Hes1 Suppresses Acute Myeloid Leukemia Development Through FLT3 Repression

ORIGINAL ARTICLE Hes1 suppresses acute myeloid leukemia development through FLT3 repression

T Kato1,2,3, M Sakata-Yanagimoto1,3, H Nishikii1, M Ueno4, Y Miyake3, Y Yokoyama1, Y Asabe3, Y Kamada3, H Muto1,3, N Obara1,3, K Suzukawa1, Y Hasegawa1,3, I Kitabayashi5, K Uchida6, A Hirao4, H Yagita7, R Kageyama8,9 and S Chiba1,2,3

In leukemogenesis, Notch signaling can be up and downregulated in a context-dependent manner. The transcription factor hairy and enhancer of split-1 (Hes1) is well-characterized as a downstream target of Notch signaling. Hes1 encodes a basic helix–loop– helix-type protein, and represses target gene expression. Here, we report that deletion of the Hes1 gene in mice promotes acute myeloid leukemia (AML) development induced by the MLL–AF9 fusion protein. We then found that Hes1 directly bound to the promoter region of the FMS-like tyrosine kinase 3 (FLT3) gene and downregulated the promoter activity. FLT3 was consequently upregulated in MLL–AF9-expressing immortalized and leukemia cells with a Hes1-orRBPJ-null background. MLL–AF9-expressing Hes1-null AML cells showed enhanced proliferation and ERK phosphorylation following FLT3 ligand stimulation. FLT3 inhibition efﬁciently abrogated proliferation of MLL–AF9-induced Hes1-null AML cells. Furthermore, an agonistic anti-Notch2 antibody induced apoptosis of MLL–AF9-induced AML cells in a Hes1-wild type but not a Hes1-null background. We also accessed two independent databases containing messenger RNA (mRNA) expression proﬁles and found that the expression level of FLT3 mRNA was negatively correlated with those of HES1 in patient AML samples. These observations demonstrate that Hes1 mediates tumor suppressive roles of Notch signaling in AML development, probably by downregulating FLT3 expression.

Leukemia (2015) 29, 576–585; doi:10.1038/leu.2014.281

INTRODUCTION of Notch signaling in development of early T lymphocytes.11 The Notch pathway, which is highly conserved from Drosophila to Subsequently, activating mutations in Notch1 were identified in 12 mammals, functions in maintenance, proliferation and differentia- mature B-cell neoplasms, including chronic lymphocytic leukemia tion of various cell types. In mammals, four receptors (Notch1–4) and mantle cell lymphoma,13 and in Notch2 in diffuse large B-cell and five ligands (Jagged1/2, Delta-like 1/3/4) have been lymphoma14 and splenic marginal zone B-cell lymphoma.15 Gain identified.1 Ligand binding initiates proteolytic cleavage of the of function mutations in Notch genes have also been identified in Notch receptor by γ-secretase, leading to nuclear translocation of non-hematologic malignancies such as breast cancer.16 the Notch intracellular domain.2 Notch intracellular domain binds In contrast, mouse genetic studies demonstrate that Notch 17 18 to the transcription factor RBPJ (also known as CSL for CBF-1, Su(H) signaling has tumor suppressive activity in skin and vascular and Lag2) and forms a transactivation complex, inducing tumors. And, in contrast with genetic evidence gathered from transcription of Notch–RBPJ target genes.3,4 analysis of T-ALL, chronic lymphocytic leukemia and some B-cell Hes1 is a commonly described Notch–RBPJ target gene in blood lymphomas, Notch signaling reportedly has a tumor-suppressive cells.5 The Hes1 gene encodes a basic helix–loop–helix transcrip- role in B-cell ALL.19 tion factor that recruits co-repressors of the transducin-like Both pro- and anti-tumorigenic roles of Notch signaling have enhancer of split (groucho) family.6 Through its basic helix– been reported in myeloid malignancies (Supplementary Table 1). loop–helix domain, Hes1 forms either heterodimers with other In mouse chronic myeloid leukemia models, myeloid blast crisis 20,21 basic helix–loop–helix transcription factors or homodimers that transition is facilitated by upregulation of Hes1, an event bind both to canonical enhancer box (E-Box) or N-box (CACNAG) reportedly triggered by expression of the RNA binding protein promoter elements.7,8 Musashi2 through downregulation of Numb, an inhibitor of Notch Many lines of evidence indicate that deregulated Notch signaling.21 Subsequently, loss-of-function mutations in multiple signaling functions in initiation, promotion and progression of components of the Notch pathway, including Notch2, have numerous cancers. Genetic evidence for that role in human been identified in chronic myelomonocytic leukemia patients.22 cancers was first reported in the case of T-cell acute lymphoblastic Relevant to acute myeloid leukemia (AML), recent studies also leukemia (T-ALL) in which activating Notch1 mutations occur at a suggest a tumor-suppressive activity: activation of Notch signaling frequency 450%,9,10 possibly reflecting the indispensable role induces apoptosis in human AML cells in vitro, and constitutively

