Drug repurposing study. To generate the Connectivity Scores between the interspecies signature and each chemical perturbagen (CP) available in the L1000 dataset (22), we used the Library of Integrated Network-based Cellular Signatures (LINCS) program (http://c3.lincscloud.org). For all analyses, we used the sig_query tool. To obtain predictions for every CP in the L1000 dataset, we set the flag --column_space to “full”. To ensure maximum coverage of our signature, which due to high stringency was relatively small, we set the flag --row_space to “full”. For all other settings we used defaults provided. Results were provided on the signature level, and thus showed the effect of a drug in one cell, at one dose, measured at one time-point. As there are multiple signatures per CP and we sought an overall assessment of CP effect in a novel condition, we first took the mean of scores within each cell line and then took the mean of those cell line means. This sequential averaging was done to avoid giving extra weight to effects in cells that had been more frequently assayed. Finally, to filter down to CPs with the most robustly assayed effects, we retained only CPs which had been measured in more than 10 different conditions. All analysis and processing of results was done using R statistical programming language (https://www.R-project.org).
Cell lines. Human wt (H1437, H1568, H2126, HCC78, H1650 and H1993) and mut KRAS (H1792, H2009, A549, H358, H23, HCC44, CP435) LUAD cell lines were used. All these cell lines but CP435, which was generated from a KRASG12C primary LUAD, were obtained from ATCC and authenticated by the Genomics Unit at CIMA using Short Tandem Repeat profiling (AmpFLSTR Identifiler Plus PCR Amplification Kit). Human cells were grown according to ATCC specifications and were tested for mycoplasma using the MycoAlert Mycoplasma Detection Kit (LONZA). Only mycoplasma negative cells were used. H1792, H2009 and A549 cell lines were exposed to increasing concentrations of Trametinib until they were completely resistant to the drug at the concentration of interest (0.5 µM) to obtain H1792-TR (Trametinib resistant), H2009-TR and A549-TR. H23 and H358 were treated with Sotorasib to generate resistant cells (H358-SR: resistant to 0,5 µM KRASi; H23: resistant to 10 µM KRASi). H2009, H23 and H358 cell lines were infected with pLENTI6-LacZ and pLENTI6-MYC as previously described (25).
Reagents. Pharmacological inhibitors to MEK1/2 (Trametinib/GSK1120212, MEKi), HDAC (Panobinostat/LBH589, HDACi) and WEE1 (Adavosertib/MK-1775, WEE1i) were acquired from Selleckchem; to MEK5-K2 (BIX02189, MEK5i) was purchased from Tocris. Lestaurtinib/L6307 (mtPKCi), Neratinib (pan-HERi) and Afatinib (EGFRi) were obtained from LC Laboratories, and Midostaurin/PKC412 (mtPKCi), Sotorasib/AMG-510 (KRASi), Darovasertib (PKCAi), Barasertib (AURKBi), Adagrasib/MRTX849 (KRASi) and MG132 (proteasome inhibitor) were purchased from MedChemExpress. Dabrafenib (RAFi) was a kind gift from Imanol Arozarena (Navarrabiomed, Spain).
RNA sequencing. RNA sequencing was carried out as previously described (59).
Western blotting. Western blot analysis was performed as previously published (25). Antibodies used: β-TUBULIN (1:2,000, sc-9104, Santa Cruz Biotechnology), GAPDH (1:5,000, ab9484, Abcam), ACTIN (1:5,000, A5441, Sigma), HSP90 (1:500, sc-69703, Santa Cruz Biotechnology), KRAS (1:1,000, WH0003845M1, Sigma), MYC (1:1,000, D84C12, #5605, Cell Signalling Technology-CST), phospho-MYC S62 (1:1000, ab185656, Abcam), phosphor-MYC T48 (1:1000, ab28842, Abcam), ERK1/2 (1:1,000, #9102, CST), p-ERK1/2 (1:1,000, #9101, CST), PARP (1:1,000, #9542, CST), AKT (1:1,000, #9272, CST), p-AKT (1:1,000, #9271, CST), p70S6K (1:1,000, #2708, CST), p-p70S6K (1:1,000, #9205, CST), EGFR (1:1,000, #2232, CST), p-EGFR (1:1,000, #2236, CST), STAT3 (1:500, #4904, CST), p-STAT3 (1:1,000, #9145, CST), cJUN (1:700, #9165, CST), SHP2 (1:1,000, #3397, CST), phospho-SHP2 (1:1,000, #3751, CST), FLT3 (1:500, #3462, CTS), and p-FLT3 (1:500, #3461, CTS)
Drug combination studies in vitro. For 2D assays, cell lines were plated at density ranging from 1,500 to 10,000 cells in 96-well plates and treated on the following day with single drugs or both. CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega) was used to determine the number of viable cells in proliferation and the potential cytotoxicity of drugs in cell lines. Experiments were read after 72 hours of exposure to the drugs according to manufacturer's instructions. CompuSyn software (www.combosyn.com) was used to determine the potential synergism of two single drugs in the pairwise drug screen. Combination Index (CI) values lower than 0.8 were considered synergistic.
