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FBW7 mutations in leukemic cells mediate NOTCH pathway activation and resistance to γ-secretase inhibitors

FBW7 mutations in leukemic cells mediate NOTCH pathway activation and resistance to γ-secretase... ARTICLE FBW7 mutations in leukemic cells mediate NOTCH pathway activation and resistance to  -secretase inhibitors 1 2 3 3 Jennifer O ’ Neil, Jonathan Grim, Peter Strack, Sudhir Rao, 4 3 5 2 Deanne Tibbitts, Christopher Winter, James Hardwick, Markus Welcker, 6 6 3 4 Jules P. Meijerink, Rob Pieters, Giulio Draetta, Rosalie Sears, 2 1,7 Bruce E. Clurman, and A. Thomas Look Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA 02115 Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, WA 98109 Merck Research Laboratories, Boston, MA 02115 Department of Molecular and Medical Genetics, Oregon Health and Sciences University, Portland, OR 97239 Merck Research Laboratories, West Point, PA 19486 Department of Pediatric Oncology/Hematology, Erasmus MC/Sophia Children ’ s Hospital, 3000 CB Rotterdam, Netherlands Division of Hematology, Children ’ s Hospital Boston, Boston, MA 02115 -secretase inhibitors (GSIs) can block NOTCH receptor signaling in vitro and therefore offer an attractive targeted therapy for tumors dependent on deregulated NOTCH activity. To clarify the basis for GSI resistance in T cell acute lymphoblastic leukemia (T-ALL), we studied T-ALL cell lines with constitutive expression of the NOTCH intracellular domain (NICD), but that lacked C-terminal truncating mutations in NOTCH1 . Each of the seven cell lines examined and 7 of 81 (8.6%) primary T-ALL samples harbored either a mutation or homozygous deletion of the gene FBW7 , a ubiquitin ligase implicated in NICD turnover. Indeed, we show that FBW7 mutants cannot bind to the NICD and defi ne the phosphode- gron region of the NICD required for FBW7 binding. Although the mutant forms of FBW7 were still able to bind to MYC, they do not target it for degradation, suggesting that stabilization of both NICD and its principle downstream target, MYC, may contribute to transformation in leukemias with FBW7 mutations. In addition, we show that all seven leukemic cell lines with FBW7 mutations were resistant to the MRK-003 GSI. Most of these resistant lines also failed to down-regulate the mRNA levels of the NOTCH targets MYC and DELTEX1 after treatment with MRK-003, implying that residual NOTCH signaling in T-ALLs with FBW7 mutations contributes to GSI resistance. The mammalian NOTCH proteins are het- that fuses the intracellular form of NOTCH1 CORRESPONDENCE A. Thomas Look: erodimeric transmembrane receptors that con- to the T cell receptor  locus in lymphoblasts Thomas_Look@dfci.harvard.edu trol cell proliferation, apoptosis, and cell fate of T-ALL patients ( 10 ). Recently, 50% of hu- OR during the development of diverse cellular lin- man T-ALL cell lines and primary patient sam- Bruce E. Clurman: bclurman@fhcrc.org eages ( 1 ). Aberrant NOTCH signaling has been ples were shown to harbor activating mutations extensively linked to cancer and development. in NOTCH1 that result in aberrant NOTCH Abbreviations used: AML, acute In mouse models, constitutive NOTCH signal- signaling ( 11 ). Although mutations that di- myeloid leukemia; CPD, Cdc ing contributes to the genesis of breast cancer, rectly activate NOTCH receptors have not phosphodegron; GSI,  -secretase inhibitor; MDS, myelodysplastic medulloblastoma, and T cell leukemia ( 2 – 9 ), been identifi ed in other types of human can- syndrome; NICD, NOTCH whereas in human cancer its role is best exem- cers, there is abundant evidence to support the intracellular domain; T-ALL, plifi ed by T cell acute lymphoblastic leukemias importance of deregulated NOTCH activity in T cell acute lymphoblastic leukemia. (T-ALLs). Human NOTCH1 was fi rst discov- the development of ovarian cancer ( 12 ), breast ered as a gene activated at the breakpoint of the cancer ( 13 ), anaplastic large cell lymphoma and t(7;9), a very rare chromosomal translocation Hodgkin disease ( 14 ), melanoma ( 15 ), gliomas ( 16 ), lung carcinomas ( 17, 18 ), and cancers of the pancreas ( 18 ) and prostate ( 19 ). Hence, J. O ’ Neil and J. Grim, and B.E. Clurman and A.T. Look contributed equally to this work. modulation of the NOTCH signaling cascade JEM © The Rockefeller University Press $15.00 Vol. 204, No. 8, August 6, 2007 1813-1824 www.jem.org/cgi/doi/ 10.1084/jem.20070876 The Journal of Experimental Medicine at one or more points could short-circuit this pathway in fi ve GSI-sensitive cell lines, indicating that MRK-003 acts by NOTCH-supported tumors, leading to clinically important inducing apoptosis as well as by blocking cell growth ( Fig. 1 B ). antitumor eff ects. Importantly, the altered proliferation, cell cycle arrest, and Blocking the intramembranous cleavage of NOTCH is increase in apoptosis could be rescued by overexpressing the an especially attractive strategy of targeted therapy. When the NICD, demonstrating that these eff ects indeed result from NOTCH receptor is recognized by its membrane-bound inhibition of the NOTCH signaling pathway ( 24 and unpub- ligand, a conformational change exposes the receptor to lished data). The cell counts, cell cycle profi les, and percent- sequential rounds of protease cleavage. Binding of the ligand ages of apoptotic cells in the remaining 15 T-ALL cell lines results in proteolytic cleavage of the receptor, fi rst outside the were not aff ected by treatment with MRK-003 ( Fig. 1, cell by TNF-  – converting enzyme and then by the  -secretase C and D, and Table I ). membrane protease complex, releasing the NOTCH intra- cellular domain (NICD), which translocates to the nucleus NICD levels are high in several T-ALL cell lines without where it regulates the expression of its target genes, including truncating mutations in NOTCH1 MYC and DELTEX1 ( 20 – 23 ). Small molecule inhibitors of Treatment with MRK-003 led to reduced levels of NICD in -secretase activity are now available that eff ectively inhibit each of the T-ALL cell lines in which the intracellular pro- NOTCH signaling in vitro. One commercial product, com- tein could be detected, indicating that the compound eff ec- pound E, induces growth arrest in several diff erent T-ALL tively inhibited NOTCH1 cleavage at the cell surface ( Fig. 1 E ). cell lines by inhibiting the NOTCH pathway ( 11 ). Recently, However, only a subset of the 20 T-ALL cell lines had a GSI- we showed that treatment of T-ALL cells with the MRK-003 sensitive cellular phenotype, indicating that a resistant pheno- -secretase inhibitor (GSI) results in prolonged cell cycle type can be observed with or without evidence for constitutive arrest followed by apoptosis ( 24 ). NOTCH signaling (i.e., without detectable expression of Despite the promise of GSI therapy for tumors driven by NICD; Table I and Fig. 1 E ). Western blot analysis also dem- aberrant NOTCH signaling, most human T-ALL cell lines onstrated that all of the cell lines with C-terminal truncating are resistant to these agents and grow normally despite GSI mutations of NOTCH1 expressed high levels of NICD treatment. Thus, to establish the molecular basis of GSI resis- ( Table I and Fig. 1 E ), refl ecting aberrant activation of the tance in tumor cells, we used T-ALL cell lines as a model NOTCH pathway. Interestingly, there were seven cell lines system to test the ability of GSI treatment to reduce cellular without C-terminal truncating mutations in NOTCH1 that levels of NICD, as well as its transcriptional targets MYC and also expressed high levels of NICD (RPMI8402, CEM, DELTEX1 . We found that mutations aff ecting the FBW7 BE13, DU528, HSB2, Jurkat, and PEER), implying that ubiquitin ligase occur in GSI-resistant cell lines and are asso- NOTCH is stabilized in these cell lines through a mechanism ciated with sustained NOTCH signaling. Moreover, we other than mutation of NOTCH1 . found FBW7 mutations in primary T-ALL samples, and the mutational spectrum suggests that they produce dominant- FBW7 is mutated in T-ALL cell lines and primary samples negative FBW7 alleles. Our fi ndings implicate FBW7 muta- Cellular levels of NICD are determined by the net eff ects of tions in both the pathogenesis of T-ALL and leukemic cell the rates of production and destruction of the protein. The resistance to GSIs. SCF-FBW7 ubiquitin ligase plays a critical role in NICD degradation that is dependent on an intact PEST domain of RESULTS NOTCH ( 25 – 30 ). Other known FBW7 substrates include MRK-003 treatment leads to Notch-dependent cyclin E, MYC, SR-EBP, and c-Jun ( 31 ). FBW7 interacts antiproliferative effects in a subset of T-ALL cell lines with its substrates via the  propeller formed by its WD40 To inhibit NOTCH-mediated signal transduction, we treated repeats after the substrate becomes phosphorylated within each of 20 T-ALL cell lines with the Merck GSI MRK-003 highly conserved Cdc phosphodegrons (CPDs) ( 32, 33 ). Human (24) at 1  M or with DMSO (vehicle control) for 7 d. To tumors harbor nonsense mutations that truncate FBW7 as study the eff ects of GSI treatment, we analyzed the cell well as missense mutations that target key arginine residues in counts, cell cycle profi les, and apoptosis at various time points the WD40 repeats. These mutations disable FBW7 – substrate after treatment. We did not observe any eff ects on prolifera- interactions and impair substrate degradation by FBW7 ( 34 – 37 ). tion or viability at 6 h, 24 h, or 3 d in any of the 20 lines. Five Mutations aff ecting CPDs in tumor-derived alleles of both of the T-ALL cell lines (DND41, Koptk1, ALL-SIL, HPB- MYC and JUN have been described ( 38 – 41 ). Thus, muta- ALL, and TALL1) were sensitive to GSI treatment, exhibit- tions removing or altering the substrate CPD can also lead to ing two- to threefold fewer live cells in the GSI-treated fl asks loss of regulation by FBW7. Because the PEST domain trun- compared with DMSO-treated fl asks on day 7 of treatment cations found in human T-ALL are predicted to disrupt ( Table I ). Cell cycle analysis at that time revealed a G /G cell FBW7 binding to NICD, we hypothesized that FBW7 mu- 0 1 cycle arrest with decreased cells in the S phase in each of the tation could represent an alternative mechanism for NOTCH fi ve lines (representative histograms are shown for one line in deregulation in human cancers. Fig. 1 A ). Annexin V staining showed a two- to fourfold in- To test this hypothesis, we sequenced the entire coding crease in the percentage of cells undergoing apoptosis in the region of FBW7 to determine whether mutations aff ecting 1814 FBW7 MUTATION IN T-ALL | O ’ Neil et al. ARTICLE Table I. FBW7 mutations in human T -ALL cell lines NOTCH1 status HD domain PEST domain NICD detected FBW7 status Sensitive Cell Lines DND41 heterozygous mutation heterozygous insertion 2444 CCSHWAPAAWRCTLFCPRRAPPCP Yes WT 1594 L → P RRCHPRWSHP*STOP HPB-ALL heterozygous mutation heterozygous insertion 2442 Yes WT 1575 L → P EGRGRCSHWAPAAWRCTLFCPRRAPPCPRRCHPRWSHP*STOP Koptk1 heterozygous mutation heterozygous deletion 2515 RVP*STOP Yes WT 1601 L → P ALL-SIL heterozygous mutation heterozygous insertion 2476 AHP*STOP Yes WT 1594 L → P TALL1 WT WT No WT Resistant Cell Lines RPMI8402 heterozygous insertion WT Yes homozygous 1584 PVELMPPE mutation 465 R → H CEM heterozygous insertion WT Yes heterozygous 1595 PRLPHNSSFHFL mutation 465 R → H BE13 homozygous mutation WT Yes homozygous 1601 L → P deletion DU528 WT WT Yes heterozygous mutation 465 R → H HSB2 WT WT Yes heterozygous mutation 505 R → C Jurkat WT WT Yes heterozygous mutation 505 R → C PF382 heterozygous mutation heterozygous insertion 2494 ASCILWTTPPATSYRCLSTPSSPRP Yes WT 1575 L → P LSPLTSGPSRPRIPTSPTGPRASPALPPACSPRSPAFRRPSSKRRAPRDP GFLSQAFGRLCALCGCQGRPEEPF*STOP Molt13 heterozygous mutation heterozygous deletion 2515 *STOP Yes WT 1601 L → P Supt7 heterozygous insertion heterozygous insertion 2429 Yes WT 1593 F → LGA AHKTYRCSSRTCSQQTSSSSKACSRHHHHHSR TLA*STOP Molt4 heterozygous mutation heterozygous deletion 2515 RVP*stop Yes WT 1601 L → P PEER WT WT Yes heterozygous mutation 505 R → C Loucy WT WT No WT Supt11 WT WT No WT Supt13 WT WT No WT Molt16 WT WT No WT Numbers correspond to amino acid