1Department of Hematology, Faculty of Medicine, University of Tsukuba, Tsukuba, Japan; 2Life Science center of Tsukuba Advanced Research Alliance, University of Tsukuba, Tsukuba, Japan; 3Department of Hematology, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba, Japan; 4Division of Molecular Genetics, Cancer and Stem Cell Research Program, Cancer Research Institute, Kanazawa University, Kanazawa, Japan; 5Molecular Oncology Division, National Cancer Center Research Institute, Tokyo, Japan; 6Department of Molecular Biological Oncology, Faculty of Medicine, University of Tsukuba, Tsukuba, Japan; 7Department of Immunology, Juntendo University School of Medicine, Tokyo, Japan; 8Institute of Virus Research, Kyoto University, Kyoto, Japan and 9World Premier International Research Initiative-Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Kyoto, Japan. Correspondence: Professor S Chiba, Department of Hematology, Faculty of Medicine, University of Tsukuba, 1-1-1, Tennodai, Tsukuba 305-8575, Japan. E-mail: [email protected] Received 13 January 2014; revised 25 August 2014; accepted 9 September 2014; accepted article preview online 19 September 2014; advance online publication, 17 October 2014 Tumor suppressive role of Hes1 in myeloid leukemia T Kato et al 577 active forms of Notch1 and Notch2 reduce leukemogenicity in N-boxes (N1, 5′-CACTAG-3′ fragment at position − 50/ − 45 and N2, transgenic mouse models.23,24 5′-CACCAG-3′ fragment at position − 425/ − 419; Supplementary Table 3). In this paper, we took advantage of an MLL–AF9-induced AML model in RBPJ-deficient mice to confirm that Notch signaling Statistics functions as a tumor suppressor in AML. We also conducted Survival of transplanted mice was analyzed statistically using the log-rank experiments with the MLL–AF9-induced AML model in Hes1-null test. Correlation of messenger RNA (mRNA) expression levels was mice and demonstrated that Hes1 is indispensable for AML statistically evaluated by calculating Pearson’s correlation coefficient. suppression. Finally, we provide evidence that FMS-like tyrosine Other data were analyzed by using Student’s t-test. P-valueso0.05 were fi kinase 3 (FLT3) tyrosine kinase signaling is hyperactivated in Hes1- considered signi cant. null AML cells. These studies identify Hes1 as the Notch effector functioning to suppress AML development and suggest that loss Study approval of Hes1 promotes oncogenesis through FLT3 upregulation. All experiments were performed according to NIH guidelines and approved by the University of Tsukuba’s Committee on Use and Care of Animals. MATERIALS AND METHODS Gene expression analysis Other experimental methods RNA was isolated from MLL–AF9-induced AML cells using an RNeasy kit Mice, transplantation, vectors, flow cytometry, preparation of recombinant (QIAGEN, Valencia, CA, USA), and complementary DNA was synthesized retroviruses, cell cycle analysis, apoptosis activity, reverse transcription and with SuperScript III (Invitrogen, Carlsbad, CA, USA). Gene expression real time PCR analysis, reporter assay, short interfering RNA interference, analysis was performed on the Mouse Genome 430 2.0 Array (Affymetrix, CRISPR-Cas9 system and immunoblotting protocols are described in Santa Clara, CA, USA). The data were analyzed with the Expression Console Supplementary Information. using Affymetrix default analysis settings and normalized with the Gene Level-RMA Sketch. We used a cutoff level of twofold for both up and downregulation in MLL–AF9/Hes1− / − AML cells compared with MLL–AF9/ RESULTS Hes1+/+ AML cells, and sorted the genes by the relative values. See RBPJ deletion accelerates MLL–AF9-induced leukemia Supplementary Experimental Procedures for details of sample preparation. development in mice To determine whether Notch signaling has a tumor-suppressive Lentivirus production and generation of stable cell lines function in MLL–AF9-induced AML, we transformed common f/f For lentivirus production, HEK293T cells were transfected with the CS–Hes1 myeloid progenitors (CMPs) purified from bone marrow of RBPJ or mock plasmid together with the psPAX2 packaging plasmid and the mice25,26 by retroviral transduction of a construct containing pMD2.G envelope plasmid, and the concentrated supernatant was used to MLL–AF9–IRES–GFP. To eliminate the RBPJ allele in these cells, infect THP1 cells. To establish stable lines inducibly expressing Hes1, green we serially infected MLL–AF9-transduced RBPJf/f cells with fluorescent protein (GFP)-expressing THP1 cells were sorted on a FACSAria Cre recombinase (iCre)–IRES-nerve growth factor receptor (BD Biosciences, San Jose, CA, USA). (Supplementary Figure 1A, See Methods). Deletion of the RBPJ locus in double-positive RBPJf/f (MLL–AF9/iCre/RBPJf/f) cells was Chromatin immunoprecipitation confirmed by genomic PCR (Supplementary Figure 1B). We found Detailed protocols for chromatin immunoprecipitation are described in that levels of mRNA transcripts encoding Hes1 and Hes5 were f/f Supplementary Methods. Immunoprecipitated DNA fragments were decreased in MLL–AF9/iCre/RBPJ compared with control cells quantified by real-time PCR with the use of two Flt3 promoter sets, N1 (Figure 1a). MLL–AF9/iCre/RBPJf/f cells proliferated more rapidly and N2, which amplify sequences including putative Hes1 binding sites, than did MLL–AF9/mock/RBPJf/f cells in vitro in the presence

6 3 150

5 5 MLL-AF9/Mock/RBPJf/f

4 2 100 MLL-AF9/iCre/RBPJf/f Gapdh 3 Gapdh expression 2 expression 1 50 Hes1 relative to relative Hes5 relative to relative 1 of cells x10 Number

0 0 0 Mock iCre Mock iCre f/f d0 d2 d3 d4 d5 RBPJf/f RBPJ Days

0.8 P =0.017 0.6

0.4 MLL-AF9/Mock/RBPJf/f (n =13) Survival rate 0.2 MLL-AF9/iCre/RBPJf/f1 0 (n =12) 0 20 40 60 80 Days after transplantation Figure 1. Deletion of RBPJ accelerates MLL–AF9-induced leukemia development. (a) Hes1 and Hes5 transcript levels in MLL–AF9/mock/RBPJf/f and MLL–AF9/iCre/RBPJf/f cells. (b) Growth of MLL–AF9/iCre-RBPJf/f and MLL–AF9/mock-RBPJf/f cells cultured in RPMI supplemented with 10% fetal calf serum and interleukin 3. About 50 000 cells were originally plated. Representative data from four independent experiments is shown; n = 3 each. (c) Survival of mice transplanted with MLL–AF9/iCre/RBPJf/f or MLL–AF9/mock/RBPJf/f cells.