For 3D-cultures, cold 96-well plates were pre-treated with 50% Matrigel Growth Factor Reduced (MG) (Corning) coating before cell seeding. Cells were resuspended in 10% Matrigel culture medium (31) and seeded at 2 x 103 cells per well. Cells were incubated overnight and drugs were added on the next day. Proliferation of 3D-cultures was measured using CellTiter-Glo® 3D Cell Viability Assay (Promega) according to manufacturer’s instructions. Organoid images were taken using an inverted microscope DMI3000 from Leica.
Clonogenic assay. Cells were seeded in triplicate into 24-well plates (range: 350 to 6,000 cells per well depending on the cell line). The next day, cells were cultured in the absence or presence of single drugs or drug combinations for 10 days. Media with or without drugs was replaced every 3 days. Remaining cells were fixed with 4% formaldehyde (Panreac) for 15 minutes at RT, stained with crystal violet solution (Sigma-Aldrich) (1% crystal violet in H2O) for 15 minutes and photographed using a digital scanner (EPSON Perfection v850 Pro). Relative growth was quantified by measuring absorbance at 570 nm in a spectrophotometer (SPECTROstar Nano – BMG Labtech) after extracting crystal violet from the stained cells using 20% of acetic acid (Sigma).
Patient derived xenograft organoids. Organoids from TP60, TP79, TP80, TP181 and TP126 PDXs derived from LUAD samples were used. All these samples were whole-exome sequenced and confirmed to carry a KRASG12C mutation. Establishment and growth of PDXs was approved by the institutional Committee on Animal Research and Ethics of Hospital Virgen del Rocío, Hospital 12 Octubre, IBIS and CNIO under the protocol references SSA/SI/MD/pdm, PROEX 084/15 and PROEX 313/19.
PDXs were minced into small fragments and enzymatically digested with collagenase in Basic medium (Advanced DMEM/F12, 1x HEPES, 1x Glutamine, 1X Primocin) at 37ºC for 60 minutes. After incubation, the digested samples were filtered using 70 µm filters and the disaggregated cells were centrifuged at 1500 rpm for 5 minutes. After two washes with Basic medium, the cells were counted and resuspended in Complete Feeding Media (Basic medium, 500 nM TGFβi/A83-01, 50 ng/mL mEGF, mNoggin 100 ng/mL, hFGF10 100 ng/mL, gastrin I 0.01 µM, N-acetylcysteine 1.25 mM, nicotinamide 10 mM, B-27 supplement 1X, R-spondin I-conditioned media 1X, Wnt3a-conditioned media 1X) with 10% of matrigel.
Apoptosis assay. Alexa Fluor 647-conjugated Annexin-V (Invitrogen) was used to perform the apoptotic cell detection assays following the manufacturer’s instructions. Cells were treated for 24 h, acquired in FACSCanto II Cytometer (BD Biosciences), and analysed using FlowJo software v9.3.
Proteosome inhibitor assay. Two million of cells were plated in 10 cm2 plates and treated on the following day with both drugs for 48 h and next, with 5 µM proteasome inhibitor MG132 for additional 6 h, before western blot analysis.
Proteomics. Cellular pellets derived from H1792 cells exposed to DMSO, Trametinib (0.5 µM), Lestaurtinib (0.625 µM) or both for 48 h, and H1792-TR grown in 0.5 µM Trametinib ± Lestaurtinib for 48 h were subjected to proteomics using an isobaric tandem mass tag (TMT) approach LC-MS/MS analysis was done using a 5600 Triple-TOF system (Sciex) (see supplementary methods for details). Raw MS/MS spectra searches were processed using the MaxQuant software (60) and searched against the Uniprot proteome reference for Homo Sapiens (Proteome ID: UP000005640_9606, February 2019). The Perseus software (version 1.5.6.0) (61) was used for statistical analysis and data visualization. The identification of significantly dysregulated regulatory/metabolic pathways across proteomic datasets was performed using Metascape (60).