residues in NOTCH1. As determined by western blot analysis. this ubiquitin ligase gene might account for the accumulation lines with increased levels of NICD but without C-terminal of NICD in T-ALL cell lines that lacked truncations of the truncating mutations in NOTCH1 (RPMI8402, CEM, NOTCH1 PEST degradation domain. Sequencing of the BE13, DU528, HSB2, Jurkat, and PEER) harbored either a entire coding region of FBW7 in 20 T-ALL cell lines re- mutation in FBW7 or homozygous deletion of the gene, thus vealed mutations in 6 of the 20 lines (30%) ( Table I ). The providing an explanation for persistent NICD expression in GSI-resistant cell line RPMI8402 harbored a homozygous these lines ( Fig. 1 E ). Conversely, none of the cell lines with arginine-to-histidine mutation at residue 465; all of the other FBW7 mutations had PEST domain mutations in NOTCH1 , mutations were heterozygous missense mutations of critical suggesting that there is no selective pressure for FBW7 muta- WD40 arginine residues (465 and 505). One additional cell tion in cells with stabilized NOTCH1. line (BE13) has a homozygous deletion of FBW7 as deter- We also sequenced exons encoding the substrate-binding mined by quantitative DNA PCR. Each of the seven cell domain (exons 7, 8, 9, 10, and 11) of FBW7 in 150 primary JEM VOL. 204, August 6, 2007 1815 Figure 1. Treatment with MRK-003 results in cell cycle arrest, apoptosis, and inhibition of NICD production in human T-ALL cell lines. (A) Cell cycle analysis of the GSI-sensitive HPB-ALL cell line. Cells were treated with DMSO (vehicle) or 1  M MRK-003 for 7 d, stained with propidium iodide, and analyzed by fl ow cytometry. (B) Annexin V – FITC/propidium iodide staining of the DND41 cell line after 7 d in DMSO or 1  M MRK-003. Numbers represent the percentage of cells in each quadrant. Similar results were observed in other GSI-sensitive cell lines. (C) Cell cycle analysis of the GSI-resistant Molt13 cell line after 7 d in DMSO (vehicle) or MRK-003. Similar results were observed for all other GSI-resistant cell lines listed in Table I. (D) Annexin V – APC/propidium iodide staining of the Molt4 cell line after 7 d in DMSO or 1  M MRK-003. Similar results were observed in all other GSI-resistant cell lines. (E) Activated NOTCH1 Western blot analysis. T-ALL cell lines were treated with 1  M MRK-003 for 3 d. Whole cell lysates were subjected to SDS- PAGE electrophoresis and immunoblotting with the NOTCH1 (V1744) antibody. acute myeloid leukemia (AML) samples, 60 primary myelo- residues critical for substrate binding ( Table II ). As in the dysplastic syndrome (MDS) samples, and 81 primary T- T-ALL cell lines, none of the primary samples with an ALL samples. Although the AML or MDS samples lacked FBW7 mutation had C-terminal truncating mutations in mutations in FBW7 , 7 of 81 (8.6%) of the T-ALL samples NOTCH1 . These results indicate that mutation of FBW7 harbored mutations in the substrate-binding domain of in primary T-ALL may represent an alternative mechanism FBW7. All of the mutations in the patient samples were of NOTCH deregulation that contributes to the pathogen- heterozygous missense mutations that altered arginine esis this disease. 1816 FBW7 MUTATION IN T-ALL | O ’ Neil et al. ARTICLE Table II. FBW7 mutations in primary T -ALL patient samples a b Sample NOTCH1 status FBW7 status HD domain PEST domain 2773 heterozygous mutation 1605 V → E WT heterozygous mutation 479 R → Q 2788 heterozygous mutation 1601 L → P WT heterozygous mutation 465 R → H 1179 WT WT heterozygous mutation 465 R → C 2748 heterozygous mutation 1586 L → P WT heterozygous mutation 479 R → Q 368 heterozygous mutation 1748 F → S WT heterozygous mutation 465 R → H 037 heterozygous mutation 1601 L → P WT heterozygous mutation 465 R → C 452 heterozygous mutation 1748 F → S WT heterozygous mutation 465 R → H The substrate binding encoding exons of FBW7 were sequenced in 81 primary T-ALL patient samples. Mutations were found in 7 out of 81 (8.6%) Samples with mutations are shown. Numbers correspond to amino acid residues in NOTCH1. The NOTCH1 T2512 region comprises mutation of another possible NOTCH1 CPD consensus mo- an FBW7 phosphodegron tif anchored around threonine 2133 (38) did not aff ect FBW7 As outlined above, loss of FBW7-mediated regulation of its binding (unpublished data). substrates can result from mutation of FBW7 itself or from mu- Because two putative CDK8 sites in the T2512 region tation of the substrate. The PEST domain of NICD is known have been previously implicated in NOTCH degradation by to regulate protein stability and is a hot spot for mutations FBW7 (S2514 and S2517), we also tested the role of these in primary human and mouse T-ALLs ( 7, 11 ). Furthermore, residues in FBW7 binding ( 26 ). We found that FBW7 bind- FBW7 binding to NOTCH is dependent on an intact PEST ing was disrupted by the S2514A mutation and that binding domain ( 27 ), strongly suggesting that the NOTCH CPD lies was restored when S2514 was replaced by a phosphomimetic within this region. Alignment of the human NOTCH1 PEST residue (S2514E) ( Fig. 2 D ). Although these data are consistent domain with other FBW7 substrates demonstrated a CPD con- with a role for S2514 phosphorylation in FBW7 binding, a sensus motif anchored around threonine 2512 (T2512) in hu- negative charge in the +2 position (relative to the central man NOTCH1. This residue is highly conserved across species threonine) is not a characteristic CPD feature. We thus mu- as well as across members of the NOTCH family ( Fig. 2 A ). tated S2514 to proline, which conforms to the CPD consen- Notably, T-ALL – associated NOTCH1 PEST mutations iden- sus, and found that S2514P also restored FBW7 binding. tifi ed in both human and mouse tumors occur upstream, or Thus, although a negative charge is tolerated in the +2 posi- very nearby, this putative CPD, suggesting that these mutations tion, it is not required for FBW7 binding. We also examined may disrupt its function ( 7, 11 ). the binding of the NICD to each of the three FBW7 isoforms, To determine if the T2512 region contains a functional which reside in distinct subcellular compartments ( 44 ). The NOTCH CPD, we examined its role in binding interactions results show that NICD binds to both FBW7  (nucleoplasmic) between the NICD and FBW7. For these studies, we used an and FBW7  (nucleolar), but not to FBW7  (cytoplasmic), expression plasmid encoding the mouse NICD, where T2487 supporting the idea that the NICD interacts with FBW7 in corresponds to human T2512 (human numbering is used to the nucleus ( Fig. 2 E ). refer to this CPD throughout this article). We fi rst mutated We next examined the role of T2512 in directing FBW7- T2512 to alanine (T2512A) and cotransfected 293 cells ( Fig. mediated endogenous NICD ubiquitination and, in accord 2 B ) or K562 erythroleukemia cells ( Fig. 2 C ) with FBW7 with our binding studies, found that the T2512A mutation and NICD. Because the interaction of FBW7 with its sub- markedly reduces NICD ubiquitination ( Fig. 2 F ). The ex- strates leads to ubiquitination and degradation by the protea- tent to which this mutation inhibited ubiquitination was some, we uncoupled substrate binding from turnover so that similar to that seen with a C-terminal PEST deletion, termed we could observe stable FBW7 – substrate interactions. To deltaCT, which is defective in ubiquitination by FBW7 ( 27 ). disrupt this pathway, we expressed two previously described The small amount of residual ubiquitination could refl ect ei- SCF mutants: (a) dominant-negative cullin-1 (dnCul1), which ther basal, T2512A-independent interactions of the NICD interacts with F-box proteins, but not ubiquitin-conjugating with FBW7, or could be due to alternative NICD ubiqui- enzymes ( 42 ), and (b) a truncated version of FBW7 (termed tination pathways. Finally, we used metabolic labeling and Fb-WD), which encodes the WD40 substrate binding do- pulse chase to compare the half-life of WT-NICD to the mains but lacks the N-terminal domains, including the F-box T2512A mutant in 293 cells. As predicted, the T2512A mu- motif, and thus is unable to associate with cullin-1 and with tant shows a prolonged half-life, refl ecting impaired degrada- the SCF ( 43 ). In both cases we found that the T2512A NICD tion in vivo ( Fig. 2 G ). Collectively, these data indicate that mutant showed markedly decreased binding to FBW7 when the T2512 region comprises a functional CPD that directs compared with WT-NICD ( Fig. 2, B and C ). In contrast, binding interactions between NOTCH and FBW7. JEM VOL. 204, August 6, 2007 1817 Figure 2. NOTCH T2512A mutant shows increased stability and decreased binding to FBW7. (A) Alignment of the four human NOTCH proteins, as well as Notch proteins from various species, shows strong conservation of the putative NOTCH CPD. Known CPDs are aligned with NOTCH for compari- son. (B) The T2512A NOTCH mutant is defi cient in binding to WT FBW7, and the tumor-derived FBW7 arginine mutant (R465C) can no longer bind WT NOTCH. Cells were transfected as indicated, and FLAG-FBW7 was immunoprecipitated. Samples were subjected to SDS-PAGE electrophoresis and immuno- blotting with anti-MYC tag (9E10) to detect transfected Notch proteins. Whole cell lysates were analyzed as indicated to verify expression of transfected constructs. (C) Co-immunoprecipitation assays in the K562 erythroleukemia cell line show that T2512A is defi cient in binding to FBW7 in hematopoietic cells. Whole cell lysates were analyzed as indicated to verify expression of transfected constructs. (D) Phosphorylation of S2514 is not required for the FBW7 – Notch ICD interaction. 293a cells were transfected as indicated and analyzed as in B above. (E) NICD preferentially associates with nuclear and nucleolar FBW7 isoforms. 293a cells were transfected with the indicated plasmids and analyzed as in B above. (F) In vivo ubiquitination assays show that the NOTCH T2512A mutant is resistant to FBW7-mediated ubiquitination. 293a cells were transfected as indicated. Cell lysates were prepared and immuno- precipitated with anti-HA antibody to pull down ubiquitinated proteins. Samples were subjected to SDS-PAGE electrophoresis and immunoblotted with 9E10 to detect MYC-tagged NOTCH proteins. Whole cell lysates were analyzed as indicated to verify expression of transfected constructs. (G) The NOTCH T2512A mutant has an extended half-life in 293a cells compared with WT NOTCH. 293a cells were transfected with either WT NICD or the T2512A mutant. 48 h later, cells were pulse labeled in vivo with S-methionine/cysteine and chased in medium with excess unlabeled methionine and cysteine for the indicated times. Transfected NOTCH proteins were immunoprecipitated with MN-1 antisera, and samples were subjected to SDS-PAGE electrophoresis. Gels were then exposed to x-ray fi lm. GSI treatment results in decreased MYC expression in a To determine if MYC protein levels are aff ected by treat- subset of T-ALL cell lines ment with MRK-003 in our panel of T-ALL cell lines, Recent studies have demonstrated that MYC is a direct tar- we performed Western blot analysis of lysates from a panel get of NOTCH in both human and mouse T-ALL ( 20 – 22 ). of 19 T-ALL cells that were either treated with vehicle 1818 FBW7 MUTATION IN T-ALL | O ’ Neil et al. ARTICLE Figure 3. GSI treatment results in decreased MYC protein levels in a subset of T-ALL cell lines. T-ALL cell lines were treated with 1  M MRK-003 or DMSO for 3 d, and whole cell lysates were prepared using RIPA lysis buffer. Western blot analysis was performed using antibodies against MYC and  -actin as a loading control. (DMSO) or 1  M MRK-003 for 3 d ( Fig. 3 ). A reduction We also examined if MYC mRNA levels responded to of MYC protein levels upon GSI treatment was apparent in GSI treatment by performing expression arrays on 18 of the 8 of the 19 lines (Koptk1, HPB-ALL, DND41, RPMI8402, T-ALL cell lines treated with DMSO or MRK-003 (1  M) Molt13, PF382, Supt7, and TALL1), including all of the for 3 d. We observed either a very modest eff ect or no eff ect GSI-sensitive lines tested (Koptk1, HPB-ALL, DND41, of GSI treatment on the levels of MYC RNA in four of the and TALL1). 