Notch an introduction l.jpg
This presentation is the property of its rightful owner.
Sponsored Links
1 / 36

Notch: an Introduction PowerPoint PPT Presentation


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

Download Presentation

Notch: an Introduction

An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -

Presentation Transcript


Notch an introduction l.jpg

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


Notch structure and activation l.jpg

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.


Notch induced signaling l.jpg

Notch-Induced Signaling


Slide5 l.jpg

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


Notch function in normal thymopoiesis l.jpg

NOTCH Function in Normal Thymopoiesis

Maillard et al, Annu Rev Immunol 2005; 23:945-974


Notch function in normal thymopoiesis7 l.jpg

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


Discovery of oncogenic notch alleles l.jpg

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


Discovery of oncogenic notch alleles9 l.jpg

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


Discovery of oncogenic notch alleles10 l.jpg

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


Discovery of oncogenic notch alleles11 l.jpg

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


Discovery of oncogenic notch alleles12 l.jpg

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


Activation of notch1 by leukemia associated mutations l.jpg

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


Activation of notch1 by leukemia associated mutations15 l.jpg

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


Activation of notch1 by leukemia associated mutations16 l.jpg

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


Activation of notch1 by leukemia associated mutations17 l.jpg

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


Normal and pathophysiologic notch1 signaling l.jpg

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


Incidence of notch1 mutations in t all l.jpg

Incidence of NOTCH1 Mutations in T-ALL

71%

56%

Mansour et al, Leukemia 2006; 20:537-539


Slide20 l.jpg

Distribution of genomic aberrations and NOTCH1 mutations in

pediatric T-ALL


Prognostic significance of notch1 mutations in t all l.jpg

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


Prognostic significance of notch1 mutations in t all22 l.jpg

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


Evidence for oncogenic activity of activated notch notch in vivo l.jpg

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


Evidence for oncogenic activity of activated notch notch in vivo24 l.jpg

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


Activating notch1 mutations are frequent in mouse models of t all l.jpg

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


Activating notch1 mutations are frequent in mouse models of t all26 l.jpg

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


Summary of findings from notch notch transgene studies l.jpg

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


Targets of activated notch c myc l.jpg

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


Targets of activated notch c myc29 l.jpg

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


Targets of activated notch nf b l.jpg

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


Effects of pharmacological inhibition of notch signaling in t all l.jpg

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


Effects of pharmacological inhibition of notch signaling in t all32 l.jpg

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


Effects of pharmacological inhibition of notch signaling in t all33 l.jpg

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


Gsis as therapeutic agents l.jpg

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


Phase i clinical trial of gsi in t all l.jpg

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)


Summary l.jpg

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


  • Login