Sample Graphs for torc2 Paper

T450A

S473A

D292A

K179M

E17K

R25C

K8R

K14R

T308A

AA107

AA131

PH Domain

Akt Crystal Structure

- Akt crystal structure PDB #3O96- AAs 430-480 modeled using I-TASSER- Image generated using CHIMERA (UCSF)

No rescue Akt1 Akt2 K179M D292A T308A

T450A T308D S473D myrΔ1-107S473A T308A/S473A

myrΔ1-107K179M

E17K R25C Δ1-131 Δ1-107

Mutational Data(HCT-116)

G0 ASY G0 ASY G0 ASY G0 ASY G0 ASY G0 ASY

G0 ASY G0 ASY G0 ASY G0 ASY G0 ASY G0 ASY

G0 ASY G0 ASY G0 ASY G0 ASY G0 ASY

Control (HCT-116 wt)Experimental (HCT-116 Akt1/2 k.o.)

Control (HCT-116 wt)Experimental (HCT-116 Akt1/2 k.o.)

G0

Asymmetry

K8R K14R

G0 ASY G0 ASY

K8/14R

G0 ASY

Mutational Analysis(HCT-116)

G0 Count

AKT1/2 k.o.

AKT rescue

ΔKinase domain

ΔCanonical phosphorylation sites

ΔUbiquitination domain

Myristylation

ΔPlekstrin homology

Akt1/2 k.o.

AKt1 rescu

e

AKt2 rescu

e

K179MD292A

T308AS4

73A

T308A/S473A

T450AT308D

S473D

K8RK14R

K8R/K14R

Myr-delPH107

Myr-delPH107(K179M)

E17KR25C

delPH107

delPH131

% o

f Wild

type

Phe

noty

pe

Genetic Construct

Asymmetry Count

Akt1/2 k.o.

AKt1 rescu

e

AKt2 rescu

e

K179MD292A

T308AS4

73A

T308A/S473A

T450AT308D

S473D

K8RK14R

K8R/K14R

Myr-delPH107

E17KR25C

delPH107

delPH131

Myr-delPH107(K179M)

AKT1/2 k.o.

AKT rescue

ΔKinase domain

ΔCanonical phosphorylation sites

ΔUbiquitination domain

Myristylation

ΔPlekstrin homology

% o

f Wild

type

Phe

noty

pe

Genetic Construct

AKt1/2i (20uM)

MK2206 (10uM)

TORIN 1 (0.5uM)

Palomid529 (10uM)

INK128 (0.05uM)

AZD8055 (0.7uM)

Rapamycin (20uM)

Rad001 (10uM)

Pharmacologic Data (HCT-116)

G0 ASY G0 ASY

G0 ASY G0 ASY G0 ASY G0 ASYG0 ASY G0 ASY

Control (HCT-116)Experimental (HCT-116 + drug.)G0

Asymmetry Control (HCT-116)Experimental (HCT-116+ drug.)

MG132 1d (1uM)

G0 ASY

Velcade 1d(0.4uM)

G0 ASY

GDC0068(50uM)

AZD5363(50uM)

G0 ASY G0 ASY

AKT PH domain inhibitors AKT kinase inhibitors

Torc1 inhibitors Torc1/2 inhibitors

Proteasomal inhibitors

Small Molecule Treatment (HCT-116)

G0 Count

AKT PHD inhibitor

AKT kinase domain inhibitor

Torc1 inhibitor

Torc1/2 inhibitor

Proteasomal inhibitor

% o

f Wild

type

Phe

noty

pe

(Log

arith

mic

Axi

s)

Small Molecule

Asymmetry Count

Akt1/2i

MK2206

GDC0063

AZD5363

Rapamycin

Rad001To

rin1

Palomid529

Ink128

AZD8055

MG132

Velcade

AKT PHD inhibitor

AKT kinase domain inhibitor

Torc1 inhibitor

Torc1/2 inhibitor

Proteasomal inhibitor

% o

f Wild

type

Phe

noty

pe

(Log

arith

mic

Axi

s)

Small Molecule

Akt1/2i

MK2206

GDC0063

AZD5363

Rapamycin

Rad001To

rin1

Palomid529

Ink128

AZD8055

MG132

Velcade

Rapamycin

Rad001To

rin1

Palomid529

Ink128

AZD8055

MG132

Velcade

Torc1 inhibitor

Torc1/2 inhibitor

Proteasomal inhibitor

% o

f Wild

type

Phe

noty

pe

(With

out A

KT in

hibi

tors

)

Rapamycin

Rad001To

rin1

Palomid529

Ink128

AZD8055

MG132

Velcade

Torc1 inhibitor

Torc1/2 inhibitor

Proteasomal inhibitor

% o

f Wild

type

Phe

noty

pe

(With

out A

KT in

hibi

tors

)

Small Molecule

Small Molecule

AKt1/2i (2uM)

MK2206 (3uM)

TORIN 1 (0.25uM)

Palomid529 (20uM)

INK128 (0.01uM)

AZD8055 (0.1uM)

TTC3 K.D.