4 of the other 10 lines (Loucy, Supt11, resistant lines that express NICD and have mutated FBW7 Supt13, and Molt16) lack activated NOTCH signaling (CEM, BE13, DU528, and HSB2) ( Fig. 5 ). Hence, the lack (i.e., NICD was not detectable by Western blot analysis; Fig. of down-regulation of MYC by GSI treatment in these lines 1 E and Table I ), and thus we would not expect GSI treat- is due to mechanisms acting upstream of MYC transcription ment to aff ect the expression of NOTCH target genes in rather than at the protein level. In the microarray expression these lines. The remaining seven cell lines (Jurkat, Molt4, analysis, other NOTCH target genes such as DELTEX1 and PEER, CEM, DU528, BE13, and HSB2) have activated SHQ1 (22) ( Fig. 5 and not depicted) were also not aff ected NOTCH signaling but do not display decreased MYC ex- by GSI treatment in the CEM, DU528, BE13, and HSB2 pression upon GSI treatment. Of note, all of the cell lines cell lines. In the PEER cell line, which also has a mutation in in which MYC expression is not decreased upon GSI treat- FBW7 , we detected residual expression of NICD after 3 d of ment are resistant to the drug, reinforcing the importance treatment with MRK-003, and quantitative RT-PCR showed of this NOTCH target gene for T-ALL cell growth. Because that MYC and DELTEX1 RNA expression levels did not over expression of MYC has been shown to rescue the decrease in this line upon GSI treatment ( Fig. 1 E and not growth-suppressive eff ects of GSIs ( 20, 21 ), lack of MYC depicted). This suggests that the FBW7 mutations (or homo- down-regulation in these T-ALL cell lines may contribute zygous deletion) found in these fi ve lines result in residual to their GSI resistance. signaling through NICD, even in the face of GSI treatment, Because MYC is also a FBW7 target, we examined MYC thus sustaining MYC transcription and promoting continued half-life in a subset of fi ve T-ALL cell lines (Molt4, DND41, cell proliferation. KOPTK1, CEM, and Jurkat) by pulse-chase analysis. Although MYC half-life was prolonged in each cell line compared with Tumor-derived FBW7 mutants are functionally impaired a B cell lymphoblastoid cell line (JY) ( Fig. 4 ), it was not in- and may act as dominant-negative mutants to prevent creased in cell lines harboring mutations in FBW7 (CEM and MYC degradation Jurkat) compared with those with WT FBW7 . This result was Because FBW7 is haploinsuffi cient for tumor suppression ( 46 ), not surprising because several lymphoblastic leukemia cell lines the single allele FBW7 mutations most commonly found in and patient samples have been shown to have aberrantly stabi- T-ALL could lead to stabilization of its substrates simply by lized MYC ( 45 ). Thus, MYC may be stabilized in T-ALL cell reducing FBW7 activity by 50%. However, if the selective pres- lines without FBW7 mutations due to mutations in other sure for these mutations consisted simply of loss-of-function components of the MYC degradation pathway. Furthermore, of one allele, we would predict that we would fi nd nonsense in some contexts, FBW7 loss may lead to MYC stabilization in mutations in the FBW7 gene ( 36, 47 ). Our fi nding of muta- subcellular compartments (e.g., the nucleolus) without grossly tional hot spots aff ecting the three key arginine residues of altering bulk MYC turnover ( 44 ). FBW7 argues against this possibility. Instead, we propose that JEM VOL. 204, August 6, 2007 1819 Figure 4. T-ALL cell lines have increased MYC half-life. (A) T-ALL cell lines with WT FBW7 (Molt4, DND41 and KOPTK1) were pulse-labeled in vivo with S-methionine/cysteine and chased in medium with excess unlabeled methionine and cysteine for the indicated times. Endogenous MYC was immunoprecipitated from an equal number of cells for each time point and analyzed by gel electrophoresis. S-labeled MYC from each sample was quantitated by phosphoimager. The rate of degradation of MYC for each cell line is represented in the graph by best-fi t exponential lines. Half-lives of MYC were calculated from exponential line equations. (B) Pulse-chase experiments were similarly performed on T-ALL cell lines with mutant FBW7 (CEM and Jurkat). All T-ALL cell lines analyzed have increased MYC half-life compared with JY cells, a B cell lymphoblastoid cell line. Figure 5. NOTCH target genes are not decreased upon GSI treatment in T-ALL cell lines with mutant FBW7 . MYC and DELTEX RNA levels in 18 T-ALL cell lines after 3 d of 1  M MRK-003 GSI treatment (compared with DMSO-treated cells) as determined by microarray gene expression analysis. Values are a log ratio of the expression level of MYC and DEL TEX in MRK-003 – treated cells compared with DMSO-treated cells. 1820 FBW7 MUTATION IN T-ALL | O ’ Neil et al. ARTICLE the restricted mutational spectrum indicates a strong selection were unable to promote MYC degradation ( Fig. 6 B ). We for expression of an FBW7 protein that is defective for pro- also examined whether a T-ALL – derived FBW7 mutant can ductive substrate interactions and/or turnover, which sug- dominantly interfere with FBW7-driven MYC degradation gests activity as a dominant-negative allele. by expressing either increasing amounts of FBW7-  or a To explore this possibility, we analyzed each of the three mixture of a fi xed amount of FBW7-  in combination with mutant FBW7 proteins identifi ed in T-ALL ( Tables I and II ) increasing amounts of FBW7-  -R465C. This analysis sug- for their ability to bind to both MYC and NOTCH. As ex- gests that the mutant impairs FBW7-driven MYC degrada- pected, the mutants were defective in NOTCH binding, al- tion in a dose-dependent manner ( Fig. 6 C ). though FBW7R479Q showed a modest amount of residual At least two mechanisms may contribute to the dominant- binding ( Fig. 6 A , right). In contrast, each of the mutant negative activities of the mutant FBW7 proteins. The fi rst FBW7 proteins bound to MYC, consistent with our recent is suggested by our fi nding that the FBW7 mutants still in- fi nding that in addition to its canonical interaction with the teract with Myc through a binding interaction independent MYC threonine 58 (T58) CPD, full-length FBW7 also binds of the MYC CPD that cannot promote MYC degradation. to MYC through a second interaction that is independent of Hence, in the case of MYC, they may simply compete with the T58 CPD ( Fig. 6 A , left, and unpublished data). Impor- WT FBW7 for MYC binding. However, a second mecha- tantly, although these FBW7 mutants bound to MYC, they nism of dominant-negative function is suggested by recent fi ndings that FBW7 (and its orthologs) form dimers ( 48 – 51 ), thus raising the possibility that WT and mutant FBW7 pro- teins form heterodimeric dominant-negative complexes that are unable to degrade MYC. However, because mutation of the FBW7 dimerization region also disrupts its noncanonical binding interaction with MYC, we cannot currently distin- guish between these two possibilities (unpublished data). We thus propose that FBW7 mutations may contribute to T-ALL pathogenesis and GSI resistance by leading to the stabilization of MYC as well as NICD. DISCUSSION The early successes of molecularly targeted therapy, such as imatinib mesylate directed against the BCR-ABL fusion pro- tein in chronic myelogenous leukemia, stimulated enormous interest in subverting abnormal signaling pathways in cancer cells. The NOTCH signaling cascade infl uences normal devel- opment by regulating diff erentiation, proliferation, and apop- tosis ( 52 ). Activation of the NOTCH signaling pathway is fi rmly established in T-ALL and is likely involved in the gene- Figure 6. Mutant FBW7 cannot bind to NICD and acts in a sis of many other tumor types ( 53 ). Thus, it is not surprising dominant-negative manner to prevent MYC degradation. (A) FBW7 arginine point mutants effi ciently co-precipitate with MYC but not with that eff orts to block NOTCH signaling as a novel therapeutic NOTCH. 293a cells were transfected with the indicated plasmids. Whole strategy are under way in T-ALL as well as in solid tumors. cell lysates were subjected to immunoprecipitation with anti-FLAG, and One of the most promising approaches has been to inhibit resulting samples were analyzed via immunoblotting for either MT- NOTCH receptor signaling using GSIs. This strategy sup- NOTCH or HA-MYC as indicated. Whole cell lysates were also analyzed presses the generation of NICD and thus, in principle, should directly by SDS-PAGE and immunoblotting for FLAG-FBW7, HA-MYC, or inhibit the downstream transcriptional events normally in- MT-NOTCH as indicated to verify expression of transfected constructs. duced by this key signaling component after it traverses to the dnCul1 was included in all transfections to block FBW7-mediated pro- nucleus. Although eff ective against some T-ALL cell lines, teasomal degradation. (B) T-ALL – associated FBW7 mutants are unable GSIs do not uniformly eliminate leukemic cells with activated to mediate MYC degradation. 293a cells were transfected as indicated. Whole cell lysates were subjected to SDS-PAGE and immunoblotting for NOTCH signaling. Understanding the mechanisms of GSI either HA-MYC or FLAG-FBW7 as indicated. (C) Tumor-derived FBW7 resistance may lead to better treatments for T-ALL. mutants dominantly inhibit MYC turnover by WT-FBW7. 293a cells We found missense FBW7 mutations or homozygous were transfected with a constant amount of HA-MYC with increasing FBW7 deletion in GSI-resistant T-ALL cell lines and in pri- amounts of either FLAG-FBW7-  or a combination of a constant amount mary T-ALL samples. Moreover, we have defi ned the NOTCH (500 ng) of FLAG-FBW7-  and increasing amounts of FLAG-FBW7-  - phosphodegron and demonstrated that the mutant forms of R465H (1 – 5  g). The ratios of mutant to WT FBW7 expression are indi- FBW7 found in T-ALL cannot bind to the NICD. Each T- cated above lanes 7 – 9. HA-cdk2, which does not affect turnover of MYC ALL cell line with constitutive NICD expression harbored by FBW7, is included as a transfection control. Whole cell lysates were either NOTCH1 PEST domain or FBW7 mutations, suggesting subjected to SDS-PAGE and immunoblotting for HA-MYC, HA-cdk2, or FLAG-FBW7 as indicated. that these two classes of mutations provide a mutually exclusive JEM VOL. 204, August 6, 2007 1821 sent and Institutional Review Board approval was obtained to use leftover means of prolonging the NICD half-life. We also show that patient material for research purposes. the expression of NOTCH target genes including DELTEX1 and MYC are not aff ected by GSI treatment in fi ve resistant Cell lines. T-ALL cell lines were obtained from American Type Culture T-ALL cell lines with mutations in FBW7 (CEM, BE13, PEER, Collection or Deutsche Sammlung von Mikroorganismen und Zellkulturen DU528, and HSB2), demonstrating that the mechanism GmbH. 293a cells were provided by S. Reed (The Scripps Research Insti- of resistance in these leukemias lies upstream of MYC and tute, La Jolla, CA). DELTEX1 transcription. It appears that in these T-ALL lines, Treatment of T-ALL cell lines with MRK-003. T-ALL cell lines were one consequence of FBW7 mutation is stabilization of the 4 5 plated at a density of 5  10 – 1.5  10 cells per ml (depending on dou- NICD resulting in sustained NOTCH signaling, and thus pro- bling time) and were treated with either DMSO or 1  M MRK-003 for moting resistance to GSIs. However, other FBW7 substrates, 0 h, 6 h, 24 h, 3 d, or 7 d. For each time point, triplicate fl asks were such as MYC, may also account for selection for FBW7 muta- analyzed by the trypan blue exclusion assay to determine cell viability. tions and for GSI resistance in T-ALL, as suggested by our For propidium iodide staining to analyze the cell cycle profi le, cells were fi nding that ALL-associated FBW7 mutations can dominantly washed with PBS, stained in a PBS buff er containing 50  g/ml propidium iodide and 200  g/ml RNase A, and analyzed on a Becton Dickinson inhibit MYC degradation. FACSCalibur Instrument. For annexin V staining to determine the per- An important question is why FBW7 mutation confers centage of cells undergoing apoptosis, the annexin V – FITC (or annexin GSI resistance, whereas NOTCH PEST domain truncations V – APC) Apoptosis detection kit I (BD Biosciences) was used according to that remove