from in vitro cultures of LT-HSC-derived (G) and GMP-derived (H) cells grown with DOX. DOX-dependent genes are in bold. See also Figure S3 and Table S2.

Remarkably, as in LT-HSC-derived cultures in vitro, about 15% of the colonies established from LT-HSC-early-derived AML formed type IV colonies (Figures 2B and 4E). In contrast, LT-HSC-late and GMP-AML blasts never formed type IV colonies (Figure 4E). Thus, LT-HSC-early-AML and LT-HSC-lateAML may originate from different HSC populations, which likely relates to the timing of iMLL-AF9-mediated transformation relative to the cellular differentiation upon BMT in recipient animals. We observed that primary LT-HSC-early-AML blasts expressed lower FcgRII/III levels than primary LT-HSC-late-AML and GMP-AML blasts (Figure S4D). We developed a protocol to enrich for type IV colony-forming cells from primary LTHSC-early-AML blasts (Figure S4E). We flow-sorted for 48 Cancer Cell 30, 43–58, July 11, 2016

c-KitHighCD34HighFcgRII/IIILowGr1Low cells (referred to as population ‘‘P1’’) or c-KitHighCD34HighFcgRII/IIIHighGr1High cells (referred to as population ‘‘P2’’) and found that in MC P1 cells formed type IV and type I colonies, whereas P2 cells mainly formed type I colonies (Figure 4G). Mice transplanted with P1 cells developed a much more invasive AML reflected by extensive and diffuse infiltration of the lungs and livers, whereas recipients of P2 cells presented with more focal organ infiltration (n = 10) (Figures 4H, S4F, and S4G). P1-derived AML cells also expressed lower FcgRII/II and exhibited a higher invasion capacity in vitro than P2-derived AML cells (Figures 4H, 4I, S4H, and S4I). We corroborated these findings by transplantation of single AML-derived colonies that also led to induction of a more

(legend on next page)

Cancer Cell 30, 43–58, July 11, 2016 49

aggressive disease by LT-HSC-early-AML type IV than GMPAML type I forming cells (Figures S4J–S4L). Thus, LT-HSCearly-AML is propagated by type IV colony-forming cells in vitro, able to reproduce the aggressive phenotype in vivo, further underlining the origin-related biological differences of iMLL-AF9 AML. LT-HSC-Derived iMLL-AF9 AML Express Cell Migration and Tissue-Invasion Genes To relate origin-associated phenotypic differences to geneexpression programs, we sequenced RNA from BM cells from LT-HSC- or GMP-derived AML mice and fluorescence-activated cell sorting (FACS)-sorted WT GMP. PCA showed that activation of iMLL-AF9 results in a partially comparable expression program in LT-HSC- and GMP-derived blasts (Figure 5A), with 417 genes more highly expressed in both AML types compared with WT GMP (Figure 5B). Among them were several classical MLL-fusion targets and some potential MLL-fusion-regulated genes (Figure 5C and Table S3). PCA analysis demonstrated cell-of-origin-related expression differences between LT-HSC- and GMP-derived AML (Figure 5A) with some variations among LT-HSC-derived AML (data not shown). As in rMLL-AF9-induced AML (Bindels et al., 2012), we observed significantly higher Evi1 transcript levels in the more aggressive LT-HSC-early-AML compared with LT-HSClate- and GMP-AML (Figures 5D and 5E). To identify markers reflecting the cellular hierarchy, we searched for genes more highly expressed in all iMLL-AF9 LT-HSC-AML compared with GMPAML, and known to have a prognostic impact on AML patients. We chose Erg, which fulfilled both criteria (Figures 5D and 5E) (Rockova et al., 2011). We propose that iMLL-AF9 AML can be classified into three cellular states, integrating cellular hierarchy (LT-HSC versus GMP, reflected by different Erg expression levels) and level of aggressiveness (among LT-HSC-derived AML) as two main variables, with Evi1 as a marker for aggressiveness (Figure 5F). We then grouped the iMLL-AF9 AML samples according to Evi1 and Erg expression and performed pairwise RNA profile comparisons. As part of the hierarchy signature, 247 genes were at least 1.5-fold more highly expressed in LT-HSC-late AML compared with GMP-AML, suggesting an HSC-like cellof-origin effect (Figure S5A and Table S3). Moreover, 55 genes more highly expressed in LT-HSC-early AML than LT-HSC-

late-AML may contribute to different degrees of aggressiveness (Figure S5B and Table S3). Finally, 572 genes were more highly expressed in LT-HSC-early-AML than GMP-AML (Figure 5G and Table S3), reflecting a combination of hierarchy and aggressiveness specific effects. Consistently, the combination signature captures 77% and 49% of the hierarchy- and aggressiveness-associated genes (Figure 5H). Importantly, comparison of both LT-HSC-derived AML types relative to GMP-AML by IPA revealed highly significant enrichments for functions such as ‘‘cell movement,’’ ‘‘invasion of cell,’’ ‘‘metastasis,’’ and ‘‘cell growth and proliferation’’ (Figure 5I and Table S3). Common to these terms, we identified genes previously linked to EMT, inflammation, or invasion such as Zeb1, Tcf4, and Trps1 (Figure 5J) and those encoding components of the transforming growth factor b (TGF-b) and nuclear factor kB (NF-kB) signaling pathways implicated in EMT. Genes such as Zeb1, Evi1, Flt3, and Itga6 were similarly differentially expressed in ex vivo established iMLL-AF9 cultures (Figure 3G). We validated 51 putative origin-related genes by comparing the expression levels in early and late LT-HSC-AML- and GMP-AML-derived blasts cultured for 24 hr with or without DOX (Figures S5C and S5D). Some genes more highly expressed in LT-HSC-AML (e.g., Zeb1, Evi1, Erg) were also abundantly expressed in WT LSK (Figure 5E), whereas others such as Mmp9, Map3k9, and Kdm6b were hardly expressed in WT LSK (Figure S5C). Other genes such as Six1 and Six4 were exclusively expressed in GMP-AML-derived blasts (Figure S5D). Thus, the aggressive iMLL-AF9 LT-HSC-derived AML expressed EMTrelated genes involved in migration and invasion. To directly link origin-related expression of known (Evi1, Hoxa9) and putative (Zeb1) iMLL-AF9 targets, we performed chromatin immunoprecipitation (ChIP)-PCR assays on LTHSC-early-AML and GMP-AML cells with an antibody for the N terminus of MLL1. While we observed robust occupancy at the Hoxa9 locus in both cell types, binding to Zeb1 and Evi1 loci was only found in LT-HSC-early-AML cells (Figures S5E and S5F). Consistently, occupancy of histone H3 lysine 4 trimethylation (H3K4me3) and H3 lysine 27 trimethylation (H3K27me3), associated with transcriptional activity and repression, respectively, strongly correlated with promoter occupancy by iMLLAF9/MLL and differential expression of the three genes in the two respective AML (Figures S5G and S5H). Thus, these findings indicate direct transcriptional regulation of Evi1 and Zeb1 by

