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High-level IGF1R expression is required for leukemia-initiating cell activity in T-ALL and is supported by Notch signaling

High-level IGF1R expression is required for leukemia-initiating cell activity in T-ALL and is... A r t i c l e High-level IGF1R expression is required for leukemia-initiating cell activity in T-ALL and is supported by Notch signaling 1 1 2 3 Hind Medyouf, Samuel Gusscott, Hongfang Wang, Jen-Chieh Tseng, 1 1 4 5 Carol Wai, Oksana Nemirovsky, Andreas Trumpp, Francoise Pflumio, 6 6 7 3,8 Joan Carboni, Marco Gottardis, Michael Pollak, Andrew L. Kung, 2 9 1 Jon C. Aster, Martin Holzenberger, and Andrew P. Weng Terry Fox Laboratory/Department of Pathology, BC Cancer Agency, Vancouver, BC, V52 1L3 Canada Department of Pathology, Brigham & Women’s Hospital/Harvard Medical School, Boston, MA 02115 Lurie Family Imaging Center, Dana-Farber Cancer Institute, Boston, MA 02115 Heidelberg Institute for Stem Cell Technology and Experimental Medicine (HI-STEM), and Division of Stem Cells and Cancer, German Cancer Research Center (DKFZ), D-69120 Heidelberg, Germany LSHL/IRCM, Institut National de la Santé et de la Recherche Médicale U967, Université Paris 7, CEA, 92265 Fontenay-aux- Roses, France Oncology Drug Discovery, Bristol-Myers Squibb Company, Princeton, NJ 08543 Department of Oncology, McGill University, Montreal, Quebec, H3T 1E2 Canada Department of Pediatric Oncology, Dana-Farber Cancer Institute and Children’s Hospital/Harvard Medical School, Boston, MA 02115 Centre de Recherche Institut National de la Santé et de la Recherche Médicale Saint-Antoine, Université Pierre-et-Marie- Curie, 75571 Paris, France T cell acute lymphoblastic leukemia (T-ALL) is an aggressive cancer of immature T cells that often shows aberrant activation of Notch1 and PI3K–Akt pathways. Although muta- tions that activate PI3K–Akt signaling have previously been identified, the relative contri - bution of growth factor-dependent activation is unclear. We show here that pharmacologic inhibition or genetic deletion of insulin-like growth factor 1 receptor (IGF1R) blocks the growth and viability of T-ALL cells, whereas moderate diminution of IGF1R signaling com- promises leukemia-initiating cell (LIC) activity as defined by transplantability in syngeneic/ congenic secondary recipients. Furthermore, IGF1R is a Notch1 target, and Notch1 signaling is required to maintain IGF1R expression at high levels in T-ALL cells. These findings suggest effects of Notch on LIC activity may be mediated in part by enhancing the responsiveness of T-ALL cells to ambient growth factors, and provide strong rationale for use of IGF1R inhibitors to improve initial response to therapy and to achieve long-term cure of patients with T-ALL. T cell acute lymphoblastic leukemia (T-ALL) is including mutation or inactivation of PTEN CORRESPONDENCE Andrew P. Weng: an aggressive cancer of immature T cell pro- (Kawamura et al., 1999; Perentesis et al., 2004; aweng@bccrc.ca genitors that often shows aberrant activation of Maser et al., 2007; Palomero et al., 2007; Silva OR NOTCH1 and PI3K–Akt pathways. Activating et al., 2008; Gutierrez et al., 2009) and muta- Hind Medyouf: h.medyouf@dkfz-heidelberg.de mutations of Notch1 occur in >50% of cases of tion of PIK3 and Akt (Kawamura et al., 1999; T-ALL (Weng et al., 2004), whereas mutations Gutierrez et al., 2009). Activation of PI3K–Akt Abbreviations used: 4-OHT, in related Notch pathway elements such as has been shown to collaborate with Notch in 4-hydroxytamoxifen; ChIP, chromatin immunoprecipita- Sel10/Fbw7 occur in 8–16% of cases (O’Neil leukemogenesis (Medyouf et al., 2010), enhance tion; LIC, leukemia-initiating et al., 2007; Thompson et al., 2007). PI3K–Akt growth of established leukemias (Chiarini et al., cell; NOD, nonobese diabetic; pathway activation occurs in >85% of cases 2009; Cullion et al., 2009; Levy et al., 2009; qPCR, quantitative PCR; (Silva et al., 2008) via diverse mechanisms, Sanda et al., 2010), and in some contexts to T-ALL, T cell acute lympho- blastic leukemia. © 2011 Medyouf et al. This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first H. Medyouf’s present address is Heidelberg Institute for Stem six months after the publication date (see http://www.rupress.org/terms). After Cell Technology and Experimental Medicine (HI-STEM), six months it is available under a Creative Commons License (Attribution–Non- and Division of Stem Cells and Cancer, German Cancer commercial–Share Alike 3.0 Unported license, as described at http://creativecom- Research Center (DKFZ), D-69120 Heidelberg, Germany mons.org/licenses/by-nc-sa/3.0/). The Rockefeller University Press $30.00 J. Exp. Med. Vol. 208 No. 9 1809-1822 www.jem.org/cgi/doi/10.1084/jem.20110121 The Journal of Experimental Medicine relieve dependence on Notch signaling (Palomero et al., samples (Weng et al., 2004; Weng et al., 2006; Medyouf 2007). For cases that lack such mutations, however, the mech- et al., 2010). For mouse cells, we examined primary leuke- anisms that support activation of the pathway are unknown. mias derived by retroviral transduction/transplantation of More generally, it is also unknown to what extent growth bone marrow with an activated form of NOTCH1 termed factor–dependent stimulation of cognate receptor tyrosine E (Pear et al., 1996). To confirm IGF1R-stimulated PI3K– kinases (RTKs) contributes to the net signaling output. Akt in these contexts, we pulsed serum-starved leukemia cells Although previous works have focused on the role of with recombinant IGF-1 and measured phospho-Akt activa- IL-7 signaling in T-ALL, including effects on downstream tion by flow cytometry. We observed that both human and PI3K–Akt activation (Dibirdik et al., 1991; Barata et al., mouse leukemia cells respond robustly to IGF-1 stimulation 2004a,b,c, 2005; González-Garcia et al., 2009; Shochat et al., under these conditions (Fig. S1). 2011; Silva et al., 2011), we considered that insulin-like growth factor (IGF)-1 receptor (IGF1R) may also play an Pharmacologic inhibition of IGF1R compromises important role. IGFs and their receptors regulate normal cell T-ALL cell growth growth and contribute to transformation and growth of ma- To assess the extent to which T-ALL cells are dependent on lignant cells in many contexts (Pollak et al., 2004). IGF1 and IGF1R signaling, we used pharmacologic IGF1R inhibitors. IGF2 bind to IGF1R, a transmembrane receptor tyrosine Most small molecule IGF1R inhibitors also affect insulin re - kinase (RTK), thereby initiating a cascade of downstream ceptor caused by their close homology and at higher doses phosphorylation events that bifurcates along both PI3K–Akt may be expected to cross react with more distantly related and Ras–Raf–MAPK pathways. PI3K–Akt activation leads receptor tyrosine kinases. One such inhibitor, BMS-536924 to enhanced cellular metabolism and protein synthesis via (Wittman et al., 2005), substantially inhibited growth/prolif- mTOR and enhanced survival via BAD/Bcl2, p53, NF-kB, eration of both mouse and human leukemia cells in vitro and FOXOs, whereas Ras–Raf–MAPK activation generally (Fig. 2 and Fig. S2). Primary leukemia cells were generally results in increased cellular proliferation (Pollak et al., 2004; more sensitive to BMS-536924 with IC50 values in the 0.1– Greer and Brunet, 2005). Signaling through IGF1R has also 1.0 µM range, whereas established cell lines required some- been implicated in self-renewal of stem cells, both in embry- what higher doses (2–4 µM) to achieve similar effects. As onic (Bendall et al., 2007) and hematopoietic (Ivanova et al., expected, BMS-536924 completely suppressed Akt activa- 2002) contexts. tion by both IGF-1 and insulin in human and mouse T-ALL cells (Fig. S1). RESULTS We next examined whether blocking antibodies against IGF1R is broadly expressed in T-ALL IGF1R, which might be expected to exhibit a higher degree To begin to address a potential role for IGF1R in T-ALL, we of specificity than small molecule kinase inhibitors, would assessed IGF1R expression in mouse and human T-ALL cells. recapitulate effects seen with BMS-536924. The IGF1R- Analysis of IGF1R by Western blot and flow cytometry re - blocking antibody IR3 substantially inhibited proliferation vealed IGF1R was expressed in all cases examined, albeit at in three of four primary human leukemias tested (Fig. 2 B, varying levels (Fig. 1). For human cells, we examined both rightmost column). Notably, the one resistant case (K419) established cell lines and xenograft-expanded primary human expressed lower levels of IGF1R compared with the others Figure 1. IGF1R is expressed broadly in human and mouse T-ALL. (A and B) Western blot and (C and D) flow cytometric analysis of total and surface IGF1R protein expression, respectively, from human cell lines (A and C), primary mouse leukemias (B) derived by retro- viral transduction/transplantation of bone marrow with an activated form of Notch1 termed E, and xenograft-expanded primary human samples (D). Western blot controls in (B) are mouse embryonic fibroblasts derived null from IGF1R mouse embryos (R) and the same cells stably transfected with an IGF1R cDNA expression construct (R+). At least 20,000 events were collected within each gate for all flow cytometry assays. Data de - picted are representative of at least two inde- pendent experiments. 1810 Notch-IGF1R signaling supports LICs in T-ALL | Medyouf et al. A r t i c l e +/+ (Fig. S3) and showed minimal response to IGF-1 stimulation IGF1R E leukemias (Fig. 3, C and D; and not depicted). neo/neo (unpublished data), suggesting the effect of BMS-536924 on Importantly, we confirmed that IGF1R E leukemias this particular tumor may be mediated by inhibition of insulin expressed reduced levels of full-length IGF1R protein (32 ± +/+ receptor. In contrast, case M69 is highly sensitive to IR3, 20% of levels observed in IGF1R E leukemias, n = 7; but largely resistant to BMS-536924, raising the possibility of Fig. 3 E and not depicted). either off-target antibody effects or inherent drug resistance. Notwithstanding these exceptions, the overall results support Reduced IGF1R signaling compromises leukemia-initiating that pharmacologic inhibition of IGF1R signaling can signif- cell (LIC) activity icantly inhibit the growth of most T-ALL cells. To explore whether the increased latency observed for neo/neo IGF1R E leukemias might be caused by delayed trans- Generation of T cell leukemias with reduced IGF1R signaling formation or a reduced net proliferative rate, we performed +/+ neo/neo We next used a genetic approach to further explore the serial transplantation of IGF1R and IGF1R E leu- effects of reduced and/or complete loss of IGF1R on T-ALL kemias into syngeneic/congenic recipients by tail vein injec- cells. Specifically, we generated primary mouse T cell leuke - tion. Unexpectedly, we observed that only a subset of neo/neo mias by transduction of bone marrow from conditional IGF1R primary leukemias was capable of transferring IGF1R mice with Notch1(E) retrovirus, followed by trans- disease to secondary recipients. Among 11 independent plantation into congenic WT recipients. The conditional primary leukemias assessed, 7 (64%) were nontransplantable, neo allele used, IGF1R , carries loxP sites flanking the third 2 (18%) were transplantable in a minority of recipients, and exon, but also retains a loxP-flanked neo cassette within the 2 (18%) were fully transplantable, including one with pro- +/+ second intron (Fig. 3 A). This neo cassette interferes with longed latency (Fig. 4 A). In contrast, all IGF1R primary normal transcript splicing and results in reduced expression leukemias were fully transplantable and exhibited short laten- neo/neo of full-length IGF1R protein in homozygous IGF1R cies. We also performed tertiary and quarternary transplants neo/neo cells (Holzenberger et al., 2000). Despite the decreased level for a subset of cases. The IGF1R leukemia with pro- of IGF1R expression, we were able to generate primary longed latency in secondary recipients (#3115) failed to neo/neo IGF1R leukemias with Notch1(E) retrovirus, albeit with produce disease in tertiary recipients, whereas the other trans- neo/neo +/+ slightly increased latency as compared with WT background plantable IGF1R leukemia (#3105) and all IGF1R (median survival 64.5 vs. 51.5 d; Fig. 3 B). All other disease leukemias produced short latency disease in all tertiary and parameters (penetrance, immunophenotype, histology, disease quarternary recipients (Table S1). Notably, injection of neo/neo distribution, and extent) were highly comparable to control IGF1R leukemia cells directly into the femoral bone marrow space of recipient mice failed to produce leukemia, suggesting that their defect in transplantability is not caused by impaired homing to the bone marrow (Fig. 4 B). Thus, these results indicate that although moder- ate levels of IGF1R signaling are suffi - cient for expansion of bulk leukemia cells, higher levels are required to Figure 2. Pharmacologic inhibition of IGF1R blocks growth of T-ALL cells. Flow cytometric