© 2015 Macmillan Publishers Limited Leukemia (2015) 576 – 585 Tumor suppressive role of Hes1 in myeloid leukemia T Kato et al 578 of interleukin 3 (Figure 1b). These results indicate successful (Supplementary Figure 2A). These results indicate that Hes1 loss knockout of the RBPJ locus in MLL–AF9-transduced cells and accelerates MLL–AF9-transduced cell proliferation (Figure 2a). − − consequent abrogation of Notch signaling, an outcome that Following the second replating, we expanded MLL–AF9/Hes1 / increased proliferation of MLL–AF9-transduced cells. and MLL–AF9/Hes1+/+ cells in liquid culture and transplanted them After deletion of RBPJ locus and following expansion of cells for into lethally irradiated syngenic mice. As anticipated, mice 2–3 weeks in the presence of interleukin 3, MLL–AF9/iCre/RBPJf/f transplanted with MLL–AF9/Hes1+/+ cells developed leukemia at and control cells exhibited a similar myelomonoblastic morphol- a median latency of 81 days.29 By comparison, mice transplanted ogy and surface antigen expression profile (Mac1+, Gr1+, c-Kit+/ − , with MLL–AF9/Hes1− / − cells developed leukemia at significantly Sca1−, CD34+/ − , B220− and CD3ε−; Supplementary Figure 1C). We shorter latencies (Po0.01, Figure 2b). Leukemia cells with both then transplanted lethally irradiated syngenic mice through the genotypes were transplantable to the secondary recipients that tail vein with these cells. Mice transplanted with MLL–AF9/iCre/ received sublethal irradiation (data not shown). We compared the RBPJf/f cells developed leukemia at shorter latencies than did MLL–AF9 mRNA expression levels between AML cells with Hes1− / − recipients of MLL–AF9/mock/RBPJf/f control cells (P = 0.017, and Hes1+/+ genotype, and found no difference (Supplementary Figure 1c). In both groups, leukemic cells were found in peripheral Figure 2B). The morphology and cell surface antigen profiles of blood, bone marrow, spleen, lungs and liver, and no differences MLL–AF9/Hes1− / − and MLL–AF9/Hes1+/+ AML cells were essen- in morphology were seen between groups (Supplementary tially the same and similar to those seen in RBPJ-deficient AML Figures 1D and E). cells (Supplementary Figures 2C–F). To exclude the possibility that shortened latency of leukemia A previous study suggested that Hes1 downregulate Bcl2 in development seen following transplantation of MLL–AF9/iCre/ AML cells, indicating that Bcl2 downregulation participates in RBPJf/f cells was attributable to enhanced homing ability, we Notch-induced apoptosis of AML cells.24 Consistent with this compared frequencies of GFP-positive cells in bone marrow 72 h report, the Bcl2 expression levels were upregulated in MLL–AF9/ after transplantation. We observed no significant difference in Hes1− / − AML cells compared with MLL–AF9/Hes1+/+ AML cells GFP-positive cell frequencies between MLL–AF9/iCre/RBPJf/f and (Supplementary Figure 2G). control cells (Supplementary Figure 1F), suggesting that shortened Taken together, all these results suggest that Hes1 functions latencies for AML development were due to mechanisms other downstream to Notch signaling for the suppression of AML than enhanced homing ability of in vitro expanded MLL–AF9/iCre/ development in mice. RBPJf/f cells. These results indicate that loss of Notch signaling causes enhanced leukemia development from MLL–AF9-trans- Expression of a dominant-negative Hes1 construct enhances AML duced cells in mice. development Next, we constructed a dominant-negative mutant of Hes1 Hes1 loss increases clonogenic potential of MLL–AF9-trandsduced (dnHes1) lacking the WRPW domain, which interacts with the fetal liver CMPs and accelerates development of MLL–AF9- transducin-like enhancer co-repressor30,31 (Figure 3a) and is induced leukemia essential for transcriptional repression. Adult bone marrow CMPs Hes1 is the best characterized target of the canonical Notch–RBPJ purified from wild-type mice were transformed by retroviral pathway in diverse cellular contexts. To determine if Hes1 was transduction of MLL–AF9–GFP and then serially infected with responsible for Notch signaling-mediated suppression of AML retrovirus expressing dnHes1–humanized Kusabira Orange (hKO) development, we employed conventional Hes1− / − mice. Although or mock-hKO. We then sorted both MLL–AF9–GFP/dnHes1–hKO Hes1 is known to repress Hes5 in a certain context,27 we found double-positive (MLL–AF9/dnHes1) and control MLL–AF9–GFP/ Hes5 expression level was unchanged in this model (data not mock-hKO double-positive (MLL–AF9/mock) cells and trans- shown). These mice die at embryonic day (E) 18–19 from defects planted them into lethally irradiated mice (Supplementary in neurogenesis.28 Therefore, we used CMPs prepared from fetal Figure 3A). Mice harboring MLL–AF9/dnHes1 cells developed liver at E14.5–15.5. CMPs were retrovirally transduced with MLL– leukemia at significantly shorter latencies than did those AF9–GFP, and GFP-positive cells were then serially plated in transplanted with MLL–AF9/mock cells (Po0.01, Figure 3b). The semisolid medium every 7 days. MLL–AF9-transduced CMPs morphology of MLL–AF9/dnHes1-induced AML cells was similar to formed colonies at multiple rounds of plating in both Hes1-null that of MLL–AF9/iCre/RBPJf/f- and MLL–AF9/Hes1− / −-induced AML and wild-type backgrounds. At the second and third replating, cells. Cell surface antigen profiles were also similar between these colony numbers of MLL–AF9-transduced cells in a Hes1-null MLL–AF9-induced AML cells, although we observed higher background (MLL–AF9/Hes1− / −) were greater than those in a expression levels of CD34 in MLL–AF9/dnHes1-induced AML cells similarly-transduced wild-type (MLL–AF9/Hes1+/+) background than others (Supplementary Figures 3B and C). This might be

1 +/+ P = 0.001 MLL-AF9/Hes1 (n =13) 600 +/+ MLL-AF9/Hes1-/- (n =14) Mock/Hes1 0.8 Mock/Hes1-/- 400 0.6 MLL-AF9/Hes1+/+ P = 0.01 0.4 MLL-AF9/Hes1-/- 200 Survival rate 0.2 P < 0.01 0

Number of colonies/1000 cells 0 1st 2nd 3rd 0 50 100 150 200 Days after transplantation Figure 2. Loss of Hes1 increases clonogenic potential of MLL–AF9-trandsduced fetal liver CMPs and accelerates development of MLL–AF9- induced leukemia. (a) The number of colonies derived from MLL–AF9/Hes1− / − cells and MLL–AF9/Hes1+/+ cells. All cells were harvested, and 1000 cells were replated every 7 days. (b) Survival of mice transplanted with MLL–AF9/Hes1+/+ or MLL–AF9/Hes1− / − cells.

wt b HLH Orange WRPW 1 P < 0.01 0.8 MLL-AF9/mock (n =10) dn b HLH Orange 0.6 MLL-AF9/dnHes1(n =10) Function of dnHes1 Corepressor 0.4

Corepressor Survival rate 0.2

Hes1 dnHes1 0 0 50 100 150 200 250 OFF ON N box N box Days after transplantation

Hes1-NGFR 1 or NGFR C57BL/6 0.8 P < 0.001

0.6 Mock-Hes1-/-MLL-AF9 (n = 7)

0.4 -/- MLL-AF9/Hes1-/- NGFR+ Sort Hes1-Hes1 MLL-AF9 (n = 8) Survival rate CMPs 0.2

