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Notch: an Introduction

Notch: an Introduction. Notch receptors participate in a conserved signaling pathway that regulates diverse cellular differentiation programs in metazoans Genetic analyses have elucidated diverse CONTEXT-dependent and DOSE-dependent functions for Notch signaling

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Notch: an Introduction

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  1. Notch: an Introduction Notch receptors participate in a conserved signaling pathway that regulates diverse cellular differentiation programs in metazoans Genetic analyses have elucidated diverse CONTEXT-dependent and DOSE-dependent functions for Notch signaling These diverse functions are mediated through a signal transduction pathway relying on the regulated proteolysis of Notch receptors

  2. Notch Structure and Activation Cleavage at site S1 during receptor maturation creates two non-covalently associated subunits protected from premature activation by NRR and the HD. Ligand binding induces a cleavage at site S2 by ADAM-type metallo- protease followed by an additional cleavage at site S3 by -secretase.

  3. Notch-Induced Signaling

  4. In mice, Notch1 has a non-redundant role during the earliest stages of T-cell development, including initial commitment to T-cell fate and subsequent progression to the DN3 stage of development There is an absolute requirement for Notch for T-cell progenitors of the  lineage to progress past the DN3  selection checkpoint

  5. NOTCH Function in Normal Thymopoiesis Maillard et al, Annu Rev Immunol 2005; 23:945-974

  6. NOTCH Function in Normal Thymopoiesis Small molecule inhibitors of -secretase, conditional knockout of CSL, or dominant-negative forms of mastermind-like-1 (MAML) recapitulate the phenotypes observed with loss of Notch1 function, implicating the canonical signaling pathway and the CSL/ICN/Mastermind complex in thymic development

  7. Discovery of Oncogenic NOTCH Alleles Human NOTCH1 was identified through its involvement by a recurring chromosomal translocation t(7;9)(q34;q34.3) in a cell line (SUPT-1) derived from a patient with T-ALL In these breakpoints, DNA sequences 3´ of the breakpoints are fused to TCR promoter/enhancer sequences, resulting in synthesis of a series of N-terminally truncated (and constitutively activated) NOTCH1 polypeptides SUPT-1 cells were growth inhibited by dominant negative MAML1 but not by GSIs, indicating that some t(7;9)-specific NOTCH1 proteins access the nucleus in a -secretase-independent fashion Although recurrent, this translocation is observed in <1% of patients with T-ALL Ellisen et al, Cell 1991; 66:649-661

  8. Discovery of Oncogenic NOTCH Alleles In 2004, Aster, Look, and colleagues discovered two types of activating mutations in NOTCH1, at least one of which is found in ~55-60% of human T-ALLs, making it the most frequent genetic alteration in this form of leukemia This came about through testing of T-ALL cell lines lacking the t(7;9) for their sensitivity (NOTCH dependency) to a -secretase Inhibitor Sequencing revealed NOTCH1 mutations in cell lines that were sensitive and many that were insensitive to the GSI Sequencing also showed HD (both HD-N and HD-C) and PEST domain mutations in a series of primary T-ALL samples from 96 children at diagnosis Importantly, NOTCH1 mutations were found in association with oncogenes of all the major molecular subtypes of T-ALL, suggesting it has the potential to influence multiple signaling pathways Weng et al, Science 2004; 306:269-271

  9. Discovery of Oncogenic NOTCH Alleles Sequencing revealed mutations involving both the HD-N (missense) and PEST domains (insertions/deletions) Weng et al, Science 2004; 306:269-271

  10. Discovery of Oncogenic NOTCH Alleles Western blot analysis revealed that cell lines with HD-N and PEST domain mutations contained a polypeptide of expected size for NTM, plus additional (smaller) polypeptides Weng et al, Science 2004; 306:269-271

  11. Discovery of Oncogenic NOTCH Alleles Using a NOTCH-sensitive reporter, HD-N mutations caused a 3- to 9-fold increase in luciferase activity and PEST domain mutations an ~1.5- to 2-fold increase When HD and PEST domain mutations were present in cis, 20- to 40-fold increases were measured Weng et al, Science 2004; 306:269-271

  12. Activation of NOTCH1 by Leukemia-Associated Mutations Schematic representation of human NOTCH1 receptor with the leukemia-associated mutations studied Malecki et al, Mol Cell Biol 2006; 26:4642-4651

  13. Activation of NOTCH1 by Leukemia-Associated Mutations HD domain mutations resulted in activation of NOTCH1 signaling in the context of both full-length NOTCH1 (A) and ligand binding- defective EGF NOTCH1 (B) Malecki et al, Mol Cell Biol 2006; 26:4642-4651