Figure 4. iMLL-AF9 Expression in LT-HSC Induces a Highly Invasive AML (A) Outline of in vivo experiments. (B) KM curves of DOX-exposed primary recipients after BMT of different numbers of naive iMLL-AF9 LT-HSC or GMP BM cells (n = 5 per group). Control mice were transplanted but never received DOX. Mean latencies ± SD per group are indicated. The statistical significances among the iMLL-AF9 LT-HSC-early and iMLL-AF9 LT-HSC-late groups were between p < 0.02 and p < 0.007 (log-rank test). (C) Differences in WBC and spleen weights between LT-HSC- and GMP-AML (***p < 0.0001; **p < 0.0075). Lines indicate the median weight values. (D) Histopathology of lung and spleen showing leukemic blast infiltration in GMP- and LT-HSC-early-AML. (E) Representative images of LT-HSC-early-AML, LT-HSC-late-AML, and GMP-AML blasts cultured in MC + DOX and quantification of colony types at subsequent platings (input of 104 cells). Mean ± SD of three experiments. (F) Average survival of secondary BMT recipients (n = 5 per group) transplanted with the indicated number of LT-HSC-AML and GMP-AML blasts, median disease latencies ± SD. (G) Morphology and quantification of colony types formed by LT-HSC-early-AML subpopulations ‘‘P1’’ (c-KitHighCD34HighFcgRII/IIILowGr1Low) and ‘‘P2’’ (c-KitHighCD34HighFcgRII/IIIHighGr1High), cultured in MC. Mean ± SD of duplicates. (H) Histopathology of lung from sick mice transplanted with LT-HSC-early-AML P1 (n = 10) or P2 subpopulations (n = 10). (I) Migration capacity of cells (4 hr) through Matrigel toward MS-5 conditioned media of P1 versus P2 LT-HSC-early-AML subpopulation (***p < 0.0001). Lines indicate the median values (% of input cells). See also Figure S4.

50 Cancer Cell 30, 43–58, July 11, 2016

Figure 5. Invasive Leukemic Phenotypes Are Linked to Origin-Related Gene-Expression Signatures (A) PCA of iMLL-AF9 LT-HSC- and GMP-AML. (B) Correlation plot showing lfc between LT-HSC-AML or GMP-AML versus WT GMP. RefSeq IDs significantly more highly expressed in both AML types are labeled in red (‘‘upregulated’’) and those more highly expressed in WT GMP in blue (‘‘downregulated’’) (adjusted p < 0.01). (legend continued on next page)

Cancer Cell 30, 43–58, July 11, 2016 51

iMLL-AF9 and/or MLL, which is likely linked to the cellular state at the point of transformation, allowing the fusion protein to access their promoters and sustain their expression only in LTHSC-early-derived AML. Zeb1 Controls Adhesion and Invasion of LT-HSC-Early-AML Blasts Next, we studied the function of EMT-related genes in iMLL-AF9expressing blasts in adhesion and invasion assays. First, we assayed the capacity of cells to migrate below a layer of MS-5 stroma cells and to generate cobblestone-area forming colonies (CAFC) (Figure 6A). LT-HSC-early-AML cells produced significantly more CAFC than GMP-AML cells (Figure 6B). Comparable differences were seen with in vitro immortalized LT-HSC- and GMP-derived cells (data not shown). LT-HSC-early-AML blasts also displayed increased adhesion to MS-5 stroma (Figure 6C) and were more potent in invading Matrigel layers than GMPderived AML cells (Figure 6D). We then tested the function of the EMT-related transcription factors, ZEB1 and TCF4, in migration and invasion of iMLLAF9 leukemic cells by small hairpin RNA (shRNA) knockdown (KD) experiments using retroviral vectors co-expressing GFP. We validated the KD efficacies of shRNAs on mRNA levels in LT-HSC-early-AML (Figure S6A) and on protein levels in Ba/F3 cells (Figures S6B and S6C). KD of ZEB1 and TCF4 significantly reduced the invasion capacity of LT-HSC-early-AML cells on MS-5 stroma (Figure 6E) and compromised invasion through Matrigel (Figure 6F). ZEB1 KD also impaired the limited migration capacity of GMP-derived AML blasts (Figure 6G). Moreover, ZEB1 KD reduced the growth of LT-HSC-early-AML cells in MC with growth factors while TCF4 KD had no effect (Figure S6D). In vivo, ZEB1 KD in LT-HSC-early AML cells significantly impaired cell infiltration in the BM and other organs (Figures 6H, 6I, and S6E–S6H). No effect on AML progression was observed upon TCF4 KD (data not shown). These data indicate that ZEB1 regulates the invasive behavior of iMLL-AF9 LTHSC-early AML cells. Cross-Species Expression Profiling Identifies Distinct Gene Sets that Correlate to Overall Survival of Human MLL-Rearranged AML To assess whether origin-dependent iMLL-AF9 targets relate to disease progression in patients, we hypothesized that EVI1 and