analysis of cell proliferation by BrdU incorporation after treatment with a small molecule IGF1R inhibitor (BMS- 536924) versus DMSO vehicle (mock), or IGF1R blocking antibody (IR3), for 48–72 h in vitro. (A) Three representative indepen- dent primary mouse Notch1(E) leukemias (#324, #327, #329). (B) Four independent xenograft-expanded primary human T-ALL samples (D115, K419, K424, and M69). (C) Two human T-ALL cell lines (ALLSIL and HPBALL). Error bars indicate standard deviation for assays performed in triplicate. Data de- picted are representative of at least three independent experiments. JEM Vol. 208, No. 9 1811 Figure 3. Primary mouse T cell leukemias are generated efficiently by activated Notch1 despite reduced IGF1R expression. neo (A) Schematic of the IGF1R allele. The re- tained neo cassette within the second intron results in reduced expression of full-length neo/neo IGF1R protein in IGF1R mice (Holzenberger et al., 2000). (B) Survival of mice transplanted with retroviral Notch1(E)- transduced bone marrow from either WT +/+ (IGF1R ; n = 6) or IGF1R hypomorph neo/neo (IGF1R ; n = 14) donor animals. ***, P < 0.0001 (Log-rank test). (C) Spleen and liver organ weights at necropsy of individual mor- bid mice transplanted with Notch1(E)- +/+ transduced bone marrow from IGF1R (n = 6) neo/neo and IGF1R (n = 11) backgrounds. Error bars indicate standard deviation. (D) Immuno- phenotypic analysis of representative primary neo/neo mouse E leukemias derived on the IGF1R background. (E) Western blot analysis of total IGF1R protein expression in representative +/+ primary mouse E leukemias on IGF1R , neo/+ neo/neo IGF1R , and IGF1R backgrounds. R+ and R mouse embryonic fibroblast staining controls and Erk2 loading control are indicated. Data depicted in D and E are repre- sentative of at least seven independent samples. compared with controls (Fig. S4, A–E). Furthermore, leukemia cells explanted from moribund, untreated animals (8 wk post-transplant) and cultured with 4-hydroxytamoxifen (4-OHT) in vitro to induce deletion of IGF1R also demonstrated growth arrest and loss of viability (Fig. S4 F). 4-OHT support LIC activity as assayed by serial transplantation in treatment showed no discernible toxic effects on control neo/neo +/+ syngeneic/congenic recipients. IGF1R Rosa26 leukemia cells. Additionally, prelimi- nary results from mice engrafted with a T-ALL cell line G12D Deletion or pharmacologic inhibition of IGF1R prevents (144CLP) derived from a mouse Kras tumor (Chiang et al., disease establishment/progression 2008) show that treatment with BMS-754807, a potent To assess the effect of complete loss of IGF1R in T-ALL IGF1R inhibitor currently in clinical development (Carboni neo/neo cells, we transduced bone marrow from IGF1R et al., 2009), significantly prolonged survival ( Fig. S5), thus CreERT Rosa26 mice with Notch1(E) retrovirus, followed by corroborating the genetic deletion results. Collectively, these transplantation into WT recipient mice. At 4 wk after trans- data support that abrogation of IGF1R signaling impedes plant, peripheral blood was assessed by flow cytometry to T-ALL cell growth/survival and interferes with disease + + + confirm engraftment by GFP CD4 CD8 leukemia cells. establishment/progression. Mice were then divided into equivalent control versus treat- neo ment groups, and the latter was fed tamoxifen-containing Transplantable IGF1R leukemias show evidence chow (1 g/kg) for a 7-d period to induce deletion of IGF1R. for downstream compensation neo/neo Mice from both groups were then sacrificed, and tissues were Our observation that a minority of IGF1R leukemias is harvested. Preliminary results show that tamoxifen-treated transplantable suggests secondary alterations may be selected mice demonstrate significantly lower levels of disease in - for in vivo that compensate for reduced IGF1R signaling. volvement in all tissues examined (bone marrow, spleen, and Despite the known frequency of PTEN loss and PIK3CA/ thymus), and leukemia cells from tamoxifen-treated animals PIK3R1 mutations in T-ALL (Maser et al., 2007; Palomero exhibited lower proliferative and higher apoptotic indices as et al., 2007; Gutierrez et al., 2009), we were unable to detect 1812 Notch-IGF1R signaling supports LICs in T-ALL | Medyouf et al. A r t i c l e Figure 4. Mouse T cell leukemias with reduced IGF1R expression are defective in serial transplantation. Survival of mice transplanted with +/+ neo/neo primary mouse E leukemia cells from either IGF1R or IGF1R backgrounds by i.v. (A) intrafemoral (IF; B) injection route. Each numbered sample represents a different primary leukemia from mice in Fig. 3 B injected into secondary recipients. Raw survival data, including numbers of animals in each cohort, are provided in Table S1. Data depicted are collated from four independent transplantation experiments. neo/neo these alterations in any of the transplantable E-IGF1R responsive to IGF-1, and are comparable to nontransplant- neo/neo leukemias (Fig. 5, A and B; and not depicted). We thus con- able IGF1R leukemias in this regard (Fig. 5 D). We also neo/neo sidered that enhanced responsiveness to growth factor stimu- noted the one IGF1R leukemia exhibiting very short lation could compensate for reduced IGF1R expression. latency in secondary recipients (#3105) had unusually high +/+ In fact, transplantable leukemias #3112 and #3115 demon- levels of pAkt at steady state (twofold greater than IGF1R strated enhanced pAkt response to serum stimulation as com- cells; Fig. 5 E). The elevated pAkt in these cells was not re- pared with nontransplantable leukemias, though not to the duced even after prolonged serum starvation (unpublished +/+ degree exhibited by IGF1R cells (Fig. 5 C). We excluded data), suggesting the presence of some constitutively activat- the possibility that IGF1R itself was up-regulated as trans- ing mutation that confers growth factor independence. In neo/neo plantable IGF1R leukemias remained only minimally contrast, we have not found similarly compelling evidence Figure 5. Transplantable clones with reduced IGF1R expression show compensatory PI3K–Akt activation. (A) Western blot and (B) flow cyto - neo metric analysis of PTEN protein expression in primary and transplantable secondary E-IGF1R leukemias. (C and D) Akt activation as measured by intra- cellular phospho-Akt(Ser473) flow cytometry in response to stimulation with FBS (C) or (D) recombinant IGF-1 × (D) 10 min after 5-h serum starvation. +/+ neo (E) Steady-state level of Akt activation as measured by pAkt(Ser473) in transplantable E-IGF1R and E-IGF1R (#3105) leukemias (n = 3 indepen- neo dent mice for each cohort). For C and D, the five E-IGF1R transplantable clones shown are (4 x #3115, 1 x #3112). Error bars indicate standard devia- tion. *, P < 0.05; **, P < 0.01 (Student’s t test). Data depicted in A and B are representative of at least three independent experiments. Data depicted in C–E are representative of two independent experiments and include at least three independent mice per cohort per experiment. JEM Vol. 208, No. 9 1813 for hyperactivation of the Ras–Raf–MAPK pathway to ex- (GSI) to inhibit Notch signaling or vehicle control (DMSO). neo/neo plain the unique transplantability of these IGF1R clones These five cell lines were selected because they undergo (unpublished data). These few examples suggest PI3K–Akt growth arrest upon GSI treatment (Weng et al., 2004; activation may potentially be more important than Ras–Raf– Palomero et al., 2007). To control for off-target effects of GSI, MAPK signaling in conferring LIC activity; however, further we also profiled cell lines that had been retrovirally trans - study is needed to clarify this issue. duced with ICN1, and then treated with GSI or DMSO. ICN1, the intracellular domain of Notch1, is unaffected by IGF1R mRNA and protein expression GSI treatment and thus maintains Notch signaling in the is up-regulated by Notch presence of GSI (Weng et al., 2003, 2004). Further, because To explore mechanisms that may support the high-level Notch1 induces transcription of c-Myc (Palomero et al., 2006b; IGF1R expression required for LIC activity, we considered a Sharma et al., 2006; Weng et al., 2006), which in turn induces potential role for Notch signaling. Notch has previously been transcription of many other genes (Fernandez et al., 2003), we shown to promote activation of PI3K–Akt in developing also prolfi ed cells that had been transduced with c-Myc and then thymocytes (Ciofani and Zuñiga-Pflucker, 2005), and we treated with GSI or DMSO to segregate Notch targets from noted in our expression profile datasets evidence for up- c-Myc targets. We identie fi d genes whose expression level most regulation of IGF1R by Notch signaling. More specic fi ally, strongly correlated with Notch activity, but not with c-Myc, and we performed microarray-based expression profiling of five noted the conspicuous presence of IGF1R on this list (Table S2) human T-ALL cell lines (ALLSIL, DND41, HPBALL, and in other published microarray datasets generated from GSI- KOPTK1, and TALL-1) treated with -secretase inhibitor treated T-ALL cells (Fig. S6; Palomero et al., 2006b). Figure 6. Inhibition of Notch signaling with GSI down-regulates IGF1R expression in human T-ALL cells. (A) qRT-PCR analysis of IGF1R mRNA in human T-ALL cell lines treated in vitro with -secretase inhibitor (GSI) to block Notch signaling versus DMSO vehicle (mock) for 2–10 d. Error bars indi- cate standard deviation for assays performed in triplicate. (B and C) Flow cytometric analysis of surface IGF1R expression by human T-ALL cell lines treated with GSI versus DMSO vehicle for 6–8 d. Data are representative of multiple replicates. (D) Flow cytometric analysis of surface IGF1R expression by xenograft-expanded primary human T-ALL cells. Cells were cultured on MS5-DL1 feeders to stimulate Notch signaling versus MS5 control feeders and then treated with 1.0 µM compound E (GSI) for 2–4 d to block Notch signaling. Flow histograms for a representative case are depicted on the left, and results from six different patient samples are summarized on the right. Error bars indicate standard deviation. **, P < 0.01 (Student’s t test). At least 20,000 gated live events were collected for all flow cytometry assays. 1814 Notch-IGF1R signaling supports LICs in T-ALL | Medyouf et al. A r t i c l e We also performed a similar expression profiling experi - ment using the human T-ALL cell line, CUTLL1, which har- bors a TcrB/Notch1 fusion gene that drives expression of a form of Notch1 resembling E (Palomero et al., 2006a). CUTLL1 cells transduced with empty retrovirus (MigR1) or dominant-negative (DN) MAML1-GFP, a specific Notch inhibitor (Weng et al., 2003; Maillard et al., 2004; Weng et al., 2004), were treated with GSI for 3 d, after which GSI was washed out to create a timed pulse of Notch signaling activity. Expression profiling was done on cells before washout and at 2 and 4 h after washout to identify likely direct Notch1 target genes. We observed GSI washout up-regulated expression of IGF1R along with other known Notch1 target genes, such as Hes1, Hes4, Hes5, NRARP, and DTX1 in MigRI control cells, and that DN-MAML1-GFP partially or completely abrogated up-regulation of the same set of genes (Fig. S7). To validate these expression profile data, we performed qRT-PCR for IGF1R mRNA. Notch-dependent T-ALL cell lines (ALLSIL, DND41, HPBALL, KOPTK1, and TALL-1) down-regulated IGF1R mRNA levels up to two- fold when treated with GSI, whereas Notch-independent lines (Jurkat, PF382, and RPMI 8402) showed 6–14-fold down-regulation of IGF1R mRNA (Fig. 6 A). GSI blockade also down-regulated IGF1R protein levels 2–3 fold on aver- age, as judged by flow cytometry and Western blotting of human T-ALL cell lines and xenograft-expanded primary samples (Fig. 6, B–D; and Fig. S8). This effect of GSI is likely specific to Notch, as DN-MAML1 also decreased IGF1R protein levels (Fig. 7 A), and the effect of GSI on IGF1R was rescued by retroviral transduction with ICN1 (Fig. 7 B). Conversely, culture of xenograft-expanded primary human T-ALL cells on MS5 stromal cells expressing the Notch ligand DL1, but not MS5 control cells, increased IGF1R levels, whereas no such change was observed for the control marker CD45 (Fig. 7 C). In addition, this effect of DL1 ligand on IGF1R levels was abrogated by treatment with GSI (Fig. 6 D). Thus, these data are consistent with a role for Notch signaling in up-regulation of IGF1R ex- pression in human T-ALL cells. ICN1/CSL binds to an intronic IGF1R enhancer Figure 7. Inhibition of Notch signaling with dominant-negative Once cleaved from the plasma membrane by -secretase, MAML1 and activation by ICN1 or DL1 ligand conr fi m IGF1R regulation ICN1 translocates to the nucleus where it forms a ternary by Notch. Flow cytometric analysis of surface IGF1R expression. (A) Human complex on DNA with the DNA-binding factor CSL and T-ALL cell lines were transduced with dominant negative Mastermind-like-1 the coactivator MAML1 to stimulate transcription of target retrovirus (DN-MAML1-GFP) or empty virus