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A Genome-Scale CRISPR Screen Identifies the ERBB and Mtor Signaling Networks As Key Determinants of Response to PI3K Inhibition in Pancreatic Cancer

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A Genome-Scale CRISPR Screen Identifies the ERBB and Mtor Signaling Networks As Key Determinants of Response to PI3K Inhibition in Pancreatic Cancer Published OnlineFirst May 5, 2020; DOI: 10.1158/1535-7163.MCT-19-1131 MOLECULAR CANCER THERAPEUTICS | SMALL MOLECULE THERAPEUTICS A Genome-scale CRISPR Screen Identifies the ERBB and mTOR Signaling Networks as Key Determinants of Response to PI3K Inhibition in Pancreatic Cancer A C Charlotte K. Milton1, Annette J. Self1, Paul A. Clarke1, Udai Banerji1,2, Federica Piccioni3, David E. Root3, and Steven R. Whittaker1 ABSTRACT ◥ KRAS mutation is a key driver of pancreatic cancer and PI3K inhibition in spheroid, stromal cocultures. Near-complete loss of pathway activity is an additional requirement for Kras-induced ribosomal S6 phosphorylation was associated with synergy. Genetic tumorigenesis. Clinical trials of PI3K pathway inhibitors in pan- alterations in the ERBB–PI3K signaling axis were associated with creatic cancer have shown limited responses. Understanding the decreased survival of patients with pancreatic cancer. Suppression molecular basis for this lack of efficacy may direct future treatment of the PI3K/mTOR axis is potentiated by dual PI3K and ERBB strategies with emerging PI3K inhibitors. We sought new thera- family or mTOR inhibition. Surprisingly, despite the presence of peutic approaches that synergize with PI3K inhibitors through oncogenic KRAS, thought to bestow independence from receptor pooled CRISPR modifier genetic screening and a drug combination tyrosine kinase signaling, inhibition of the ERBB family blocks screen. ERBB family receptor tyrosine kinase signaling and mTOR downstream pathway activation and synergizes with PI3K inhibi- signaling were key modifiers of sensitivity to alpelisib and pictilisib. tors. Further exploration of these therapeutic combinations is Inhibition of the ERBB family or mTOR was synergistic with PI3K warranted for the treatment of pancreatic cancer. Introduction dent on PI3Ka (14, 15), but not PI3Kb (15), or Craf (14) for tumorigenesis. Consequently, PI3K signaling is an attractive thera- The 10-year survival rate for pancreatic ductal adenocarcinoma peutic target for PDAC. However, clinical trials of allosteric mTOR (PDAC) has remained at just 3% for the past 40 years (1). Activation of inhibitors, including temsirolimus (7), or everolimus (16), have shown the oncogene, KRAS, is one of the earliest genetic alterations detected limited activity in patients with gemcitabine-refractory, metastatic in the development of PDAC and KRAS mutations are found in over pancreatic cancer, likely due to loss of negative feedback on IRS1 and 90% of cases (2, 3). Transgenic mouse models expressing oncogenic reactivation of PI3K (16). Multiple oncogenic pathways are engaged KrasG12D have demonstrated that mutant KRAS is an important driver downstream of KRAS (17, 18), so it is perhaps unsurprising that in pancreatic cancer, as switching off Kras signaling results in tumor targeting a single downstream effector may not be enough to affect cell regression (4). Recently discovered inhibitors of KRAS12C have further viability. We hypothesize that PI3K inhibition selects for compensa- validated the dependency of pancreatic, colon, and lung tumor models tory mechanisms sufficient to maintain tumor cell survival. on oncogenic KRAS and demonstrated promising early clinical This study aimed to elucidate the mechanisms of intrinsic resistance activity (5, 6). to PI3K inhibition in pancreatic cancer and identify rational drug There is a strong rationale for targeting PI3K in PDAC. Activation combinations to overcome them. Functional genomic screens have of the PI3K pathway is commonly observed in PDAC patient successfully identified loss-of-function events that drive drug resis- samples (7–9), regulates cell metabolism, growth, and survival, and tance, finding NF1 loss to be a key driver of resistance to RAF is commonly implicated as a driver of human cancer (10). Importantly, inhibition in melanoma (19). We therefore employed a genome- phosphorylation of PI3K signaling markers, including AKT (Ser473), scale synthetic lethal CRISPR screen to find loss-of-function events mTOR (Ser2448), and GSK3b (Ser9; ref. 11) or low expression of that could modulate sensitivity to PI3K inhibition. We discovered that PTEN, a suppressor of PI3K signaling (12), is predictive of poor the ERBB and mTOR signaling networks regulate response to PI3K survival in pancreatic cancer. Moreover, the interaction between Ras inhibition in PDAC. Furthermore, we used a combination drug screen and PI3Ka is essential for KrasG12D-induced tumorigenesis in to prioritize clinically relevant targeted agents that synergize with PI3K mice (13). Notably, KrasG12D-driven murine PDAC tumors are depen- inhibition to improve therapeutic response. 