ERG expression may also reflect the cellular origin of human AML (Figures 5F and 7A). Classification of MLL-rearranged AML cases (11q23+, n = 43) according to EVI1 and ERG expression, was indeed associated with differential overall survival (OS) (Figure S7A). We then compared gene expression of presumptive HSCderived AML with high EVI1 and ERG expression (EVI1high ERGhigh) with presumptive GMP-derived AML with low expression (EVI1lowERGlow), and found 2,027 probe sets more highly expressed in the first group and 1,711 probe sets in the latter (Figure 7B and Table S4). Among the 2,027 probes, many genes have been implicated in invasion, EMT, inflammation, and NF-kB signaling as in the mouse LT-HSC-early AML (Table S4). By comparing the transcriptional profiles between species, we found 111 genes more highly expressed in EVI1highERGhigh AML samples as in Evi1highErghigh iMLL-AF9 LT-HSC-early AML, referred to as ‘‘cluster I.’’ Moreover, 40 genes were more highly expressed in EVI1lowERGlow AML samples as in iMLL-AF9 GMPderived AML, referred to as ‘‘cluster II’’ (Figure 7C and Table S4). Among the 111 genes in cluster I, 39 have previously been associated with EMT in solid cancers, including ZEB1, TCF4, PLAUR, and KDM5B (Table S5). Correlation analysis showed a significant anti-correlation of gene expression between the two clusters of genes (Figure S7B). Next, we analyzed how these gene clusters relate to OS of patients with 11q23+ AML (Figure 7D). Patients with high expression of cluster I genes were associated with poor OS. In contrast, patients expressing high levels of cluster II genes showed variable OS with a propensity of poor disease outcome with coexpression of a substantial fraction of cluster I genes. These data demonstrate that genes characteristic for LT-HSC-derived iMLL-AF9 AML characterize patients with 11q23-altered AML with poor outcome. Notably, many cluster I genes, characterizing presumptive HSC-derived AML, are implicated in cell migration, invasion, EMT, and inflammation, such as ETS2, EIF4E3, LSP1, MAP7, STK17B, and TRPS1, which were significantly associated with poor OS in patients with 11q23+ AML (Figures 7E and S7C). In contrast, most surviving patients were characterized by low expression of cluster I genes and high expression of cluster II genes, suggesting that in analogy to the iMLL-AF9 model such AML most likely represents a GMPderived disease.

(C) Heatmap showing mean log2 normalized counts per million (cpm) of RefSeq IDs, significantly upregulated in LT-HSC-AML and GMP-AML compared with WT GMP samples (adjusted p < 0.01). (D) Evi1 and Erg expression levels (in cpm) in LT-HSC-early AML (Evi1highErghigh, purple box) (n = 5), LT-HSC-late AML (Evi1lowErghigh, dashed purple box) (n = 3), and GMP-AML (Evi1lowErglow, orange box) (n = 3). (E) mRNA expression of Evi1, Erg, Zeb1, and Pbx1 in BM cells isolated from naive GMP and LSK progenitors and LT-HSC-early-AML, LT-HSC-late-AML, and GMP-AML cultured in vitro for 4 hr + DOX or overnight DOX. Relative expression levels were normalized to Gapdh mRNA level and to expression in naive murine GMP. Mean ± SD of triplicates per AML group (n = 3 per group). (F) Classification of AML according to Evi1 and Erg expression levels, reflecting aggressive and moderate leukemic stem cell states and moderate leukemic progenitor states. ‘‘Hierarchy,’’ ‘‘aggressiveness,’’ and ‘‘combination’’ refer to expression signatures of differentially expressed RefSeq IDs between different leukemic states (see H). (G) Correlation plot showing mean expression values of RefSeq IDs in iMLL-AF9 LT-HSC-early-AML versus GMP-AML. RefSeq IDs significantly more highly expressed in LT-HSC-early-AML are in purple and those more highly expressed in GMP-AML in orange (adjusted p < 0.01). (H) Venn diagram showing the number of genes more highly expressed in LT-HSC-late-AML versus GMP-AML (‘‘hierarchy signature’’), in LT-HSC-early-AML versus LT-HSC-late-AML (‘‘aggressiveness’’), and in LT-HSC-early-AML versus GMP-AML (‘‘combination’’) (adjusted p < 0.01). (I) p Value for enrichments of Ingenuity gene sets for hierarchy, aggressiveness, and combination signatures. (J) Heatmap showing expression values of selected RefSeq IDs of the combination signature. See also Figure S5 and Table S3.

52 Cancer Cell 30, 43–58, July 11, 2016

Figure 6. Knockdown of EMT Regulators Compromises Migration of Aggressive LT-HSC-Derived iMLL-AF9 Leukemic Cells (A) Giemsa-stained CAFC and differentiating cells from co-cultures of LT-HSC-early-AML blasts on MS-5 stromal cells, + DOX. (B) CAFC formation of LT-HSC-early-AML and GMP-AML blasts assessed in limited dilutions in co-cultures with MS-5 stromal cells (mean ± SD of duplicates, n = 2 experiments). (C) Adhesion/invasion of blasts of different cellular origins measured 4 hr after co-culture with MS-5 stromal cells (LT-HSC-early-AML, n = 5 and GMP-AML, n = 4). Lines indicate the median values (% of input cells) (*p < 0.02). (D) Differential invasion through a layer of Matrigel toward MS-5 conditioned media. Mean ± SD of three experiments (*p < 0.02, **p < 0.002). (legend continued on next page)