control (MigRI). (B) Human T-ALL genes (Aster et al., 2008). To define the regulatory elements cell lines were transduced with ICN retrovirus (Mig ICN) and then treated with through which Notch1 up-regulates target genes in an unbi- 1.0 µM compound E (GSI) for 4 d (HPBALL and TALL-1) or 8 d (PF382) to block endogenous Notch signaling or DMSO vehicle (mock). Retrovirally transduced ased fashion, we performed chromatin immunoprecipitation cells in A and B were discriminated from nontransduced cells by gating for GFP. (ChIP)-Seq analysis on the human T-ALL cell line, CUTLL1, Data depicted in A and B are representative of multiple replicates. (C) Xenograft- using antibodies directed against Notch1 and CSL (Wang expanded primary human T-ALL cells were cultured in vitro on MS5-DL1 feeders et al., 2011). Alignment of sequencing reads from duplicate to stimulate Notch signaling versus control MS5 feeders. CD45 expression levels CSL and Notch1 libraries identified one high-confidence were also assessed simultaneously with IGF1R by o fl w cytometry. Flow histo - ICN1/CSL-binding site within intron 20 of IGF1R at a grams for a representative case are depicted on the left, and results from six position >250 kb 3 of the proximal promoter (Fig. 8 A). different patient samples are summarized on the right. Error bars indicate The next nearest ICN1/CSL-binding site lies 10.5 Mbp 5 standard deviation. *, P < 0.05 (Student’s t test). At least 20,000 live events of the proximal promoter, making this 3 binding site the were collected within each gate for all o fl w cytometry assays. JEM Vol. 208, No. 9 1815 only likely candidate response element for Notch1 regula- rapid reloading, indicating that ICN1/CSL occupancy is tion. Motif analysis using recently developed algorithms dynamic (Fig. 8 D). derived from protein-binding microarrays (Del Bianco et al., The distant location of the putative ICN1/CSL response 2010) showed that the center of the region under the ICN1/ element in IGF1R suggested that it represents an enhancer. CSL-binding peak contains high (CATGGGAA) and mod- In support of this possibility, additional ChIP analyses docu- erate (GCTGAGAA) an ffi ity CSL sites oriented head-to-head mented recruitment of the histone acetyltransferases CREB- and separated by a 17-bp spacer (Fig. 8 B). This architecture binding protein (CBP) and p300, as well as RNA polymerase II, is typical of a sequence-paired site, a special type of Notch to this site (Fig. S9 A; Hatzis and Talianidis, 2002; Wang response element first identified in Drosophila enhancer of et al., 2005). Moreover, we observed enrichment of histone split (E[spl]) locus that is present in mammalian E(spl) homo- H3K4 mono- and dimethylation chromatin marks (charac- logues such as Hes1 (Jarriault et al., 1995) and other genes teristic of enhancers) relative to H3K4 trimethylation marks such as preT (Liu et al., 2010). Loading of ICN1 onto this (a feature of promoters; Heintzman et al., 2007), and low site was confirmed by ChIP/quantitative PCR (qPCR) levels of the repressive trimethyl histone H3K27 chromatin analyses, performed on CUTLL1 cells and two additional mark (Fig. S9 B; Kirmizis et al., 2004). Finally, additional human T-ALL cell lines, HPBALL and KOPTK1 (Fig. 8 C). ChIP-Seq data showed a local decrease in H3K4 methyl- In addition, GSI treatment of CUTLL1 cells depleted both ation at the precise site of the intronic IGF1R sequence- CSL and ICN1 from this site (in line with work from Bray’s paired site, indicating that this site lies in accessible chromatin group indicating that ICN stabilizes CSL interactions with that has been depleted of nucleosomes (Fig. S9 C). Collec- DNA; Krejcí and Bray, 2007), and GSI washout resulted in tively, these data support the presence of an intronic Figure 8. ICN1/CSL binds dynamically to a site within intron 20 of human IGF1R. (A) Alignment of sequencing reads over intron 20 of the IGF1R locus from ChIP libraries prepared from the human T-ALL cell line, CUTLL1, with antibodies specific for Notch1 and CSL as compared with input cells. (B) Genomic DNA sequence from human IGF1R intron 20 (NT_010274.17: 14461410–14461610) with sequence-paired ICN1/CSL binding sites highlighted in bold and 17-bp spacer underlined. (C) ChIP/qPCR analysis of IGF1R intron 20 from three different human T-ALL cell lines using a Notch1-specific antibody as compared with preimmune antiserum. (D) ChIP/qPCR analysis of IGF1R intron 20 from CUTLL1 cells using antibodies specific for Notch1 and CSL com - pared with control rabbit IgG. Cells were treated with GSI for 3 d to block Notch signaling (GSI x 3d) versus DMSO vehicle (DMSO). GSI was washed out, and cells were harvested 4 h later (GSI x 3d, then wash 4 h). Quantitation of immunoprecipitated DNA is expressed relative to input DNA (% input). Error bars indicate standard deviation for qPCR assays performed in triplicate. Numbers above bars in D indicate relative enrichment over control. ChIP libraries were prepared in duplicate, and local ChIP/qPCR analyses were performed twice. Representative results are shown. 1816 Notch-IGF1R signaling supports LICs in T-ALL | Medyouf et al. A r t i c l e Notch1-responsive enhancer in human IGF1R that is retroviral transduction only partially rescued the pAkt re- active in human T-ALL cells. sponse to IGF1 in HPBALL cells (Fig. 9 C), whereas rescue null was complete in PTEN PF382 cells (Fig. 9 D; Palomero Physiological relevance of Notch-induced et al., 2007). Importantly, we confirmed retroviral IGF1R up-regulation of IGF1R restored expression in GSI-treated cells to levels comparable Notch signaling is likely one of several factors that influence to parental cells. Thus, although Notch inhibition can affect IGF1R expression in T-ALL, as Notch inhibition results in both IGF1R and PTEN expression, Notch contributes to only a two- to threefold decrease in surface IGF1R expres- supporting IGF1R expression at high levels, thereby allow- sion, on average. To assess whether this two- to threefold ing T-ALL cells to respond more robustly to ambient levels change in IGF1R protein levels has a significant effect on of IGF-1/2. downstream signaling, we measured levels of intracellular phosphorylated Akt (pAkt) after pulsing serum-starved leu- DISCUSSION kemia cells with recombinant IGF-1. We observed GSI- We have presented data showing that IGF1R mediates im- treated cells to be 20-fold less responsive to IGF-1 than portant growth/survival signals in T-ALL cells, and that vehicle-treated control cells (Fig. 9 A). Although some of this although moderate levels of signaling are adequate for main- effect is presumably caused by GSI effects on IGF1R expres - tenance of the bulk cell population, high levels are required sion, it has also been reported that Notch represses PTEN via for maintenance of LIC activity as indicated by serial trans- HES-1 (Palomero et al., 2007), which could also impact the plantation assay. The effect of reduced IGF1R signaling on pAkt response. Indeed, GSI treatment induced PTEN ex- disease transplantability could be caused by (a) a quantitative pression in HPBALL cells; however, there was no detectable decrease in the number of leukemia stem cells; (b) a qualita- change in ALLSIL or DND41 cells (Fig. 9 B). Accordingly, tive defect in self-renewal, engraftment ability, and/or restoration of IGF1R expression in GSI-treated cells by immune resistance of leukemia stem cells; or (c) a reduced Figure 9. Notch-induced IGF1R expression enhances PI3K–Akt signaling in response to IGF1. (A) Flow cytometric analysis of intracellular phos- pho-Akt levels. Cells were treated with GSI versus DMSO vehicle for 6–8 d, serum starved overnight, and then pulsed with recombinant IGF1 for 10 min before assay. (B) Western blot analysis of PTEN protein expression in cells treated with GSI versus mock for 4 d. (C and D) Flow cytometric analysis for intracellular phospho-Akt levels (left) and surface IGF1R levels (right). Cells were retrovirally transduced with Mig IGF1R, treated with GSI versus DMSO vehicle for 4 d, serum starved overnight, and then pulsed with recombinant IGF1 for 10 min before phospho-Akt assay. Transduced versus nontransduced cells within the same culture were distinguished by gating for GFP. Surface IGF1R expression level was also assessed by flow cytometry immediately before stimulation with IGF1. Filled grey histograms represent second antibody staining controls. At least 20,000 events were collected within each gate for all flow cytometry assays. Data depicted are representative of at least two independent experiments. JEM Vol. 208, No. 9 1817 probabilistic likelihood of engraftment/propagation of bulk inhibition of IGF1R signaling blocks growth/survival of bulk leukemia cells. Notably, the last possibility does not presume leukemia cells confirms previous results with PI3K–Akt/ the existence of leukemia stem cells in this model; however, mTOR inhibitors, and further illustrates that IGF1-dependent we have generated data that supports that Notch T-ALLs activation contributes in a substantive way to net PI3K–Akt indeed contain bona fide leukemia stem cells (unpublished signaling output. Perhaps most intriguing, however, is our data). Nonetheless, further studies will be required to distin- observation that moderately decreased IGF1R signaling (as neo/neo guish between these possibilities. modeled by IGF1R leukemias) results in selective loss We have also shown that Notch directly up-regulates of LIC activity, yet leaves the growth and survival of bulk IGF1R expression in human T-ALL cells to an extent that cells relatively unaffected. These findings suggest that LICs in substantially enhances their sensitivity to ambient ligand. It is T-ALL may be uniquely sensitive to inhibition of IGF1R worthwhile to note that we have found no evidence to sup- signaling, and raise the possibility that pharmacologic IGF1R port that Notch regulates IGF1R in mouse T-ALL cells. In inhibitors now in clinical development could, in combina- fact, the sequence-paired ICN1/CSL-binding site within tion with standard regimens, enhance initial response to ther- intron 20 of human IGF1R is not conserved in the mouse, apy and reduce rates of disease relapse. Importantly, normal suggesting that sequence divergence has decoupled this point hematopoietic stem cell function appears not to be affected in neo/neo of interaction between the Notch1 and IGF1R pathways. IGF1R mice (unpublished data), suggesting a therapeu- This is perhaps not surprising given that 40–90% of transcrip- tic window exists in which IGF1R inhibitors block LIC tion factor binding events are not conserved between mouse activity yet have minimal effects on normal hematopoiesis. and human (Odom et al., 2007), underscoring the impor- tance of studying human cells and animal models in parallel. MATERIALS AND METHODS The existence of leukemia stem cells in human T-ALL is neo Mice. The IGF1R line contains a PGK-neo-poly(A) expression cassette supported by xenograft transplantation assays (Cox et al., integrated within the second intron, resulting in decreased expression of 2007; Chiu et al., 2010; Gerby et al., 2011) and also in mouse neo full-length IGF1R (Holzenberger et al., 2000). The IGF1R allele has been / models of T-ALL by transplantation into SCID or Rag1 backcrossed onto the C57BL/6 background for over 20 generations. CreERT immunocompromised recipients (Guo et al., 2008; Tremblay Rosa26 mice were obtained from The Jackson Laboratory. All trans- et al., 2010). The precise cellular compartment most enriched plants involving mouse cells were performed using C57BL/6J (B6) or c-2J B6(Cg)-Tyr /J (“B6-albino”) recipient mice (The Jackson Laboratory). for LIC activity appears to vary from patient to patient and Transplants involving human cells were performed using NOD-Scid/ between different animal models, and likely depends on the / IL2Rc recipient mice. Animals were housed under specific pathogen– particular complement of genetic alterations present in the free conditions at the BC Cancer Agency or Dana-Farber Cancer Institute leukemic clone and the particular transplant recipient used animal facilities, and experimental studies performed under protocols ap- (Cox et al., 2007; Quintana et al., 2008; Chiu et al., 2010; proved by the University of British Columbia or Dana-Farber Institutional Gerby et al., 2011). Nonetheless, it is interesting to note that Review Boards, respectively. IGF1R genotyping was performed by multi- plex assay with primers YNex (5-CCATGGGTGTTA AATGTTAAT- Notch signaling has been shown to contribute to human GGC-3), YNvl (5-ATGAATGCTGGTGAGGGTTGTCTT-3) and T-ALL LIC activity in nonobese diabetic (NOD)/Scid trans- YNmt2 (5-ATCTTGGAGTGGTGGGTCTGTTTC-3), as previously plantation assays (Armstrong et al., 2009). As Notch signaling described (Leneuve et al., 2001). Rosa26 genotyping was performed with is mediated through transcriptional activation, identification primers RcreFW (5-AAAGTCGCTCTGAGTTGTTAT-3) and RcreRV of downstream target genes has received much attention. In (5-GCGAAGAGTTTGTCCTCAACC-3) for the CreERT allele, and with RwtFW (5-GCACTTGCTCTCCCAAAGTC-3) and RwtRV several such studies, c-Myc has been identified as a direct (5-GGCGGATCACAAGCAATAAT-3) for the WT allele. transcriptional target of Notch1 (Palomero et al., 2006b; Sharma et al., 2006; Weng et al., 2006), and may constitute Plasmids. Retroviral vectors all used the MSCV-IRES-GFP (Mig) backbone. part of a self-renewal genetic program similar to that demon- Mig ICN1, Mig E, and MSCV-DN-MAML1-GFP have been described pre- strated in induced pluripotent stem cells (Takahashi and viously (Aster et al., 1997; Weng et al., 2003). Mig IGF1R was generated by Yamanaka, 2006; Takahashi et al., 2007). Notch may also subcloning a 4.2-kb NotI-BamHI fragment containing the human IGF1R repress PTEN (Palomero et al., 2007), and thus potentiate cDNA from pBABE-bleo IGF1R (Addgene) into the Mig vector. PI3K–Akt/mTOR signaling which may also promote LIC Retroviral transduction/bone marrow transplantation. High titer, activity (Yilmaz et al., 2006). replication-defective retroviral supernatants were produced by transient Most prior studies have focused on signaling via IL-7 transfection of PlatE cells as described previously (Medyouf et al., 2010). (Dibirdik et al., 1991; Barata et al., 2004a,b,c, 2005; González- MSCV-based retroviral expression vectors included IRES-GFP cassettes Garcia et al., 2009; Shochat et al., 2011; Silva et al., 2011) or for fluorescent tagging of transduced cells. Retroviral transduction of mutational activation of intermediates in the PI3K–Akt path- 5-u fl orouracil-treated or lineage-depleted (CD4, CD8, CD11b, B220, Ter119, and Gr1) bone marrow cells was performed as described previously (Medyouf way (Palomero et al., 2007; Gutierrez et al., 2009). The im- et al., 2010). For primary transplants, WT C57BL/6 recipient mice were portance of PI3K–Akt activation in T-ALL is underscored by lethally irradiated (810 rads), and then injected intravenously with 30,000– multiple studies showing that PI3K–Akt/mTOR inhibitors + 5 45,000 transduced GFP cells along with a minimum of 10 syngeneic whole block growth/survival of T-ALL cells (Avellino et al., 2005; bone marrow cells to ensure hematopoietic reconstitution. For serial transplants, 6 + Wei et al., 2006; Palomero et al., 2007; Chiarini et al., 2009; 0.5–1.0 × 10 GFP leukemia cells (from spleen or bone marrow of a moribund Cullion et al., 2009). Notably, our observation that complete leukemic mouse) were injected into the tail vein (i.v.) or intramedullary 1818 Notch-IGF1R signaling supports LICs in T-ALL | Medyouf et al. A r t i c l e space of the femur (intrafemoral [IF]) of nonirradiated C57BL/6 recipients. (Altromin) and mice were allowed to feed ad libitum. For induction of Cre- We define LIC activity here as the ability to produce aggressive disease in ERT activity in vitro, 4-OHT (Sigma-Aldrich) was dissolved in ethanol and syngeneic/congenic secondary recipients under these transplant conditions added to culture media at 50 nM final concentration. within 20 wk. Expression profiling. Total RNA was isolated by TRIzol (Invitrogen) ex- Human leukemia samples. Cryopreserved lymphoblast samples were traction or RNeasy Mini kit (QIAGEN) and submitted to the McGill Uni- provided by collaborating institutions. Primary samples were obtained at ini- versity/Génome Québec Innovation Centre or Harvard Medical School tial diagnosis with informed consent from patients or their legal guardians Biopolymers Core for expression profiling using Affymetrix HG-U133 Plus or as discarded pathological material under approved Institutional Review 2.0 GeneChips. Data were analyzed using dChip software (Schadt et al., Board protocols at the Karmanos Cancer Institute, Hôpital Armand-Trousseau, 2001). Microarray data are available from the Gene Expression Omnibus Hopital Saint-Louis, BC Cancer Agency, and BC Children’s and Women’s accession no. GSE29959. Hospital following guidelines established by the Declaration of Helsinki. Ex- / pansion of primary human T-ALL cells in irradiated NOD-Scid/IL2Rc Quantitative real-time PCR. Total RNA was extracted after cell lysis in mice has been described previously (Medyouf et al., 2010). TRIzol reagent (Invitrogen). First-strand cDNA was generated by reverse transcription with SuperScript III (Invitrogen) using a mix of random 15-mer In vitro culture of primary T-ALL cells. Primary human T-ALL cells and anchored oligo(dT)20+1 primers, and then amplified using Platinum were cultured on MS5-DL1/MS5 stromal feeder cells as previously de- SYBR Green qPCR SuperMix-UDG (Invitrogen) and the following specific scribed (Medyouf et al., 2010). Primary mouse T-ALL cells were cultured primer sets: hIGF1R forward 5-ACTTACTCGGACGTCTGGTCCTTC-3, without feeders in complete media with supplemental cytokines. hIGF1R reverse 5-ATCTTGGGGTTATACTGCCAGCAC-3 for IGF1R, and hActB RT5 5-CGCGAGAAGATGACCCAGAT-3plus hActB RT3 Cell lines. IGF1R knockout mouse embryo fibroblasts (R-cells) that were 5-GAT AGC ACA GCC TGG ATA GCA AC-3 for -actin. Each sample engineered to re-express human IGF1R (R+) were a gift from P. Sorensen was assayed in triplicate using a Dyad Disciple thermal cycler equipped with (BC Cancer Agency, Vancouver, British Columbia, Canada). All established Chromo4 optical head (Bio-Rad Laboratories). Expression levels were cal- T-ALL cell lines were grown in RPMI 1640 medium supplemented with culated by the Ct method with normalization to -actin. 10% FCS, 1 mM sodium pyruvate, 2 mM L-glutamine, and antibiotics. ChIP. ChIP was performed with the ChIP Assay kit (Millipore). In brief, Ligand stimulation assay. Leukemia cells were serum starved overnight, cells were cross-linked with 1% formaldehyde for 10 min at 37°C, lysed in and then stimulated for 10 min with either recombinant IGF-1 (PeproTech), 1% SDS, and 10 mM EDTA, 50 mM Tris, pH 8.1, and then sonicated to recombinant insulin (Sigma-Aldrich), or fetal calf serum (Invitrogen), at in- obtain DNA fragments from 200–600 bp. Chromatin was then immuno- dicated doses. Cells were then fixed immediately by addition of formalde - precipitated with the following antibodies: Notch1(Tc; Aster et al., 1997), hyde (Electron Microscopy Services) to 1.5%, and permeabilized with CSL (gift from E. Kieff, Brigham and Women’s Hospital, Boston, MA), ice-cold methanol for at least 1 h before analysis by flow cytometry. H3K4me1 (ab8895; Abcam), H3K4me2 (07–030; Millipore), H3K4me3 (07–745; Millipore), CBP (ab10489; Abcam), p300 (sc-585; Santa Cruz In vitro proliferation/apoptosis assays. Cells were pulsed with 10 µM Biotechnology, Inc.), RNA pol2 (05623B; Millipore), and mouse IgG BrdU for 1 h at 37°C, and then fixed/stained with anti-BrdU antibody (12–371; Millipore) as control. After overnight incubation with antisera according to manufacturer instructions (FITC or APC BrdU Flow kit; BD) at 4°C, immunoprecipitated chromatin was captured with Protein A-agarose and analyzed by flow cytometry with gating for mouse and human leukemia beads, washed, and eluted. After reversal of cross-links, DNA was pu- cells by GFP- and hCD45-positive events, respectively. Cell growth was also rified using the QIAquick PCR purification kit (QIAGEN). Input con - measured by CellTiter-Blue assay (Promega) or Ki67-Alexa Fluor 647/ trol DNA was also prepared in parallel, omitting the immunoprecipitation Hoechst staining (BD Biosciences). Apoptotic cells were identified by stain - steps. Primers used for qPCR after ChIP were as follows: IGF1Rchip for- ing with Annexin V-FITC/7-aminoactinomycin or active caspase-3-PE ward 5-GGTGGGTGAGGGAGAGCGGT-3 and IGF1Rchip reverse (BD). Cell viability was assessed by propidium iodide exclusion and analysis 5-GGCTGCGTCCCAGGCAGTTT-3. by flow cytometry. ChIP-Seq and data analysis. ChIP-Seq libraries were prepared according Antibody reagents. Antibodies directed against the following proteins to the Illumina ChIP DNA library preparation kit. After addition of adap- were used: IGF1R (sc-712, Santa Cruz Biotechnology), PTEN (Y184, tors, libraries were amplified by 18 cycles of PCR, size selected (150–250 bp) Abcam), and ERK2 (sc-154, Santa Cruz Biotechnology) for Western blots, by electrophoresis, and purified using a QIAGEN gel extraction kit. After and hCD45 (eBioscience), CD4 (L3T4; eBioscience), CD8a (53–6.7, Bio- quality control testing on an Agilent 2100 Bioanalyzer, the library was sub- Legend), IGF1Ra (IR3; EMD), and P-AKT (S473; Cell Signaling Tech- jected to deep sequencing using an Illumina Genome Analyzer II in the nology) for flow cytometry. Harvard Medical School Biopolymers Core facility. Sequencing reads were aligned to human genome build hg18 and analyzed using CisGenome Flow cytometry. Acquisition was performed on FACSCalibur or LSRII (Ji et al., 2008). One-sample and two-sample analyses were performed using cytometers (BD) and data analyzed using FlowJo software (Tree Star, Inc.). 100-bp windows and reads of >10 bp. Drugs. IGF1R inhibitors BMS-536924 and BMS-754807 were obtained Online supplemental material. Fig. S1 shows Akt response to stimula- under Material Transfer Agreement from the manufacturer. BMS-536924 tion with IGF1 and insulin and effects of BMS-536924 inhibitor in T-ALL was resuspended in DMSO at 10 mM, then serially diluted in Hank’s Bal- cells. Fig. S2 shows effects of the BMS-536924 inhibitor on T-ALL cell anced Salt Solution (Invitrogen) before addition to culture media. BMS- growth and viability in vitro. Fig. S3 shows resistance to growth inhibi- 754807 was resuspended in PEG400:H O (80:20 by volume) at 5 mg/ml tion by IGF1R blocking antibody correlates with reduced surface IGF1R final concentration before administration to mice. An azide-free preparation expression. Fig. S4 shows effects of IGF1R deletion on T-ALL develop - of IR3 antibody was used for IGF1R blocking studies (EMD). -Secretase ment in vivo and cell growth/viability in vitro. Fig. S5 demonstrates in vivo inhibitor XXI (compound E) was used at 1.0 µM final concentration for all efficacy of the IGF1R inhibitor BMS-754807 in a mouse T-ALL model. studies (EMD). GSI washout was performed by washing cells twice with Fig. S6 shows that IGF1R is a Notch target gene in an independent ex- prewarmed, complete culture medium. For induction of CreERT activity in pression profiling dataset. Fig. S7 shows IGF1R is a Notch target gene in vivo, 1 g tamoxifen (Sigma-Aldrich) was admixed per 1 kg mouse chow the human T-ALL cell line, CUTLL1. Fig. S8 shows Western blot analysis JEM Vol. 208, No. 9 1819 of total IGF1R expression after GSI treatment of human T-ALL cell lines. Barata, J.T., A.A. Cardoso, and V.A. Boussiotis. 2005. Interleukin-7 in Fig. S9 shows chromatin modification marks over the human IGF1R locus T-cell acute lymphoblastic leukemia: an extrinsic factor support- in the vicinity of the Notch/CSL binding site. Table S1 shows survival data ing leukemogenesis? Leuk. Lymphoma. 46:483–495. doi:10.1080/ for all secondary, tertiary, and quarternary transplant experiments. Table S2 Bendall, S.C., M.H. Stewart, P. Menendez, D. George, K. Vijayaragavan, lists genes whose mRNA expression level decreases most after inhibition of T. Werbowetski-Ogilvie, V. Ramos-Mejia, A. Rouleau, J. Yang, M. Notch signaling. Online supplemental material is available at http://www Bossé, et al. 2007. IGF and FGF cooperatively establish the regulatory .jem.org/cgi/content/full/jem.20110121/DC1. stem cell niche of pluripotent human cells in vitro. Nature. 448:1015– 1021. doi:10.1038/nature06027 We thank Amina Kariminia (BC Children’s Hospital, Vancouver) for sample Carboni, J.M., M. Wittman, Z. Yang, F. Lee, A. Greer, W. Hurlburt, S. preparation, Drs. Kirk Schultz (BC Children’s Hospital, Vancouver), Larry H. Matherly Hillerman, C. Cao, G.H. Cantor, J. Dell-John, et al. 2009. BMS-754807, (Karmanos Cancer Institute, Detroit), Paola Ballerini (Hôpital Armand-Trousseau, a small molecule inhibitor of insulin-like growth factor-1R/IR. Mol. Paris), and Thierry Leblanc (Hopital Saint-Louis, Paris) generously contributed Cancer Ther. 8:3341–3349. doi:10.1158/1535-7163.MCT-09-0499 human T-ALL samples. Chiang, M.Y., L. Xu, O. Shestova, G. Histen, S. L’heureux, C. Romany, This work was funded by grants from the Canadian Cancer Society Research M.E. Childs, P.A. Gimotty, J.C. Aster, and W.S. Pear. 2008. Leukemia- Institute/Terry Fox Foundation, US National Cancer Institute (K22CA112538, associated NOTCH1 alleles are weak tumor initiators but accelerate P01CA119070), Leukemia and Lymphoma Society of Canada, Lymphoma Foundation K-ras-initiated leukemia. J. Clin. 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Growth suppression of pre-T acute lymphoblastic doi:10.1038/nature04703 1822 Notch-IGF1R signaling supports LICs in T-ALL | Medyouf et al. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Experimental Medicine Pubmed Central

High-level IGF1R expression is required for leukemia-initiating cell activity in T-ALL and is supported by Notch signaling

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Abstract

