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Sample Graphs for torc2 Paper. Akt Crystal Structure. S473A. T450A. D292A. T308A. K179M. R25C. K8R. AA131. E17K. AA107. K14R. PH Domain. - Akt crystal structure PDB #3O96 - AAs 430-480 modeled using I-TASSER - Image generated using CHIMERA (UCSF). Mutational Data (HCT-116).

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slide2

Akt Crystal Structure

S473A

T450A

D292A

T308A

K179M

R25C

K8R

AA131

E17K

AA107

K14R

PH Domain

- Akt crystal structure PDB #3O96

- AAs 430-480 modeled using I-TASSER

- Image generated using CHIMERA (UCSF)

slide3

Mutational Data

(HCT-116)

Control (HCT-116 wt)

G0

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

Control (HCT-116 wt)

Asymmetry

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

Akt1

Akt2

No rescue

D292A

T308A

K179M

G0

ASY

G0

ASY

G0

ASY

G0

ASY

G0

ASY

G0

ASY

S473A

T308A/S473A

T450A

T308D

S473D

myrΔ1-107

G0

ASY

G0

ASY

G0

ASY

G0

ASY

G0

ASY

G0

ASY

E17K

R25C

myrΔ1-107

K179M

Δ1-131

Δ1-107

G0

ASY

G0

ASY

G0

ASY

G0

ASY

G0

ASY

K8R

K14R

K8/14R

G0

ASY

G0

ASY

G0

ASY

mutational analysis hct 116
Mutational Analysis(HCT-116)

G0 Count

AKT1/2 k.o.

AKT rescue

ΔKinase domain

ΔCanonical phosphorylation sites

ΔUbiquitination domain

Myristylation

ΔPlekstrin homology

% of Wildtype Phenotype

E17K

R25C

delPH107

delPH131

Akt1/2 k.o.

AKt1 rescue

AKt2 rescue

K179M

D292A

T308A

S473A

T308A/S473A

T450A

T308D

S473D

K8R

K14R

K8R/K14R

Myr-delPH107

Myr-delPH107(K179M)

Genetic Construct

Asymmetry Count

AKT1/2 k.o.

AKT rescue

ΔKinase domain

ΔCanonical phosphorylation sites

ΔUbiquitination domain

Myristylation

ΔPlekstrin homology

% of Wildtype Phenotype

E17K

R25C

delPH107

delPH131

Akt1/2 k.o.

AKt1 rescue

AKt2 rescue

K179M

D292A

T308A

S473A

T308A/S473A

T450A

T308D

S473D

K8R

K14R

K8R/K14R

Myr-delPH107

Myr-delPH107(K179M)

Genetic Construct

slide5

Pharmacologic Data (HCT-116)

Control (HCT-116)

G0

Experimental (HCT-116 + drug.)

Control (HCT-116)

Asymmetry

Experimental (HCT-116+ drug.)

AKT PH domain inhibitors

AKT kinase inhibitors

AKt1/2i (20uM)

MK2206 (10uM)

AZD5363

(50uM)

GDC0068

(50uM)

G0

ASY

G0

ASY

G0

ASY

G0

ASY

Torc1 inhibitors

Torc1/2 inhibitors

Rapamycin (20uM)

Rad001 (10uM)

INK128 (0.05uM)

AZD8055 (0.7uM)

Palomid529 (10uM)

TORIN 1 (0.5uM)

G0

ASY

G0

ASY

G0

ASY

G0

ASY

G0

ASY

G0

ASY

Proteasomal inhibitors

MG132 1d (1uM)

Velcade 1d

(0.4uM)

G0

ASY

G0

ASY

small molecule treatment hct 116
Small Molecule Treatment (HCT-116)

G0 Count

AKT PHD inhibitor

Torc1 inhibitor

AKT kinase domain inhibitor

Torc1/2 inhibitor

Torc1 inhibitor

Proteasomal inhibitor

Torc1/2 inhibitor

Proteasomal inhibitor

% of Wildtype Phenotype (Logarithmic Axis)

% of Wildtype Phenotype (Without AKT inhibitors)

Akt1/2i

MK2206

GDC0063

AZD5363

Rapamycin

Rad001

Torin1

Palomid529

Ink128

AZD8055

MG132

Velcade

Rapamycin

Rad001

Torin1

Palomid529

Ink128

AZD8055

MG132

Velcade

Small Molecule

Small Molecule

Asymmetry Count

AKT PHD inhibitor

Torc1 inhibitor

AKT kinase domain inhibitor

Torc1/2 inhibitor

Torc1 inhibitor

Proteasomal inhibitor

Torc1/2 inhibitor

Proteasomal inhibitor

% of Wildtype Phenotype (Logarithmic Axis)

% of Wildtype Phenotype (Without AKT inhibitors)

Rapamycin

Rad001

Torin1

Palomid529

Ink128

AZD8055

MG132

Velcade

Akt1/2i

MK2206

GDC0063

AZD5363

Rapamycin

Rad001

Torin1

Palomid529

Ink128

AZD8055

MG132

Velcade

Small Molecule

Small Molecule

slide7

Pharmacologic Data (MCF-7)

Control (MCF-7)

G0

Experimental (MCF-7 + drug.)