Cancer Cell 30, 43–58, July 11, 2016 53

We then asked whether the iMLL-AF9 model was instrumental in identifying migration- and invasion-related genes differentially expressed between EVI1highERGhigh and EVI1lowERGlow human AML. Extensive statistical analyses indeed showed that the mouse-to-human transcriptomic comparisons strongly facilitated the identification of genes involved in migration and invasion in mouse and human AML (Figures S7D–S7G and Table S6). EVI1highERGhigh AML with a Presumptive HSC Origin Is Not Limited to Those with 11q23 Alterations Lastly, we explored whether origin-related genes identified in the iMLL-AF9 model could subclassify AML with other genetic alterations. All AML cases with 3q26 alterations (n = 14) with known elevated EVI1 expression also expressed high levels of ERG. In contrast, all AML cases with PML-RARa (n = 25) or with core binding factor alterations t(8; 21) (n = 46) and inv(16) (n = 50) were characterized by high ERG but low EVI1 expression (Figure 8A). These data suggest that while MLL-rearranged AML originate from stem, early progenitor, or committed progenitor cells, the majority of other AML likely originate from stem or early progenitor cells rather than from committed progenitors as proposed before (Figure 8B) (Welch et al., 2012). Interestingly, besides EVI1 and ERG, we identified several cluster I genes including ALCAM, CEACAM1, ETS2, ITGA6, LSP1, MAP7, MMP8, TCF4, TRPS1, and ZEB1 that were associated with poor OS in many patients of the AML cohort (Figure 8C). Intermediate to high EVI1 expression was also observed in some AML patients without any known cytogenetic aberrations (Figure S8), showing a trend to poorer OS (p = 0.076). These observations suggest that EVI1 expression and possibly other cluster I genes may drive the aggressiveness of AML independently of any MLL1 or major 3q26 alterations. DISCUSSION The impact of cellular origin on the biology and clinical outcome of cancers of the hematopoietic system is poorly understood. We generated a transgenic mouse model to show that cellular origin determines iMLL-AF9-induced immortalization in vitro and AML induction in vivo. Moreover, we discovered that invasive iMLL-AF9 LT-HSC-derived AML cells expressed genes linked to EMT in solid cancers, many of which were associated with poor OS in AML patients. A subset of iMLL-AF9 immortalized LT-HSC displayed a dispersed clonogenic growth in vitro (‘‘type IV colonies’’) while rMLL-AF9-immortalized hematopoietic progenitors formed compact type I colonies. Replating cells of single type IV colonies resulted in type IV and I colonies, whereas type I cells formed type I colonies only. Such behavior likely reflects differ-

ential developmental potentials according to origin. Comparably, iMLL-AF9 induction in transplanted naive LT-HSC or GMP resulted in AML with distinct latencies, tissue invasion, ex vivo clonogenic activity, Ara-C sensitivity, and genetic signatures. About 20% of iMLL-AF9 LT-HSC recipients developed a particularly aggressive AML. Such selectivity may result from iMLL-AF9-mediated transformation of either a rare LT-HSC subpopulation upon engraftment (Copley et al., 2012) or from the timing of transformation in relation to HSC differentiation. Transcriptomic analyses indicate that iMLL-AF9- and rMLLAF9-driven AML partly share a gene-expression program irrespective of cellular origin (Krivtsov et al., 2006; Somervaille and Cleary, 2006; Wang et al., 2011). Nonetheless, LT-HSC-early, LT-HSC-late, and GMP-derived iMLL-AF9 AML showed clear phenotypic differences. Integrating Erg and Evi1 expression levels enabled us to classify AML according to cellular hierarchy and aggressiveness. During normal hematopoiesis, Evi1 is more highly expressed in LSK than GMP, and high Evi1 expression marks HSC with long-term multilineage repopulating potential (Kataoka et al., 2011). Evi1 is a known transcriptional target and mediator of oncogenic MLL fusions in murine LSK (Bindels et al., 2012). Consistently, we measured DOX-dependent Evi1 expression in iMLL-AF9 LT-HSC grown ex vivo. Erg is a critical transcriptional regulator of hematopoiesis promoting HSC maintenance (Knudsen et al., 2015; Loughran et al., 2008). In human AML, EVI1 and ERG have been proposed as biomarkers of AML patients with poor prognosis (Rockova et al., 2011). The Evi1/Erg expression-based classification of different iMLL-AF9-induced AML enabled the identification of genes involved in cell movement, invasion, metastasis, and EMT. In solid tumors, EMT is a feature of transformed epithelial cells undergoing loss of cellular polarity and gaining mesenchymal characteristics (Tam and Weinberg, 2013). EMT is in part controlled by the transcription factor ZEB1 in such tumors (Hill et al., 2013). In turn, ZEB1 is regulated by a negative feedback loop with the miR-200 microRNA family and was found to be a target of b-catenin/TCF4 (Sanchez-Tillo et al., 2013). Interleukin1b-derived signals and KDM5B-controlled miR-200 were shown to drive ZEB1 expression and to promote EMT (Enkhbaatar et al., 2013; Li et al., 2012). In iMLL-AF9-mediated AML, Zeb1, Tcf4, Il1b, and Kdm5b are more highly expressed in LT-HSC-early- than GMP-derived AML. shRNA-mediated KD of ZEB1 and TCF4 significantly impaired cell invasion in vitro. KD of ZEB1 but not of TCF4 affected invasiveness in vivo, assigning relevance to Zeb1 in regulating aggressiveness of LT-HSC-derived AML. The relevance of TCF4, as well as of IL1b and KDM5B, in transcriptional and posttranscriptional regulation of Zeb1 in AML awaits further investigation. ChIP-PCR assays revealed binding of iMLL-AF9