A r t i c l e High-level IGF1R expression is required for leukemia-initiating cell activity in T-ALL and is supported by Notch signaling 1 1 2 3 Hind Medyouf, Samuel Gusscott, Hongfang Wang, Jen-Chieh Tseng, 1 1 4 5 Carol Wai, Oksana Nemirovsky, Andreas Trumpp, Francoise Pflumio, 6 6 7 3,8 Joan Carboni, Marco Gottardis, Michael Pollak, Andrew L. Kung, 2 9 1 Jon C. Aster, Martin Holzenberger, and Andrew P. Weng Terry Fox Laboratory/Department of Pathology, BC Cancer Agency, Vancouver, BC, V52 1L3 Canada Department of Pathology, Brigham & Women’s Hospital/Harvard Medical School, Boston, MA 02115 Lurie Family Imaging Center, Dana-Farber Cancer Institute, Boston, MA 02115 Heidelberg Institute for Stem Cell Technology and Experimental Medicine (HI-STEM), and Division of Stem Cells and Cancer, German Cancer Research Center (DKFZ), D-69120 Heidelberg, Germany LSHL/IRCM, Institut National de la Santé et de la Recherche Médicale U967, Université Paris 7, CEA, 92265 Fontenay-aux- Roses, France Oncology Drug Discovery, Bristol-Myers Squibb Company, Princeton, NJ 08543 Department of Oncology, McGill University, Montreal, Quebec, H3T 1E2 Canada Department of Pediatric Oncology, Dana-Farber Cancer Institute and Children’s Hospital/Harvard Medical School, Boston, MA 02115 Centre de Recherche Institut National de la Santé et de la Recherche Médicale Saint-Antoine, Université Pierre-et-Marie- Curie, 75571 Paris, France T cell acute lymphoblastic leukemia (T-ALL) is an aggressive cancer of immature T cells that often shows aberrant activation of Notch1 and PI3K–Akt pathways. Although muta- tions that activate PI3K–Akt signaling have previously been identified, the relative contri - bution of growth factor-dependent activation is unclear. We show here that pharmacologic inhibition or genetic deletion of insulin-like growth factor 1 receptor (IGF1R) blocks the growth and viability of T-ALL cells, whereas moderate diminution of IGF1R signaling com- promises leukemia-initiating cell (LIC) activity as defined by transplantability in syngeneic/ congenic secondary recipients. Furthermore, IGF1R is a Notch1 target, and Notch1 signaling is required to maintain IGF1R expression at high levels in T-ALL cells. These findings suggest effects of Notch on LIC activity may be mediated in part by enhancing the responsiveness of T-ALL cells to ambient growth factors, and provide strong rationale for use of IGF1R inhibitors to improve initial response to therapy and to achieve long-term cure of patients with T-ALL. T cell acute lymphoblastic leukemia (T-ALL) is including mutation or inactivation of PTEN CORRESPONDENCE Andrew P. Weng: an aggressive cancer of immature T cell pro- (Kawamura et al., 1999; Perentesis et al., 2004; aweng@bccrc.ca genitors that often shows aberrant activation of Maser et al., 2007; Palomero et al., 2007; Silva OR NOTCH1 and PI3K–Akt pathways. Activating et al., 2008; Gutierrez et al., 2009) and muta- Hind Medyouf: h.medyouf@dkfz-heidelberg.de mutations of Notch1 occur in >50% of cases of tion of PIK3 and Akt (Kawamura et al., 1999; T-ALL (Weng et al., 2004), whereas mutations Gutierrez et al., 2009). Activation of PI3K–Akt Abbreviations used: 4-OHT, in related Notch pathway elements such as has been shown to collaborate with Notch in 4-hydroxytamoxifen; ChIP, chromatin immunoprecipita- Sel10/Fbw7 occur in 8–16% of cases (O’Neil leukemogenesis (Medyouf et al., 2010), enhance tion; LIC, leukemia-initiating et al., 2007; Thompson et al., 2007). PI3K–Akt growth of established leukemias (Chiarini et al., cell; NOD, nonobese diabetic; pathway activation occurs in >85% of cases 2009; Cullion et al., 2009; Levy et al., 2009; qPCR, quantitative PCR; (Silva et al., 2008) via diverse mechanisms, Sanda et al., 2010), and in some contexts to T-ALL, T cell acute lympho- blastic leukemia. © 2011 Medyouf et al. This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first H. Medyouf’s present address is Heidelberg Institute for Stem six months after the publication date (see http://www.rupress.org/terms). After Cell Technology and Experimental Medicine (HI-STEM), six months it is available under a Creative Commons License (Attribution–Non- and Division of Stem Cells and Cancer, German Cancer commercial–Share Alike 3.0 Unported license, as described at http://creativecom- Research Center (DKFZ), D-69120 Heidelberg, Germany mons.org/licenses/by-nc-sa/3.0/). The Rockefeller University Press $30.00 J. Exp. Med. Vol. 208 No. 9 1809-1822 www.jem.org/cgi/doi/10.1084/jem.20110121 The Journal of Experimental Medicine relieve dependence on Notch signaling (Palomero et al., samples (Weng et al., 2004; Weng et al., 2006; Medyouf 2007). For cases that lack such mutations, however, the mech- et al., 2010). For mouse cells, we examined primary leuke- anisms that support activation of the pathway are unknown. mias derived by retroviral transduction/transplantation of More generally, it is also unknown to what extent growth bone marrow with an activated form of NOTCH1 termed factor–dependent stimulation of cognate receptor tyrosine E (Pear et al., 1996). To confirm IGF1R-stimulated PI3K– kinases (RTKs) contributes to the net signaling output. Akt in these contexts, we pulsed serum-starved leukemia cells Although previous works have focused on the role of with recombinant IGF-1 and measured phospho-Akt activa- IL-7 signaling in T-ALL, including effects on downstream tion by flow cytometry. We observed that both human and PI3K–Akt activation (Dibirdik et al., 1991; Barata et al., mouse leukemia cells respond robustly to IGF-1 stimulation 2004a,b,c, 2005; González-Garcia et al., 2009; Shochat et al., under these conditions (Fig. S1). 2011; Silva et al., 2011), we considered that insulin-like growth factor (IGF)-1 receptor (IGF1R) may also play an Pharmacologic inhibition of IGF1R compromises important role. IGFs and their receptors regulate normal cell T-ALL cell growth growth and contribute to transformation and growth of ma- To assess the extent to which T-ALL cells are dependent on lignant cells in many contexts (Pollak et al., 2004). IGF1 and IGF1R signaling, we used pharmacologic IGF1R inhibitors. IGF2 bind to IGF1R, a transmembrane receptor tyrosine Most small molecule IGF1R inhibitors also affect insulin re - kinase (RTK), thereby initiating a cascade of downstream ceptor caused by their close homology and at higher doses phosphorylation events that bifurcates along both PI3K–Akt may be expected to cross react with more distantly related and Ras–Raf–MAPK pathways. PI3K–Akt activation leads receptor tyrosine kinases. One such inhibitor, BMS-536924 to enhanced cellular metabolism and protein synthesis via (Wittman et al., 2005), substantially inhibited growth/prolif- mTOR and enhanced survival via BAD/Bcl2, p53, NF-kB, eration of both mouse and human leukemia cells in vitro and FOXOs, whereas Ras–Raf–MAPK activation generally (Fig. 2 and Fig. S2). Primary leukemia cells were generally results in increased cellular proliferation (Pollak et al., 2004; more sensitive to BMS-536924 with IC50 values in the 0.1– Greer and Brunet, 2005). Signaling through IGF1R has also 1.0 µM range, whereas established cell lines required some- been implicated in self-renewal of stem cells, both in embry- what higher doses (2–4 µM) to achieve similar effects. As onic (Bendall et al., 2007) and hematopoietic (Ivanova et al., expected, BMS-536924 completely suppressed Akt activa- 2002) contexts. tion by both IGF-1 and insulin in human and mouse T-ALL cells (Fig. S1). RESULTS We next examined whether blocking antibodies against IGF1R is broadly expressed in T-ALL IGF1R, which might be expected to exhibit a higher degree To begin to address a potential role for IGF1R in T-ALL, we of specificity than small molecule kinase inhibitors, would assessed IGF1R expression in mouse and human T-ALL cells. recapitulate effects seen with BMS-536924. The IGF1R- Analysis of IGF1R by Western blot and flow cytometry re - blocking antibody IR3 substantially inhibited proliferation vealed IGF1R was expressed in all cases examined, albeit at in three of four primary human leukemias tested (Fig. 2 B, varying levels (Fig. 1). For human cells, we examined both rightmost column). Notably, the one resistant case (K419) established cell lines and xenograft-expanded primary human expressed lower levels of IGF1R compared with the others Figure 1. IGF1R is expressed broadly in human and mouse T-ALL. (A and B) Western blot and (C and D) flow cytometric analysis of total and surface IGF1R protein expression, respectively, from human cell lines (A and C), primary mouse leukemias (B) derived by retro- viral transduction/transplantation of bone marrow with an activated form of Notch1 termed E, and xenograft-expanded primary human samples (D). Western blot controls in (B) are mouse embryonic fibroblasts derived null from IGF1R mouse embryos (R) and the same cells stably transfected with an IGF1R cDNA expression construct (R+). At least 20,000 events were collected within each gate for all flow cytometry assays. Data de - picted are representative of at least two inde- pendent experiments. 1810 Notch-IGF1R signaling supports LICs in T-ALL | Medyouf et al. A r t i c l e +/+ (Fig. S3) and showed minimal response to IGF-1 stimulation IGF1R E leukemias (Fig. 3, C and D; and not depicted). neo/neo (unpublished data), suggesting the effect of BMS-536924 on Importantly, we confirmed that IGF1R E leukemias this particular tumor may be mediated by inhibition of insulin expressed reduced levels of full-length IGF1R protein (32 ± +/+ receptor. In contrast, case M69 is highly sensitive to IR3, 20% of levels observed in IGF1R E leukemias, n = 7; but largely resistant to BMS-536924, raising the possibility of Fig. 3 E and not depicted). either off-target antibody effects or inherent drug resistance. Notwithstanding these exceptions, the overall results support Reduced IGF1R signaling compromises leukemia-initiating that pharmacologic inhibition of IGF1R signaling can signif- cell (LIC) activity icantly inhibit the growth of most T-ALL cells. To explore whether the increased latency observed for neo/neo IGF1R E leukemias might be caused by delayed trans- Generation of T cell leukemias with reduced IGF1R signaling formation or a reduced net proliferative rate, we performed +/+ neo/neo We next used a genetic approach to further explore the serial transplantation of IGF1R and IGF1R E leu- effects of reduced and/or complete loss of IGF1R on T-ALL kemias into syngeneic/congenic recipients by tail vein injec- cells. Specifically, we generated primary mouse T cell leuke - tion. Unexpectedly, we observed that only a subset of neo/neo mias by transduction of bone marrow from conditional IGF1R primary leukemias was capable of transferring IGF1R mice with Notch1(E) retrovirus, followed by trans- disease to secondary recipients. Among 11 independent plantation into congenic WT recipients. The conditional primary leukemias assessed, 7 (64%) were nontransplantable, neo allele used, IGF1R , carries loxP sites flanking the third 2 (18%) were transplantable in a minority of recipients, and exon, but also retains a loxP-flanked neo cassette within the 2 (18%) were fully transplantable, including one with pro- +/+ second intron (Fig. 3 A). This neo cassette interferes with longed latency (Fig. 4 A). In contrast, all IGF1R primary normal transcript splicing and results in reduced expression leukemias were fully transplantable and exhibited short laten- neo/neo of full-length IGF1R protein in homozygous IGF1R cies. We also performed tertiary and quarternary transplants neo/neo cells (Holzenberger et al., 2000). Despite the decreased level for a subset of cases. The IGF1R leukemia with pro- of IGF1R expression, we were able to generate primary longed latency in secondary recipients (#3115) failed to neo/neo IGF1R leukemias with Notch1(E) retrovirus, albeit with produce disease in tertiary recipients, whereas the other trans- neo/neo +/+ slightly increased latency as compared with WT background plantable IGF1R leukemia (#3105) and all IGF1R (median survival 64.5 vs. 51.5 d; Fig. 3 B). All other disease leukemias produced short latency disease in all tertiary and parameters (penetrance, immunophenotype, histology, disease quarternary recipients (Table S1). Notably, injection of neo/neo distribution, and extent) were highly comparable to control IGF1R leukemia cells directly into the femoral bone marrow space of recipient mice failed to produce leukemia, suggesting that their defect in transplantability is not caused by impaired homing to the bone marrow (Fig. 4 B). Thus, these results indicate that although moder- ate levels of IGF1R signaling are suffi - cient for expansion of bulk leukemia cells, higher levels are required to Figure 2. Pharmacologic inhibition of IGF1R blocks growth of T-ALL cells. Flow cytometric analysis of cell proliferation by BrdU incorporation after treatment with a small molecule IGF1R inhibitor (BMS- 536924) versus DMSO vehicle (mock), or IGF1R blocking antibody (IR3), for 48–72 h in vitro. (A) Three representative indepen- dent primary mouse Notch1(E) leukemias (#324, #327, #329). (B) Four independent xenograft-expanded primary human T-ALL samples (D115, K419, K424, and M69). (C) Two human T-ALL cell lines (ALLSIL and HPBALL). Error bars indicate standard deviation for assays performed in triplicate. Data de- picted are representative of at least three independent experiments. JEM Vol. 208, No. 9 1811 Figure 3. Primary mouse T cell leukemias are generated efficiently by activated Notch1 despite reduced IGF1R expression. neo (A) Schematic of the IGF1R allele. The re- tained neo cassette within the second intron results in reduced expression of full-length neo/neo IGF1R protein in IGF1R mice (Holzenberger et al., 2000). (B) Survival of mice transplanted with retroviral Notch1(E)- transduced bone marrow from either WT +/+ (IGF1R ; n = 6) or IGF1R hypomorph neo/neo (IGF1R ; n = 14) donor animals. ***, P < 0.0001 (Log-rank test). (C) Spleen and liver organ weights at necropsy of individual mor- bid mice transplanted with Notch1(E)- +/+ transduced bone marrow from IGF1R (n = 6) neo/neo and IGF1R (n = 11) backgrounds. Error bars indicate standard deviation. (D) Immuno- phenotypic analysis of representative primary neo/neo mouse E leukemias derived on the IGF1R background. (E) Western blot analysis of total IGF1R protein expression in representative +/+ primary mouse E leukemias on IGF1R , neo/+ neo/neo IGF1R , and IGF1R backgrounds. R+ and R mouse embryonic fibroblast staining controls and Erk2 loading control are indicated. Data depicted in D and E are repre- sentative of at least seven independent samples. compared with controls (Fig. S4, A–E). Furthermore, leukemia cells explanted from moribund, untreated animals (8 wk post-transplant) and cultured with 4-hydroxytamoxifen (4-OHT) in vitro to induce deletion of IGF1R also demonstrated growth arrest and loss of viability (Fig. S4 F). 