Cancer Biology Pathway Class – March 18, 2014
Her2/neu Tyrosine Kinase Signaling
Ron Bose, MD PhD
Division of Oncology, Department of Medicine
Department of Cell Biology and Physiology
Tips for Success in Science
The most exciting phrase to hear in science,
the one that heralds the most discoveries, is
not “Eureka” (I found it) but “That’s funny . . .”
- Isaac Asimov, Ph.D.
Key point: An important observation can
open up new directions for science!
1. Big Ideas often Start Small.
“What if…”, “Why not…”, “That’s funny…”
2. Try out new Ideas!
Don’t talk yourself out of a good idea!
3. Don’t Fall in Love with Ideas!
Discard ideas when there is evidence that
they are not correct!
It is amateurs who have one big, bright, beautiful
idea that they can never abandon.
Professionals know that they have to produce
theory (idea) after theory before they are likely to
hit the jackpot. The very process of abandoning
one theory (idea) for another gives them a degree
of critical detachment that is almost essential if
they are to succeed.
-Sir Francis Crick, Nobel Laureate.
“What Mad Pursuit: A Personal View of Scientific Discovery”
The ErbB family of Receptor Tyrosine Kinases
(RTK’s)
EGFR
Her2/neu
I
II
I
III
Kinase
Domain
{
I
II
III
IV
Her3
II
I
III
IV
Her4
II
III
IV
IV
ErbB3
HER3
ErbB4
HER4
ErbB1
HER1
EGFR
ErbB2
HER2
Neu
N
N
N
C
C
C
C-terminal tail
Kinase Domain
Inactive
Zahnow, C.A., Expert Rev Mol Med. 2006
Extracellular
Transmembrane
Cytoplasmic
Ligands for the ErbB receptors
EGF
TGF α
HB-EGF
Amphiregulin
Neuregulins
NRG 1Α
NRG 1β
NRG 2α
NRG 2β
Betacellulin
Epiregulin
I
II
I
III
III
IV
Kinase
Domain
{
I
II
II
Epiregulin
Betacellulin
NRG’s
I
III
IV
HB-EGF
Tomoregulin
II
III
IV
IV
ErbB3
HER3
ErbB4
HER4
ErbB1
HER1
EGFR
ErbB2
HER2
Neu
N
N
N
C
C
C
C-terminal tail
Zahnow, C.A., Expert Rev Mol Med. 2006
Extracellular
Transmembrane
Cytoplasmic
Ligands for the ErbB receptors
EGF
TGF α
HB-EGF
Amphiregulin
Neuregulins
HB-EGF
Tomoregulin
NRG 1Α
NRG 1β
Epiregulin
Her2 functions
as a Co-Receptor andNRG
does2α
not have Betacellulin
any ligands of its own !
Betacellulin
NRG 2β
Epiregulin
NRG’s
I
II
I
III
III
IV
Kinase
Domain
{
I
II
II
I
III
IV
II
III
IV
IV
ErbB3
HER3
ErbB4
HER4
ErbB1
HER1
EGFR
ErbB2
HER2
Neu
N
N
N
C
C
C
C-terminal tail
Zahnow, C.A., Expert Rev Mol Med. 2006
Extracellular
Transmembrane
Cytoplasmic
Dimerization
Arm
EGF
I
III
I
II
I
II
III
IV
Inactive
ErbB
III
III
IV
Active
IV
IV
ErbB
2
1
1
1
N
N
N
N
C
C
C
CC
I
Extracellular
II
Transmembrane
Cytoplasmic
I
II
III
III
IV
Zahnow, C.A., Expert Rev Mol Med. 2006
I
II
II
IV
1
2
N
N
C
C
ErbB Homo- and Hetero-dimerization
Overexpressed
Her2/neu
Ligand Dependent
Signaling
Ligand Independent
Signaling
Adapted from Hynes & Lane, Nature Reviews Cancer 2005
ErbB Receptor Homo- or
Heterodimerization
from Marmor, Skaria, and Yarden 200
Activation of the MAPK pathway by RTK’s
(C)
Ras-GTP
Raf (MAPKKK)
MEK (MAPKK)
Erk 1 & 2 (MAPK)
Figure 6.12 The Biology of Cancer (© Garland Science 2007)
Activation of the PI 3-kinase pathway by RTK’s
AKT
From Engelman, Luo, and Cantley, Nature Reviews Genetics 2006
AKT
Activation of the Phospholipase C by RTK’s
from Marmor, Skaria, and Yarden 2004
STAT transcription factors and RTK’s
I
II
I
II
III
III
IV
IV
1
2
N
N
C
C
Src
STAT
STAT
Cytoplasm
STAT
Nucleus
Her2/neu and Breast Cancer
• Her2 was first identified as an oncogene from a
carcinogen-induced rat brain tumor model.