(E) Adhesion/invasion assay of LT-HSC-early-AML blasts 48 hr after retroviral transduction with control vector and shRNA constructs against TCF4 and ZEB1, detected by co-expressed GFP, 4 hr after co-culture with MS-5 stromal cells. Mean ± SD of three experiments in duplicates (**p < 0.001, ***p < 0.0002). (F) Invasion assay toward MS-5 conditioned media through thin Matrigel of LT-HSC-early-AML cells detected by co-expressed GFP blasts 48 hr after retroviral transduction with control vector and shRNA constructs against TCF4 and ZEB1. Mean ± SD of three experiments in duplicates (**p < 0.001). (G) Invasion assay with LT-HSC-early-AML and GMP-AML cells retrovirally transduced with control vector or shRNA against ZEB1. Mean ± SD of two experiments in duplicates (**p < 0.001, ***p < 0.0002). (H and I) Quantification (H, GFP+) and representative biopsies (I) of LT-HSC-AML cells in the BM retrovirally expressing ZEB1 shRNA or the control vector 1 week post BMT (**p < 0.002). Lines indicate the median values (% GFP+). See also Figure S6.

54 Cancer Cell 30, 43–58, July 11, 2016

(legend on next page)

Cancer Cell 30, 43–58, July 11, 2016 55

Figure 8. EVI1highERGhigh AML Are Not Limited to Patients with 11q23 Alterations (A and B) EVI1 and ERG expression levels in human AML with known major cytogenetic aberrations (A) or without detectable cytogenetic aberrations (B). (C) KM curves of OS and p value for significantly modulated cluster I probe sets of the indicated genes in all AML cohorts analyzed (n = 662; adjusted p < 0.05). Patients were classified in higher and lower expression groups as described in Experimental Procedures. See also Figure S8.

and/or MLL1 as well as occupancy for H3K4me3, a modification associated with transcriptional activity, at the Zeb1 and Evi1 loci in LT-HSC-early-AML only. In GMP-AML, however, these loci were marked by H3K27me3, a repressive modification mediated by the Polycomb repressive complex 2 (PRC2). These data strongly support a direct role for MLL-AF9 in driving Zeb1 expression in LT-HSC-early-AML while the locus is actively repressed by PRC2 in GMP-AML. Akin to these observations,

basal CD44high human breast cancer cells expressing ZEB1 displayed active H3K4me3 and H3K79me2 marks at the ZEB1 promoter, whereas basal CD44low and luminal cells exhibited bivalent H3K4me3/H3K27me3 patterns or only H3K27me3 repressive marks, respectively, controlling differential responses to external EMT-inducing signals (Chaffer et al., 2013). We hypothesize that the differential chromatin marking at Zeb1 in different iMLL-AF9 AML reflects cellular origin, possibly controlling heritability of Zeb1 activity or repression during leukemic transformation. In solid tumors, ZEB1 is a master regulator of EMT-related signaling pathways linked to stemness, growth, survival, and drug resistance (Siebzehnrubl et al., 2013). A major target of ZEB1 is CDH1 encoding for E-cadherin, an epithelial specific transmembrane adhesion protein that is repressed during EMT (Lamouille et al., 2014). In iMLLAF9-driven AML tumors, however, Cdh1 is not expressed and other downstream effectors of ZEB1 are unknown. Future ChIP-sequencing studies will demonstrate whether, e.g., genes bound by ZEB1 in non-hematopoietic cells such as Evi1 and Klf2 (Gubelmann et al., 2014), which are more highly expressed in LTHSC-early-AML than GMP-AML cells, are direct ZEB1 targets regulating the invasive phenotype of leukemic cells.

Figure 7. Cross-Species Comparison Identifies Genes Characteristic for EVI1highERGhigh, Cluster I, and EVI1lowERGlow, Cluster II, Human AML Associated with Differential Overall Survival (A) Classification of human 11q23+ AML according to EVI1 and ERG expression. EVI1highERGhigh (purple box), EVI1lowERGhigh (dashed purple box), EVI1lowERGlow (orange box). (B) Correlation of mean expression values of probe sets in 11q23+ AML characterized by EVI1highERGhigh (n = 14) versus EVI1lowERGlow (n = 11) expression. Probe sets more highly expressed in EVI1highERGhigh AML are in purple while those more highly expressed in EVI1lowERGlow are in orange (lfc > 0.585; adjusted p < 0.05). (C) Comparison of lfc between RefSeq IDs of mouse LT-HSC-early AML (Evi1highErghigh) versus GMP-AML (Evi1lowErglow) and probe sets of human EVI1highERGhigh AML versus EVI1lowERGlow AML. Mouse RefSeq IDs and human probe sets significantly more highly expressed in Evi1highErghigh and EVI1highERGhigh AML are in purple (mouse-human combination signature) and those more highly expressed in Evi1lowErglow and EVI1lowERGlow AML are in orange (mouse: adjusted p < 0.01; human: lfc > 0.585; adjusted p < 0.05). (D) Classification of 11q23+ patients into two OS groups according to expression values of 154 genes identified by mouse-human expression comparisons. Yellow and blue boxes (top) refer to patients alive or deceased at the end of study, respectively. Genes in bold were linked to invasion, EMT, or metastasis. (E) KM curves and p value for cluster I and II probe sets of ETS2, EIF43E, LPS1, MAP7, STK17B, and TRPS1 in MLL-AF9 and/or 11q23+ cohorts analyzed. Patients were classified in higher and lower expression groups as described in Experimental Procedures. See also Figure S7 and Tables S4, S5, and S6.