4-OHT support LIC activity as assayed by serial transplantation in treatment showed no discernible toxic effects on control neo/neo +/+ syngeneic/congenic recipients. IGF1R Rosa26 leukemia cells. Additionally, prelimi- nary results from mice engrafted with a T-ALL cell line G12D Deletion or pharmacologic inhibition of IGF1R prevents (144CLP) derived from a mouse Kras tumor (Chiang et al., disease establishment/progression 2008) show that treatment with BMS-754807, a potent To assess the effect of complete loss of IGF1R in T-ALL IGF1R inhibitor currently in clinical development (Carboni neo/neo cells, we transduced bone marrow from IGF1R et al., 2009), significantly prolonged survival ( Fig. S5), thus CreERT Rosa26 mice with Notch1(E) retrovirus, followed by corroborating the genetic deletion results. Collectively, these transplantation into WT recipient mice. At 4 wk after trans- data support that abrogation of IGF1R signaling impedes plant, peripheral blood was assessed by flow cytometry to T-ALL cell growth/survival and interferes with disease + + + confirm engraftment by GFP CD4 CD8 leukemia cells. establishment/progression. Mice were then divided into equivalent control versus treat- neo ment groups, and the latter was fed tamoxifen-containing Transplantable IGF1R leukemias show evidence chow (1 g/kg) for a 7-d period to induce deletion of IGF1R. for downstream compensation neo/neo Mice from both groups were then sacrificed, and tissues were Our observation that a minority of IGF1R leukemias is harvested. Preliminary results show that tamoxifen-treated transplantable suggests secondary alterations may be selected mice demonstrate significantly lower levels of disease in - for in vivo that compensate for reduced IGF1R signaling. volvement in all tissues examined (bone marrow, spleen, and Despite the known frequency of PTEN loss and PIK3CA/ thymus), and leukemia cells from tamoxifen-treated animals PIK3R1 mutations in T-ALL (Maser et al., 2007; Palomero exhibited lower proliferative and higher apoptotic indices as et al., 2007; Gutierrez et al., 2009), we were unable to detect 1812 Notch-IGF1R signaling supports LICs in T-ALL | Medyouf et al. A r t i c l e Figure 4. Mouse T cell leukemias with reduced IGF1R expression are defective in serial transplantation. Survival of mice transplanted with +/+ neo/neo primary mouse E leukemia cells from either IGF1R or IGF1R backgrounds by i.v. (A) intrafemoral (IF; B) injection route. Each numbered sample represents a different primary leukemia from mice in Fig. 3 B injected into secondary recipients. Raw survival data, including numbers of animals in each cohort, are provided in Table S1. Data depicted are collated from four independent transplantation experiments. neo/neo these alterations in any of the transplantable E-IGF1R responsive to IGF-1, and are comparable to nontransplant- neo/neo leukemias (Fig. 5, A and B; and not depicted). We thus con- able IGF1R leukemias in this regard (Fig. 5 D). We also neo/neo sidered that enhanced responsiveness to growth factor stimu- noted the one IGF1R leukemia exhibiting very short lation could compensate for reduced IGF1R expression. latency in secondary recipients (#3105) had unusually high +/+ In fact, transplantable leukemias #3112 and #3115 demon- levels of pAkt at steady state (twofold greater than IGF1R strated enhanced pAkt response to serum stimulation as com- cells; Fig. 5 E). The elevated pAkt in these cells was not re- pared with nontransplantable leukemias, though not to the duced even after prolonged serum starvation (unpublished +/+ degree exhibited by IGF1R cells (Fig. 5 C). We excluded data), suggesting the presence of some constitutively activat- the possibility that IGF1R itself was up-regulated as trans- ing mutation that confers growth factor independence. In neo/neo plantable IGF1R leukemias remained only minimally contrast, we have not found similarly compelling evidence Figure 5. Transplantable clones with reduced IGF1R expression show compensatory PI3K–Akt activation. (A) Western blot and (B) flow cyto - neo metric analysis of PTEN protein expression in primary and transplantable secondary E-IGF1R leukemias. (C and D) Akt activation as measured by intra- cellular phospho-Akt(Ser473) flow cytometry in response to stimulation with FBS (C) or (D) recombinant IGF-1 × (D) 10 min after 5-h serum starvation. +/+ neo (E) Steady-state level of Akt activation as measured by pAkt(Ser473) in transplantable E-IGF1R and E-IGF1R (#3105) leukemias (n = 3 indepen- neo dent mice for each cohort). For C and D, the five E-IGF1R transplantable clones shown are (4 x #3115, 1 x #3112). Error bars indicate standard devia- tion. *, P < 0.05; **, P < 0.01 (Student’s t test). Data depicted in A and B are representative of at least three independent experiments. Data depicted in C–E are representative of two independent experiments and include at least three independent mice per cohort per experiment. JEM Vol. 208, No. 9 1813 for hyperactivation of the Ras–Raf–MAPK pathway to ex- (GSI) to inhibit Notch signaling or vehicle control (DMSO). neo/neo plain the unique transplantability of these IGF1R clones These five cell lines were selected because they undergo (unpublished data). These few examples suggest PI3K–Akt growth arrest upon GSI treatment (Weng et al., 2004; activation may potentially be more important than Ras–Raf– Palomero et al., 2007). To control for off-target effects of GSI, MAPK signaling in conferring LIC activity; however, further we also profiled cell lines that had been retrovirally trans - study is needed to clarify this issue. duced with ICN1, and then treated with GSI or DMSO. ICN1, the intracellular domain of Notch1, is unaffected by IGF1R mRNA and protein expression GSI treatment and thus maintains Notch signaling in the is up-regulated by Notch presence of GSI (Weng et al., 2003, 2004). Further, because To explore mechanisms that may support the high-level Notch1 induces transcription of c-Myc (Palomero et al., 2006b; IGF1R expression required for LIC activity, we considered a Sharma et al., 2006; Weng et al., 2006), which in turn induces potential role for Notch signaling. Notch has previously been transcription of many other genes (Fernandez et al., 2003), we shown to promote activation of PI3K–Akt in developing also prolfi ed cells that had been transduced with c-Myc and then thymocytes (Ciofani and Zuñiga-Pflucker, 2005), and we treated with GSI or DMSO to segregate Notch targets from noted in our expression profile datasets evidence for up- c-Myc targets. We identie fi d genes whose expression level most regulation of IGF1R by Notch signaling. More specic fi ally, strongly correlated with Notch activity, but not with c-Myc, and we performed microarray-based expression profiling of five noted the conspicuous presence of IGF1R on this list (Table S2) human T-ALL cell lines (ALLSIL, DND41, HPBALL, and in other published microarray datasets generated from GSI- KOPTK1, and TALL-1) treated with -secretase inhibitor treated T-ALL cells (Fig. S6; Palomero et al., 2006b). Figure 6. Inhibition of Notch signaling with GSI down-regulates IGF1R expression in human T-ALL cells. (A) qRT-PCR analysis of IGF1R mRNA in human T-ALL cell lines treated in vitro with -secretase inhibitor (GSI) to block Notch signaling versus DMSO vehicle (mock) for 2–10 d. Error bars indi- cate standard deviation for assays performed in triplicate. (B and C) Flow cytometric analysis of surface IGF1R expression by human T-ALL cell lines treated with GSI versus DMSO vehicle for 6–8 d. Data are representative of multiple replicates. (D) Flow cytometric analysis of surface IGF1R expression by xenograft-expanded primary human T-ALL cells. Cells were cultured on MS5-DL1 feeders to stimulate Notch signaling versus MS5 control feeders and then treated with 1.0 µM compound E (GSI) for 2–4 d to block Notch signaling. Flow histograms for a representative case are depicted on the left, and results from six different patient samples are summarized on the right. Error bars indicate standard deviation. **, P < 0.01 (Student’s t test). At least 20,000 gated live events were collected for all flow cytometry assays. 1814 Notch-IGF1R signaling supports LICs in T-ALL | Medyouf et al. A r t i c l e We also performed a similar expression profiling experi - ment using the human T-ALL cell line, CUTLL1, which har- bors a TcrB/Notch1 fusion gene that drives expression of a form of Notch1 resembling E (Palomero et al., 2006a). CUTLL1 cells transduced with empty retrovirus (MigR1) or dominant-negative (DN) MAML1-GFP, a specific Notch inhibitor (Weng et al., 2003; Maillard et al., 2004; Weng et al., 2004), were treated with GSI for 3 d, after which GSI was washed out to create a timed pulse of Notch signaling activity. Expression profiling was done on cells before washout and at 2 and 4 h after washout to identify likely direct Notch1 target genes. We observed GSI washout up-regulated expression of IGF1R along with other known Notch1 target genes, such as Hes1, Hes4, Hes5, NRARP, and DTX1 in MigRI control cells, and that DN-MAML1-GFP partially or completely abrogated up-regulation of the same set of genes (Fig. S7). To validate these expression profile data, we performed qRT-PCR for IGF1R mRNA. Notch-dependent T-ALL cell lines (ALLSIL, DND41, HPBALL, KOPTK1, and TALL-1) down-regulated IGF1R mRNA levels up to two- fold when treated with GSI, whereas Notch-independent lines (Jurkat, PF382, and RPMI 8402) showed 6–14-fold down-regulation of IGF1R mRNA (Fig. 6 A). GSI blockade also down-regulated IGF1R protein levels 2–3 fold on aver- age, as judged by flow cytometry and Western blotting of human T-ALL cell lines and xenograft-expanded primary samples (Fig. 6, B–D; and Fig. S8). This effect of GSI is likely specific to Notch, as DN-MAML1 also decreased IGF1R protein levels (Fig. 7 A), and the effect of GSI on IGF1R was rescued by retroviral transduction with ICN1 (Fig. 7 B). Conversely, culture of xenograft-expanded primary human T-ALL cells on MS5 stromal cells expressing the Notch ligand DL1, but not MS5 control cells, increased IGF1R levels, whereas no such change was observed for the control marker CD45 (Fig. 7 C). In addition, this effect of DL1 ligand on IGF1R levels was abrogated by treatment with GSI (Fig. 6 D). Thus, these data are consistent with a role for Notch signaling in up-regulation of IGF1R ex- pression in human T-ALL cells. ICN1/CSL binds to an intronic IGF1R enhancer Figure 7. Inhibition of Notch signaling with dominant-negative Once cleaved from the plasma membrane by -secretase, MAML1 and activation by ICN1 or DL1 ligand conr fi m IGF1R regulation ICN1 translocates to the nucleus where it forms a ternary by Notch. Flow cytometric analysis of surface IGF1R expression. (A) Human complex on DNA with the DNA-binding factor CSL and T-ALL cell lines were transduced with dominant negative Mastermind-like-1 the coactivator MAML1 to stimulate transcription of target retrovirus (DN-MAML1-GFP) or empty virus control (MigRI). (B) Human T-ALL genes (Aster et al., 2008). To define the regulatory elements cell lines were transduced with ICN retrovirus (Mig ICN) and then treated with through which Notch1 up-regulates target genes in an unbi- 1.0 µM compound E (GSI) for 4 d (HPBALL and TALL-1) or 8 d (PF382) to block endogenous Notch signaling or DMSO vehicle (mock). Retrovirally transduced ased fashion, we performed chromatin