Determining the role of the PI3K/AKT pathway

in KRAS mutant pancreatic cancer

07.24.2023
Merissa R. Smith
SURP 2023
Dr. Aaron Hobbs
1
 RAS is commonly mutated in cancer
RAS mutation frequency Estimated US cancer deaths
% Cancer Site Deaths %
97 Pancreatic ductal adenocarcinoma Lung & bronchus 135,720 22.4
52 Colorectal adenocarcinoma Colon & rectum 53,200 8.8
43 Multiple myeloma Pancreas 47,050 7.8
32 Lung adenocarcinoma Breast 42,690 7.0
28 Skin cutaneous melanoma Prostate 33,330 5.5
25 Uterine corpus endometrioid carcinoma Liver & intrahepatic bile duct 30,160 5.0
13 Thyroid carcinoma Non-Hodgkin lymphoma 19,940 3.3
13 Uterine carcinosarcoma Brain & nervous system 18,020 3.0
12 Stomach adenocarcinoma Urinary bladder 17,980 3.0
11 Acute myeloid leukaemia Esophagus 16,170 2.7
11 Bladder urothelial carcinoma Kidney & renal pelvis 14,830 2.4
8 Cervical adenocarcinoma Ovary 13,940 2.3
6 Head & neck squamous cell carcinoma Myeloma 12,830 2.1

Cox et al (2014) Nat Rev Drug Discov 13:828 Siegel et al (2020) CA Cancer J Clin 70:9
Why do we study pancreatic cancer?

• KRAS is mutated in nearly 100% of cases

• Initiating mutation

• We have preclinical models that can help us transition

research findings to the clinic
• GEMMs; PDX tumors; organoids; cell lines

• The current treatment consists of conventional

cytotoxic drugs and is ineffective
• 5-year survival of 12%; when diagnosed at Stage 1, 37%
• No effective targeted therapies
 KRASG12R mutations are rare in cancer overall
but common in pancreatic cancer
KRASG12R mutation frequency in cancer
G12R
(3%) Cancer % KRAS
Pancreatic ductal adenocarcinoma* 18.4%
G12D
G12V Colorectal cancer* 1.6%
G13D Lung adenocarcinoma* 0.5%
G12D G12C All cancers 3.3%
34% G12A
G12C G12S
12% Other (~5%)
G12R
Q61H
G13D G13C
13% A146T G12R
G12V G12D
Q61L ~20%
23% G12D G12V
G13S ~45% G12R
Other
Other
G12V
~30%
All cancers
(COSMIC)
Pancreatic cancer
(TCGA, 2017, Cancer Cell 32:185) *cBioPortal
 KRASG12R-mutant PDAC may be sensitive to
isoform-specific PI3K inhibition
GDP inactive
inactive GDP GDP
Pi KRAS
KRAS GTP Pi KRAS
Pi GTP GTP

GAP G12D GEF GAP

GAP WT GEF KRASG12R GEF
GTP
GTP GDP GTP
GDP KRAS GDP
KRAS KRAS
active active ROS
PTEN
RAL PI3Ka
RAL PI3Ka RAL
RAF
RAF RAF

PI3Ka PI3Kg PI3Kd

Unchecked
proliferation/growth proliferation/growth
? ?
Unchecked
proliferation/growth
All four Class I PI3K isoforms are
expressed in PDAC
KRASG12D KRASG12R

p110α, p110β:
ubiquitous

TCC-PAN2

VMP-395T
VMP-654

VMP-366

VMP-188
HPAF-II
AsPC-1
P110δ:

Pa16C

PSN-1
HPAC

PK-8

KP2
leukocytes p110a

p110b

p110γ: p110d

leukocytes p110g

Vinculin
Question: What is the role of Class 1 PI3K
isoforms in promoting PDAC proliferation?