Preclinical in vivo models. All experiments in mice were performed following ARRIVE guidelines and approved by the institutional Committee on Animal Research and Ethics of CIMA and CNIO under the protocol numbers 057 − 18 and PROEX 316/19. Mice were randomized to get a similar average tumor size across treatment groups at treatment start. Animals were treated without knowledge of anticipated outcomes and blind treatments were followed. For xenograft experiments 3 x 106 cells (H1792, A549, H1792-TR, H358-SR, H358-LacZ, H358-MYC and T1) were suspended in 100 µL of DPBS and injected subcutaneously into the two lower flanks of 8–12 weeks-old female immune-deficient Rag2−/−; Il2γr−/− mice (Jackson Laboratories) or immunocompetent F1 Sv129xC57bl/6 mice. Beginning 1-week post-injection, tumor dimensions were measured every 3 days using a Digital caliper (DIN862, Ref 112-G, SESA Tools) and tumor volume was calculated by the formula: Volume = π/6 × length × width2. When tumors reached an average volume of 100 mm3, administration of pharmacological inhibitors was carried out: Trametinib (1 mg/kg), Lestaurtinib (30 mg/kg), Midostaurin (25 mg/kg), Sotorasib (10 or 30 mg/kg) or dual administration was done by oral gavage daily 5 days per week for 3 weeks. KRasFSFG12C; Trp53FRT/FRT mice in a mixed C57Bl6/Sv129 background were infected with AdFlp (106 p.f.u) and aged for 6–8 months until mice had developed 1–4 tumors per lung. Then, tumors were treated with vehicle, Midostaurin (25 mg/kg), Sotorasib (100 mg/kg) or both by oral gavage for 6 weeks. Tumor follow up was done using microCT scans. In brief, lung images were acquired using SuperArgus COMPACT (Sedecal) microCT scanner. Image processing, analysis and 3D rendering was performed using the 3D Slicer Viewer Software.
Immunohistochemistry. Immunohistochemical staining was performed using the EnVision TM + System (K400311-2, Agilent) according to the manufacture’s recommendations. Antigen retrieval was performed for 30 min at 95 ºC in Tris-EDTA, pH 9.0. The following rabbit primary antibodies were used: MYC (Abcam, ab32072) and CD8 (Cell Signalling, 98941).
Real time PCR. RT-PCR was performed using SYBR® GreenER™Select Master Mix method (Applied Biosystems) on a QuantStudio 3 Real-Time PCR machine (ThermoScientific) following the manufacturer’s instructions. GAPDH was used as a housekeeping gene. Primers used for RT-PCR were as follows: ‘MYC forward’ is 5’-GCTGCTTAGACGCTGGATTT-3’, ‘MYC reverse’ is 5’- TAACGTTGAGGGGCATCG-3’, ‘GAPDH forward’ is 5’- GAGTCAACGGATTTGGTCGT-3’ and ‘GAPDH reverse’ is 5’- AAGTGAAGGGGTCATTGATGG-3’.
Lentiviral infections. A MYC cDNA in a pDONR221 vector was provided by Alejandro Sweet-Cordero (University of California San Francisco, USA) and cloned into a pLenti6/V5-DEST using the Gateway system (Thermofisher). Lentivirus was produced by transfection into 293FT cells as previously described (25), filtered and applied directly to cells for infection at a MOI lower than 1.
Statistical analyses. Statistical analyses were performed using GraphPad Prism 8 (GraphPad Software, Inc). For in vitro experiments, at least 3 independent experiments were carried out with 2-6 replicates per experiment. Sample size was chosen using http://www.biomath.info/power/ttest.htm or based on similar experiments previously published by the authors. For comparisons of two groups, samples were explored for normality (Shapiro-Wilk test) and variance (Levene test). Groups with normal distribution of samples followed a t-test. Non-normal samples were analysed using a Mann–Whitney test (equal variances) or a Median test (unequal variances). All analyses were two-tailed. For multiple comparisons of normally distributed variables, ANOVA and posterior Tukey tests were carried out. In the case of multiple comparisons of non-normally distribution variables, Kruskal-Wallis and posterior Tukey Adjusted-Mann-Whitney U tests were used. Statistical significance was defined as significant (p < 0.05), very significant (p < 0.01) and highly significant (p < 0.001). Error bars correspond to either standard deviation (n < 8) or standard error of the mean (n ≥ 8), as indicated for each experiment. No data points were removed as outliers.