Rapamycin (20uM)

Rad001 (5uM)

Pharmacologic Data (MCF-7)

G0 ASY G0 ASY

G0 ASY G0 ASY G0 ASY G0 ASYG0 ASY G0 ASY

G0 ASY

Control (MCF-7)Experimental (MCF-7 + drug.)G0

Asymmetry Control (MCF-7)Experimental (MCF-7 + drug.)

TTC3hp3 K.D.

G0 ASY

MG132 1d (10uM)

G0 ASY

Velcade 1d (4uM)

G0 ASY

GDC0068(5uM)

AZD5363(5uM)

G0 ASY G0 ASY

AKT PH domain inhibitors AKT kinase inhibitors

Torc1 inhibitors Torc1/2 inhibitors

Proteasomal inhibitors

Small Molecule Treatment (MCF7)

G0 Count

AKT PHD inhibitor

AKT kinase domain inhibitor

Torc1 inhibitor

Torc1/2 inhibitor

Proteasomal inhibitor

% o

f Wild

type

Phe

noty

pe

(Log

arith

mic

Axi

s)

Small Molecule

Asymmetry Count

Akt1/2i

MK2206

GDC0063

AZD5363

Rapamycin

Rad001To

rin1

Palomid529

Ink128

AZD8055

MG132

Velcade

AKT PHD inhibitor

AKT kinase domain inhibitor

Torc1 inhibitor

Torc1/2 inhibitor

Proteasomal inhibitor

% o

f Wild

type

Phe

noty

pe

(Log

arith

mic

Axi

s)

Small Molecule

Akt1/2i

MK2206

GDC0063

AZD5363

Rapamycin

Rad001To

rin1

Palomid529

Ink128

AZD8055

MG132

Velcade

Rapamycin

Rad001To

rin1

Palomid529

Ink128

AZD8055

MG132

Velcade

Torc1 inhibitor

Torc1/2 inhibitor

Proteasomal inhibitor

% o

f Wild

type

Phe

noty

pe

(With

out A

KT in

hibi

tors

)

Rapamycin

Rad001To

rin1

Palomid529

Ink128

AZD8055

MG132

Velcade

Torc1 inhibitor

Torc1/2 inhibitor

Proteasomal inhibitor

% o

f Wild

type

Phe

noty

pe

(With

out A

KT in

hibi

tors

)

Small Molecule

Small Molecule

Empty Vector shRNA-225915

shRNA-120392 shRNA-120389 shRNA-38014

shRNA Rictor Data (HCT-116)

G0 ASY G0 ASY G0 ASY G0 ASY G0 ASY

Control (HCT-116 + non-silencing shRNA)Experimental (HCT-116 + hairpin)G0

Asymmetry Control (HCT-116 + non-silencing shRNA)Experimental (HCT-116 + hairpin)

(Control = non-silencing)

Non-Silencing

shRNA-225915

shRNA-120392 shRNA-120389 shRNA-38014

G0 ASY G0 ASY G0 ASY G0 ASY G0 ASY

(Control = empty-vector)

Live-cell Imaging Results2/18/12

• HCT-116 shRictor variants– +/- 1ug/mL doxy

Boxplot of Cell Cycle Time(HCT116 shRNA-Rictor 225915)

No doxy 1ug/mL doxy

Alpha Plot comparison

No doxy1ug/mL doxy

Live-cell Imaging Results3/12/12

• 3 conditions:– HCT-116 shNon-silencing + 2ug/mL

doxy • (~700 events)

– HCT-116 shRictor225915 No doxy • (~1300 events)

– HCT-116 shRictor225915 No +2ug/mL doxy • (~1300 events)

Cell Fate – Overall Comparison

shRNA225915 No Rx

shRNA225915 2 ug/mL Doxy

Mitosis (55%)Lost (24%)

Death (20%)Other (1%)

Mitosis (55%)Lost (26%)

Death (18%)

Other (1%)

Non-silencing 2 ug/mL Doxy

Mitosis (52%)Lost (28%)

Death (20%)

Alpha Plot comparison

225915 + No doxy225915 + doxy

Non-silencing + doxy

Boxplot of Cell Cycle Time(HCT116 shRNA-Rictor 225915)

225915 No doxy225915

2ug/mL doxy

Non-silencing 2ug/mL doxy

225915 2ug/mL doxy

Beta Plot comparison

225915 + No doxy225915 + doxy

Non-silencing + doxy

Boxplot of Sibling-sibling Cell Cycle Time

(HCT116 shRNA-Rictor 225915)

225915 No doxy225915

2ug/mL doxy

Non-silencing 2ug/mL doxy

225915 2ug/mL doxy