0 20 40 60 80 100 120 Days after transplantation Figure 3. Dominant-negative Hes1 accelerates MLL–AF9-induced leukemia development and re-introduction of wild-type Hes1 into Hes1- deficient AML cells represses AML development. (a) Structure of the dominant-negative mutant of Hes1 (dnHes1). bHLH, basic helix–loop– helix domain; dn, dominant-negative Hes1; orange, orange domain; WRPW, WRPW motif; WT, wild-type Hes1. (b) Survival of mice transplanted with MLL–AF9/dnHes1 or MLL–AF9/mock cells. (c) MLL–AF9/Hes1− / − cells were serially infected with Hes1-nerve growth factor receptor (Hes1- NGFR). NGFR-positive (Hes1/MLL–AF9/Hes1− / −) and control (mock/MLL–AF9/Hes1− / −) cells were sorted and injected into lethally irradiated mice. (d) Survival of mice transplanted with Hes1-transduced MLL–AF9/Hes1− / − and mock-transduced MLL–AF9/Hes1− / − cells. reflected by the interference with the formation of heterodimers Flt3 mRNA was validated by quantitative reverse transcription-PCR comprising Hes1 and basic helix–loop–helix proteins independent in both MLL–AF9/Hes1− / − cells maintained in liquid culture (data of Notch signaling. MLL–AF9/dnHes1-induced AML cells devel- not shown) and in cells recovered from mice that developed AML oped in the primary recipient mice were transplantable to the (Figure 4b). Flt3 mRNA was also upregulated in MLL–AF9/iCre/ secondary recipients, which were sublethally irradiated (Data not RBPJf/f cells and cells expressing dnHes1 (Supplementary shown). These findings provide further support that Hes1 is Figure 4A). Cell surface FLT3 expression was also higher in MLL– − − indeed the Notch effector that functions to suppress AML AF9/Hes1 / cells compared with respective controls (Figure 4c). development in this context. Expression levels of Flt3 mRNA and cell surface FLT3 protein in Hes1-transduced MLL–AF9/Hes1− / − cells decreased relative to – − / − Re-induction of wild-type Hes1 rescues shortened latency of AML those seen in mock-transduced MLL AF9/Hes1 cells, and the − − – − / − development seen in MLL–AF9/Hes1 / cells proliferative capacity of Hes1-transduced MLL AF9/Hes1 cells in the presence of FLT3 ligand was lower than that observed in To determine whether shortened latency of AML phenotypes in − − mock-transduced cells (Figure 4d, Supplementary Figures 4B and C). MLL–AF9/Hes1 / cells could be reversed, we reintroduced Hes1 There are two N-boxes on the Flt3 promoter region. We into these cells (Figure 3c). Lethally irradiated mice transplanted − − performed chromatin immunoprecipitation assay using a human with Hes1-transduced MLL–AF9/Hes1 / cells developed leukemia − − AML cell line, THP1, in which Hes1 was expressed in an inducible at longer latencies than did mock-transduced MLL–AF9/Hes1 / manner with doxycycline, and showed that Hes1 directly bound to cells (Po0.01, Figure 3d). These phenotypes indicate that the Flt3 promoter region (Figure 4e). We also performed reporter aberrations seen following Hes1 deletion were reverted by the assay, and showed that Hes1, but not dnHes1, repressed the Flt3 exogenous expression of Hes1. promoter activity (Figure 4f). When N-box sites on Flt3 promoter were mutated, repression of the Flt3 promoter activity by Hes1 Hyperactivation of FLT3 signaling underlies enhanced proliferation was mitigated (Figure 4f). Furthermore, FLT3 was phosphorylated of RBPJ- and Hes1-deficient AML cells following stimulation with FLT3 ligand specifically in MLL–AF9/ To elucidate signaling downstream of the Notch–RBPJ–Hes1 axis Hes1− / − cells (Figure 4g). Stimulation of cells expressing wild-type in AML cells, we compared mRNA expression profiles between FLT3 with FLT3 ligand activates ERK signaling,34 while signaling − − MLL–AF9/Hes1 / and MLL–AF9/Hes1+/+ AML cells using micro- through FLT3-internal tandem duplication mutants aberrantly array analysis (www.ncbi.nlm.nih.gov/geo, accession number activates other downstream signaling pathways, such as STAT and GSE50234). Using a cutoff level of twofold, of 35 079 genes, 552 AKT, in addition to ERK. In MLL–AF9/Hes1− / − leukemic cells, ERK and 376 were up and downregulated, respectively, in MLL–AF9/ phosphorylation was enhanced by FLT3 ligand stimulation, an − − Hes1 / AML cells compared with control cells (Figure 4a). Among effect much weakly seen in MLL–AF9/Hes1+/+ cells (Figure 4g). We upregulated genes, we focused on Flt3, which encodes a receptor- did not detect differences in phosphorylation of STAT or AKT in type tyrosine kinase, because hyperactivation of FLT3 signaling by MLL–AF9/Hes1− / − and MLL–AF9/Hes1+/+ leukemic cells (Figure 4g). internal tandem duplication mutations or mRNA overexpression is These results suggest that FLT3–ERK signaling is activated a known indicator of poor prognosis of AML.32,33 Upregulation of through FLT3 upregulation specifically in the absence of Hes1.

MLL-AF9 10 +/+ -/- 60 P = 0.03 +/+ -/- Hes1 Hes1 Hes1 Hes1 Flt3

BM1 BM2 BM1 BM2 Gapdh 5.6% 41.1% 40 5 cells (%) +

transcripts/ 20 FLT3

Flt3 0 Hes1+/+ Hes1-/- FLT3 0 Leukemic mice BM Hes1+/+ Hes1-/- MLL-AF9

MLL-AF9/Hes1-/- Mock Hes1 Control Line1 Lline3 Line3 Analysis of 35,056 genes 78.6% 3.9% 4.9% 7.5%

FLT3

Hes1 +/+ -/- +/+ -/-

1.2 FLT3L --++ Mock 1 IP FLT3 0.8 Hes1 FLT3 0.6 pTyr 0.4 Hes1 +/+-/- +/+ -/- 0.2 FLT3L --++ 0 ERK relative Flt3 promoter activity Hes1 Hes1 mock pERK dnHes1 AKT WT WT WT Mut pAKT

STAT5

pSTAT5

Actin

Figure 4. Activation of FLT3 signaling underlies enhanced proliferation of RBPJ- and Hes1-deﬁcient AML cells. (a) Microarray analysis of leukemic bone marrow cells from mice transplanted with MLL–AF9/Hes1+/+ or MLL–AF9/Hes1− / − cells. The colors represent the absolute expression levels. (b) Flt3 transcript levels in leukemic bone marrow cells. Cells were prepared from two mice of each genotype. (c) Cell-surface FLT3 expression of leukemic BM cells. Representative histograms are shown (left). (d) Cell-surface FLT3 expression of Hes1- or mock- transduced MLL–AF9/Hes1− / − cells after serum starvation. (e) Chromatin immunoprecipitation analysis for Flag–Hes1 or mock-expressing THP1 cells that used the indicated antibodies (n = 3). Error bars indicate ± s.d.; *Po0.05. (f) Relative luciferase activity of Flt3 promoter and its N-box mutant in HEK293T cells expressing wild-type Hes1 or dnHes1 protein. Error bars indicate ± s.d.; *Po0.05. A representative result from three independent experiments is shown. Mut, N-box mutants on Flt3 promoter; WT, wild type. (g) FLT3 total protein levels and levels of phosphorylated FLT3. MLL–AF9/Hes1+/+ and MLL–AF9/Hes1− / − cells were treated with or without FLT3 ligand. Cell lysates were prepared and immunoprecipitated with anti-FLT3 antibody and then immunoblotted with anti-FLT3 or anti-phosphotyrosine antibodies (4G10).

MLL–AF9/Hes1− / − cells, but not MLL–AF9/Hes1+/+ cells, showed AC220, reduced the number of colonies derived from MLL–AF9/ higher proliferative capacity by the FLT3 ligand stimulation than Hes1− / −cells with or without Flt3 ligand to the level comparable to did without this cytokine stimulation (Figure 5a). This effect was the number of colonies derived from wild-type cells (Figure 5d). blocked by treatment of cells with the FLT3 tyrosine kinase The Flt3 inhibitor did not affect the number of colonies derived inhibitor KRN383 (ref. 35; Figures 5b and c). Similarly, stimulation from MLL–AF9/Hes1+/+ cells (Figure 5d). These data indicate that of MLL–AF9/iCre/RBPJf/f cells with FLT3 ligand enhanced their there is a weak autocrine loop for FLT3 signaling, surrounding proliferation (data not shown), while treatment of these cells with colonies. The increase in the colony number with MLL–AF9/Hes1− / − KRN383 in the presence of FLT3 ligand reversed this proliferative cells in the second and tertiary platings without exogenous FLT3 effect (Supplementary Figure 4D). Flt3 ligand also increased the ligand shown in Figure 2a, therefore, implies the combinatorial number of colonies derived from MLL–AF9/Hes1− / − cells, but not effect of exogenous interleukin 3 and autocrine FLT3 signaling in MLL–AF9/Hes1+/+ cells. In contrast, an FLT3 kinase inhibitor, Hes1-null background.

