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-0063794 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-0063794 (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-0063794 alone or in combination with ABT-737. ABT-737 caused sensitivity to PI-103 and GDC-0941 but not MK-2206 or KU-0063794. This suggests that sensitisation is via a PI3K dependent, AKT and mTOR independent pathway.