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Neratinib (nM). SKBR3. Supplementary figure S4. A) Response to lapatinib. Cell survival assay using HER2-positive breast cancer cells expressing equal ...
A

B

C

Supplementary figure S1. A) Localization of EGFR mutations. Mutations (cyan) are superimposed onto wild-type (magenta) on the model for EGFR bound to lapatinip (stick model with carbons in salmon). The initial crystallographic model (1XKK) has been completed by homology modeling. Lower panels show details of the top panel. For G857E, the left figure shows the mutation in the inactive, lapatinib-bound kinase form. The right figure shows G857E in the active kinase conformation. B) Localization of HER2 mutations. Top left shows the mutations projected onto HER2 in an inactive conformation. Top right shows mutations projected onto HER2 in an active conformation (with the activator kinase shown in gray). Lower panels show details of the top panels. For L726F the left panel shows the mutation projected onto the HER2 model produced from the lapatinib-bound EGFR structure (1XKK). The right panel shows the model based on the HER2 kinase in its TKI-bound active-like conformation (3PP0). For V794M, the top left panel shows the model based on activated EGFR kinase (2GS6), lower left panel shows model based on the ‘receiver’ molecule of EGFR (K721M) in an inactive conformation (3GOP); top left shows stick model, bottom left the corresponding van der Waals sphere model. ‘activator’ molecule is in gray, ‘receiver’ in green. Right V794M panel shows model built on HER2 in active-like conformation (open conformation, but with the αC helix in an inactive conformation; 3PP0). C) Localization of HER4 mutations. Lower panels show details of the top panel. For details see Supplementary Information.

MDA-MB-175

MCF10A

cmyc

cmyc

Her2

Her2

Actin

Actin

SKBR3

cmyc

BT474-m1

cmyc

Her2

Her2

Actin

Actin

Supplementary figure S2. Expression levels of exogenous HER2 wt and mutants in each cell line are comparable.

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A

3.5

Cell number (10^6)

3

pLVX WT

2.5

L726F

2

V794M D808N

1.5 1 0.5 0 0

1

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Days

2.50

B Cell number (10^6)

2.00 pLVX 1.50

WT L726F V794M

1.00

D808N 0.50

0.00 0

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Days

Supplementary figure S3. Proliferation of MCF10A cells in complete growth medium (A) and in serum-starved conditions (B).

A

SKBR3

BT474-m1 100 80 60 40 20 0 0

0.05

0.1

0.25

0.5

1

Survival assay % of untreated control

Survival assay % of untreated control

Survival assay % of untreated control

MDA-MB-175 120

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100 80 60 40 20 0

B

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0.05 0.1 0.25 0.5

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80 60 40

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MDA-MB-175

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**

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Lapatinib (µM)

Survival assay % of untreated control

Survival assay % of untreated control

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BT474-m1

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*

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Lapatinib (µM)

**

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Lapatinib (µM)

Survival assay % of untreated control

***

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***

0 0

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3 4 5 6 7 Neratinib (nM)

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3 4 5 6 7 Neratinib (nM)

C

Supplementary figure S4. A) Response to lapatinib. Cell survival assay using HER2-positive breast cancer cells expressing equal amounts of exogenous HER2 wt or L726F and incubated with lapatinib. IC50 calculated using GraphPad Prism 6 : BT474-m1/WT=0.09 µM and BT474-m1/L726F=0.41 µM ; SKBR3/WT=0.19 µM and SKBR3/L726F=1.03 µM ; MDA-MB-175/WT=0.16 µM and MDA-MB175/L726F=0.68 µM. B) Response to neratinib. Cell survival assay using HER2-positive breast cancer cells expressing equal amounts of exogenous HER2 wt or L726F and incubated with neratinib. C) Lapatinib and neratinib bind similarly to the kinase active site. Comparison of the experimental structures of EGFR bound to lapatinib (kinase: cyan; compound: pale orange; PDB 1XKK) and to neratinib (kinase: green; compound: magenta; PDB 2JIV).

lapatinib (µM)

MDA-MB-175

SKBR3

0 .01 .025 .05 .1 .25 .5 1

0 .01 .025 .05 .1 .25 .5 1

cmyc (wt) cmyc (L726F) Akt (wt) Akt (L726F) Erk1/2 (wt) Erk1/2 (L726F)

Supplementary figure S5: Protein levels from figure 5C.

MDA-MB-175

MCF10A

P-1248 Her2

P-1248 Her2

cmyc

cmyc

Her2

Her2

Actin

Actin

SKBR3

BT474-m1

P-1248 Her2

P-1248 Her2

cmyc

cmyc

Her2

Her2

Actin

Actin

Supplementary figure S6: Underphosphorylation of HER2 L726F was consistent in all cell lines.

WCL

IP

IP

Dimers

Monomers

Low exposition time

High exposition time

Supplementary figure S7: The underphosphorylated state of HER2 L726F is not due to an impaired homodimerization of HER2.

MDA-MB-175

Primary Tumor

L726F

WT

MCF0A

Supplementary figure S8: HER2 localization is impaired in both breast cancer cells and tissues.

Mutation EGFR. L792F EGFR. V843I EGFR. G857E EGFR. E804D EGFR. G735S EGFR. N842I HER2. L726F HER2. V794M HER2. D808N HER4. M887I HER4. R838Q HER4. G785S

MT peak/WT peak estimate (%) ~50% ~15% ~40% ~15% ~30% ~10% ~95% 100% ~30% ~20% ~25% ~15%

Supplementary figure S9: Estimated mutant to wild-type peak ratios for all the variants detected .

WT

17AAG (nM)

0

L726F

50 75 100 150 200 0 50 75 100 150 200

P-1248 Her2

cmyc

Actin

Supplementary figure S10: HER2 WT and L726F expression after 48 hours incubation with 17AAG in MCF10A.