the FBW7 interaction domain do not. In each the manufacturer ’ s instructions. cell line with PEST mutations, only one allele is aff ected by these heterozygous mutations, leaving the remaining normal Microarray gene expression analysis. Total RNA isolated from cultured cells was used to make fl uorescently labeled cRNA that was hybridized to allele, which encodes an NICD that is stabilized in the pres- DNA oligonucleotide microarrays as described previously ( 55, 56 ). Human ence of mutant FBW7. Thus, although the amount of in- microarrays contained oligonucleotide probes corresponding to  21,000 creased NOTCH activity resulting from the single allele genes. All oligonucleotide probes on the microarrays were synthesized in situ PEST mutations may be suffi cient to underlie the primary with inkjet technology (Agilent Technologies) ( 55 ). After hybridization, selection for these mutations, disruption of FBW7 function arrays were scanned and fl uorescence intensities for each probe were recorded. may be more active in sustaining NOTCH signaling and also Ratios of transcript abundance (experimental to control) were obtained after normalization and correction of the array intensity data. Gene expression may prolong the half-life of MYC (as well as other substrates), data were analyzed with the Rosetta Resolver gene expression analysis soft- thus promoting GSI drug resistance. ware (version 6.0; Rosetta Biosoftware). Microarray data have been depos- The association between FBW7 mutations and resistance ited in the GEO database under accession number GSE8416. to GSIs has implications for clinical testing of these agents in patients whose cancers show deregulation of the NOTCH Western blotting. For analysis of endogenous protein expression, T-ALL pathway. Molecular analysis of the FBW7 gene, as well as cell lines were treated with MRK-003 for 3 d and lysed in RIPA buff er. genes encoding the relevant NOTCH receptors and other Blots were probed with 9E10 anti-MYC antibody (1:500; Santa Cruz Bio- technology, Inc.), the cleaved NOTCH1 (Val1744) antibody (1:1,000; Cell key components of the NOTCH signaling pathway, such as Signaling Technology) as described previously ( 24 ), and anti –  -actin NUMB, may contribute to identifi cation of patients likely to (1:7,000; Abcam) for the loading control. Signal was detected with Super- be most responsive to GSI therapy. Because Myc has recently Signal West Femto ECL reagent (Pierce Chemical Co.). For transfections, been shown to be an important target of Notch in mammary MYC-tagged proteins were detected with 9E10 hybridoma supernatant tumorigenesis as well as in T-ALL ( 5 ), it might also be possi- (1:4), HA-tagged proteins were detected with HA-11 (1:1,000), and FLAG- tagged proteins were detected with M2 anti-FLAG (1:4,000; Sigma-Aldrich). ble to overcome GSI resistance by combining GSIs with other These same antibodies were conjugated to protein G sepharose and used for drugs that block the MYC pathway to synergistically reduce immunoprecipitations as described previously ( 57 ). MYC levels and block tumor cell growth. One attractive candidate is TMPyP4, a cationic porphyrin that binds to and Mutation detection. The entire coding region of FBW7 was sequenced in stabilizes guanine quadruplexes in DNA. MYC contains a se- genomic DNA isolated from the 20 T-ALL cell lines. Exons 7, 8, 9, 10, and quence in its promoter that forms a guanine quadruplex, and 11 of FBW7 were sequenced in primary patient samples. All sequencing was performed at Agencourt Bioscience Corporation. Residues are numbered TMPyP4 has been shown to inhibit MYC transcription and according to their location in the  isoform of FBW7 (GenBank RefSeq the growth of tumor cells in vivo ( 54 ). Thus, TMPyP4 or NM_033632). other agents that inhibit MYC transcription may be useful in combination with GSIs to overcome resistance in patients Plasmids and transient transfections. The following expression plasmids harboring FBW7 mutations. Pharmacologic or genetic strat- have been described (43, 44): pFLAG-FBW7-  , -  , and -  ; pFLAG-FbWD; egies that restore the normal function of FBW7 in tumor cells pFLAG-FBW7-  -R465H (also termed R298H); and HA-MYC, HA-dncul1, could also be therapeutically useful. and HA-ubiquitin. All mutagenesis were performed by the QuickChange method (Stratagene) and confi rmed by sequencing. Point mutations in the FBW7 common region R465H, R479Q, and R505C were generated by MATERIALS AND METHODS site-directed mutagenesis using pFLAG-FBW7 vectors as template. Although Patient samples. Bone marrow or peripheral blood samples were collected numbering is diff erent between isoforms, the  numbering is used for simplic- at diagnosis from T-ALL patients at the Sophia Children ’ s Hospital/Erasmus ity. CS2-mNICD-MT was provided by R. Kopan (Washington University, MC and the Dutch Childhood Oncology Group, as well as from AML, St. Louis, MO). It encodes the mouse Notch1 intracellular domain from MDS, and T-ALL patients at Dana-Farber Cancer Institute. Informed con- Val1744 to the end of the WT protein and includes six in-frame MYC epitope 1822 FBW7 MUTATION IN T-ALL | O ’ Neil et al. ARTICLE tags at the C terminus. Mutagenesis of this construct was used to generate the 6 . Pear , W.S. , J.C. Aster , M.L. Scott , R.P. Hasserjian , B. Soff er , J. Sklar , T2512A, S2514A, S2514E, and S2514P mutants. The deltaCT mutation, and D. Baltimore . 1996 . Exclusive development of T cell neoplasms in mice transplanted with bone marrow expressing activated Notch alleles. generated by PCR, deletes the PEST domain but maintains the C-terminal J. Exp. Med. 183 : 2283 – 2291 . MYC tags. 293A cells were transfected by the calcium phosphate precipita- 7 . O ’ Neil , J. , J. Calvo , K. McKenna , V. Krishnamoorthy , J.C. Aster , tion method as described previously ( 57 ). Cells were harvested at 36– 48 h C.H. Bassing , F.W. Alt , M. Kelliher , and A.T. Look . 2006 . Activating after transfection using TENT buff er (50 MM Tris-Cl, pH 8.0, 2 mM EDTA, Notch1 mutations in mouse models of T-ALL. Blood . 107 : 781 – 785 . 150 mM NaCl, 1% Triton X-100) ( 30 ) with protease and phosphatase inhib- 8 . Mantha , S. , M. Ward , J. McCaff erty , A. Herron , T. Palomero , A. itors and analyzed by immunoprecipitation and Western blotting as described Ferrando , A. Bank , and C. Richardson . 2007 . Activating Notch1 mu- previously ( 57 ). tations are an early event in T-cell malignancy of Ikaros point mutant Plastic/+ mice. Leuk. Res. 31 : 321 – 327 . Metabolic labeling and pulse chase. NICD pulse-chase assays were per- 9 . Lin , Y.W. , R.A. Nichols , J.J. Letterio , and P.D. Aplan . 2006 . Notch1 formed as described previously ( 43 ). In brief, 293a cells were transfected mutations are important for leukemic transformation in murine models with plasmids encoding MYC-tagged NICD or NICD T2512A. At 36 h af- of precursor-T leukemia/lymphoma. Blood . 107 : 2540 – 2543 . ter transfection, cells were preincubated with media lacking cysteine and 10 . Ellisen , L.W. , J. Bird , D.C. West , A.L. Soreng , T.C. Reynolds , methionine, pulsed with media containing Trans 35S label, and chased with S.D. Smith , and J. Sklar . 1991 . TAN-1, the human homolog of the media with cold methionine. Samples were obtained at the indicated time Drosophila notch gene, is broken by chromosomal translocations in points and immunoprecipitated with anti-Notch serum MN1, a mouse T lymphoblastic neoplasms. Cell . 66 : 649 – 661 . 11 . Weng , A.P. , A.A. Ferrando , W. Lee , J.P. Morris , L.B. Silverman , C. monoclonal antibody recognizing the NICD (provided by I. Bernstein, Fred Sanchez-Irizarry , S.C. Blacklow , A.T. Look , and J.C. Aster . 2004 . Hutchinson Cancer Research Center, Seattle, WA). MYC pulse-chase ex- Activating mutations of NOTCH1 in human T cell acute lymphoblastic periments were performed as described previously ( 45 ). leukemia. Science . 306 : 269 – 271 . 12 . Park , J.T. , M. Li , K. Nakayama , T.L. Mao , B. Davidson , Z. Zhang , R.J. Ubiquitination assays. Cell-based ubiquitination assays were performed as Kurman , C.G. Eberhart , M. Shih Ie , and T.L. Wang . 2006 . Notch3 described previously ( 58 ). In brief, transiently transfected 293a cells were gene amplifi cation in ovarian cancer. Cancer Res. 66 : 6312 – 6318 . harvested at 48 h after transfection. Cells were trypsinized, rinsed once with 13 . Pece , S. , M. Serresi , E. Santolini , M. Capra , E. Hulleman , V. Galimberti , PBS, and lysed in 100  l 2% SDS in TBS (10 mM Tris-HCl, pH 8.0, 150 S. Zurrida , P. Maisonneuve , G. Viale , and P.P. Di Fiore . 2004 . Loss of mM NaCl) with boiling for 10 min. These samples were diluted at 1:10 with negative regulation by Numb over Notch is relevant to human breast 1% triton in TBS containing protease inhibitors, 5 mM N-ethymaleimide, carcinogenesis. J. Cell Biol. 167 : 215 – 221 . and 5 mM MG132, sonicated, precleared with protein G sepharose, and 14 . Jundt , F. , I. Anagnostopoulos , R. Forster , S. Mathas , H. Stein , and B. centrifuged to pellet-insoluble debris. Aliquots were removed for Western Dorken . 2002 . Activated Notch1 signaling promotes tumor cell pro- blot analysis, and the remaining lysates were immunoprecipitated with anti- liferation and survival in Hodgkin and anaplastic large cell lymphoma. HA antisera prebound to protein G sepharose. Samples were analyzed by Blood . 99 : 3398 – 3403 . SDS-PAGE and probed with 9E10 to visualize MYC-tagged ubiquitinated 15 . Balint , K. , M. Xiao , C.C. Pinnix , A. Soma , I. Veres , I. Juhasz , E.J. Notch proteins. Brown , A.J. Capobianco , M. Herlyn , and Z.J. Liu . 2005 . Activation of Notch1 signaling is required for beta-catenin-mediated human primary melanoma progression. J. Clin. Invest. 115 : 3166 – 3176 . The authors wish to thank Theresa Zhang, Leslie Carlini, and Xudong Dai for helpful 16 . Purow , B.W. , R.M. Haque , M.W. Noel , Q. Su , M.J. Burdick , J. Lee , T. discussions regarding mRNA profi ling; Hellen Kim for discussions about Western Sundaresan , S. Pastorino , J.K. Park , I. Mikolaenko , et al . 2005 . Expression blotting methods; Keith McKenna and Chitra Raghunathan for technical assistance; of Notch-1 and its ligands, Delta-like-1 and Jagged-1, is critical for glioma and John Gilbert for editorial review. cell survival and proliferation. Cancer Res. 65 : 2353 – 2363 . This work was funded by a sponsored research agreement from Merck Research 17 . Haruki , N. , K.S. Kawaguchi , S. Eichenberger , P.P. Massion , S. Olson , Laboratories (to A.T. Look), an American Society of Hematology Fellow Scholar A. Gonzalez , D.P. Carbone , and T.P. Dang . 2005 . Dominant-negative Award and by the National Institutes of Health (1K08CA109124-01A2; to J. Grim), Notch3 receptor inhibits mitogen-activated protein kinase pathway and National Institutes of Health grants (R01CA84069 and R01CA102742) and the the growth of human lung cancers. Cancer Res. 65 : 3555 – 3561 . Burroughs Welcome Foundation (to B.E. Clurman), as well as a Harvard Medical 18 . Collins , B.J. , W. Kleeberger , and D.W. Ball . 2004 . Notch in lung de- School Hematology Training Grant (to J. O ’ Neil). velopment and lung cancer. Semin. Cancer Biol. 14 : 357 – 364 . The authors have no confl icting fi nancial interests. 19 . Santagata , S. , F. Demichelis , A. Riva , S. Varambally , M.D. Hofer , J.L. Kutok , R. Kim , J. Tang , J.E. Montie , A.M. Chinnaiyan , et al . 2004 . Submitted: 2 May 2007 JAGGED1 expression is associated with prostate cancer metastasis and Accepted: 28 June 2007 recurrence. Cancer Res. 64 : 6854 – 6857 . 20 . Weng , A.P. , J.M. Millholland , Y. Yashiro-Ohtani , M.L. Arcangeli , A. REFERENCES Lau , C. Wai , C. Del Bianco , C.G. Rodriguez , H. Sai , J. Tobias , et al . 1 . Wilson , A. , and F. Radtke . 2006 . Multiple functions of Notch signaling 2006 . c-Myc is an important direct target of Notch1 in T-cell acute in self-renewing organs and cancer. FEBS Lett. 580 : 2860 – 2868 . lymphoblastic leukemia/lymphoma. Genes Dev. 20 : 2096 – 2109 . 2 . Gallahan , D. , and R. 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FBW7 mutations in leukemic cells mediate NOTCH pathway activation and resistance to γ-secretase inhibitors

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Pubmed Central