  14. Activation of NOTCH1 by Leukemia-Associated Mutations GSI treatment abrogated the stimulatory effect of HD mutations in full-length NOTCH1 (A) and these mutations caused increased S2 and S3 cleavage Malecki et al, Mol Cell Biol 2006; 26:4642-4651

  15. Activation of NOTCH1 by Leukemia-Associated Mutations Classification of leukemia- associated HD mutations Class I: dissociation or reduced stability of heterodimer Class II: no effect on hetero- dimer stability but a repositioning of S2 site away from protective residues Malecki et al, Mol Cell Biol 2006; 26:4642-4651

  16. Normal and Pathophysiologic NOTCH1 Signaling Biochemical and genetic data suggest that PEST domain mutations increase Notch signaling by enhancing the STABILITY of the ICN, while mutations in HD-N and HD-C are predicted to enhance PRODUCTION of the ICN by destabilizing intersubunit association Pear and Aster, Curr Opin Hematol 2004; 11:426-433

  17. Incidence of NOTCH1 Mutations in T-ALL 71% 56% Mansour et al, Leukemia 2006; 20:537-539

  18. Distribution of genomic aberrations and NOTCH1 mutations in pediatric T-ALL

  19. Prognostic Significance of NOTCH1 Mutations in T-ALL In a study of 77 patents with T-ALL, 32 mutations identified in 29 off 77 patients (38% incidence) NOTCH1 mutation was more frequent in patients with high white counts and was associated with shorter survival (both relapse-free and overall survival) The association between mutation and survival was significant only in adult patients This negative effect on prognosis was potentiated by some oncogenes (HOX11L2) and attenuated by others (HOX11) Zhu et al, Clin Cancer Res 2006; 12:3043-3049

  20. Prognostic Significance of NOTCH1 Mutations in T-ALL In a study of 157 pediatric patients with T-ALL treated uniformly, NOTCH1 mutations were found in 52%: 67% in HD, 16% in the PEST domain, and 17% in both The presence of mutation correlated significantly with an excellent early response to therapy (good prednisone response, favorable MRD kinetics) Activating NOTCH1 mutations associated with better event-free survival (lower rate of relapse than in patients with germline NOTCH) This raises question of how molecularly targeted therapy can be best tested in the context of and/or integrated into current therapy Breit et al, Blood 2006; 108:1151-1157

  21. Evidence for Oncogenic Activity of Activated Notch/NOTCH In Vivo Development of T-cell neoplasms in mice transplanted with bone marrow expressing activated Notch/NOTCH alleles: Pear et al, J Exp Med 1996; 183:2283-2291 Development of T-cell neoplasms in mice bearing activated Notch transgenes: Robey et al 1996; Cell 87:483-492. Development of T-cell neoplasms in zebrafish bearing an activated NOTCH transgene: Chen et al, Leukemia 2007; 21:462-471

  22. Evidence for Oncogenic Activity of Activated Notch/NOTCH In Vivo It is likely that the tranforming actions of NOTCH reflect its normal roles in T-cell development, i.e. drive pluripotent bone marrow cells toward T-cell fate and expand the pool of immature T cell progenitors NOTCH1 mutations could occur in very immature T lineage cells or uncommitted pluripotent marrow progenitors In this model, NOTCH1 mutations are EARLY events that set the stage for acquisition of other genetic aberrations

  23. Activating Notch1 Mutations are Frequent in Mouse Models of T-ALL Notch1 is frequently mutated in murine models of T-ALL 68% of cell lines + 59% of tumors from TAL1/LMO1, OLIG2/LMO1, OLIG2, LMO1, NUP98/HOXD13, and p27-/-/SMAD3+/- mice (Lin etal, Blood 2006; 107:2540-2543) 74% of tumors from TAL1, TAL1/HEB+/-, and TAL1/Ink4a/Arf+/- mice and 31% of tumors from mice deficient for various combinations of H2AX, p53, and Rag2 (O’Neill et al, Blood 2006; 107:781-785) Mutations were observed in HD (most single-base substitutions) and in the PEST domain (most insertions or deletions) These were acquired relatively early in the process of leukemic transformation Cell lines derived from these tumors underwent G0/G1 arrest and apoptosis when treated with a GSI

  24. Activating Notch1 Mutations are Frequent in Mouse Models of T-ALL The frequent association of Notch1 mutations with other oncogenes and the capacity of activated Notch1 alleles to cooperate with several genes in mouse models of T-AL are reminiscent of the association of NOTCH1 mutations with many molecular subtypes of human T-ALL There may be a specific, critical set of NOTCH1-dependent signals not easily created otherwise or NOTCH1 may be able to activate multiple pro-transforming pathways simultaneously