• Her2 is gene amplified in about 25% of human breast
cancers.
• Overexpression of Her2 in the mammary gland of
transgenic mice causes breast cancer.
• Herceptin, a monoclonal antibody to Her2/neu,
effectively treats Her2 gene amplified human breast
cancer.
Her2/neu and Breast Cancer
• 1987 – Southern blots of
genomic DNA from breast
cancer patients shows Her2
gene amplification.
–
–
–
–
Patient Survival
100%
Sample 3 & 4: normal level
Sample 1 & 2: 2-5 x normal
Sample 6 & 26: >5 x normal
Sample 18: > 20 x normal
• Correlation between Her2
gene copy number and patient
survival
80%
60%
40%
20%
Slamon, et al., Science 1987
0%
Time (months)
Her2/neu and Breast Cancer
• Transgenic mice bearing the
MMTV-Her2/neu construct
develop breast cancer in all 5
pairs of mouse mammary
glands.
• Tumor formation with Her2 in
this tg model is more rapid
than with the Myc oncogene.
Muller et al., Cell 1988
Successful treating Her2 amplified Breast Cancer
• The combination of chemotherapy (AC→T) plus Herceptin markedly
improves patient survival as compared to chemotherapy alone.
• Treatment of women with Herceptin has saved THOUSANDS of lives.
New England Journal of Medicine 2005
Drugs used to Treat HER2 positive
Breast Cancer
FDA- Approved Drugs
Antibody based:
1. Trastuzumab
2. Pertuzumab
3. T-DM1 (antibody-drug
conjugate based on
trastuzumab)
ATP-mimetic, kinase inhibitors:
4. Lapatinib
Kinase inhibitors in clinical trials
for Her2+ breast cancer:
1. Neratinib
2. Afatinib
WJ Gradishar. New England Journal of Medicine, 2012.
“Activated” EGFR and HER2 have Similar
Structure
EGF
Dimerization
Contact
Surfaces
EGFR:EGF
Her2/neu
Receptor Dimer:
the Extracellular Domains
Dimerization
Contact
Surfaces
Burgess et al., Mol Cell 2003
Therapeutic Antibodies Target ErbB2
ErbB2
ErbB2
Pertuzumab
Herceptin
Cho et al. (2003) Nature
Franklin et al. (2004) Cancer Cell
Structure of T-DM1
an antibody-drug conjugate
Trastuzumab
Maytansinoids
(Microtubule depolymerizing agent)
Nonreducible, thioether linkage (MCC)
Stoichiometry: 3 to 3.6 Maytansinoids per Ab.
Lysosomal degradation produces lysine-NεMCC-DM1
Structure of the EGFR Kinase Domain
Erlotinib
Stamos et al., JBC 2002
HER2 tyrosine kinase inhibitors
Alterations of EGFR receptors in Cancer
EGFR Overexpression/Gene Amplification
Many cancers including lung, GI, & brain
Truncation
EGFR vIII mutation in Glioblastomas
EGFR Activating Mutations
EGFR del E746-A750
EGFR L858R
Both found in Gefitinib sensitive Non-small
cell lung cancer
Alterations of ErbB receptors in Cancer
HER2 Mutations in Non-Small Cell Lung Cancer
G776YVMA insertion
G776V, insertion C or G776L, insertion C
P780GSP insertion
HER2 mutations in HER2 negative breast
cancer: ACOSOG Z1031 Clinical Trial
Eligibility Criteria
Postmenopausal
Clinical Stage II or III
ER+ (Allred 6-8)
HER2 negative
B
I
O
P
S
Y
R
Letrozole
Anastrozole
Genome Sequencing
Somatic HER2
mutation
V777L
del.755-759
S
U
R
G
E
R
Y
Exemestane
Genome Sequencing
Stage
ER
PR
HER2
IIB
IIB
+
+
+
+
Negative
Negative
PI: Matthew Ellis
HER2 Somatic Mutations identified by
TCGA Breast Cancer Project
Somatic HER2 Stage ER PR
mutations
HER2
Status
HER2
IHC
G309A
R678Q and
L755W
IIB
IIB
+
+
+
+
Negative
Negative
1+
1+
L755S
L755S
D769H
V777L
V842I
IIA
I
IIB
IIIA
IIIB
+
+
+
+
+ Negative*
- Negative*
- Positive
+ Negative
+ Negative
2+
2+
3+
0
1+
HER2 FISH
Ratio
Not available
2.05
* Confirmed by SNP chip and/or exome sequencing read number.
25 Patients with HER2 Somatic Mutations
• Each blue circle represents a patient.
• Sequencing data from 1,499 patients. (8 published studies)
• 20% of affected patients have mutations at a.a. 309-310.