Is any single
PI3K isoform
essential in
PDAC?
Single-Agent Class I PI3K Isoforms Inhibition fails to
subside PDAC Proliferation
Area Under Curve
500
Aspc1
100
Cell survival (%) relative to day 0

A818

Total Proliferation
75 400 HUPT3
50 PK8
300
PSN1
25
200 TCC-PAN
0 VMP 395T
100
-25 VMP 366
-9 -8 -7 -6 -5 -4 0
[Inhibitor] (logM) VMP 188

b
Pi 63
49
b

1
22

si
i
lis

0
I-5

ili
X-

I-3
e

ct
IP
lp

TG

IP
A PI3K Inhibitors
 Single-Agent Class I PI3K Isoforms Inhibition fails to
subside PDAC Proliferation
Area Under Curve
Published Cellular IC50 values
500
Aspc1
Alpelisiba TGX- IPI- IPI- Pictilisibc A818
221 3063 549b

Total Proliferation
400 HUPT3
p110α 5 nM 5000 1901 250 3 nM *
nM nM nM PK8
300
p110β 1200 nM 7 nM 102 240 33 nM PSN1
nM nM 200 TCC-PAN
p110γ 250 nM >350 419 1.2 75 nM VMP 395T
0 nM nM nM 100
VMP 366
p110δ 290 nM 100 0.1 180 3 nM
0 VMP 188
nM nM nM

b
Pi 63
49
b

1
22

si
i
lis

0
I-5

ili
X-

I-3
e

ct
IP
lp

TG

IP
A
PI3K Inhibitors
Single-Agent Class I PI3K Isoforms Inhibition fails to
subside PDAC Proliferation
Area Under Curve
TCC-PAN2
500
Aspc1
A818
Alpelisib 100nM

Pictilisib 1.5uM
IPI3063 100nM
TGX 221 50nM
IPI 549 300nM

Total Proliferation
400 HUPT3
PK8
Veh

300
pAKT S473
PSN1
pAKT T308 200 TCC-PAN
AKT VMP 395T
100
pPras40
VMP 366
Pras40
0 VMP 188
Vinculin

b
Pi 63
49
b

1
22

si
i
lis

0
I-5

ili
X-

I-3
e

ct
IP
lp

TG

IP
A
What about the negative regulator of PI3K signaling?
PI3K Inhibitors
Non-reducing SDS-PAGE detects oxidized PTEN

ASPC1 A818 HUPT3 PK8 PSN1 TCC-PAN VMP 366

PTEN

VINC

If PTEN is oxidized (inactivated), then PTEN knockdown should not effect cell proliferation
 PTEN knockdown has minimal effect on cell proliferation
PTEN knockdown
500
Non-Specific

Normalized Cell proliferation (%)

400 siPTEN

300

200
ASPC-1 A818.4 HuP-T3 PK-8 PSN-1
100
siRNA NS PTEN NS PTEN NS PTEN NS PTEN NS PTEN
0
PTEN

.4

-1
-1

T3

-8
18

N
C

PK
P-

PS
P

A8

U
AS

H
pPRAS PDAC Cell lines

ASPC-1 A818.4 HuP-T3 PK-8 PSN-1

PRAS

VINC NS

siPTEN

N=1
 Conclusion

• Inhibiting one Isoform of the Class I PI3K doesn’t have a

significant impact on cell proliferation.
• All Isoforms of PI3K need to be inhibited to stop
proliferation.
• We propose that PTEN oxidation mimics PTEN loss
 Future Directions:
Lentiviral Insertion of mutant-PTEN in PDAC
ASPC1 HUPT3 PK8 PSN1

EV PTEN C71A C124A EV PTEN C71A C124A EV PTEN C71A C124A EV PTEN C71A C124A

PTEN
HA

VINC

PTEN constructs:
PTEN Overexpression

C124A
C71A
WT
EV
1.5
EV
Normalized Cell proliferation (%)

WT ASPC-1

1.0 C71A
C124A
HuP-T3
0.5

PK-8
0.0
-1
-1

T3

-8

N
C

PK

PSN-1
P-

PS
P

U
AS

PDAC Cell lines

Acknowledgements
MUSC
Hobbs Lab Mike Ostrowski
Aaron Hobbs Sam Saberikashani
Kamala Sundararaj Sudu Sharma
Rachel Burge Lu Han
Lucas Bialousow Dennis Guttridge
Clara Errard Toros Dincman
John O’Bryan
Lauren Ball
Martin Romero
Silvia Vaena
Collaborators
Tim Barnoud
Dave Kashatus, UVA
Todd Bauer, UVA
Sharon Campbell, UNC
Chris Counter, Duke
Udo Rudloff, NCI
Questions?