  25. Summary of Findings from Notch/NOTCH Transgene Studies T-cell malignancy was induced with high penetrance and with a shorter latency than observed with other oncogenes (e.g. TAL1, LMO2) The immunophenotype of mouse tumors was similar to that in human leukemias with NOTCH activation (e.g. CD4+/CD8+ double positive) Even when a promoter generally active in hematopoietic cells was used, T-cell neoplasms developed exclusively Mice doubly transgenic for activated Notch and another T-cell oncogene (e.g.myc) developed tumors with much shorter latency than singly transgenic mice

  26. Targets of Activated NOTCH: c-MYC Using gene expression profiling and chromatin immunoprecipitation approaches in conjunction with GSI treatment, c-MYC was identified as a direct and essential target of Notch signaling: c-myc RNA levels increased in tumor cells that contain Notch1/NOTCH1 mutations Notch1/NOTCH1 inhibition decreases c-myc levels Inhibitors of c-myc interfere with proproliferative effects of activated Notch1/NOTCH1 Retroviral expression of c-myc (as well as ICN) rescues growth arrest and apoptosis induced by GSI or Notch1 inhibition

  27. Targets of Activated NOTCH: c-MYC Activated Notch1 can rescue the effects of withdrawal of c-myc in murine T-ALL cells. This is associated with upregulation of endogenous c-myc and its downstream targets ICN and MAM can be recruited to the c-myc promoter Sharma et al, Mol Cell Biol 2006; 26:8022-8031 Weng et al, Genes Develop 2006; 20:2096-2109 Palomero et al, Proc Natl Acad Sci USA 2006; 103:18261-18266

  28. Targets of Activated NOTCH: NF-B Activated Notch1 upregulates the NF-B pathway transcriptionally (RELB and NFKB2) and via the IB kinase complex The NF-B pathway is activated in human T-ALL Human T-ALL lines are susceptible to NF-B inhibition (with IKK kinase inhibitor BMS-345541) NF-B is important for NotchIC-induced T-cell leukemia NF-B ctivation is not sufficient for T-cell leukemia in the absence of activated Notch1 Vilimas et al, Nat Med 2007; 13:70-77

  29. Effects of Pharmacological Inhibition of Notch Signaling in T-ALL GS!-mediated G0/G1 arrest in five human T-ALL cell lines: abrogated by retroviral tranduction of ICN and phenocopied by dominant negative Mastermind-like-1 Weng et al, Science 2004; 306:269-271

  30. Effects of Pharmacological Inhibition of Notch Signaling in T-ALL GSI treatment of a novel T-ALL cell line (CUTLL1) bearing the t(7;9) blocked Notch processing and caused rapid clearance of activated ICN domain In contrast to the originally characterized t(7;9)(q34;q34) in SUPT1 cells, this rearrangement generated a fusion transcript encoding a truncated but membrane-bound form of Notch Loss of Notch activity led to downregulation of Notch target genes, G1 cell cycle arrest, and apoptosis Palomero et al, Leukemia 2006; 20:1279-1287

  31. Effects of Pharmacological Inhibition of Notch Signaling in T-ALL Pharmacological inhibition of -secretase activty led to decreased Notch signaling, as measured by NIC domain formation, in a T-ALL cell line Inhibition of  -secretase activity was associated with decreased viability, which correlated with G0/G1 cell cycle block The anti-proliferative and pro-apoptotic effects of the inhibitor could be rescued by exogenous expression of NIC domain A less active enantiomer of the GSI showed reduced biological activity Lewis et al, Chem Biol 2007; 14:209-219

  32. GSIs as Therapeutic Agents Biological potency: determined by the “tumor dependence” on activated Notch, the extent to which their actions are cytotoxic vs. cytostatic, and their effects on leukemic stem cells Selectivity: GSIs affect proteolytic activation of all four Notch receptors Specificity: -secretase has many substrates in addition to Notch ligands Toxicity: e.g. from undesired effects on gut epithelial differentiation, lymphocyte development Drug resistance

  33. Phase I Clinical Trial of GSI in T-ALL Six adult and two pediatric patients with T-ALL and AML were enrolled in Phase I (dose escalation) study of potent GSI MK-0752 (IC50 ~50 nM) 4/7 patients with T-ALL had activating mutations of Notch1 Dose-limiting toxicity was diarrhea, observed at highest of four doses tested (300 mg/m2) Plasma concentrations of MK-0752 at all of the tested doses were sufficient to inhibit -secretase activity and would be predicted to inhibit Notch signaling Deangelo et al, J Clin Oncol 2006; 24(Suppl):357a (abstract 6585)

  34. Summary Activation of NOTCH is a frequent event in T-ALL NOTCH mutations are found in >50% of pediatric and adult T-ALLs Inhibition of -secretase activity results in inhibition of leukemia cell NOTCH signaling Use of -secretase inhibitors is a rational approach to the therapy of T-ALL

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