1.5 MLL-AF9/Hes1+/+ 12 0 nM +/+ +/+ MLL-AF9/Hes1 with FLT3 ligand MLL-AF9/Hes1-/- 10 10 nM MLL-AF9/Hes1 6 5 -/- 30 nM MLL-AF9/Hes1 with FLT3 ligand 1 0 nM 8 +/+ MLL-AF9/Hes1 10 nM MLL-AF9/Hes1-/- MLL-AF9/Hes1-/- 5 30 nM 4 0.5 Treated OD relative to untreated (450nM) Number of cells ×10 Number of cells x10 0 0 0 d0 d2 d3 d4 0102030 0 0h 48h 96h KRN383 (nM)

250000 500 MLL-AF9/Hes1+/+ sh1 MLL-AF9/Hes1-/- 400 200000 sh2 sh3 300 150000 sh4 200 100000 cont 100 50000 0 Number of colonies/ 500 cells 0 0h 24h 72h

350000 +/+ Flt3 control -/- MLL-AF9/Hes1 300000 -/- Flt3+/+ Flt3-/- Flt3 Line1 250000 -/- Control Line1 Line2 Line3 Line4 Line5 Flt3 Line2 50.4% 0.1% 0.0% 1.0% 2.6% 0.7% -/- 200000 Flt3 Line3 * -/- 150000 Flt3 Line4 -/- Flt3 Line5 100000 FLT3 50000

0 0h 24h 72h Figure 5. Upregulated FLT3 is biologically functional. (a) Growth of MLL–AF9/Hes1− / − or MLL–AF9/Hes1+/+ cells with or without FLT3 ligand in liquid medium containing low interleukin 3 (IL3) levels. Shown are representative results (n = 3 each). (b) Effect of KRN383 on cell viability. MLL–AF9/Hes1+/+ or MLL–AF9/Hes1− / − cells were incubated with 10% fetal calf serum and IL3 containing KRN383 at indicated concentrations. A fractional growth referenced to untreated controls at 48 h is shown. The line represents a ﬁt of data to the Hill equation. (c) Growth of MLL– AF9/Hes1+/+ or MLL–AF9/Hes1− / − cells in liquid medium in the presence of KRN383. (d) The number of colonies derived from MLL–AF9/Hes1− / − and MLL–AF9/Hes1+/+ cells using either FLT3 ligand or AC220 (FLT3 kinase inhibitor) or both. Error bars indicate ± s.d.; *Po0.05. (e) Growth of MLL–AF9/Hes1− / − cells introduced with shRNA for Flt3. Representative data from four independent experiments is shown. n = 4 each. (f) Cell-surface FLT3 expression of Flt3-deleted MLL–AF9/Hes1− / − cells by CRISPR-Cas9. Blue line, Flt3+/+ control cells; Red line, Flt3− / − cells. (g) Growth of MLL–AF9/Hes1− / − cells with deleted Flt3 by CRISPR/CAS9. Representative data from three independent experiments is shown; n = 4 each.

Then, we performed Flt3 knockdown experiments using short mice transplanted with 10 000 FLT3-positive cells developed hairpin RNA (shRNA; Supplementary Figure 4E). The growth of the leukemia in the same latency as those transplanted with FLT3- cells introduced with each Flt3 shRNA was significantly reduced negative cells, the mice transplanted with 1000 or 100 FLT3- compared with the control shRNA-introduced MLL–AF9/Hes1− / − positive cells developed leukemia in significantly shorter latencies cells (Figure 5e). We further established Flt3 knockout cell lines by than those transplanted with the same number of FLT3-negative using the CRISPR/CAS9 technology. We evaluated five indepen- cells (Figure 6). These data indicate that the FLT3-positive fraction dent clones with the Flt3 genome edited, accompanying robust contains leukemia-initiating cells at a greater frequency than the reduction of FLT3 expression (Figure 5f). Each Flt3-edited clone FLT3-negative fraction, consequently suggesting that loss of Hes1 showed significantly reduced growth compared with the clone increases the number of leukemia-initiating cells. retaining Flt3 genome and expression retained (Figure 5g). Expression of FLT3 mRNA is negatively correlated with that of HES1 Hes1 loss increases the frequency of leukemia-initiating cells or NOTCH2 in AML patient samples Based on the increase in the FLT3-positive cell frequency in MLL– We next assessed databases containing mRNA expression profiles AF9/Hes1− / − AML cells, we performed secondary transplantation derived from microarray analysis of 285 AML patient samples by infusing serially diluted FLT3-positive or -negative MLL–AF9/ (www.ncbi.nlm.nih.gov/geo, accession number GSE1159; ref. 36) Hes1− / − AML cells prepared from the primary mice. Although the and found that 13 samples exhibited MLL fusions. In these

P = 0.160 P = 0.027

P = 0.017 Survival rate

Figure 6. Upregulated FLT3 marks leukemia-initiating cells. (a) Survival of mice secondary transplanted with diluted numbers of FLT3-positive or -negative MLL–AF9/Hes1− / − AML cells prepared from the primary mice.

MLL-associated AML AML with normal karyotype AML with t(8;21) AML with t(15;17) 100 100 100 100 P =0.002 P =0.270 P =0.440 P =0.820

50 50 50 50 HES1 HES1 HES1 HES1

0 0 0 0 0 1000 2000 0 1000 2000 0 1000 2000 0 1000 2000 FLT3 FLT3 FLT3 FLT3

MLL-associated AML

10 10 P < 0.043 P < 0.001

5 5 HES1 NOTCH2

0 0 0102001020 FLT3 FLT3 Figure 7. FLT3 expression is negatively correlated with that of HES1 in MLL-related AML samples. (a) Re-analysis of the GSE1159 Gene Expression Omnibus database of AML tumor samples (deposited by Valk et al. 36). Relative expression levels of FLT3 and HES1 in 13 MLL-related AML samples, 116 AML samples with normal karyotype, 18 AML samples with t(15;17) and 22 AML samples with t(8;21). (b) Reanalysis of the GSE19577 database (deposited by Pigazzi et al.37). Relative expression levels of FLT3 and NOTCH2, and FLT3 and HES1 in 42 MLL-related AML samples.

samples, FLT3 expression levels were negatively correlated with and observed similar relationships between expression levels of those of HES1 and NOTCH2. In contrast, expression levels of HES1 FLT3, NOTCH2 and HES1 (Figure 7b). and FLT3 showed no correlation in the MLL fusion-negative AML sub-cohort, although expression of NOTCH2 and FLT3 showed a negative correlation also in this population (Figure 7a and A Notch agonist induces apoptosis of MLL–AF9-transduced cells Supplementary Figure 5). We also assessed a different database dependently on Hes1 derived from microarray analysis of 42 MLL-related AML samples Flow cytometric analysis indicated that Notch2 is highly expressed (www.ncbi.nlm.nih.gov/geo, accession number GSE19577; ref. 37) on the surface of MLL–AF9-transduced cells, whereas other Notch