• 68% of affected patients have mutations at a.a. 755-781.
Copy Number and RNA Expression of HER2
Mutations in TCGA patients
Gene Expression from Microarray data
A. Exome seq. gene copy no.
HER2
mutation
D769H
R678Q+ L755S
L755W
V842I
V777L
G309A
L755S
L755S
HER2 +
Patients
HER2
Negative
Patients
1
B. RNA Seq read counts
2
4
8
16
32
HER2 Gene Copy Number
Mutant WT
Allele Allele
reads reads
864
745
%
Mutant
Allele
53.7%
L755S
D769H
509
2099
164
1280
75.6%
62.1%
V777L
253
143
63.9%
V842I
N/A
N/A
N/A
G309A
80
416
16.1%
569
538
608
585
51.4% for
R678Q
51.0% for
L755W
R678Q +
L755W
Protein Structure Visualizations
of HER2 Somatic Mutations
EC Domain Mutations
Kinase Domain Mutations
I767
D769 N-lobe
V777
L755
755-759
αC helix
780781
S310
G309
HER2
EGFR
ECD Dimer
Arm
Y835
EGFR
ECD
V842
R896
C-lobe
Protein Structure Visualizations
of HER2 Somatic Mutations
L755
6.6 & 7.0 Å
Kinase
Inhibitor
Kinase Domain Mutations
I767
D769 N-lobe
V777
L755
αC
helix
755-759
αC helix
780781
Y835
V842
R896
C-lobe
In vitro Kinase Activity of 3 HER2 Mutations
Specific Activity
(pmol product/µg Her2/min)
250
Monomer
Dimer
33-fold
200
22-fold
150
20-fold
100
7.5-fold
50
5-fold
2.3-fold
3.3-fold
0
WT
D769H
V777L
V842I
Fold increase is relative to WT HER2 monomer specific activity
Increased Signaling by HER2 mutants
KD
JM ECD
MW
[kDa]
phospho-HER2
KD = Kinase domain
JM = Juxtamembrane
domain
ECD = Extracellular
domain
150
phospho-EGFR
150
Substrate
phospho-PLC-γ
150
Total HER2
150
Total EGFR
150
Total PLC-γ
150
Cell Line: MCF10A. Identical results were also obtained in MCF7 and NIH3T3 cells.
Inhibition of Signaling by Neratinib and Lapatinib
WT
V777L
L755S
MW
[kDa]
Lapatinib (µM)
phospho-HER2
150
phospho-PLC-γ
150
Total HER2
150
Total PLC-γ
150
WT
Neratinib (µM)
phospho-HER2
phospho-PLC-γ
Total HER2
Total PLC-γ
L755S
MW
[kDa]
150
150
150
150
KD
WT
V777L
D769H
V842I
L755S
Del.
755-759
WT
L755S
Del.
755-759
+100 µg/ml
Trastuzumab
+ 0.5 µM
Lapatinib
Colony Morphology
in Matrigel Culture
+ 0.5 µM
Neratinib
JM
ECD
R678Q
G309A
HER2 mutations Increase Xenograft Growth
G309A D769H
Tumor Volume (mm3)
V777L
WT
Empty vector
Time (Days)
Cell Growth Inhibition by Neratinib and Lapatinib
IC50 (nM)
Neratinib
Lapatinib
<2
400 ± 60
G309A
<2
470 ± 50
V777L
<2
1,040 ± 570
D769H
<2
980 ± 950
V842I
<2
650 ± 210
2.1 ± 0.2
660 ± 90
15 ± 6
> 10,000
BT474 cells
<2
31 ± 2
MCF7 cells
> 3,000
> 10,000
MCF10A - Her2 WT
Del755-759
L755S
HER2 mutations - Conclusions
1. HER2 mutations predominantly occur in HER2 gene
amplification negative patients.
2. The majority of HER2 mutations are activating events that
cause oncogenic transformation, thus they are highly likely
to be driver events in these breast cancers.
3. Neratinib is a highly potent, irreversible pan-ErbB tyrosine
kinase inhibitor for all of the HER2 mutations.
4. A multi-institution, phase II trial of neratinib treatment for
metastatic breast cancer with HER2 somatic mutations has
been launched.
Acknowledgements
Washington University
Breast Cancer Program
Matthew Ellis
Cynthia Ma
Shyam Kavuri
Adam Searleman
John Monsey
Wei Shen
Shunqiang Li
Timothy Collier
Tom Kitchens
Adam Aronson
Genome Institute
Elaine Mardis
Rick Wilson
Li Ding
Dong Shen
Dan Koboldt
TCGA Project
Charles Perou
Katherine Hoadley
Kenna Shaw (NCI)
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