1Division of Cancer Therapeutics, The Institute of Cancer Research, London, Materials and Methods United Kingdom. 2The Royal Marsden NHS Foundation Trust, London, United Kingdom. 3The Broad Institute, Cambridge, Massachusetts. Cell lines and cell culture Pancreatic cancer cell lines were a kind gift from Dr. Anguraj Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). Sadanandam (The Institute of Cancer Research, London, United Kingdom), with the exception of PANC1, PATU8902, MIAPACA2, Corresponding Author: Steven R. Whittaker, The Institute of Cancer Research, London SW7 3RP, United Kingdom. Phone: 4420-8722-4220; Fax: 4420-8770- YAPC, and HEK293T cells, which were obtained from the ATCC. 7899; E-mail: [email protected] T47D cells were from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ). All cells were cultured in DMEM (Sigma) Mol Cancer Ther 2020;19:1423–35 supplemented with 10 % FBS (FBS Good, Pan Biotech), with the doi: 10.1158/1535-7163.MCT-19-1131 exception of MIAPACA2, which was supplemented with 20% FBS. Ó2020 American Association for Cancer Research. Human pancreatic stellate cells (PSC) were obtained from ScienCell AACRJournals.org | 1423 Downloaded from mct.aacrjournals.org on October 1, 2021. © 2020 American Association for Cancer Research. Published OnlineFirst May 5, 2020; DOI: 10.1158/1535-7163.MCT-19-1131 Milton et al. laboratories. Recombinant growth factors were obtained from Bio- Cell lysis and Western blotting Techne. Cell lines were tested for Mycoplasma using the MycoAlert After the desired treatment, cells were washed with cold PBS and Mycoplasma Detection Kit (Lonza). Cell line authentication was not lysed in NP40 buffer [0.5% NP40, 150 mmol/L NaCl, 50 mmol/L Tris performed. pH 7.5, Pierce Protease and Phosphatase Inhibitor Mini Tablets (Life Technologies)]. Where detection of KRAS was necessary, cells were Small-molecule inhibitors lysed in SDS buffer (1 % SDS, 10 mmol/L EDTA, 50 mmol/L Tris, All small-molecule inhibitors were purchased from Selleck Chemi- pH 8). Bicinchoninic acid (Sigma) was used to determine protein cals: BYL719 (S2814), GDC0941 (S1065), pelitinib (S1392), everolimus concentration. Equal amounts of protein were separated by gel elec- (S1120), AZD8055 (S1555), AZD2014 (S2783), and BEZ235 (S1009). trophoresis, using NuPAGE polyacrylamide gels (Life Technologies). Stock solutions were prepared in dimethyl sulfoxide (DMSO) and Proteins were transferred to a nitrocellulose membrane using the iBlot stored at À20C. 2 system (Life Technologies) and then blocked with LI-COR blocking buffer (LI-COR Biosciences). Membranes were incubated with the Cell proliferation assays primary antibodies overnight at 4C, followed by IRdye-conjugated For GI50 determination, cells were seeded in 96-well plates. The next secondary antibodies (LI-COR Biosciences) and detected using an day, cells were treated with increasing concentrations of inhibitor or Odyssey Fc imaging system (LI-COR Biosciences). Quantification of with DMSO alone. After a 72-hour incubation period, cell proliferation Western blots was performed using Image Studio Lite (LI-COR was quantified using CellTiter-Blue reagent (Promega) and normal- Biosciences). Details of the antibodies used can be found in Supple- ized to DMSO-treated wells. GI50 values were calculated using non- mentary Table S1. linear regression analysis in GraphPad Prism software. For population doubling experiments, cells were seeded at an initial density of 1 Â 107 Lentiviral production cells/flask in 225 cm2 flasks. Cells were allowed to proliferate to 80%– HEK293T cells were seeded at a density of 2.4 Â 106 cells/plate in 90% confluence before they were counted and then reseeded at the 10-cm plates. The next day cells were transfected with shRNA/sgRNA same initial density. Population doublings (PD) were calculated plasmid (3 mg) and the packaging plasmids psPAX2 (2.1 mg) and according to the equation below. pmD2.G (0.9 mg) using 30 mL lipofectamine per transfection. Cells were incubated for 72 hours at 37C, after which the supernatant was LogðÞ cellcount=initialcell number collected and stored in 0.5 mL aliquots at À80C for future experi- Population doublings ¼ Log ð2Þ ments. Each batch of lentivirus was titrated on cells to determine concentration needed for 100% infection efficiency. For determination of maximum excess above bliss, cells were treated with a matrix of increasing concentrations of two inhibitors or DMSO. shRNA After a 72-hour incubation period, cell proliferation was quantified MISSION shRNA plasmids (pLKO.1) were obtained from Sigma- using CellTiter-Blue reagent and normalized to the DMSO-treated Aldrich. The pLKO.1-LacZ and -Luciferase targeting shRNA plasmids well. The Bliss independence model (20) was used to calculate synergy. were from
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