+/+ -/- +/+ -/- MLL-AF9/Hes1 MLL-AF9/Hes1 NC MLL-AF9/Hes1 MLL-AF9/Hes1 0.1% 0.8% 0 1.1 0.2 8.90.4 12.2

Notch1 91.4 7.5 IgG 82.8 8.179.8 7.6

IgG PI 0.6 36.20.1 9.4 95% 94% 0 1.2 Notch2 N2 PI 92.5 6.4 47.2 16.082.2 8.3

0.4% 1.4% N2 6ug/ml AnnexinV 0 20.8 Notch3 P < 0.01 P = 0.01 P < 0.01 P = 0.01 30 60 66.1 13.0

20 40 Cells (%) - Cells (%)

+ 0.4% 1.5% PI

AnnexinV + Notch4 10 20 AnnexinV AnnexinV 0 0 APC Hes1 +/+ -/-+/+ -/- Hes1 +/+ -/-+/+ -/- IgG N2 IgG N2

15.0 P = 0.045

10.0

5.0

GFP+ 7AAD- cells in BM (%) 0.0 IgG(N=5) α N2(N=6) Figure 8. A Notch2 agonistic antibody induces apoptosis in MLL–AF9-transduced cells. (a) Cell-surface expression of Notch1–4 in MLL–AF9/ Hes1− / − or MLL/AF9–Hes1+/+ cells. Representative histograms are shown from three independent experiments. (b) Apoptosis of MLL–AF9 leukemic cells following treatment with anti-Notch2 agonistic antibody. Shown is a representative result from three independent experiments. (c) Apoptosis of MLL–AF9-transduced cells following Notch2 stimulation requires Hes1. (Left) A representative flow cytometric pattern from three independent experiments. (Right) Summary of three independent experiments. (d)Therapeutic AML mouse model using an agonistic anti-Notch2 antibody. Hamster anti-mouse Notch2 (HMN2–29, 100 μg/mouse) or hamster immunoglobulin-G (IgG; 100 μg/mouse) was injected on days 1, 3, 7 and 10 after transplantation of MLL–AF9/Hes1+/+-transduced cells. The engraftment ratio, which is shown by the GFP- positive cell ratio in bone marrow, was measured by flow cytometry at day 14 after transplantation. αN2, anti-Notch2 antibody. family receptors were either undetectable or detectable at low model experiment using a Notch2 agonistic antibody, and found levels (Figure 8a). Thus, to evaluate the effect of Notch2 signaling that the antibody was also effective in vivo. The frequencies of we treated MLL–AF9-transduced cells with a hamster anti-mouse leukemic cells, which is shown by the GFP-positive cell ratio in Notch2 agonistic antibody or control immunoglobulin-G.38 Notch2 bone marrow were significantly reduced in mice treated with the antibody treatment significantly induced apoptosis in MLL–AF9 Notch2 agonistic antibody compared with immunoglobulin-G cells compared with immunoglobulin-G-treated cells (Figure 8b), (Figure 8d). which was abrogated in a Hes1-null background (Figure 8c). This effect was also blocked when MLL–AF9-transduced cells were treated with the Notch2 agonistic antibody in the presence of a DISCUSSION γ-secretase inhibitor, DAPT, which inhibits Notch signaling AML is characterized by clonal expansion of myeloid progenitor (Supplementary Figure 6), indicating that apoptosis requires cells in bone marrow. MLL fusion genes are detected in ~ 5% of Notch cleavage. Furthermore we performed a therapeutic animal AML patients and have an unfavorable impact on the prognosis.