56 Cancer Cell 30, 43–58, July 11, 2016

Several other genes linked to EMT or cell invasion were differentially expressed in iMLL-AF9 AML of different origin. These include genes encoding components of the TGF-b (e.g., Tgfb1, Tgfbi, Tgfbr1, and Tgfbr2) and NF-kB (Nfkbia and Nfkbid) signaling pathways, integrins (e.g., Itga6, Itgam, and Itgb2), matrix proteins (Fn1 and Vim), matrix metalloproteinases (Mmp8 and Mmp9), CD87 (Plaur), the FOS (Fos) and JUN (Jun) transcription factors and their putative effector proteins including transcription factors, chromatin-modifying enzymes, signaling components, and adhesion proteins (Erg, Padi4, Csrnp1, Nfkb2, Tnfaip3, Kdm5b, and Alcam) (Lamouille et al., 2014). Hematopoietic cells do not need to exit an epithelial state to acquire mesenchymal properties associated with migration and invasion. Further studies are thus required resolve the function of these genes in the aggressive AML and to elucidate the molecular regulatory circuitries between them. Cross-species comparison revealed higher gene expression in 111 genes and lower expression in 40 genes in EVI1highERGhigh versus EVI1lowERGlow human (11q23+) and the respective mouse iMLL-AF9 AML. Expression levels of 67 genes such as TRPS1, LSP1, or ETS2 were significantly associated with poor OS in cohorts of patients with t(9; 11) AML. 12 genes including EVI1, ERG, ETS2, LSP, and TRPS1 were associated with poor OS in a cohort of >600 AML patients. Several of these genes have previously been linked to EMT or invasion in solid cancers but never associated with AML (Bindels et al., 2012; Chen et al., 2008; Krivtsov et al., 2006). Together, origin-related gene-expression signatures obtained from the iMLL-AF9 model relate to clinical heterogeneity of human AML, and may provide a rich source of potential novel biomarkers and targets for future personalized therapeutic strategies. EXPERIMENTAL PROCEDURES Establishment of rtTA; MLL-AF9 Transgenic Mice The human MLL-AF9 cDNA was cloned into p2Lox and electroporated into A2Lox-Cre ES cells (Iacovino et al., 2011). Standard procedures were used to generate a transgenic line that was backcrossed to C57BL/6 (>10 generations). All experiments were in adherence to Swiss animal welfare laws and approved by the Swiss Cantonal Veterinary Office of Basel-Stadt. BM Reconstitution Experiments BM (106) or sorted progenitor cells were tail-vein injected in lethally irradiated (137Cs, 9 Gy) 8-week-old C57BL/6 mice (Charles River Laboratories). In vivo serial dilution BMT experiments were done in sublethally irradiated (4.5 Gy) recipients injected with BM cells from primary diseased mice. Chemoresistance was tested in sublethally irradiated secondary recipients injected with 105 leukemic cells and treated with 5 3 100 mg/kg/day Ara-C starting 13 days post BMT. DOX (400 mg/ml, Sigma) was provided in the water with 5% sucrose or as impregnated food pellets (Harlan Laboratories) corresponding to 1.2 mg/day. Morphologic analysis of peripheral blood, BM, and spleen cells, and histologic analysis were performed according to standard procedures. Flow Cytometry, Colony Assays, Cell Culture, Invasion/Migration Assays, Knockdown Experiments, RT-PCR Analysis, Immunoblotting, and ChIP Assays Sorting of HSC and progenitor cells was performed using protocols described in Supplemental Experimental Procedures, which also provide details of protocols for colony assays, cell culture, invasion and migration assays, primers for target validation by RT and qPCR analysis, and shRNA vectors used for KD experiments. For immunoblotting and ChIP assays, we used standard protocols and antibodies described in Supplemental Experimental Procedures.

Transcriptional Analyses For RNA isolation and transcriptional profiling experiments on in vitro cultured LT-HSC- and GMP-derived cells, and BM cells from leukemic mice, we used standard RNA isolation, microarray, and RNA-sequencing approaches. We analyzed transcriptomic data generated from BM aspirates or peripheral blood samples collected at diagnosis from 662 adult de novo AML patients. Patients were treated according to Dutch-Belgian Hemato-Oncology Cooperative Group and the Swiss Group for Clinical Cancer Research (HOVON/SAKK) AML-04, -04A, -29, -32, -42, -42A, -43, and -92 protocols (available at http://www.hovon.nl). All patients provided written informed consent in accordance to the Declaration of Helsinki for collection and use of sample material for research purposes at the Erasmus Medical Center (MEC-2015-155). The HOVON study and the use of patient samples were approved by the ethical board of the Erasmus Medical Center. ACCESSION NUMBERS Raw data of mouse samples are available from the Gene Expression Omnibus (GEO: GSE65384). The raw Affymetrix Human Genome U133 Plus 2.0 Microarray data of the 662 adult AML cases are available from the ArrayExpress database (www.ebi.ac.uk/arrayexpress) under accession number E-MTAB-3444. SUPPLEMENTAL INFORMATION Supplemental Information includes Supplemental Experimental Procedures, eight figures, and six tables and can be found with this article online at http://dx.doi.org/10.1016/j.ccell.2016.05.011. AUTHOR CONTRIBUTIONS V.S., S.K., A.H.F.M.P., and J.S. conceived and designed experiments. V.S. established the transgenic mouse line and performed the in vivo, cell, and molecular biology experiments with assistance of L.B., S.J., S.M., and J.S. S.K. performed computational analyses on mouse expression datasets and generated KD vectors. M.A.S. performed computational analyses on human samples with advice by P.J.M.V. A.T. and J.S. performed the histological analyses. I.-J.L. performed ChIP experiments with advice by T.A.M. H.R. performed RNA-sequencing analysis. M.K. and M.I. provided critical reagents. A.H.F.M.P., J.S., V.S., and S.K. wrote the original draft with input from M.A.S. and P.J.M.V. V.S., J.S., and A.H.F.M.P. revised and edited the manuscript. ACKNOWLEDGMENTS We thank Dr. J.L. Hess (Ann Arbor, USA) for the pMSCV-MLL-AF9 plasmid, and E. Traunecker, T. Krebs, U. Schneider and his team for FACS and animal husbandry support. We acknowledge the FMI members: M.B. Stadler for computational support, T. Roloff and his team for transcriptional profiling, J.F. Spetz, P. Kopp, and B. Kuchemann for generating iMLL-AF9 mice, and J. Tjeertes for discussions. A.H.F.M.P.’s laboratory was supported by: Novartis Research Foundation, SystemsX.ch (Cell plasticity), and EMBO YIP program. J.S.’s laboratory was supported by: Swiss National Science Foundation (SNF-31003A_130661 & 31003A_149714/1), Swiss Cancer League (OCS2357-02-2009, OCS-02778-02-2011, KFS-3019-08-2012), Wilhelm Sander Foundation (Munich), Novartis Research Foundation, Swiss Bridge Foundation, and the Gertrude Von Meissner Foundation Basel. H.R. thanks the FP7PEOPLE-2013-IEF grant. T.A.M. was supported by a Medical Research Council (UK) Molecular Hematology Unit grant MC_UU_12009/6 and I.-J.L. by an MRC UK Clinical Research Training Fellowship MR/M003221/1. T.A.M. is a founding shareholder of Oxstem Oncology, a subsidiary company of OxStem Ltd. Received: March 30, 2015 Revised: March 22, 2016 Accepted: May 23, 2016 Published: June 23, 2016