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Copyright © 2007, The Rockefeller University Press
ISSN
0022-1007
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1540-9538
DOI
10.1084/jem.20070876
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Abstract

ARTICLE FBW7 mutations in leukemic cells mediate NOTCH pathway activation and resistance to  -secretase inhibitors 1 2 3 3 Jennifer O ’ Neil, Jonathan Grim, Peter Strack, Sudhir Rao, 4 3 5 2 Deanne Tibbitts, Christopher Winter, James Hardwick, Markus Welcker, 6 6 3 4 Jules P. Meijerink, Rob Pieters, Giulio Draetta, Rosalie Sears, 2 1,7 Bruce E. Clurman, and A. Thomas Look Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA 02115 Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, WA 98109 Merck Research Laboratories, Boston, MA 02115 Department of Molecular and Medical Genetics, Oregon Health and Sciences University, Portland, OR 97239 Merck Research Laboratories, West Point, PA 19486 Department of Pediatric Oncology/Hematology, Erasmus MC/Sophia Children ’ s Hospital, 3000 CB Rotterdam, Netherlands Division of Hematology, Children ’ s Hospital Boston, Boston, MA 02115 -secretase inhibitors (GSIs) can block NOTCH receptor signaling in vitro and therefore offer an attractive targeted therapy for tumors dependent on deregulated NOTCH activity. To clarify the basis for GSI resistance in T cell acute lymphoblastic leukemia (T-ALL), we studied T-ALL cell lines with constitutive expression of the NOTCH intracellular domain (NICD), but that lacked C-terminal truncating mutations in NOTCH1 . Each of the seven cell lines examined and 7 of 81 (8.6%) primary T-ALL samples harbored either a mutation or homozygous deletion of the gene FBW7 , a ubiquitin ligase implicated in NICD turnover. Indeed, we show that FBW7 mutants cannot bind to the NICD and defi ne the phosphode- gron region of the NICD required for FBW7 binding. Although the mutant forms of FBW7 were still able to bind to MYC, they do not target it for degradation, suggesting that stabilization of both NICD and its principle downstream target, MYC, may contribute to transformation in leukemias with FBW7 mutations. In addition, we show that all seven leukemic cell lines with FBW7 mutations were resistant to the MRK-003 GSI. Most of these resistant lines also failed to down-regulate the mRNA levels of the NOTCH targets MYC and DELTEX1 after treatment with MRK-003, implying that residual NOTCH signaling in T-ALLs with FBW7 mutations contributes to GSI resistance. The mammalian NOTCH proteins are het- that fuses the intracellular form of NOTCH1 CORRESPONDENCE A. Thomas Look: erodimeric transmembrane receptors that con- to the T cell receptor  locus in lymphoblasts Thomas_Look@dfci.harvard.edu trol cell proliferation, apoptosis, and cell fate of T-ALL patients ( 10 ). Recently, 50% of hu- OR during the development of diverse cellular lin- man T-ALL cell lines and primary patient sam- Bruce E. Clurman: bclurman@fhcrc.org eages ( 1 ). Aberrant NOTCH signaling has been ples were shown to harbor activating mutations extensively linked to cancer and development. in NOTCH1 that result in aberrant NOTCH Abbreviations used: AML, acute In mouse models, constitutive NOTCH signal- signaling ( 11 ). Although mutations that di- myeloid leukemia; CPD, Cdc ing contributes to the genesis of breast cancer, rectly activate NOTCH receptors have not phosphodegron; GSI,  -secretase inhibitor; MDS, myelodysplastic medulloblastoma, and T cell leukemia ( 2 – 9 ), been identifi ed in other types of human can- syndrome; NICD, NOTCH whereas in human cancer its role is best exem- cers, there is abundant evidence to support the intracellular domain; T-ALL, plifi ed by T cell acute lymphoblastic leukemias importance of deregulated NOTCH activity in T cell acute lymphoblastic leukemia. (T-ALLs). Human NOTCH1 was fi rst discov- the development of ovarian cancer ( 12 ), breast ered as a gene activated at the breakpoint of the cancer ( 13 ), anaplastic large cell lymphoma and t(7;9), a very rare chromosomal translocation Hodgkin disease ( 14 ), melanoma ( 15 ), gliomas ( 16 ), lung carcinomas ( 17, 18 ), and cancers of the pancreas ( 18 ) and prostate ( 19 ). Hence, J. O ’ Neil and J. Grim, and B.E. Clurman and A.T. Look contributed equally to this work. modulation of the NOTCH signaling cascade JEM © The Rockefeller University Press $15.00 Vol. 204, No. 8, August 6, 2007 1813-1824 www.jem.org/cgi/doi/ 10.1084/jem.20070876 The Journal of Experimental Medicine at one or more points could short-circuit this pathway in fi ve GSI-sensitive cell lines, indicating that MRK-003 acts by NOTCH-supported tumors, leading to clinically important inducing apoptosis as well as by blocking cell growth ( Fig. 1 B ). antitumor eff ects. Importantly, the altered proliferation, cell cycle arrest, and Blocking the intramembranous cleavage of NOTCH is increase in apoptosis could be rescued by overexpressing the an especially attractive strategy of targeted therapy. When the NICD, demonstrating that these eff ects indeed result from NOTCH receptor is recognized by its membrane-bound inhibition of the NOTCH signaling pathway ( 24 and unpub- ligand, a conformational change exposes the receptor to lished data). The cell counts, cell cycle profi les, and percent- sequential rounds of protease cleavage. Binding of the ligand ages of apoptotic cells in the remaining 15 T-ALL cell lines results in proteolytic cleavage of the receptor, fi rst outside the were not aff ected by treatment with MRK-003 ( Fig. 1, cell by TNF-  – converting enzyme and then by the  -secretase C and D, and Table I ). membrane protease complex, releasing the NOTCH intra- cellular domain (NICD), which translocates to the nucleus NICD levels are high in several T-ALL cell lines without where it regulates the expression of its target genes, including truncating mutations in NOTCH1 MYC and DELTEX1 ( 20 – 23 ). Small molecule inhibitors of Treatment with MRK-003 led to reduced levels of NICD in -secretase activity are now available that eff ectively inhibit each of the T-ALL cell lines in which the intracellular pro- NOTCH signaling in vitro. One commercial product, com- tein could be detected, indicating that the compound eff ec- pound E, induces growth arrest in several diff erent T-ALL tively inhibited NOTCH1 cleavage at the cell surface ( Fig. 1 E ). cell lines by inhibiting the NOTCH pathway ( 11 ). Recently, However, only a subset of the 20 T-ALL cell lines had a GSI- we showed that treatment of T-ALL cells with the MRK-003 sensitive cellular phenotype, indicating that a resistant pheno- -secretase inhibitor (GSI) results in prolonged cell cycle type can be observed with or without evidence for constitutive arrest followed by apoptosis ( 24 ). NOTCH signaling (i.e., without detectable expression of Despite the promise of GSI therapy for tumors driven by NICD; Table I and Fig. 1 E ). Western blot analysis also dem- aberrant NOTCH signaling, most human T-ALL cell lines onstrated that all of the cell lines with C-terminal truncating are resistant to these agents and grow normally despite GSI mutations of NOTCH1 expressed high levels of NICD treatment. Thus, to establish the molecular basis of GSI resis- ( Table I and Fig. 1 E ), refl ecting aberrant activation of the tance in tumor cells, we used T-ALL cell lines as a model NOTCH pathway. Interestingly, there were seven cell lines system to test the ability of GSI treatment to reduce cellular without C-terminal truncating mutations in NOTCH1 that levels of NICD, as well as its transcriptional targets MYC and also expressed high levels of NICD (RPMI8402, CEM, DELTEX1 . We found that mutations aff ecting the FBW7 BE13, DU528, HSB2, Jurkat, and PEER), implying that ubiquitin ligase occur in GSI-resistant cell lines and are asso- NOTCH is stabilized in these cell lines through a mechanism ciated with sustained NOTCH signaling. Moreover, we other than mutation of NOTCH1 . found FBW7 mutations in primary T-ALL samples, and the mutational spectrum suggests that they produce dominant- FBW7 is mutated in T-ALL cell lines and primary samples negative FBW7 alleles. Our fi ndings implicate FBW7 muta- Cellular levels of NICD are determined by the net eff ects of tions in both the pathogenesis of T-ALL and leukemic cell the rates of production and destruction of the protein. The resistance to GSIs. SCF-FBW7 ubiquitin ligase plays a critical role in NICD degradation that is dependent on an intact PEST domain of RESULTS NOTCH ( 25 – 30 ). Other known FBW7 substrates include MRK-003 treatment leads to Notch-dependent cyclin E, MYC, SR-EBP, and c-Jun ( 31 ). FBW7 interacts antiproliferative effects in a subset of T-ALL cell lines with its substrates via the  propeller formed by its WD40 To inhibit NOTCH-mediated signal transduction, we treated repeats after the substrate becomes phosphorylated within each of 20 T-ALL cell lines with the Merck GSI MRK-003 highly conserved Cdc phosphodegrons (CPDs) ( 32, 33 ). Human (24) at 1  M or with DMSO (vehicle control) for 7 d. To tumors harbor nonsense mutations that truncate FBW7 as study the eff ects of GSI treatment, we analyzed the cell well as missense mutations that target key arginine residues in counts, cell cycle profi les, and apoptosis at various time points the WD40 repeats. These mutations disable FBW7 – substrate after treatment. We did not observe any eff ects on prolifera- interactions and impair substrate degradation by FBW7 ( 34 – 37 ). tion or viability at 6 h, 24 h, or 3 d in any of the 20 lines. Five Mutations aff ecting CPDs in tumor-derived alleles of both of the T-ALL cell lines (DND41, Koptk1, ALL-SIL, HPB- MYC and JUN have been described ( 38 – 41 ). Thus, muta- ALL, and TALL1) were sensitive to GSI treatment, exhibit- tions removing or altering the substrate CPD can also lead to ing two- to threefold fewer live cells in the GSI-treated fl asks loss of regulation by FBW7. Because the PEST domain trun- compared with DMSO-treated fl asks on day 7 of treatment cations found in human T-ALL are predicted to disrupt ( Table I ). Cell cycle analysis at that time revealed a G /G cell FBW7 binding to NICD, we hypothesized that FBW7 mu- 0 1 cycle arrest with decreased cells in the S phase in each of the tation could represent an alternative mechanism for NOTCH fi ve lines (representative histograms are shown for one line in deregulation in human cancers. Fig. 1 A ). Annexin V staining showed a two- to fourfold in- To test this hypothesis, we sequenced the entire coding crease in the percentage of cells undergoing apoptosis in the region of FBW7 to determine whether mutations aff ecting 1814 FBW7 MUTATION IN T-ALL | O ’ Neil et al. ARTICLE Table I. FBW7 mutations in human T -ALL cell lines NOTCH1 status HD domain PEST domain NICD detected FBW7 status Sensitive Cell Lines DND41 heterozygous mutation heterozygous insertion 2444 CCSHWAPAAWRCTLFCPRRAPPCP Yes WT 1594 L → P RRCHPRWSHP*STOP HPB-ALL heterozygous mutation heterozygous insertion 2442 Yes WT 1575 L → P EGRGRCSHWAPAAWRCTLFCPRRAPPCPRRCHPRWSHP*STOP Koptk1 heterozygous mutation heterozygous deletion 2515 RVP*STOP Yes WT 1601 L → P ALL-SIL heterozygous mutation heterozygous insertion 2476 AHP*STOP Yes WT 1594 L → P TALL1 WT WT No WT Resistant Cell Lines RPMI8402 heterozygous insertion WT Yes homozygous 1584 PVELMPPE mutation 465 R → H CEM heterozygous insertion WT Yes heterozygous 1595 PRLPHNSSFHFL mutation 465 R → H BE13 homozygous mutation WT Yes homozygous 1601 L → P deletion DU528 WT WT Yes heterozygous mutation 465 R → H HSB2 WT WT Yes heterozygous mutation 505 R → C Jurkat WT WT Yes heterozygous mutation 505 R → C PF382 heterozygous mutation heterozygous insertion 2494 ASCILWTTPPATSYRCLSTPSSPRP Yes WT 1575 L → P LSPLTSGPSRPRIPTSPTGPRASPALPPACSPRSPAFRRPSSKRRAPRDP GFLSQAFGRLCALCGCQGRPEEPF*STOP Molt13 heterozygous mutation heterozygous deletion 2515 *STOP Yes WT 1601 L → P Supt7 heterozygous insertion heterozygous insertion 2429 Yes WT 1593 F → LGA AHKTYRCSSRTCSQQTSSSSKACSRHHHHHSR TLA*STOP Molt4 heterozygous mutation heterozygous deletion 2515 RVP*stop Yes WT 1601 L → P PEER WT WT Yes heterozygous mutation 505 R → C Loucy WT WT No WT Supt11 WT WT No WT Supt13 WT WT No WT Molt16 WT WT No WT Numbers