Notch: an Introduction

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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
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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.
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Notch-Induced Signaling
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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
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NOTCH Function in Normal Thymopoiesis
Maillard et al, Annu Rev Immunol 2005 23945-974
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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
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Discovery of Oncogenic NOTCH Alleles
Human NOTCH1 was identified through its
involvement by a recurring chromosomal
translocation t(79)(q34q34.3) 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(79)-specific
NOTCH1 proteins access the nucleus in a
?-secretase-independent fashion Although
recurrent, this translocation is observed in lt1
of patients with T-ALL
Ellisen et al, Cell 1991 66649-661
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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(79) 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 306269-271
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Discovery of Oncogenic NOTCH Alleles
Sequencing revealed mutations involving both the
HD-N (missense) and PEST domains
(insertions/deletions)
Weng et al, Science 2004 306269-271
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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 306269-271
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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 306269-271
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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 264642-4651
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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 264642-4651
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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 264642-4651
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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 264642-4651
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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 11426-433
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Incidence of NOTCH1 Mutations in T-ALL
71
56
Mansour et al, Leukemia 2006 20537-539
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Distribution of genomic aberrations and NOTCH1
mutations in pediatric T-ALL
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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 123043-3049
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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 1081151-1157
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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 1832283-2291 Development of T-cell
neoplasms in mice bearing activated Notch
transgenes Robey et al 1996 Cell
87483-492. Development of T-cell neoplasms in
zebrafish bearing an activated NOTCH transgene
Chen et al, Leukemia 2007 21462-471
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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
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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 et
al, Blood 2006 1072540-2543) 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
(ONeill et al, Blood 2006 107781-785) Mutation
s 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
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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
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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
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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/NOTCH
1 Retroviral expression of c-myc (as well as
ICN) rescues growth arrest and apoptosis induced
by GSI or Notch1 inhibition
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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
268022-8031 Weng et al, Genes Develop 2006
202096-2109 Palomero et al, Proc Natl Acad Sci
USA 2006 10318261-18266
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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 1370-77
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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 306269-271
32
Effects of Pharmacological Inhibition of Notch
Signaling in T-ALL
GSI treatment of a novel T-ALL cell line (CUTLL1)
bearing the t(79) blocked Notch processing and
caused rapid clearance of activated ICN domain
In contrast to the originally characterized
t(79)(q34q34) 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 201279-1287
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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 14209-219
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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
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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)
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Summary
Activation of NOTCH is a frequent event in
T-ALL NOTCH mutations are found in gt50 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