Cancer Cell 30, 43–58, July 11, 2016 57

REFERENCES

termines clinically relevant subtypes of MLL-rearranged AML. Leukemia 27, 852–860.

Armstrong, S.A., Staunton, J.E., Silverman, L.B., Pieters, R., den Boer, M.L., Minden, M.D., Sallan, S.E., Lander, E.S., Golub, T.R., and Korsmeyer, S.J. (2002). MLL translocations specify a distinct gene expression profile that distinguishes a unique leukemia. Nat. Genet. 30, 41–47.

Lamouille, S., Xu, J., and Derynck, R. (2014). Molecular mechanisms of epithelial-mesenchymal transition. Nat. Rev. Mol. Cell Biol. 15, 178–196.

Bindels, E.M., Havermans, M., Lugthart, S., Erpelinck, C., Wocjtowicz, E., Krivtsov, A.V., Rombouts, E., Armstrong, S.A., Taskesen, E., Haanstra, J.R., et al. (2012). EVI1 is critical for the pathogenesis of a subset of MLL-AF9-rearranged AMLs. Blood 119, 5838–5849. Brown, A.L., Wilkinson, C.R., Waterman, S.R., Kok, C.H., Salerno, D.G., Diakiw, S.M., Reynolds, B., Scott, H.S., Tsykin, A., Glonek, G.F., et al. (2006). Genetic regulators of myelopoiesis and leukemic signaling identified by gene profiling and linear modeling. J. Leukoc. Biol. 80, 433–447. Chaffer, C.L., Marjanovic, N.D., Lee, T., Bell, G., Kleer, C.G., Reinhardt, F., D’Alessio, A.C., Young, R.A., and Weinberg, R.A. (2013). Poised chromatin at the ZEB1 promoter enables breast cancer cell plasticity and enhances tumorigenicity. Cell 154, 61–74. Chen, W., Kumar, A.R., Hudson, W.A., Li, Q., Wu, B., Staggs, R.A., Lund, E.A., Sam, T.N., and Kersey, J.H. (2008). Malignant transformation initiated by MllAF9: gene dosage and critical target cells. Cancer Cell 13, 432–440. Copley, M.R., Beer, P.A., and Eaves, C.J. (2012). Hematopoietic stem cell heterogeneity takes center stage. Cell Stem Cell 10, 690–697. Enkhbaatar, Z., Terashima, M., Oktyabri, D., Tange, S., Ishimura, A., Yano, S., and Suzuki, T. (2013). KDM5B histone demethylase controls epithelial-mesenchymal transition of cancer cells by regulating the expression of the microRNA-200 family. Cell Cycle 12, 2100–2112. Gubelmann, C., Schwalie, P.C., Raghav, S.K., Roder, E., Delessa, T., Kiehlmann, E., Waszak, S.M., Corsinotti, A., Udin, G., Holcombe, W., et al. (2014). Identification of the transcription factor ZEB1 as a central component of the adipogenic gene regulatory network. Elife 3, e03346. Hill, L., Browne, G., and Tulchinsky, E. (2013). ZEB/miR-200 feedback loop: at the crossroads of signal transduction in cancer. Int. J. Cancer 132, 745–754. Hosen, N., Park, C.Y., Tatsumi, N., Oji, Y., Sugiyama, H., Gramatzki, M., Krensky, A.M., and Weissman, I.L. (2007). CD96 is a leukemic stem cell-specific marker in human acute myeloid leukemia. Proc. Natl. Acad. Sci. USA 104, 11008–11013. Huntly, B.J., Shigematsu, H., Deguchi, K., Lee, B.H., Mizuno, S., Duclos, N., Rowan, R., Amaral, S., Curley, D., Williams, I.R., et al. (2004). MOZ-TIF2, but not BCR-ABL, confers properties of leukemic stem cells to committed murine hematopoietic progenitors. Cancer Cell 6, 587–596. Iacovino, M., Bosnakovski, D., Fey, H., Rux, D., Bajwa, G., Mahen, E., Mitanoska, A., Xu, Z., and Kyba, M. (2011). Inducible cassette exchange: a rapid and efficient system enabling conditional gene expression in embryonic stem and primary cells. Stem Cells 29, 1580–1588. Kataoka, K., Sato, T., Yoshimi, A., Goyama, S., Tsuruta, T., Kobayashi, H., Shimabe, M., Arai, S., Nakagawa, M., Imai, Y., et al. (2011). Evi1 is essential for hematopoietic stem cell self-renewal, and its expression marks hematopoietic cells with long-term multilineage repopulating activity. J. Exp. Med. 208, 2403–2416. Knudsen, K.J., Rehn, M., Hasemann, M.S., Rapin, N., Bagger, F.O., Ohlsson, E., Willer, A., Frank, A.K., Sondergaard, E., Jendholm, J., et al. (2015). ERG promotes the maintenance of hematopoietic stem cells by restricting their differentiation. Genes Dev. 29, 1915–1929. Krivtsov, A.V., Twomey, D., Feng, Z., Stubbs, M.C., Wang, Y., Faber, J., Levine, J.E., Wang, J., Hahn, W.C., Gilliland, D.G., et al. (2006). Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9. Nature 442, 818–822. Krivtsov, A.V., Figueroa, M.E., Sinha, A.U., Stubbs, M.C., Feng, Z., Valk, P.J., Delwel, R., Dohner, K., Bullinger, L., Kung, A.L., et al. (2013). Cell of origin de-