correspond to amino acid residues in NOTCH1. As determined by western blot analysis. this ubiquitin ligase gene might account for the accumulation lines with increased levels of NICD but without C-terminal of NICD in T-ALL cell lines that lacked truncations of the truncating mutations in NOTCH1 (RPMI8402, CEM, NOTCH1 PEST degradation domain. Sequencing of the BE13, DU528, HSB2, Jurkat, and PEER) harbored either a entire coding region of FBW7 in 20 T-ALL cell lines re- mutation in FBW7 or homozygous deletion of the gene, thus vealed mutations in 6 of the 20 lines (30%) ( Table I ). The providing an explanation for persistent NICD expression in GSI-resistant cell line RPMI8402 harbored a homozygous these lines ( Fig. 1 E ). Conversely, none of the cell lines with arginine-to-histidine mutation at residue 465; all of the other FBW7 mutations had PEST domain mutations in NOTCH1 , mutations were heterozygous missense mutations of critical suggesting that there is no selective pressure for FBW7 muta- WD40 arginine residues (465 and 505). One additional cell tion in cells with stabilized NOTCH1. line (BE13) has a homozygous deletion of FBW7 as deter- We also sequenced exons encoding the substrate-binding mined by quantitative DNA PCR. Each of the seven cell domain (exons 7, 8, 9, 10, and 11) of FBW7 in 150 primary JEM VOL. 204, August 6, 2007 1815 Figure 1. Treatment with MRK-003 results in cell cycle arrest, apoptosis, and inhibition of NICD production in human T-ALL cell lines. (A) Cell cycle analysis of the GSI-sensitive HPB-ALL cell line. Cells were treated with DMSO (vehicle) or 1  M MRK-003 for 7 d, stained with propidium iodide, and analyzed by fl ow cytometry. (B) Annexin V – FITC/propidium iodide staining of the DND41 cell line after 7 d in DMSO or 1  M MRK-003. Numbers represent the percentage of cells in each quadrant. Similar results were observed in other GSI-sensitive cell lines. (C) Cell cycle analysis of the GSI-resistant Molt13 cell line after 7 d in DMSO (vehicle) or MRK-003. Similar results were observed for all other GSI-resistant cell lines listed in Table I. (D) Annexin V – APC/propidium iodide staining of the Molt4 cell line after 7 d in DMSO or 1  M MRK-003. Similar results were observed in all other GSI-resistant cell lines. (E) Activated NOTCH1 Western blot analysis. T-ALL cell lines were treated with 1  M MRK-003 for 3 d. Whole cell lysates were subjected to SDS- PAGE electrophoresis and immunoblotting with the NOTCH1 (V1744) antibody. acute myeloid leukemia (AML) samples, 60 primary myelo- residues critical for substrate binding ( Table II ). As in the dysplastic syndrome (MDS) samples, and 81 primary T- T-ALL cell lines, none of the primary samples with an ALL samples. Although the AML or MDS samples lacked FBW7 mutation had C-terminal truncating mutations in mutations in FBW7 , 7 of 81 (8.6%) of the T-ALL samples NOTCH1 . These results indicate that mutation of FBW7 harbored mutations in the substrate-binding domain of in primary T-ALL may represent an alternative mechanism FBW7. All of the mutations in the patient samples were of NOTCH deregulation that contributes to the pathogen- heterozygous missense mutations that altered arginine esis this disease. 1816 FBW7 MUTATION IN T-ALL | O ’ Neil et al. ARTICLE Table II. FBW7 mutations in primary T -ALL patient samples a b Sample NOTCH1 status FBW7 status HD domain PEST domain 2773 heterozygous mutation 1605 V → E WT heterozygous mutation 479 R → Q 2788 heterozygous mutation 1601 L → P WT heterozygous mutation 465 R → H 1179 WT WT heterozygous mutation 465 R → C 2748 heterozygous mutation 1586 L → P WT heterozygous mutation 479 R → Q 368 heterozygous mutation 1748 F → S WT heterozygous mutation 465 R → H 037 heterozygous mutation 1601 L → P WT heterozygous mutation 465 R → C 452 heterozygous mutation 1748 F → S WT heterozygous mutation 465 R → H The substrate binding encoding exons of FBW7 were sequenced in 81 primary T-ALL patient samples. Mutations were found in 7 out of 81 (8.6%) Samples with mutations are shown. Numbers correspond to amino acid residues in NOTCH1. The NOTCH1 T2512 region comprises mutation of another possible NOTCH1 CPD consensus mo- an FBW7 phosphodegron tif anchored around threonine 2133 (38) did not aff ect FBW7 As outlined above, loss of FBW7-mediated regulation of its binding (unpublished data). substrates can result from mutation of FBW7 itself or from mu- Because two putative CDK8 sites in the T2512 region tation of the substrate. The PEST domain of NICD is known have been previously implicated in NOTCH degradation by to regulate protein stability and is a hot spot for mutations FBW7 (S2514 and S2517), we also tested the role of these in primary human and mouse T-ALLs ( 7, 11 ). Furthermore, residues in FBW7 binding ( 26 ). We found that FBW7 bind- FBW7 binding to NOTCH is dependent on an intact PEST ing was disrupted by the S2514A mutation and that binding domain ( 27 ), strongly suggesting that the NOTCH CPD lies was restored when S2514 was replaced by a phosphomimetic within this region. Alignment of the human NOTCH1 PEST residue (S2514E) ( Fig. 2 D ). Although these data are consistent domain with other FBW7 substrates demonstrated a CPD con- with a role for S2514 phosphorylation in FBW7 binding, a sensus motif anchored around threonine 2512 (T2512) in hu- negative charge in the +2 position (relative to the central man NOTCH1. This residue is highly conserved across species threonine) is not a characteristic CPD feature. We thus mu- as well as across members of the NOTCH family ( Fig. 2 A ). tated S2514 to proline, which conforms to the CPD consen- Notably, T-ALL – associated NOTCH1 PEST mutations iden- sus, and found that S2514P also restored FBW7 binding. tifi ed in both human and mouse tumors occur upstream, or Thus, although a negative charge is tolerated in the +2 posi- very nearby, this putative CPD, suggesting that these mutations tion, it is not required for FBW7 binding. We also examined may disrupt its function ( 7, 11 ). the binding of the NICD to each of the three FBW7 isoforms, To determine if the T2512 region contains a functional which reside in distinct subcellular compartments ( 44 ). The NOTCH CPD, we examined its role in binding interactions results show that NICD binds to both FBW7  (nucleoplasmic) between the NICD and FBW7. For these studies, we used an and FBW7  (nucleolar), but not to FBW7  (cytoplasmic), expression plasmid encoding the mouse NICD, where T2487 supporting the idea that the NICD interacts with FBW7 in corresponds to human T2512 (human numbering is used to the nucleus ( Fig. 2 E ). refer to this CPD throughout this article). We fi rst mutated We next examined the role of T2512 in directing FBW7- T2512 to alanine (T2512A) and cotransfected 293 cells ( Fig. mediated endogenous NICD ubiquitination and, in accord 2 B ) or K562 erythroleukemia cells ( Fig. 2 C ) with FBW7 with our binding studies, found that the T2512A mutation and NICD. Because the interaction of FBW7 with its sub- markedly reduces NICD ubiquitination ( Fig. 2 F ). The ex- strates leads to ubiquitination and degradation by the protea- tent to which this mutation inhibited ubiquitination was some, we uncoupled substrate binding from turnover so that similar to that seen with a C-terminal PEST deletion, termed we could observe stable FBW7 – substrate interactions. To deltaCT, which is defective in ubiquitination by FBW7 ( 27 ). disrupt this pathway, we expressed two previously described The small amount of residual ubiquitination could refl ect ei- SCF mutants: (a) dominant-negative cullin-1 (dnCul1), which ther basal, T2512A-independent interactions of the NICD interacts with F-box proteins, but not ubiquitin-conjugating with FBW7, or could be due to alternative NICD ubiqui- enzymes ( 42 ), and (b) a truncated version of FBW7 (termed tination pathways. Finally, we used metabolic labeling and Fb-WD), which encodes the WD40 substrate binding do- pulse chase to compare the half-life of WT-NICD to the mains but lacks the N-terminal domains, including the F-box T2512A mutant in 293 cells. As predicted, the T2512A mu- motif, and thus is unable to associate with cullin-1 and with tant shows a prolonged half-life, refl ecting impaired degrada- the SCF ( 43 ). In both cases we found that the T2512A NICD tion in vivo ( Fig. 2 G ). Collectively, these data indicate that mutant showed markedly decreased binding to FBW7 when the T2512 region comprises a functional CPD that directs compared with WT-NICD ( Fig. 2, B and C ). In contrast, binding interactions between NOTCH and FBW7. JEM VOL. 204, August 6, 2007 1817 Figure 2. NOTCH T2512A mutant shows increased stability and decreased binding to FBW7. (A) Alignment of the four human NOTCH proteins, as well as Notch proteins from various species, shows strong conservation of the putative NOTCH CPD. Known CPDs are aligned with NOTCH for compari- son. (B) The T2512A NOTCH mutant is defi cient in binding to WT FBW7, and the tumor-derived FBW7 arginine mutant (R465C) can no longer bind WT NOTCH. Cells were transfected as indicated, and FLAG-FBW7 was immunoprecipitated. Samples were subjected to SDS-PAGE electrophoresis and immuno- blotting with anti-MYC tag (9E10) to detect transfected Notch proteins. Whole cell lysates were analyzed as indicated to verify expression of transfected constructs. (C) Co-immunoprecipitation assays in the K562 erythroleukemia cell line show that T2512A is defi cient in binding to FBW7 in hematopoietic cells. Whole cell lysates were analyzed as indicated to verify expression of transfected constructs. (D) Phosphorylation of S2514 is not required for the FBW7 – Notch ICD interaction. 293a cells were transfected as indicated and analyzed as in B above. (E) NICD preferentially associates with nuclear and nucleolar FBW7 isoforms. 293a cells were transfected with the indicated plasmids and analyzed as in B above. (F) In vivo ubiquitination assays show that the NOTCH T2512A mutant is resistant to FBW7-mediated ubiquitination. 293a cells were transfected as indicated. Cell lysates were prepared and immuno- precipitated with anti-HA antibody to pull down ubiquitinated proteins. Samples were subjected to SDS-PAGE electrophoresis and immunoblotted with 9E10 to detect MYC-tagged NOTCH proteins. Whole cell lysates were analyzed as indicated to verify expression of transfected constructs. (G) The NOTCH T2512A mutant has an extended half-life in 293a cells compared with WT NOTCH. 293a cells were transfected with either WT NICD or the T2512A mutant. 48 h later, cells were pulse labeled in vivo with S-methionine/cysteine and chased in medium with excess unlabeled methionine and cysteine for the indicated times. Transfected NOTCH proteins were immunoprecipitated with MN-1 antisera, and samples were subjected to SDS-PAGE electrophoresis. Gels were then exposed to x-ray fi lm. GSI treatment results in decreased MYC expression in a To determine if MYC protein levels are aff ected by treat- subset of T-ALL cell lines ment with MRK-003 in our panel of T-ALL cell lines, Recent studies have demonstrated that MYC is a direct tar- we performed Western blot analysis of lysates from a panel get of NOTCH in both human and mouse T-ALL ( 20 – 22 ). of 19 T-ALL cells that were either treated with vehicle 1818 FBW7 MUTATION IN T-ALL | O ’ Neil et al. ARTICLE Figure 3. GSI treatment results in decreased MYC protein levels in a subset of T-ALL cell lines. T-ALL cell lines were treated with 1  M MRK-003 or DMSO for 3 d, and whole cell lysates were prepared using RIPA lysis buffer. Western blot analysis was performed using antibodies against MYC and  -actin as a loading control. (DMSO) or 1  M MRK-003 for 3 d ( Fig. 3 ). A reduction We also examined if MYC mRNA levels responded to of MYC protein levels upon GSI treatment was apparent in GSI treatment by performing expression arrays on 18 of the 8 of the 19 lines (Koptk1, HPB-ALL, DND41, RPMI8402, T-ALL cell lines treated with DMSO or MRK-003 (1  M) Molt13, PF382, Supt7, and TALL1), including all of the for 3 d. We observed either a very modest eff ect or no eff ect GSI-sensitive lines tested (Koptk1, HPB-ALL, DND41, of GSI treatment on the levels of MYC RNA in four of the and TALL1). 