immunoprecipitation cells in A and B were discriminated from nontransduced cells by gating for GFP. (ChIP)-Seq analysis on the human T-ALL cell line, CUTLL1, Data depicted in A and B are representative of multiple replicates. (C) Xenograft- using antibodies directed against Notch1 and CSL (Wang expanded primary human T-ALL cells were cultured in vitro on MS5-DL1 feeders et al., 2011). Alignment of sequencing reads from duplicate to stimulate Notch signaling versus control MS5 feeders. CD45 expression levels CSL and Notch1 libraries identified one high-confidence were also assessed simultaneously with IGF1R by o fl w cytometry. Flow histo - ICN1/CSL-binding site within intron 20 of IGF1R at a grams for a representative case are depicted on the left, and results from six position >250 kb 3 of the proximal promoter (Fig. 8 A). different patient samples are summarized on the right. Error bars indicate The next nearest ICN1/CSL-binding site lies 10.5 Mbp 5 standard deviation. *, P < 0.05 (Student’s t test). At least 20,000 live events of the proximal promoter, making this 3 binding site the were collected within each gate for all o fl w cytometry assays. JEM Vol. 208, No. 9 1815 only likely candidate response element for Notch1 regula- rapid reloading, indicating that ICN1/CSL occupancy is tion. Motif analysis using recently developed algorithms dynamic (Fig. 8 D). derived from protein-binding microarrays (Del Bianco et al., The distant location of the putative ICN1/CSL response 2010) showed that the center of the region under the ICN1/ element in IGF1R suggested that it represents an enhancer. CSL-binding peak contains high (CATGGGAA) and mod- In support of this possibility, additional ChIP analyses docu- erate (GCTGAGAA) an ffi ity CSL sites oriented head-to-head mented recruitment of the histone acetyltransferases CREB- and separated by a 17-bp spacer (Fig. 8 B). This architecture binding protein (CBP) and p300, as well as RNA polymerase II, is typical of a sequence-paired site, a special type of Notch to this site (Fig. S9 A; Hatzis and Talianidis, 2002; Wang response element first identified in Drosophila enhancer of et al., 2005). Moreover, we observed enrichment of histone split (E[spl]) locus that is present in mammalian E(spl) homo- H3K4 mono- and dimethylation chromatin marks (charac- logues such as Hes1 (Jarriault et al., 1995) and other genes teristic of enhancers) relative to H3K4 trimethylation marks such as preT (Liu et al., 2010). Loading of ICN1 onto this (a feature of promoters; Heintzman et al., 2007), and low site was confirmed by ChIP/quantitative PCR (qPCR) levels of the repressive trimethyl histone H3K27 chromatin analyses, performed on CUTLL1 cells and two additional mark (Fig. S9 B; Kirmizis et al., 2004). Finally, additional human T-ALL cell lines, HPBALL and KOPTK1 (Fig. 8 C). ChIP-Seq data showed a local decrease in H3K4 methyl- In addition, GSI treatment of CUTLL1 cells depleted both ation at the precise site of the intronic IGF1R sequence- CSL and ICN1 from this site (in line with work from Bray’s paired site, indicating that this site lies in accessible chromatin group indicating that ICN stabilizes CSL interactions with that has been depleted of nucleosomes (Fig. S9 C). Collec- DNA; Krejcí and Bray, 2007), and GSI washout resulted in tively, these data support the presence of an intronic Figure 8. ICN1/CSL binds dynamically to a site within intron 20 of human IGF1R. (A) Alignment of sequencing reads over intron 20 of the IGF1R locus from ChIP libraries prepared from the human T-ALL cell line, CUTLL1, with antibodies specific for Notch1 and CSL as compared with input cells. (B) Genomic DNA sequence from human IGF1R intron 20 (NT_010274.17: 14461410–14461610) with sequence-paired ICN1/CSL binding sites highlighted in bold and 17-bp spacer underlined. (C) ChIP/qPCR analysis of IGF1R intron 20 from three different human T-ALL cell lines using a Notch1-specific antibody as compared with preimmune antiserum. (D) ChIP/qPCR analysis of IGF1R intron 20 from CUTLL1 cells using antibodies specific for Notch1 and CSL com - pared with control rabbit IgG. Cells were treated with GSI for 3 d to block Notch signaling (GSI x 3d) versus DMSO vehicle (DMSO). GSI was washed out, and cells were harvested 4 h later (GSI x 3d, then wash 4 h). Quantitation of immunoprecipitated DNA is expressed relative to input DNA (% input). Error bars indicate standard deviation for qPCR assays performed in triplicate. Numbers above bars in D indicate relative enrichment over control. ChIP libraries were prepared in duplicate, and local ChIP/qPCR analyses were performed twice. Representative results are shown. 1816 Notch-IGF1R signaling supports LICs in T-ALL | Medyouf et al. A r t i c l e Notch1-responsive enhancer in human IGF1R that is retroviral transduction only partially rescued the pAkt re- active in human T-ALL cells. sponse to IGF1 in HPBALL cells (Fig. 9 C), whereas rescue null was complete in PTEN PF382 cells (Fig. 9 D; Palomero Physiological relevance of Notch-induced et al., 2007). Importantly, we confirmed retroviral IGF1R up-regulation of IGF1R restored expression in GSI-treated cells to levels comparable Notch signaling is likely one of several factors that influence to parental cells. Thus, although Notch inhibition can affect IGF1R expression in T-ALL, as Notch inhibition results in both IGF1R and PTEN expression, Notch contributes to only a two- to threefold decrease in surface IGF1R expres- supporting IGF1R expression at high levels, thereby allow- sion, on average. To assess whether this two- to threefold ing T-ALL cells to respond more robustly to ambient levels change in IGF1R protein levels has a significant effect on of IGF-1/2. downstream signaling, we measured levels of intracellular phosphorylated Akt (pAkt) after pulsing serum-starved leu- DISCUSSION kemia cells with recombinant IGF-1. We observed GSI- We have presented data showing that IGF1R mediates im- treated cells to be 20-fold less responsive to IGF-1 than portant growth/survival signals in T-ALL cells, and that vehicle-treated control cells (Fig. 9 A). Although some of this although moderate levels of signaling are adequate for main- effect is presumably caused by GSI effects on IGF1R expres - tenance of the bulk cell population, high levels are required sion, it has also been reported that Notch represses PTEN via for maintenance of LIC activity as indicated by serial trans- HES-1 (Palomero et al., 2007), which could also impact the plantation assay. The effect of reduced IGF1R signaling on pAkt response. Indeed, GSI treatment induced PTEN ex- disease transplantability could be caused by (a) a quantitative pression in HPBALL cells; however, there was no detectable decrease in the number of leukemia stem cells; (b) a qualita- change in ALLSIL or DND41 cells (Fig. 9 B). Accordingly, tive defect in self-renewal, engraftment ability, and/or restoration of IGF1R expression in GSI-treated cells by immune resistance of leukemia stem cells; or (c) a reduced Figure 9. Notch-induced IGF1R expression enhances PI3K–Akt signaling in response to IGF1. (A) Flow cytometric analysis of intracellular phos- pho-Akt levels. Cells were treated with GSI versus DMSO vehicle for 6–8 d, serum starved overnight, and then pulsed with recombinant IGF1 for 10 min before assay. (B) Western blot analysis of PTEN protein expression in cells treated with GSI versus mock for 4 d. (C and D) Flow cytometric analysis for intracellular phospho-Akt levels (left) and surface IGF1R levels (right). Cells were retrovirally transduced with Mig IGF1R, treated with GSI versus DMSO vehicle for 4 d, serum starved overnight, and then pulsed with recombinant IGF1 for 10 min before phospho-Akt assay. Transduced versus nontransduced cells within the same culture were distinguished by gating for GFP. Surface IGF1R expression level was also assessed by flow cytometry immediately before stimulation with IGF1. Filled grey histograms represent second antibody staining controls. At least 20,000 events were collected within each gate for all flow cytometry assays. Data depicted are representative of at least two independent experiments. JEM Vol. 208, No. 9 1817 probabilistic likelihood of engraftment/propagation of bulk inhibition of IGF1R signaling blocks growth/survival of bulk leukemia cells. Notably, the last possibility does not presume leukemia cells confirms previous results with PI3K–Akt/ the existence of leukemia stem cells in this model; however, mTOR inhibitors, and further illustrates that IGF1-dependent we have generated data that supports that Notch T-ALLs activation contributes in a substantive way to net PI3K–Akt indeed contain bona fide leukemia stem cells (unpublished signaling output. Perhaps most intriguing, however, is our data). Nonetheless, further studies will be required to distin- observation that moderately decreased IGF1R signaling (as neo/neo guish between these possibilities. modeled by IGF1R leukemias) results in selective loss We have also shown that Notch directly up-regulates of LIC activity, yet leaves the growth and survival of bulk IGF1R expression in human T-ALL cells to an extent that cells relatively unaffected. These findings suggest that LICs in substantially enhances their sensitivity to ambient ligand. It is T-ALL may be uniquely sensitive to inhibition of IGF1R worthwhile to note that we have found no evidence to sup- signaling, and raise the possibility that pharmacologic IGF1R port that Notch regulates IGF1R in mouse T-ALL cells. In inhibitors now in clinical development could, in combina- fact, the sequence-paired ICN1/CSL-binding site within tion with standard regimens, enhance initial response to ther- intron 20 of human IGF1R is not conserved in the mouse, apy and reduce rates of disease relapse. Importantly, normal suggesting that sequence divergence has decoupled this point hematopoietic stem cell function appears not to be affected in neo/neo of interaction between the Notch1 and IGF1R pathways. IGF1R mice (unpublished data), suggesting a therapeu- This is perhaps not surprising given that 40–90% of transcrip- tic window exists in which IGF1R inhibitors block LIC tion factor binding events are not conserved between mouse activity yet have minimal effects on normal hematopoiesis. and human (Odom et al., 2007), underscoring the impor- tance of studying human cells and animal models in parallel. MATERIALS AND METHODS The existence of leukemia stem cells in human T-ALL is neo Mice. The IGF1R line contains a PGK-neo-poly(A) expression cassette supported by xenograft transplantation assays (Cox et al., integrated within the second intron, resulting in decreased expression of 2007; Chiu et al., 2010; Gerby et al., 2011) and also in mouse neo full-length IGF1R (Holzenberger et al., 2000). The IGF1R allele has been / models of T-ALL by transplantation into SCID or Rag1 backcrossed onto the C57BL/6 background for over 20 generations. CreERT immunocompromised recipients (Guo et al., 2008; Tremblay Rosa26 mice were obtained from The Jackson Laboratory. All trans- et al., 2010). The precise cellular compartment most enriched plants involving mouse cells were performed using C57BL/6J (B6) or c-2J B6(Cg)-Tyr /J (“B6-albino”) recipient mice (The Jackson Laboratory). for LIC activity appears to vary from patient to patient and Transplants involving human cells were performed using NOD-Scid/ between different animal models, and likely depends on the / IL2Rc recipient mice. Animals were housed under specific pathogen– particular complement of genetic alterations present in the free conditions at the BC Cancer Agency or Dana-Farber Cancer Institute leukemic clone and the particular transplant recipient used animal facilities, and experimental studies performed under protocols ap- (Cox et al., 2007; Quintana et al., 2008; Chiu et al., 2010; proved by the University of British Columbia or Dana-Farber Institutional Gerby et al., 2011). Nonetheless, it is interesting to note that Review Boards, respectively. IGF1R genotyping was performed by multi- plex assay with primers YNex (5-CCATGGGTGTTA AATGTTAAT- Notch signaling has been shown to contribute to human GGC-3), YNvl (5-ATGAATGCTGGTGAGGGTTGTCTT-3) and T-ALL LIC activity in nonobese diabetic (NOD)/Scid trans- YNmt2 (5-ATCTTGGAGTGGTGGGTCTGTTTC-3), as previously plantation assays (Armstrong et al., 2009). As Notch signaling described (Leneuve et al., 2001). Rosa26 genotyping was performed with is mediated through transcriptional activation, identification primers RcreFW (5-AAAGTCGCTCTGAGTTGTTAT-3) and RcreRV of downstream target genes has received much attention. In (5-GCGAAGAGTTTGTCCTCAACC-3) for the CreERT allele, and with RwtFW (5-GCACTTGCTCTCCCAAAGTC-3) and RwtRV several such studies, c-Myc has been identified as a direct (5-GGCGGATCACAAGCAATAAT-3) for the WT allele. transcriptional target of Notch1 (Palomero et al., 2006b; Sharma et al., 2006; Weng et al., 2006), and may constitute Plasmids. Retroviral vectors all used the MSCV-IRES-GFP (Mig) backbone. part of a self-renewal genetic program similar to that demon- Mig ICN1, Mig E, and