58 Cancer Cell 30, 43–58, July 11, 2016

Lavallee, V.P., Baccelli, I., Krosl, J., Wilhelm, B., Barabe, F., Gendron, P., Boucher, G., Lemieux, S., Marinier, A., Meloche, S., et al. (2015). The transcriptomic landscape and directed chemical interrogation of MLL-rearranged acute myeloid leukemias. Nat. Genet. 47, 1030–1037. Li, Y., Wang, L., Pappan, L., Galliher-Beckley, A., and Shi, J. (2012). IL-1beta promotes stemness and invasiveness of colon cancer cells through Zeb1 activation. Mol. Cancer 11, 87. Loughran, S.J., Kruse, E.A., Hacking, D.F., de Graaf, C.A., Hyland, C.D., Willson, T.A., Henley, K.J., Ellis, S., Voss, A.K., Metcalf, D., et al. (2008). The transcription factor Erg is essential for definitive hematopoiesis and the function of adult hematopoietic stem cells. Nat. Immunol. 9, 810–819. Meyer, C., Hofmann, J., Burmeister, T., Groger, D., Park, T.S., Emerenciano, M., Pombo de Oliveira, M., Renneville, A., Villarese, P., Macintyre, E., et al. (2013). The MLL recombinome of acute leukemias in 2013. Leukemia 27, 2165–2176. Miller, C.A., Wilson, R.K., and Ley, T.J. (2013). Genomic landscapes and clonality of de novo AML. N. Engl. J. Med. 369, 1473. Muntean, A.G., and Hess, J.L. (2012). The pathogenesis of mixed-lineage leukemia. Annu. Rev. Pathol. 7, 283–301. Rockova, V., Abbas, S., Wouters, B.J., Erpelinck, C.A., Beverloo, H.B., Delwel, R., van Putten, W.L., Lowenberg, B., and Valk, P.J. (2011). Risk stratification of intermediate-risk acute myeloid leukemia: integrative analysis of a multitude of gene mutation and gene expression markers. Blood 118, 1069–1076. Sanchez-Tillo, E., Liu, Y., de Barrios, O., Siles, L., Fanlo, L., Cuatrecasas, M., Darling, D.S., Dean, D.C., Castells, A., and Postigo, A. (2012). EMT-activating transcription factors in cancer: beyond EMT and tumor invasiveness. Cell Mol. Life Sci. 69, 3429–3456. Sanchez-Tillo, E., de Barrios, O., Siles, L., Amendola, P.G., Darling, D.S., Cuatrecasas, M., Castells, A., and Postigo, A. (2013). ZEB1 Promotes invasiveness of colorectal carcinoma cells through the opposing regulation of uPA and PAI-1. Clin. Cancer Res. 19, 1071–1082. Siebzehnrubl, F.A., Silver, D.J., Tugertimur, B., Deleyrolle, L.P., Siebzehnrubl, D., Sarkisian, M.R., Devers, K.G., Yachnis, A.T., Kupper, M.D., Neal, D., et al. (2013). The ZEB1 pathway links glioblastoma initiation, invasion and chemoresistance. EMBO Mol. Med. 5, 1196–1212. Somervaille, T.C., and Cleary, M.L. (2006). Identification and characterization of leukemia stem cells in murine MLL-AF9 acute myeloid leukemia. Cancer Cell 10, 257–268. Somervaille, T.C., Matheny, C.J., Spencer, G.J., Iwasaki, M., Rinn, J.L., Witten, D.M., Chang, H.Y., Shurtleff, S.A., Downing, J.R., and Cleary, M.L. (2009). Hierarchical maintenance of MLL myeloid leukemia stem cells employs a transcriptional program shared with embryonic rather than adult stem cells. Cell Stem Cell 4, 129–140. Tam, W.L., and Weinberg, R.A. (2013). The epigenetics of epithelial-mesenchymal plasticity in cancer. Nat. Med. 19, 1438–1449. Tamai, H., and Inokuchi, K. (2010). 11q23/MLL acute leukemia: update of clinical aspects. J. Clin. Exp. Hematop. 50, 91–98. Wang, Q.F., Wu, G., Mi, S., He, F., Wu, J., Dong, J., Luo, R.T., Mattison, R., Kaberlein, J.J., Prabhakar, S., et al. (2011). MLL fusion proteins preferentially regulate a subset of wild-type MLL target genes in the leukemic genome. Blood 117, 6895–6905. Welch, J.S., Ley, T.J., Link, D.C., Miller, C.A., Larson, D.E., Koboldt, D.C., Wartman, L.D., Lamprecht, T.L., Liu, F., Xia, J., et al. (2012). The origin and evolution of mutations in acute myeloid leukemia. Cell 150, 264–278.