4 of the other 10 lines (Loucy, Supt11, resistant lines that express NICD and have mutated FBW7 Supt13, and Molt16) lack activated NOTCH signaling (CEM, BE13, DU528, and HSB2) ( Fig. 5 ). Hence, the lack (i.e., NICD was not detectable by Western blot analysis; Fig. of down-regulation of MYC by GSI treatment in these lines 1 E and Table I ), and thus we would not expect GSI treat- is due to mechanisms acting upstream of MYC transcription ment to aff ect the expression of NOTCH target genes in rather than at the protein level. In the microarray expression these lines. The remaining seven cell lines (Jurkat, Molt4, analysis, other NOTCH target genes such as DELTEX1 and PEER, CEM, DU528, BE13, and HSB2) have activated SHQ1 (22) ( Fig. 5 and not depicted) were also not aff ected NOTCH signaling but do not display decreased MYC ex- by GSI treatment in the CEM, DU528, BE13, and HSB2 pression upon GSI treatment. Of note, all of the cell lines cell lines. In the PEER cell line, which also has a mutation in in which MYC expression is not decreased upon GSI treat- FBW7 , we detected residual expression of NICD after 3 d of ment are resistant to the drug, reinforcing the importance treatment with MRK-003, and quantitative RT-PCR showed of this NOTCH target gene for T-ALL cell growth. Because that MYC and DELTEX1 RNA expression levels did not over expression of MYC has been shown to rescue the decrease in this line upon GSI treatment ( Fig. 1 E and not growth-suppressive eff ects of GSIs ( 20, 21 ), lack of MYC depicted). This suggests that the FBW7 mutations (or homo- down-regulation in these T-ALL cell lines may contribute zygous deletion) found in these fi ve lines result in residual to their GSI resistance. signaling through NICD, even in the face of GSI treatment, Because MYC is also a FBW7 target, we examined MYC thus sustaining MYC transcription and promoting continued half-life in a subset of fi ve T-ALL cell lines (Molt4, DND41, cell proliferation. KOPTK1, CEM, and Jurkat) by pulse-chase analysis. Although MYC half-life was prolonged in each cell line compared with Tumor-derived FBW7 mutants are functionally impaired a B cell lymphoblastoid cell line (JY) ( Fig. 4 ), it was not in- and may act as dominant-negative mutants to prevent creased in cell lines harboring mutations in FBW7 (CEM and MYC degradation Jurkat) compared with those with WT FBW7 . This result was Because FBW7 is haploinsuffi cient for tumor suppression ( 46 ), not surprising because several lymphoblastic leukemia cell lines the single allele FBW7 mutations most commonly found in and patient samples have been shown to have aberrantly stabi- T-ALL could lead to stabilization of its substrates simply by lized MYC ( 45 ). Thus, MYC may be stabilized in T-ALL cell reducing FBW7 activity by 50%. However, if the selective pres- lines without FBW7 mutations due to mutations in other sure for these mutations consisted simply of loss-of-function components of the MYC degradation pathway. Furthermore, of one allele, we would predict that we would fi nd nonsense in some contexts, FBW7 loss may lead to MYC stabilization in mutations in the FBW7 gene ( 36, 47 ). Our fi nding of muta- subcellular compartments (e.g., the nucleolus) without grossly tional hot spots aff ecting the three key arginine residues of altering bulk MYC turnover ( 44 ). FBW7 argues against this possibility. Instead, we propose that JEM VOL. 204, August 6, 2007 1819 Figure 4. T-ALL cell lines have increased MYC half-life. (A) T-ALL cell lines with WT FBW7 (Molt4, DND41 and KOPTK1) were pulse-labeled in vivo with S-methionine/cysteine and chased in medium with excess unlabeled methionine and cysteine for the indicated times. Endogenous MYC was immunoprecipitated from an equal number of cells for each time point and analyzed by gel electrophoresis. S-labeled MYC from each sample was quantitated by phosphoimager. The rate of degradation of MYC for each cell line is represented in the graph by best-fi t exponential lines. Half-lives of MYC were calculated from exponential line equations. (B) Pulse-chase experiments were similarly performed on T-ALL cell lines with mutant FBW7 (CEM and Jurkat). All T-ALL cell lines analyzed have increased MYC half-life compared with JY cells, a B cell lymphoblastoid cell line. Figure 5. NOTCH target genes are not decreased upon GSI treatment in T-ALL cell lines with mutant FBW7 . MYC and DELTEX RNA levels in 18 T-ALL cell lines after 3 d of 1  M MRK-003 GSI treatment (compared with DMSO-treated cells) as determined by microarray gene expression analysis. Values are a log ratio of the expression level of MYC and DEL TEX in MRK-003 – treated cells compared with DMSO-treated cells. 1820 FBW7 MUTATION IN T-ALL | O ’ Neil et al. ARTICLE the restricted mutational spectrum indicates a strong selection were unable to promote MYC degradation ( Fig. 6 B ). We for expression of an FBW7 protein that is defective for pro- also examined whether a T-ALL – derived FBW7 mutant can ductive substrate interactions and/or turnover, which sug- dominantly interfere with FBW7-driven MYC degradation gests activity as a dominant-negative allele. by expressing either increasing amounts of FBW7-  or a To explore this possibility, we analyzed each of the three mixture of a fi xed amount of FBW7-  in combination with mutant FBW7 proteins identifi ed in T-ALL ( Tables I and II ) increasing amounts of FBW7-  -R465C. This analysis sug- for their ability to bind to both MYC and NOTCH. As ex- gests that the mutant impairs FBW7-driven MYC degrada- pected, the mutants were defective in NOTCH binding, al- tion in a dose-dependent manner ( Fig. 6 C ). though FBW7R479Q showed a modest amount of residual At least two mechanisms may contribute to the dominant- binding ( Fig. 6 A , right). In contrast, each of the mutant negative activities of the mutant FBW7 proteins. The fi rst FBW7 proteins bound to MYC, consistent with our recent is suggested by our fi nding that the FBW7 mutants still in- fi nding that in addition to its canonical interaction with the teract with Myc through a binding interaction independent MYC threonine 58 (T58) CPD, full-length FBW7 also binds of the MYC CPD that cannot promote MYC degradation. to MYC through a second interaction that is independent of Hence, in the case of MYC, they may simply compete with the T58 CPD ( Fig. 6 A , left, and unpublished data). Impor- WT FBW7 for MYC binding. However, a second mecha- tantly, although these FBW7 mutants bound to MYC, they nism of dominant-negative function is suggested by recent fi ndings that FBW7 (and its orthologs) form dimers ( 48 – 51 ), thus raising the possibility that WT and mutant FBW7 pro- teins form heterodimeric dominant-negative complexes that are unable to degrade MYC. However, because mutation of the FBW7 dimerization region also disrupts its noncanonical binding interaction with MYC, we cannot currently distin- guish between these two possibilities (unpublished data). We thus propose that FBW7 mutations may contribute to T-ALL pathogenesis and GSI resistance by leading to the stabilization of MYC as well as NICD. DISCUSSION The early successes of molecularly targeted therapy, such as imatinib mesylate directed against the BCR-ABL fusion pro- tein in chronic myelogenous leukemia, stimulated enormous interest in subverting abnormal signaling pathways in cancer cells. The NOTCH signaling cascade infl uences normal devel- opment by regulating diff erentiation, proliferation, and apop- tosis ( 52 ). Activation of the NOTCH signaling pathway is fi rmly established in T-ALL and is likely involved in the gene- Figure 6. Mutant FBW7 cannot bind to NICD and acts in a sis of many other tumor types ( 53 ). Thus, it is not surprising dominant-negative manner to prevent MYC degradation. (A) FBW7 arginine point mutants effi ciently co-precipitate with MYC but not with that eff orts to block NOTCH signaling as a novel therapeutic NOTCH. 293a cells were transfected with the indicated plasmids. Whole strategy are under way in T-ALL as well as in solid tumors. cell lysates were subjected to immunoprecipitation with anti-FLAG, and One of the most promising approaches has been to inhibit resulting samples were analyzed via immunoblotting for either MT- NOTCH receptor signaling using GSIs. This strategy sup- NOTCH or HA-MYC as indicated. Whole cell lysates were also analyzed presses the generation of NICD and thus, in principle, should directly by SDS-PAGE and immunoblotting for FLAG-FBW7, HA-MYC, or inhibit the downstream transcriptional events normally in- MT-NOTCH as indicated to verify expression of transfected constructs. duced by this key signaling component after it traverses to the dnCul1 was included in all transfections to block FBW7-mediated pro- nucleus. Although eff ective against some T-ALL cell lines, teasomal degradation. (B) T-ALL – associated FBW7 mutants are unable GSIs do not uniformly eliminate leukemic cells with activated to mediate MYC degradation. 293a cells were transfected as indicated. Whole cell lysates were subjected to SDS-PAGE and immunoblotting for NOTCH signaling. Understanding the mechanisms of GSI either HA-MYC or FLAG-FBW7 as indicated. (C) Tumor-derived FBW7 resistance may lead to better treatments for T-ALL. mutants dominantly inhibit MYC turnover by WT-FBW7. 293a cells We found missense FBW7 mutations or homozygous were transfected with a constant amount of HA-MYC with increasing FBW7 deletion in GSI-resistant T-ALL cell lines and in pri- amounts of either FLAG-FBW7-  or a combination of a constant amount mary T-ALL samples. Moreover, we have defi ned the NOTCH (500 ng) of FLAG-FBW7-  and increasing amounts of FLAG-FBW7-  - phosphodegron and demonstrated that the mutant forms of R465H (1 – 5  g). The ratios of mutant to WT FBW7 expression are indi- FBW7 found in T-ALL cannot bind to the NICD. Each T- cated above lanes 7 – 9. HA-cdk2, which does not affect turnover of MYC ALL cell line with constitutive NICD expression harbored by FBW7, is included as a transfection control. Whole cell lysates were either NOTCH1 PEST domain or FBW7 mutations, suggesting subjected to SDS-PAGE and immunoblotting for HA-MYC, HA-cdk2, or FLAG-FBW7 as indicated. that these two classes of mutations provide a mutually exclusive JEM VOL. 204, August 6, 2007 1821 sent and Institutional Review Board approval was obtained to use leftover means of prolonging the NICD half-life. We also show that patient material for research purposes. the expression of NOTCH target genes including DELTEX1 and MYC are not aff ected by GSI treatment in fi ve resistant Cell lines. T-ALL cell lines were obtained from American Type Culture T-ALL cell lines with mutations in FBW7 (CEM, BE13, PEER, Collection or Deutsche Sammlung von Mikroorganismen und Zellkulturen DU528, and HSB2), demonstrating that the mechanism GmbH. 293a cells were provided by S. Reed (The Scripps Research Insti- of resistance in these leukemias lies upstream of MYC and tute, La Jolla, CA). DELTEX1 transcription. It appears that in these T-ALL lines, Treatment of T-ALL cell lines with MRK-003. T-ALL cell lines were one consequence of FBW7 mutation is stabilization of the 4 5 plated at a density of 5  10 – 1.5  10 cells per ml (depending on dou- NICD resulting in sustained NOTCH signaling, and thus pro- bling time) and were treated with either DMSO or 1  M MRK-003 for moting resistance to GSIs. However, other FBW7 substrates, 0 h, 6 h, 24 h, 3 d, or 7 d. For each time point, triplicate fl asks were such as MYC, may also account for selection for FBW7 muta- analyzed by the trypan blue exclusion assay to determine cell viability. tions and for GSI resistance in T-ALL, as suggested by our For propidium iodide staining to analyze the cell cycle profi le, cells were fi nding that ALL-associated FBW7 mutations can dominantly washed with PBS, stained in a PBS buff er containing 50  g/ml propidium iodide and 200  g/ml RNase A, and analyzed on a Becton Dickinson inhibit MYC degradation. FACSCalibur Instrument. For annexin V staining to determine the per- An important question is why FBW7 mutation confers centage of cells undergoing apoptosis, the annexin V – FITC (or annexin GSI resistance, whereas NOTCH PEST domain truncations V – APC) Apoptosis detection kit I (BD Biosciences) was used