Control (MCF-7)

Asymmetry

Experimental (MCF-7 + drug.)

AKT PH domain inhibitors

AKT kinase inhibitors

MK2206 (3uM)

AZD5363

(5uM)

AKt1/2i (2uM)

GDC0068

(5uM)

G0

ASY

G0

ASY

G0

ASY

G0

ASY

Torc1 inhibitors

Torc1/2 inhibitors

Rapamycin (20uM)

Rad001 (5uM)

INK128 (0.01uM)

AZD8055 (0.1uM)

Palomid529 (20uM)

TORIN 1 (0.25uM)

G0

ASY

G0

ASY

G0

ASY

G0

ASY

G0

ASY

G0

ASY

Proteasomal inhibitors

MG132 1d (10uM)

Velcade 1d

(4uM)

TTC3 K.D.

TTC3hp3 K.D.

G0

ASY

G0

ASY

G0

ASY

G0

ASY

small molecule treatment mcf7
Small Molecule Treatment (MCF7)

G0 Count

AKT PHD inhibitor

Torc1 inhibitor

AKT kinase domain inhibitor

Torc1/2 inhibitor

Torc1 inhibitor

Proteasomal inhibitor

Torc1/2 inhibitor

Proteasomal inhibitor

% of Wildtype Phenotype (Logarithmic Axis)

% of Wildtype Phenotype (Without AKT inhibitors)

Akt1/2i

MK2206

GDC0063

AZD5363

Rapamycin

Rad001

Torin1

Palomid529

Ink128

AZD8055

MG132

Velcade

Rapamycin

Rad001

Torin1

Palomid529

Ink128

AZD8055

MG132

Velcade

Small Molecule

Small Molecule

Asymmetry Count

AKT PHD inhibitor

Torc1 inhibitor

AKT kinase domain inhibitor

Torc1/2 inhibitor

Torc1 inhibitor

Proteasomal inhibitor

Torc1/2 inhibitor

Proteasomal inhibitor

% of Wildtype Phenotype (Logarithmic Axis)

% of Wildtype Phenotype (Without AKT inhibitors)

Rapamycin

Rad001

Torin1

Palomid529

Ink128

AZD8055

MG132

Velcade

Akt1/2i

MK2206

GDC0063

AZD5363

Rapamycin

Rad001

Torin1

Palomid529

Ink128

AZD8055

MG132

Velcade

Small Molecule

Small Molecule

slide9

shRNA Rictor Data (HCT-116)

Control (HCT-116 + non-silencing shRNA)

G0

Experimental (HCT-116 + hairpin)

Control (HCT-116 + non-silencing shRNA)

Asymmetry

Experimental (HCT-116 + hairpin)

(Control = non-silencing)

Empty Vector

shRNA-225915

shRNA-120392

shRNA-120389

shRNA-38014

G0

ASY

G0

ASY

G0

ASY

G0

ASY

G0

ASY

(Control = empty-vector)

Non-Silencing

shRNA-225915

shRNA-120392

shRNA-120389

shRNA-38014

G0

ASY

G0

ASY

G0

ASY

G0

ASY

G0

ASY

live cell imaging results 2 18 12
Live-cell Imaging Results2/18/12
  • HCT-116 shRictor variants
    • +/- 1ug/mL doxy
alpha plot comparison
Alpha Plot comparison

No doxy

1ug/mL doxy

live cell imaging results 3 12 12
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)
slide15

Cell Fate – Overall Comparison

Death (20%)

Non-silencing 2 ug/mL Doxy

Mitosis (52%)

Lost (28%)

Other (1%)

Death (20%)

shRNA225915 No Rx

Mitosis (55%)

Lost (24%)

Other (1%)

Death (18%)

Mitosis (55%)

shRNA225915 2 ug/mL Doxy

Lost (26%)

slide16

Alpha Plot comparison

Non-silencing + doxy

225915 + No doxy

225915 + doxy

boxplot of cell cycle time hct116 shrna rictor 2259151
Boxplot of Cell Cycle Time(HCT116 shRNA-Rictor 225915)

Non-silencing 2ug/mL doxy

225915 2ug/mL doxy

225915 2ug/mL doxy

225915 No doxy

slide18

Beta Plot comparison

Non-silencing + doxy

225915 + No doxy

225915 + doxy

boxplot of sibling sibling cell cycle time hct116 shrna rictor 225915
Boxplot of Sibling-sibling Cell Cycle Time(HCT116 shRNA-Rictor 225915)

Non-silencing 2ug/mL doxy

225915 2ug/mL doxy

225915 2ug/mL doxy

225915 No doxy