© 2015 Macmillan Publishers Limited Leukemia (2015) 576 – 585 Tumor suppressive role of Hes1 in myeloid leukemia T Kato et al 584 Development of new treatment strategies requires better under- Six1 potentiates Eya1’s transforming capacity.49 Jun, an standing of the molecular pathogenesis of MLL-related AML. important component of the JNK pathway, has not been In hematopoietic malignancies, Notch signaling has both described as a direct MLL–AF9 target. These data suggest that tumor-promoting and -suppressive roles depending on context. the Notch–RBPJ–Hes1 axis suppresses MLL–AF9 leukemia by Although the significance of Notch signaling in myeloid malig- modulating direct and indirect targets of MLL–AF9. nancies remains controversial, our results, together with previous – – 23,24 In summary, we have demonstrated that the Notch RBPJ Hes1 reports, strongly suggest that Notch signaling physiologically axis functions as a tumor suppressor in AML, probably via, at least suppresses development of a broad range of myeloid leukemias, in part, repression of FLT3. Our results provide insight into AML including AML. In our model, Notch stimulation resulted in the pathogenesis and may suggest novel therapeutic approaches to growth suppression of AML cells, implying that Notch agonistic the disease. agents could serve as treatment modalities, as previously suggested by others.24 Notch signal inhibitors, such as γ-secretase inhibitors, have been developed as drugs for T-ALL.39,40 If either CONFLICT OF INTEREST Notch-activating or Notch-suppressive drugs prove effective, The authors declare no conflict of interest. accurate diagnosis would be critical, making it necessary to identify new biomarkers that could precisely predict indications to each medicine. ACKNOWLEDGEMENTS The dichotomous functions of Notch signaling may result from differences in downstream targets. The proto-oncogene MYC was We thank Drs T Machino and T Enami (University of Tsukuba) for discussion; shown to be a direct transcriptional target of Notch–RBPJ and Drs A Yokoyama (Kyoto University), H Nakauchi (University of Tokyo/Stanford University), and, M Onodera (National Research Institute for Child Health and contribute to tumor progression in T-ALL,41 and Hes1 has been Development) for vectors. We also thank T Takahashi for mouse experiments. We are demonstrated to have a major role in downstream Notch 42 also grateful to Kyowa Hakko Kirin Co., Ltd. for KRN383. This work was supported by signaling for T-ALL promotion. Nevertheless, Hes1 was subse- Grants-in-Aid for Scientific Research (KAKENHI) from the Ministry of Education, quently suggested to be a downstream mediator of Notch 19 Culture, Sports, Science and Technology of Japan (25860778 to TK; 25461407 to signaling in suppressing B-cell ALL cell growth. Although several MS-Y; and 25112703, 24390241, 23118503 and 22130002 to SC) and supported by groups have indicated possible tumor-suppressive function of the Sagawa Cancer Foundation, the Naito Foundation, the Kato Memorial Bioscience Notch–Hes1 axis, also in AML, the work described here is the first Foundation and the YASUDA Medical Foundation to MS-Y. genetic evidence showing that Hes1 actually has an essential role as a tumor suppressor downstream to Notch signaling. The observation that Hes1 is activated in varying contexts REFERENCES implies that distinct downstream regulatory networks are utilized 1 Artavanis-Tsakonas S, Rand MD, Lake RJ. Notch signaling: cell fate control and to promote or suppress a wide range of hematologic malig- signal integration in development. Science 1999; 284: 770–776. nancies. For T-ALL development, repression of the tumor 2 De Strooper B, Annaert W, Cupers P, Saftig P, Craessaerts K, Mumm JS et al. suppressors CYLD and PTEN is proposed to mediate the Notch– A presenilin-1-dependent gamma-secretase-like protease mediates release of 43,44 RBPJ–Hes1 pathway, while in a very different context, poly Notch intracellular domain. Nature 1999; 398:518–522. ADP-ribose polymerase1 is activated by interaction with Hes1 and 3 Kageyama R, Ohtsuka T, Tomita K. The bHLH gene Hes1 regulates differentiation functions to induce apoptosis of B-ALL cells.19 of multiple cell types. Mol Cells 2000; 10:1–7. We identified FLT3 downregulation as a target of Hes1 in 4 Oswald F, Tauber B, Dobner T, Bourteele S, Kostezka U, Adler G et al. p300 acts as suppressing AML development. Activating mutations in FLT3 gene a transcriptional coactivator for mammalian Notch-1. Mol Cell Biol 2001; 21: – are seen in up to 30% of AML patients.45 In particular, FLT3- 7761 7774. internal tandem duplication mutations are associated with poor 5 Bigas A, Espinosa L. Hematopoietic stem cells: to be or Notch to be. Blood 2012; 119: 3226–3235. survival when AML patients are treated with standard chemother- – 6 Grbavec D, Stifani S. Molecular interaction between TLE1 and the carboxyl- apy. In addition, recent studies indicate that ~ 10 15% of AML terminal domain of HES-1 containing the WRPW motif. Biochem Biophys Res 46 patients display high expression of wild-type FLT3. High FLT3 Commun 1996; 223:701–705. expression has a negative impact on overall and event-free 7 Ito T, Udaka N, Okudela K, Yazawa T, Kitamura H. Mechanisms of neuroendocrine survival in cytogenetically normal AML patients lacking FLT3 differentiation in pulmonary neuroendocrine cells and small cell carcinoma. mutations.47 This fact might be relevant to the enhanced AML Endocr Pathol 2003; 14:133–139. development and increased FLT3 expression we report here in a 8 Ito T, Udaka N, Yazawa T, Okudela K, Hayashi H, Sudo T et al. Basic helix-loop-helix mouse model: in AML seen in patients or mouse models, transcription factors regulate the neuroendocrine differentiation of fetal mouse increased tyrosine kinase activity is likely to be a key. Although pulmonary epithelium. Development 2000; 127:3913–3921. FLT3 inhibitors have been developed for clinical use,48 it is not 9 Weng AP, Ferrando AA, Lee W, Morris JP 4th, Silverman LB, Sanchez-Irizarry C et al. Activating mutations of NOTCH1 in human T cell acute lymphoblastic leukemia. known whether high FLT3 levels could serve as a biomarker to 306 – fi Science 2004; :269 271. predict ef cacy or resistance to those inhibitors. 10 Suzuki T, Chiba S. Notch signaling in hematopoietic stem cells. Int J Hematol 2005; Expression levels of FLT3 and HES1 were negatively correlated 82:285–294. only in MLL fusion-associated AML, as determined by analysis of 11 Radtke F, Wilson A, Stark G, Bauer M, van Meerwijk J, MacDonald HR et al. public databases, implying that FLT3 upregulation by the Notch– Deficient T cell fate specification in mice with an induced inactivation of Notch1. RBPJ–Hes1 pathway is confined to just MLL–AF9-induced AML, Immunity 1999; 10: 547–558. rather than universal to AML of diverse molecular backgrounds or 12 Puente XS, Pinyol M, Quesada V, Conde L, Ordonez GR, Villamor N et al. confined to just MLL–AF9-induced AML. Nevertheless, Lobry Whole-genome sequencing identifies recurrent mutations in chronic lymphocytic et al.23 reported that Notch signaling has a tumor suppressive leukaemia. Nature 2011; 475:101–105. role in a broad range of AML cells. Thus, whether distinct effectors 13 Kridel R, Meissner B, Rogic S, Boyle M, Telenius A, Woolcock B et al. Whole transcriptome sequencing reveals recurrent NOTCH1 mutations in mantle cell operate downstream of Notch in MLL fusion-positive and 119 – -negative AMLs needs to be clarified. In this regard, our microarray lymphoma. Blood 2012; : 1963 1971. fi fi 14 Lee SY, Kumano K, Nakazaki K, Sanada M, Matsumoto A, Yamamoto G et al. analysis of Hes1-de cient AML cells identi ed, in addition to Flt3, Gain-of-function mutations and copy number increases of Notch2 in diffuse large several other candidate genes such as Eya1, Six1 and Jun (data not B-cell lymphoma. Cancer Sci 2009; 100:920–926. shown). Eya1 and Six1 are known to be direct transcriptional 15 Kiel MJ, Velusamy T, Betz BL, Zhao L, Weigelin HG, Chiang MY et al. 49 targets of MLL–AF9. Eya1 overexpression immortalizes Whole-genome sequencing identifies recurrent somatic NOTCH2 mutations in hematopoietic progenitor cells and its co-transduction with splenic marginal zone lymphoma. J Exp Med 2012; 209: 1553–1565.

