1. PI3K inhibition does not Effect the BH3 Profile of SW620 Cells
PHOSPHOINOSITIDE 3-KINASE INHIBITION SENSITISES COLORECTAL CANCER CELLS TO THE BH3 MIMETIC ABT-737 VIA AN AKT AND mTOR INDEPENDENT MECHANISM
Danielle Potter, Caroline Dive and Christopher Morrow. Clinical and Experimental Pharmacology
Introduction
Project Objectives
BH3 Profiling assay
Colorectal cancer (CRC) is the third most common cancer and is a leading cause of morbidity worldwide. The five year survival rate of patients with advanced or metastatic CRC is less than 5%, identifying the need for new and improved therapies in the treatment of advanced and metastatic CRC. Cancer cells can resist apoptosis by upregulation of survival pathways. The phosphoinositide 3-kinase (PI3K) pathway regulates numerous downstream targets involved in survival but PI3K inhibitors do not induce apoptosis in CRC cell lines. Components of this pathway such as PIK3CA, PIK3R1 and PTEN are commonly mutated in CRC making it an attractive therapeutic drug target. The BCL-2 family proteins regulate the intrinsic apoptotic pathway. CRC cell lines are relatively resistant to the BH3 mimetic, ABT-737 which is modeled on the pro-death BCL-2 family proteins, BAD. ABT-737 antagonises anti-apoptotic BCL-2 family members, BCL-2, BCL-xL and BCL-w, interfering with their interactions with pro-apoptotic BCL-2 family members, inducing apoptosis. When CRC cells were treated with PI3K inhibitors sensitivity towards ABT-737 induced apoptosis was increased. This sensitization is PI3K dependent but AKT and mTOR independent. To further elucidate this pathway, a siRNA library screen was carried out to identify downstream effectors of PI3K that sensitise to ABT-737. PI3K produces PtdIns(3,4,5)P3 which interacts with downstream effectors. This screen targeted the mRNA of proteins with a pleckstrin homology domains that interact with PtdIns(3,4,5)P3 and therefore have the potential to be regulated by PI3K.
Cells
Mix Together
Add to plate
Analyse
Staining Buffer
JC1
BH3-only peptides
monomers
aggregates
ABT-737
PIP2
PIP3
PI3K ?
TOP DOWN
PI-103
BOTTOM UP
BAK/BAX
BCL - 2 xL w
MCL 1 A1
BIM
BID
BAD
PUMA
NOXA
BMF
HRK
Anti
apoptotic
Pro
Two complementary approaches
Pro-death BH3-only
BIM
BID
BAD
NOXA
HRK
BCL-2
BCL-xL
MCL-1
No binding
Anti-death
Binding
Activators
(activate BAX/BAK)
Sensitizers
PI3K inhibition does not Effect the BH3 Profile of SW620 Cells
Objective 2 Identify changes to the BCL-2 family caused by PI3K inhibition
Objective 1 Identify signalling effectors downstream of PI3K
PI-103 Caused Inhibition of PI3K Signalling and Increased ABT-737 Sensitivity
siRNA Screen for PI3K Downstream Effectors that Sensitise to ABT-737
SW620 cell were transfected in a 96 well plate with siRNA library
[PI-103] (mM)
0.125
0.25
0.5 1 2
pS473 AKT
Total AKT
pT246 PRAS40
Total PRAS40
60 kDa
40 kDa
SW620 cells were either treated with DMSO or 2 μM PI-103 for 8 hours prior to BH3 profiling. Permeabilized and JC1 stained cells were incubated with specific peptides. Loss of JC1 red fluorescence was measured every 5 minutes for 3 hours. Loss of JC-1 red fluorescence indicates mitochondria outer membrane permeability.
Treated with DMSO
Treated with ABT-737
48 hours after transfection cells were either drugged with DMSO or ABT-737
BCL-2, BCL-xL and MCL-1 RNAi Sensitised to PI3K but not AKT or mTOR Inhibitors
SW62O cells a CRC cell line were treated with increasing concentration of ABT-737 alone or in combination with PI-103. Treatment with PI-103 caused a left shift in concentration response to ABT-737 demonstrating sensitisation. PI-103 caused inhibition on the PI3K pathway in a concentration dependent manner, indicated by decrease in phosphorylation of AKT and PRAS40 which are downstream effectors of this pathway.
72 hours after drugging, SRB assay was carried out on cells so cellular biomass could be analysed
Potential Sensitizers to ABT-737
MCL-1
BCL-2
BCL-xL
siNT
siMCL-1
siBCL-2
siBCL-xL
40 kDa
30 kDa
25 kDa
GAPDH
ABT-737 Sensitised to PI3K inhibitors but not AKT or mTOR inhibitors
HITS
Desensitizers:
PIK3CB
PIK3R1
STAT1
Sensitizers:
SOS1
PLEKHB2
BMX
STAT2
SGK1
MK-2206
GDC-0941
PI-103 KU
PI3K
AKT
mTORC1
mTORC2
PDK1
S6K ?
Conclusion
PI3K inhibition enhanced the efficacy of ABT-737 in CRC cell lines
MK-2206 (AKT inhibitor) and KU (mTOR inhibitor) do not cause sensitivity to ABT-737 suggesting sensitization is caused by a PI3K dependent, AKT and mTOR independent pathway
A siRNA library screen of potential PI3K downstream effectors revealed 5 candidate hits that cause sensitivity to ABT-737: SOS1, PLEKHB2, BMX, STAT2 and SGK1. These must be validated as bona-fide ABT-737 sensitizers
CRC cells are reliant on BCL-2, BCL-xL and MCL-1 to resist PI3K inhibitor induced apoptosis as knock down of all three sensitise to PI3K inhibitors
The rational drug combination of PI3K inhibitors with BH3 mimetics could provide a novel therapeutic strategy in the treatment of advanced and metastatic CRC
Can I K/D hits using RNAi
Deconvolved SMARTpool siRNA to verify K/D of hit.
Validation of hits
Does RNAi cause sensitivity to ABT-737
ABT-737 concentration response curve using hit RNAi cells or NT RNAi cells
Does RNAi cause sensitivity to ABT-737 in expanded CRC cell panel (3 cell lines)
At this point if everything is yes then pretty confident we have a hit No
Yes
SW620 cells were treated with increasing concentration of either PI-103, GDC-0941, MK-2206 or KU alone or in combination with ABT-737. ABT-737 caused sensitivity to PI-103 and GDC-0941 but not MK-2206 or KU This suggests that sensitisation is via a PI3K dependent, AKT and mTOR independent pathway.