according to that remove the FBW7 interaction domain do not. In each the manufacturer ’ s instructions. cell line with PEST mutations, only one allele is aff ected by these heterozygous mutations, leaving the remaining normal Microarray gene expression analysis. Total RNA isolated from cultured cells was used to make fl uorescently labeled cRNA that was hybridized to allele, which encodes an NICD that is stabilized in the pres- DNA oligonucleotide microarrays as described previously ( 55, 56 ). Human ence of mutant FBW7. Thus, although the amount of in- microarrays contained oligonucleotide probes corresponding to  21,000 creased NOTCH activity resulting from the single allele genes. All oligonucleotide probes on the microarrays were synthesized in situ PEST mutations may be suffi cient to underlie the primary with inkjet technology (Agilent Technologies) ( 55 ). After hybridization, selection for these mutations, disruption of FBW7 function arrays were scanned and fl uorescence intensities for each probe were recorded. may be more active in sustaining NOTCH signaling and also Ratios of transcript abundance (experimental to control) were obtained after normalization and correction of the array intensity data. Gene expression may prolong the half-life of MYC (as well as other substrates), data were analyzed with the Rosetta Resolver gene expression analysis soft- thus promoting GSI drug resistance. ware (version 6.0; Rosetta Biosoftware). Microarray data have been depos- The association between FBW7 mutations and resistance ited in the GEO database under accession number GSE8416. to GSIs has implications for clinical testing of these agents in patients whose cancers show deregulation of the NOTCH Western blotting. For analysis of endogenous protein expression, T-ALL pathway. Molecular analysis of the FBW7 gene, as well as cell lines were treated with MRK-003 for 3 d and lysed in RIPA buff er. genes encoding the relevant NOTCH receptors and other Blots were probed with 9E10 anti-MYC antibody (1:500; Santa Cruz Bio- technology, Inc.), the cleaved NOTCH1 (Val1744) antibody (1:1,000; Cell key components of the NOTCH signaling pathway, such as Signaling Technology) as described previously ( 24 ), and anti –  -actin NUMB, may contribute to identifi cation of patients likely to (1:7,000; Abcam) for the loading control. Signal was detected with Super- be most responsive to GSI therapy. Because Myc has recently Signal West Femto ECL reagent (Pierce Chemical Co.). For transfections, been shown to be an important target of Notch in mammary MYC-tagged proteins were detected with 9E10 hybridoma supernatant tumorigenesis as well as in T-ALL ( 5 ), it might also be possi- (1:4), HA-tagged proteins were detected with HA-11 (1:1,000), and FLAG- tagged proteins were detected with M2 anti-FLAG (1:4,000; Sigma-Aldrich). ble to overcome GSI resistance by combining GSIs with other These same antibodies were conjugated to protein G sepharose and used for drugs that block the MYC pathway to synergistically reduce immunoprecipitations as described previously ( 57 ). MYC levels and block tumor cell growth. One attractive candidate is TMPyP4, a cationic porphyrin that binds to and Mutation detection. The entire coding region of FBW7 was sequenced in stabilizes guanine quadruplexes in DNA. MYC contains a se- genomic DNA isolated from the 20 T-ALL cell lines. Exons 7, 8, 9, 10, and quence in its promoter that forms a guanine quadruplex, and 11 of FBW7 were sequenced in primary patient samples. All sequencing was performed at Agencourt Bioscience Corporation. Residues are numbered TMPyP4 has been shown to inhibit MYC transcription and according to their location in the  isoform of FBW7 (GenBank RefSeq the growth of tumor cells in vivo ( 54 ). Thus, TMPyP4 or NM_033632). other agents that inhibit MYC transcription may be useful in combination with GSIs to overcome resistance in patients Plasmids and transient transfections. The following expression plasmids harboring FBW7 mutations. Pharmacologic or genetic strat- have been described (43, 44): pFLAG-FBW7-  , -  , and -  ; pFLAG-FbWD; egies that restore the normal function of FBW7 in tumor cells pFLAG-FBW7-  -R465H (also termed R298H); and HA-MYC, HA-dncul1, could also be therapeutically useful. and HA-ubiquitin. All mutagenesis were performed by the QuickChange method (Stratagene) and confi rmed by sequencing. Point mutations in the FBW7 common region R465H, R479Q, and R505C were generated by MATERIALS AND METHODS site-directed mutagenesis using pFLAG-FBW7 vectors as template. Although Patient samples. Bone marrow or peripheral blood samples were collected numbering is diff erent between isoforms, the  numbering is used for simplic- at diagnosis from T-ALL patients at the Sophia Children ’ s Hospital/Erasmus ity. CS2-mNICD-MT was provided by R. Kopan (Washington University, MC and the Dutch Childhood Oncology Group, as well as from AML, St. Louis, MO). It encodes the mouse Notch1 intracellular domain from MDS, and T-ALL patients at Dana-Farber Cancer Institute. Informed con- Val1744 to the end of the WT protein and includes six in-frame MYC epitope 1822 FBW7 MUTATION IN T-ALL | O ’ Neil et al. ARTICLE tags at the C terminus. Mutagenesis of this construct was used to generate the 6 . Pear , W.S. , J.C. Aster , M.L. Scott , R.P. Hasserjian , B. Soff er , J. Sklar , T2512A, S2514A, S2514E, and S2514P mutants. The deltaCT mutation, and D. Baltimore . 1996 . Exclusive development of T cell neoplasms in mice transplanted with bone marrow expressing activated Notch alleles. generated by PCR, deletes the PEST domain but maintains the C-terminal J. Exp. Med. 183 : 2283 – 2291 . MYC tags. 293A cells were transfected by the calcium phosphate precipita- 7 . O ’ Neil , J. , J. Calvo , K. McKenna , V. Krishnamoorthy , J.C. Aster , tion method as described previously ( 57 ). Cells were harvested at 36– 48 h C.H. Bassing , F.W. Alt , M. Kelliher , and A.T. Look . 2006 . Activating after transfection using TENT buff er (50 MM Tris-Cl, pH 8.0, 2 mM EDTA, Notch1 mutations in mouse models of T-ALL. Blood . 107 : 781 – 785 . 150 mM NaCl, 1% Triton X-100) ( 30 ) with protease and phosphatase inhib- 8 . Mantha , S. , M. Ward , J. McCaff erty , A. Herron , T. Palomero , A. itors and analyzed by immunoprecipitation and Western blotting as described Ferrando , A. Bank , and C. Richardson . 2007 . Activating Notch1 mu- previously ( 57 ). tations are an early event in T-cell malignancy of Ikaros point mutant Plastic/+ mice. Leuk. Res. 31 : 321 – 327 . Metabolic labeling and pulse chase. NICD pulse-chase assays were per- 9 . Lin , Y.W. , R.A. Nichols , J.J. Letterio , and P.D. Aplan . 2006 . Notch1 formed as described previously ( 43 ). In brief, 293a cells were transfected mutations are important for leukemic transformation in murine models with plasmids encoding MYC-tagged NICD or NICD T2512A. At 36 h af- of precursor-T leukemia/lymphoma. Blood . 107 : 2540 – 2543 . ter transfection, cells were preincubated with media lacking cysteine and 10 . Ellisen , L.W. , J. Bird , D.C. West , A.L. Soreng , T.C. Reynolds , methionine, pulsed with media containing Trans 35S label, and chased with S.D. Smith , and J. Sklar . 1991 . TAN-1, the human homolog of the media with cold methionine. Samples were obtained at the indicated time Drosophila notch gene, is broken by chromosomal translocations in points and immunoprecipitated with anti-Notch serum MN1, a mouse T lymphoblastic neoplasms. Cell . 66 : 649 – 661 . 11 . Weng , A.P. , A.A. Ferrando , W. Lee , J.P. Morris , L.B. Silverman , C. monoclonal antibody recognizing the NICD (provided by I. Bernstein, Fred Sanchez-Irizarry , S.C. Blacklow , A.T. Look , and J.C. Aster . 2004 . Hutchinson Cancer Research Center, Seattle, WA). MYC pulse-chase ex- Activating mutations of NOTCH1 in human T cell acute lymphoblastic periments were performed as described previously ( 45 ). leukemia. Science . 306 : 269 – 271 . 12 . Park , J.T. , M. Li , K. Nakayama , T.L. Mao , B. Davidson , Z. Zhang , R.J. Ubiquitination assays. Cell-based ubiquitination assays were performed as Kurman , C.G. Eberhart , M. Shih Ie , and T.L. Wang . 2006 . Notch3 described previously ( 58 ). In brief, transiently transfected 293a cells were gene amplifi cation in ovarian cancer. Cancer Res. 66 : 6312 – 6318 . harvested at 48 h after transfection. Cells were trypsinized, rinsed once with 13 . Pece , S. , M. Serresi , E. Santolini , M. Capra , E. Hulleman , V. Galimberti , PBS, and lysed in 100  l 2% SDS in TBS (10 mM Tris-HCl, pH 8.0, 150 S. Zurrida , P. Maisonneuve , G. Viale , and P.P. Di Fiore . 2004 . Loss of mM NaCl) with boiling for 10 min. These samples were diluted at 1:10 with negative regulation by Numb over Notch is relevant to human breast 1% triton in TBS containing protease inhibitors, 5 mM N-ethymaleimide, carcinogenesis. J. Cell Biol. 167 : 215 – 221 . and 5 mM MG132, sonicated, precleared with protein G sepharose, and 14 . Jundt , F. , I. Anagnostopoulos , R. Forster , S. Mathas , H. Stein , and B. centrifuged to pellet-insoluble debris. Aliquots were removed for Western Dorken . 2002 . Activated Notch1 signaling promotes tumor cell pro- blot analysis, and the remaining lysates were immunoprecipitated with anti- liferation and survival in Hodgkin and anaplastic large cell lymphoma. HA antisera prebound to protein G sepharose. Samples were analyzed by Blood . 99 : 3398 – 3403 . SDS-PAGE and probed with 9E10 to visualize MYC-tagged ubiquitinated 15 . Balint , K. , M. Xiao , C.C. Pinnix , A. Soma , I. Veres , I. Juhasz , E.J. Notch proteins. Brown , A.J. Capobianco , M. Herlyn , and Z.J. Liu . 2005 . Activation of Notch1 signaling is required for beta-catenin-mediated human primary melanoma progression. J. Clin. Invest. 115 : 3166 – 3176 . The authors wish to thank Theresa Zhang, Leslie Carlini, and Xudong Dai for helpful 16 . Purow , B.W. , R.M. Haque , M.W. Noel , Q. Su , M.J. Burdick , J. Lee , T. discussions regarding mRNA profi ling; Hellen Kim for discussions about Western Sundaresan , S. Pastorino , J.K. Park , I. Mikolaenko , et al . 2005 . Expression blotting methods; Keith McKenna and Chitra Raghunathan for technical assistance; of Notch-1 and its ligands, Delta-like-1 and Jagged-1, is critical for glioma and John Gilbert for editorial review. cell survival and proliferation. Cancer Res. 65 : 2353 – 2363 . This work was funded by a sponsored research agreement from Merck Research 17 . Haruki , N. , K.S. Kawaguchi , S. Eichenberger , P.P. Massion , S. Olson , Laboratories (to A.T. Look), an American Society of Hematology Fellow Scholar A. Gonzalez , D.P. Carbone , and T.P. Dang . 2005 . Dominant-negative Award and by the National Institutes of Health (1K08CA109124-01A2; to J. Grim), Notch3 receptor inhibits mitogen-activated protein kinase pathway and National Institutes of Health grants (R01CA84069 and R01CA102742) and the the growth of human lung cancers. Cancer Res. 65 : 3555 – 3561 . Burroughs Welcome Foundation (to B.E. Clurman), as well as a Harvard Medical 18 . Collins , B.J. , W. Kleeberger , and D.W. Ball . 2004 . Notch in lung de- School Hematology Training Grant (to J. O ’ Neil). velopment and lung cancer. Semin. Cancer Biol. 14 : 357 – 364 . The authors have no confl icting fi nancial interests. 19 . Santagata , S. , F. Demichelis , A. Riva , S. Varambally , M.D. Hofer , J.L. Kutok , R. Kim , J. Tang , J.E. Montie , A.M. Chinnaiyan , et al . 2004 . Submitted: 2 May 2007 JAGGED1 expression is associated with prostate cancer metastasis and Accepted: 28 June 2007 recurrence. Cancer Res. 64 : 6854 – 6857 . 20 . Weng , A.P. , J.M. Millholland , Y. Yashiro-Ohtani , M.L. Arcangeli , A. REFERENCES Lau , C. Wai , C. Del Bianco , C.G. Rodriguez , H. Sai , J. Tobias , et al . 1 . Wilson , A. , and F. Radtke . 2006 . Multiple functions of Notch signaling 2006 . c-Myc is an important direct target of Notch1 in T-cell acute in self-renewing organs and cancer. FEBS Lett. 580 : 2860 – 2868 . lymphoblastic leukemia/lymphoma. Genes Dev. 20 : 2096 – 2109 . 2 . Gallahan , D. , and R. 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Journal

The Journal of Experimental MedicinePubmed Central

Published: Aug 6, 2007

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