Leukemia (2015) 576 – 585 © 2015 Macmillan Publishers Limited Tumor suppressive role of Hes1 in myeloid leukemia T Kato et al 585 16 Robinson DR, Kalyana-Sundaram S, Wu YM, Shankar S, Cao X, Ateeq B et al. 34 Hayakawa F, Towatari M, Kiyoi H, Tanimoto M, Kitamura T, Saito H et al. Functionally recurrent rearrangements of the MAST kinase and Notch gene Tandem-duplicated Flt3 constitutively activates STAT5 and MAP kinase and families in breast cancer. Nat Med 2011; 17: 1646–1651. introduces autonomous cell growth in IL-3-dependent cell lines. Oncogene 2000; 17 Nicolas M, Wolfer A, Raj K, Kummer JA, Mill P, van Noort M et al. Notch1 functions 19:624–631. as a tumor suppressor in mouse skin. Nat Genet 2003; 33:416–421. 35 Komeno Y, Kurokawa M, Imai Y, Takeshita M, Matsumura T, Kubo K et al. 18 Yan M, Callahan CA, Beyer JC, Allamneni KP, Zhang G, Ridgway JB et al. Chronic Identification of Ki23819, a highly potent inhibitor of kinase activity of mutant DLL4 blockade induces vascular neoplasms. Nature 2010; 463:E6–E7. FLT3 receptor tyrosine kinase. Leukemia 2005; 19:930–935. 19 Kannan S, Fang W, Song G, Mullighan CG, Hammitt R, McMurray J et al. 36 Valk PJ, Verhaak RG, Beijen MA, Erpelinck CA, Barjesteh van Waalwijk van Doorn- fi Notch/HES1-mediated PARP1 activation: a cell type-speci c mechanism for tumor Khosrovani S, Boer JM et al. Prognostically useful gene-expression profiles in 117 – suppression. Blood 2011; : 2891 2900. acute myeloid leukemia. N Engl J Med 2004; 350:1617–1628. 20 Nakahara F, Sakata-Yanagimoto M, Komeno Y, Kato N, Uchida T, Haraguchi K et al. 37 Pigazzi M, Masetti R, Bresolin S, Beghin A, Di Meglio A, Gelain S et al. Hes1 immortalizes committed progenitors and plays a role in blast crisis transition MLL partner genes drive distinct gene expression profiles and genomic altera- in chronic myelogenous leukemia. Blood 2010; 115: 2872–2881. tions in pediatric acute myeloid leukemia: an AIEOP study. Leukemia 2011; 25: 21 Ito T, Kwon HY, Zimdahl B, Congdon KL, Blum J, Lento WE et al. Regulation of 560–563. myeloid leukaemia by the cell-fate determinant Musashi. Nature 2010; 466: 38 Moriyama Y, Sekine C, Koyanagi A, Koyama N, Ogata H, Chiba S et al. Delta-like 1 is 765–768. essential for the maintenance of marginal zone B cells in normal mice but not in 22 Klinakis A, Lobry C, Abdel-Wahab O, Oh P, Haeno H, Buonamici S et al. A novel autoimmune mice. Int Immunol 2008; 20: 763–773. tumour-suppressor function for the Notch pathway in myeloid leukaemia. Nature 2011; 473: 230–233. 39 Paganin M, Ferrando A. Molecular pathogenesis and targeted therapies for 25 – 23 Lobry C, Ntziachristos P, Ndiaye-Lobry D, Oh P, Cimmino L, Zhu N et al. Notch NOTCH1-induced T-cell acute lymphoblastic leukemia. Blood Rev 2011; :83 90. pathway activation targets AML-initiating cell homeostasis and differentiation. 40 Samon JB, Castillo-Martin M, Hadler M, Ambesi-Impiobato A, Paietta E, Racevskis J J Exp Med 2013; 210: 301–319. et al. Preclinical analysis of the gamma-secretase inhibitor PF-03084014 in com- 24 Kannan S, Sutphin RM, Hall MG, Golfman LS, Fang W, Nolo RM et al. Notch bination with glucocorticoids in T-cell acute lymphoblastic leukemia. Mol Cancer activation inhibits AML growth and survival: a potential therapeutic approach. Ther 2012; 11: 1565–1575. J Exp Med 2013; 210: 321–337. 41 Weng AP, Millholland JM, Yashiro-Ohtani Y, Arcangeli ML, Lau A, Wai C et al. c-Myc 25 Han H, Tanigaki K, Yamamoto N, Kuroda K, Yoshimoto M, Nakahata T et al. is an important direct target of Notch1 in T-cell acute lymphoblastic leukemia/ Inducible gene knockout of transcription factor recombination signal binding lymphoma. Genes Dev 2006; 20: 2096–2109. protein-J reveals its essential role in T versus B lineage decision. Int Immunol 2002; 42 Wendorff AA, Koch U, Wunderlich FT, Wirth S, Dubey C, Brüning JC et al. 14: 637–645. Hes1 is a critical but context-dependent mediator of canonical notch signaling in 26 Tanigaki K, Han H, Yamamoto N, Tashiro K, Ikegawa M, Kuroda K et al. Notch-RBP-J lymphocyte development and transformation. Immunity 2010; 33: 671–684. signaling is involved in cell fate determination of marginal zone B cells. Nat 43 Espinosa L, Cathelin S, D'Altri T, Trimarchi T, Statnikov A, Guiu J et al. The Notch/ Immunol 2002; 3: 443–450. Hes1 pathway sustains NF-kappaB activation through CYLD repression in T cell 27 Guiu J, Shimizu R, D'Altri T, Fraser ST, Hatakeyama J, Bresnick EH et al. Hes leukemia. Cancer Cell 2010; 18: 268–281. repressors are essential regulators of hematopoietic stem cell development 44 Palomero T, Sulis ML, Cortina M, Real PJ, Barnes K, Ciofani M et al. Mutational loss 210 – downstream of Notch signaling. J Exp Med 2013; :71 84. of PTEN induces resistance to NOTCH1 inhibition in T-cell leukemia. Nat Med 2007; 28 Tomita K, Ishibashi M, Nakahara K, Ang SL, Nakanishi S, Guillemot F et al. Mam- 13: 1203–1210. malian hairy and Enhancer of split homolog 1 regulates differentiation of retinal 45 Frohling S, Schlenk RF, Breitruck J, Benner A, Kreitmeier S, Tobis K et al. Prognostic neurons and is essential for eye morphogenesis. Neuron 1996; 16: 723–734. significance of activating FLT3 mutations in younger adults (16 to 60 years) with 29 Somervaille TC, Cleary ML. Identification and characterization of leukemia stem acute myeloid leukemia and normal cytogenetics: a study of the AML Study cells in murine MLL-AF9 acute myeloid leukemia. Cancer Cell 2006; 10:257–268. Group Ulm. Blood 2002; 100: 4372–4380. 30 Sang L, Coller HA, Roberts JM. Control of the reversibility of cellular quiescence by 46 Riccioni R, Pelosi E, Riti V, Castelli G, Lo-Coco F, Testa U. Immunophenotypic the transcriptional repressor HES1. Science 2008; 321:1095–1100. 31 McLarren KW, Theriault FM, Stifani S. Association with the nuclear matrix and features of acute myeloid leukaemia patients exhibiting high FLT3 expression not 153 – interaction with Groucho and RUNX proteins regulate the transcription repression associated with mutations. Br J Haematol 2011; :33 42. activity of the basic helix loop helix factor Hes1. J Biol Chem 2001; 276: 47 Kuchenbauer F, Kern W, Schoch C, Kohlmann A, Hiddemann W, Haferlach T et al. 1578–1584. Detailed analysis of FLT3 expression levels in acute myeloid leukemia. 90 – 32 Kang HJ, Lee JW, Kho SH, Kim MJ, Seo YJ, Kim H et al. High transcript level of FLT3 Haematologica 2005; :1617 1625. associated with high risk of relapse in pediatric acute myeloid leukemia. J Korean 48 Kindler T, Lipka DB, Fischer T. FLT3 as a therapeutic target in AML: still challenging Med Sci 2010; 25: 841–845. after all these years. Blood 2010; 116:5089–5102. 33 Ozeki K, Kiyoi H, Hirose Y, Iwai M, Ninomiya M, Kodera Y et al. Biologic and clinical 49 Wang QF, Wu G, Mi S, He F, Wu J, Dong J et al. MLL fusion proteins preferentially significance of the FLT3 transcript level in acute myeloid leukemia. Blood 2004; regulate a subset of wild-type MLL target genes in the leukemic genome. Blood 103: 1901–1908. 2011; 117: 6895–6905.

Supplementary Information accompanies this paper on the Leukemia website (http://www.nature.com/leu)