MSCV-DN-MAML1-GFP have been described pre- strated in induced pluripotent stem cells (Takahashi and viously (Aster et al., 1997; Weng et al., 2003). Mig IGF1R was generated by Yamanaka, 2006; Takahashi et al., 2007). Notch may also subcloning a 4.2-kb NotI-BamHI fragment containing the human IGF1R repress PTEN (Palomero et al., 2007), and thus potentiate cDNA from pBABE-bleo IGF1R (Addgene) into the Mig vector. PI3K–Akt/mTOR signaling which may also promote LIC Retroviral transduction/bone marrow transplantation. High titer, activity (Yilmaz et al., 2006). replication-defective retroviral supernatants were produced by transient Most prior studies have focused on signaling via IL-7 transfection of PlatE cells as described previously (Medyouf et al., 2010). (Dibirdik et al., 1991; Barata et al., 2004a,b,c, 2005; González- MSCV-based retroviral expression vectors included IRES-GFP cassettes Garcia et al., 2009; Shochat et al., 2011; Silva et al., 2011) or for fluorescent tagging of transduced cells. Retroviral transduction of mutational activation of intermediates in the PI3K–Akt path- 5-u fl orouracil-treated or lineage-depleted (CD4, CD8, CD11b, B220, Ter119, and Gr1) bone marrow cells was performed as described previously (Medyouf way (Palomero et al., 2007; Gutierrez et al., 2009). The im- et al., 2010). For primary transplants, WT C57BL/6 recipient mice were portance of PI3K–Akt activation in T-ALL is underscored by lethally irradiated (810 rads), and then injected intravenously with 30,000– multiple studies showing that PI3K–Akt/mTOR inhibitors + 5 45,000 transduced GFP cells along with a minimum of 10 syngeneic whole block growth/survival of T-ALL cells (Avellino et al., 2005; bone marrow cells to ensure hematopoietic reconstitution. For serial transplants, 6 + Wei et al., 2006; Palomero et al., 2007; Chiarini et al., 2009; 0.5–1.0 × 10 GFP leukemia cells (from spleen or bone marrow of a moribund Cullion et al., 2009). Notably, our observation that complete leukemic mouse) were injected into the tail vein (i.v.) or intramedullary 1818 Notch-IGF1R signaling supports LICs in T-ALL | Medyouf et al. A r t i c l e space of the femur (intrafemoral [IF]) of nonirradiated C57BL/6 recipients. (Altromin) and mice were allowed to feed ad libitum. For induction of Cre- We define LIC activity here as the ability to produce aggressive disease in ERT activity in vitro, 4-OHT (Sigma-Aldrich) was dissolved in ethanol and syngeneic/congenic secondary recipients under these transplant conditions added to culture media at 50 nM final concentration. within 20 wk. Expression profiling. Total RNA was isolated by TRIzol (Invitrogen) ex- Human leukemia samples. Cryopreserved lymphoblast samples were traction or RNeasy Mini kit (QIAGEN) and submitted to the McGill Uni- provided by collaborating institutions. Primary samples were obtained at ini- versity/Génome Québec Innovation Centre or Harvard Medical School tial diagnosis with informed consent from patients or their legal guardians Biopolymers Core for expression profiling using Affymetrix HG-U133 Plus or as discarded pathological material under approved Institutional Review 2.0 GeneChips. Data were analyzed using dChip software (Schadt et al., Board protocols at the Karmanos Cancer Institute, Hôpital Armand-Trousseau, 2001). Microarray data are available from the Gene Expression Omnibus Hopital Saint-Louis, BC Cancer Agency, and BC Children’s and Women’s accession no. GSE29959. Hospital following guidelines established by the Declaration of Helsinki. Ex- / pansion of primary human T-ALL cells in irradiated NOD-Scid/IL2Rc Quantitative real-time PCR. Total RNA was extracted after cell lysis in mice has been described previously (Medyouf et al., 2010). TRIzol reagent (Invitrogen). First-strand cDNA was generated by reverse transcription with SuperScript III (Invitrogen) using a mix of random 15-mer In vitro culture of primary T-ALL cells. Primary human T-ALL cells and anchored oligo(dT)20+1 primers, and then amplified using Platinum were cultured on MS5-DL1/MS5 stromal feeder cells as previously de- SYBR Green qPCR SuperMix-UDG (Invitrogen) and the following specific scribed (Medyouf et al., 2010). Primary mouse T-ALL cells were cultured primer sets: hIGF1R forward 5-ACTTACTCGGACGTCTGGTCCTTC-3, without feeders in complete media with supplemental cytokines. hIGF1R reverse 5-ATCTTGGGGTTATACTGCCAGCAC-3 for IGF1R, and hActB RT5 5-CGCGAGAAGATGACCCAGAT-3plus hActB RT3 Cell lines. IGF1R knockout mouse embryo fibroblasts (R-cells) that were 5-GAT AGC ACA GCC TGG ATA GCA AC-3 for -actin. Each sample engineered to re-express human IGF1R (R+) were a gift from P. Sorensen was assayed in triplicate using a Dyad Disciple thermal cycler equipped with (BC Cancer Agency, Vancouver, British Columbia, Canada). All established Chromo4 optical head (Bio-Rad Laboratories). Expression levels were cal- T-ALL cell lines were grown in RPMI 1640 medium supplemented with culated by the Ct method with normalization to -actin. 10% FCS, 1 mM sodium pyruvate, 2 mM L-glutamine, and antibiotics. ChIP. ChIP was performed with the ChIP Assay kit (Millipore). In brief, Ligand stimulation assay. Leukemia cells were serum starved overnight, cells were cross-linked with 1% formaldehyde for 10 min at 37°C, lysed in and then stimulated for 10 min with either recombinant IGF-1 (PeproTech), 1% SDS, and 10 mM EDTA, 50 mM Tris, pH 8.1, and then sonicated to recombinant insulin (Sigma-Aldrich), or fetal calf serum (Invitrogen), at in- obtain DNA fragments from 200–600 bp. Chromatin was then immuno- dicated doses. Cells were then fixed immediately by addition of formalde - precipitated with the following antibodies: Notch1(Tc; Aster et al., 1997), hyde (Electron Microscopy Services) to 1.5%, and permeabilized with CSL (gift from E. Kieff, Brigham and Women’s Hospital, Boston, MA), ice-cold methanol for at least 1 h before analysis by flow cytometry. H3K4me1 (ab8895; Abcam), H3K4me2 (07–030; Millipore), H3K4me3 (07–745; Millipore), CBP (ab10489; Abcam), p300 (sc-585; Santa Cruz In vitro proliferation/apoptosis assays. Cells were pulsed with 10 µM Biotechnology, Inc.), RNA pol2 (05623B; Millipore), and mouse IgG BrdU for 1 h at 37°C, and then fixed/stained with anti-BrdU antibody (12–371; Millipore) as control. After overnight incubation with antisera according to manufacturer instructions (FITC or APC BrdU Flow kit; BD) at 4°C, immunoprecipitated chromatin was captured with Protein A-agarose and analyzed by flow cytometry with gating for mouse and human leukemia beads, washed, and eluted. After reversal of cross-links, DNA was pu- cells by GFP- and hCD45-positive events, respectively. Cell growth was also rified using the QIAquick PCR purification kit (QIAGEN). Input con - measured by CellTiter-Blue assay (Promega) or Ki67-Alexa Fluor 647/ trol DNA was also prepared in parallel, omitting the immunoprecipitation Hoechst staining (BD Biosciences). Apoptotic cells were identified by stain - steps. Primers used for qPCR after ChIP were as follows: IGF1Rchip for- ing with Annexin V-FITC/7-aminoactinomycin or active caspase-3-PE ward 5-GGTGGGTGAGGGAGAGCGGT-3 and IGF1Rchip reverse (BD). Cell viability was assessed by propidium iodide exclusion and analysis 5-GGCTGCGTCCCAGGCAGTTT-3. by flow cytometry. ChIP-Seq and data analysis. ChIP-Seq libraries were prepared according Antibody reagents. Antibodies directed against the following proteins to the Illumina ChIP DNA library preparation kit. After addition of adap- were used: IGF1R (sc-712, Santa Cruz Biotechnology), PTEN (Y184, tors, libraries were amplified by 18 cycles of PCR, size selected (150–250 bp) Abcam), and ERK2 (sc-154, Santa Cruz Biotechnology) for Western blots, by electrophoresis, and purified using a QIAGEN gel extraction kit. After and hCD45 (eBioscience), CD4 (L3T4; eBioscience), CD8a (53–6.7, Bio- quality control testing on an Agilent 2100 Bioanalyzer, the library was sub- Legend), IGF1Ra (IR3; EMD), and P-AKT (S473; Cell Signaling Tech- jected to deep sequencing using an Illumina Genome Analyzer II in the nology) for flow cytometry. Harvard Medical School Biopolymers Core facility. Sequencing reads were aligned to human genome build hg18 and analyzed using CisGenome Flow cytometry. Acquisition was performed on FACSCalibur or LSRII (Ji et al., 2008). One-sample and two-sample analyses were performed using cytometers (BD) and data analyzed using FlowJo software (Tree Star, Inc.). 100-bp windows and reads of >10 bp. Drugs. IGF1R inhibitors BMS-536924 and BMS-754807 were obtained Online supplemental material. Fig. S1 shows Akt response to stimula- under Material Transfer Agreement from the manufacturer. BMS-536924 tion with IGF1 and insulin and effects of BMS-536924 inhibitor in T-ALL was resuspended in DMSO at 10 mM, then serially diluted in Hank’s Bal- cells. Fig. S2 shows effects of the BMS-536924 inhibitor on T-ALL cell anced Salt Solution (Invitrogen) before addition to culture media. BMS- growth and viability in vitro. Fig. S3 shows resistance to growth inhibi- 754807 was resuspended in PEG400:H O (80:20 by volume) at 5 mg/ml tion by IGF1R blocking antibody correlates with reduced surface IGF1R final concentration before administration to mice. An azide-free preparation expression. Fig. S4 shows effects of IGF1R deletion on T-ALL develop - of IR3 antibody was used for IGF1R blocking studies (EMD). -Secretase ment in vivo and cell growth/viability in vitro. Fig. S5 demonstrates in vivo inhibitor XXI (compound E) was used at 1.0 µM final concentration for all efficacy of the IGF1R inhibitor BMS-754807 in a mouse T-ALL model. studies (EMD). GSI washout was performed by washing cells twice with Fig. S6 shows that IGF1R is a Notch target gene in an independent ex- prewarmed, complete culture medium. For induction of CreERT activity in pression profiling dataset. Fig. S7 shows IGF1R is a Notch target gene in vivo, 1 g tamoxifen (Sigma-Aldrich) was admixed per 1 kg mouse chow the human T-ALL cell line, CUTLL1. Fig. S8 shows Western blot analysis JEM Vol. 208, No. 9 1819 of total IGF1R expression after GSI treatment of human T-ALL cell lines. Barata, J.T., A.A. Cardoso, and V.A. Boussiotis. 2005. Interleukin-7 in Fig. S9 shows chromatin modification marks over the human IGF1R locus T-cell acute lymphoblastic leukemia: an extrinsic factor support- in the vicinity of the Notch/CSL binding site. Table S1 shows survival data ing leukemogenesis? Leuk. Lymphoma. 46:483–495. doi:10.1080/ for all secondary, tertiary, and quarternary transplant experiments. Table S2 Bendall, S.C., M.H. Stewart, P. Menendez, D. George, K. Vijayaragavan, lists genes whose mRNA expression level decreases most after inhibition of T. Werbowetski-Ogilvie, V. Ramos-Mejia, A. Rouleau, J. Yang, M. Notch signaling. Online supplemental material is available at http://www Bossé, et al. 2007. IGF and FGF cooperatively establish the regulatory .jem.org/cgi/content/full/jem.20110121/DC1. stem cell niche of pluripotent human cells in vitro. Nature. 448:1015– 1021. doi:10.1038/nature06027 We thank Amina Kariminia (BC Children’s Hospital, Vancouver) for sample Carboni, J.M., M. Wittman, Z. Yang, F. Lee, A. Greer, W. Hurlburt, S. preparation, Drs. Kirk Schultz (BC Children’s Hospital, Vancouver), Larry H. Matherly Hillerman, C. Cao, G.H. Cantor, J. Dell-John, et al. 2009. BMS-754807, (Karmanos Cancer Institute, Detroit), Paola Ballerini (Hôpital Armand-Trousseau, a small molecule inhibitor of insulin-like growth factor-1R/IR. Mol. Paris), and Thierry Leblanc (Hopital Saint-Louis, Paris) generously contributed Cancer Ther. 8:3341–3349. doi:10.1158/1535-7163.MCT-09-0499 human T-ALL samples. Chiang, M.Y., L. Xu, O. Shestova, G. Histen, S. L’heureux, C. Romany, This work was funded by grants from the Canadian Cancer Society Research M.E. Childs, P.A. Gimotty, J.C. Aster, and W.S. Pear. 2008. Leukemia- Institute/Terry Fox Foundation, US National Cancer Institute (K22CA112538, associated NOTCH1 alleles are weak tumor initiators but accelerate P01CA119070), Leukemia and Lymphoma Society of Canada, Lymphoma Foundation K-ras-initiated leukemia. J. Clin. Invest. 118:3181–3194. doi:10.1172/ Canada, Cancer Research Society, and Agence Nationale de la Recherche (NT05-3 JCI35090 42491). H. Medyouf is supported by a Human Frontier Science Program Fellowship. Chiarini, F., F. Falà, P.L. Tazzari, F. Ricci, A. Astolfi, A. Pession, P. Pagliaro, J.C. Aster is supported by grants from the Leukemia and Lymphoma Society and the J.A. McCubrey, and A.M. Martelli. 2009. Dual inhibition of class IA William Lawrence Foundation. A.P. Weng is a Michael Smith Foundation for Health phosphatidylinositol 3-kinase and mammalian target of rapamycin as a Research Scholar. new therapeutic option for T-cell acute lymphoblastic leukemia. Cancer J. Carboni and M. Gottardis are employees of Bristol-Myers Squibb Company. Res. 69:3520–3528. doi:10.1158/0008-5472.CAN-08-4884 The remaining authors have no competing interests to declare. Chiu, P.P., H. Jiang, and J.E. Dick. 2010. 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Published: Aug 29, 2011

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