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Cell culture and reagents

Most cell lines were obtained from ATCC (listed in Supplementary Methods). Pa14C and Pa16C cells were a gift from A. Maitra, and the MEF cell lines were obtained from the NIH. AsPC-1 CYPA-knockout (KO), NCI-H441 CYPA-KO and eCT26 KRAS^G12C/G12C ABCB1^−/− cells were generated by Synthego (eCT26 KRAS^G12C/G12C ABCB1^−/− was engineered from the mouse CT26 KRAS^G12D/G12D tumour cell line; P-glycoprotein (PGP) drug transporter was knocked out to eliminate any confounds due to potential interaction of the test article with PGP). All cells were grown in recommended medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin and maintained at 37 °C in a humidified incubator at 5% CO₂. The sanglifehrin A-competitive CYPA inhibitor¹⁷ was synthesized at WuXi AppTec. Other tool inhibitors were acquired from Selleckchem or MedChemExpress.

Protein production

His₆-TEV-KRAS4B^WT (residues 1–169), His₆-TEV-KRAS4B^G12A (residues 1–169), His₆-TEV-KRAS4B^G12C (residues 1–169), His₆-TEV-KRAS4B^G12D (residues 1–169), His₆-TEV-KRAS4B^G12R (residues 1–169), His₆-TEV-KRAS4B^G12S (residues 1–169), His₆-TEV-KRAS4B^G12V (residues 1–169), His₆-TEV-HRAS^WT (residues 1–166), His₆-TEV-NRAS^WT (residues 1–172), His₆-TEV-AviTag-KRAS4B^G12C (residues 1–169), His₆-TEV-CYPA (full-length), His₆-TEV-AviTag-CYPA (full-length) and GST-TEV-BRAF (residues 155–229) were expressed from a pET28 vector in BL21(DE3) Escherichia coli and purified as described¹².

Crystallography

Conditions, data collection, and refinement protocols are provided in the Supplementary Methods.

RAS–RAF and RAS–CYPA TR-FRET

Time-resolved fluorescence resonance energy transfer (TR-FRET) was used as previously described to assess disruption of the interactions between wild-type RAS or the mutant oncogenic RAS proteins and the RAS-binding domain of BRAF, and to assess the induction of interactions between the RAS proteins and CYPA¹².

CYPA binding affinity

The binding affinity of compounds for CYPA (K_d1) was assessed by SPR on a Biacore 8K instrument as previously described¹².

RAS binding affinity

The binding affinity of compound-bound CYPA for the mutant oncogenic RAS proteins (K_d2) mentioned was assessed by SPR on a Biacore 8 K instrument. AviTag-RAS [residues 1–169] was immobilized on a streptavidin sensor chip, and varying compound concentrations were flowed over the chip in assay buffer (10 mM HEPES-NaOH pH 7.4, 150 mM NaCl, 0.005% v/v surfactant P20, 2% v/v DMSO, 25 μM CYPA). The SPR sensorgrams were fit using either a steady state affinity model or a 1:1 binding (kinetic) model to assess the dissociation constant (K_d) for RAS binding.

AlphaLISA and MSD analysis of cellular ERK phosphorylation

Cells were seeded in 384- or 96-well tissue culture-treated plates in 2D and incubated overnight before exposure to serial dilutions of compound or DMSO control (0.1% v/v). Cells were lysed and the levels of ERK phosphorylation determined using the AlphaLISA SureFire Ultra pERK1/2 (T202/Y204) Assay kit (Perkin Elmer ALSU-PERK-A50K) or MesoScale Discovery (MSD) Multi-Array Assay Systems for Phospho/Total ERK1/2 Whole Cell Lysate Kit (K15107D) according to manufacturers’ protocols. Signal was detected using a Perkin Elmer Envision with standard AlphaLISA settings or a Meso QuickPlex SQ120 reader. For AlphaLISA, raw signal was normalized to vehicle control and a low-signal control compound ((sample signal – average signal of low control)/(average signal for vehicle – average signal for low control) × 100%). The MSD signal of pERK1/2 was divided by the MSD signal for total ERK1/2. The ratio was normalized to vehicle pERK/total ERK (%) = ((pERK/total ERK in treatment condition)/(ratio pERK/total ERK in DMSO control)) × 100%. Data were plotted as a function of log [compound (M)] with a sigmoidal concentration response (variable slope) model fitted to the data to estimate the inhibitor EC₅₀ in Prism 9 (GraphPad).

Cell proliferation analysis

Cells were seeded in 384- or 96-well tissue culture-treated plates in 2D and incubated overnight. Alternatively, cells were seeded in round-bottom ultra-low attachment 96-well plates, centrifuged at 1,000 rpm for 10 min to pellet the cells, and incubated overnight or up to 72 h to allow for 3D spheroid formation. Cells were exposed to serial dilutions of compound or DMSO control (0.1% v/v) for 120 h. Cell viability was determined by CellTiter-Glo 2.0 reagent (2D CTG) (Promega, G9243) or 3D CellTiter-Glo reagent (3D CTG) (Promega, G9683) according to the manufacturer’s protocols. Luminescence was detected using a SpectraMax M5 Plate Reader (Molecular Devices) of Perkin Elmer Enspire. Luminescence signal was normalized to vehicle-treated wells (normalized signal (%) = (luminescence (treated)/mean luminescence (vehicle)) × 100%). For PSN1 and HUPT3, raw signal was normalized to vehicle control and a low-signal control compound ((sample signal – average low control signal)/(average vehicle signal – average low control signal) × 100%). For NCI-H441 and AsPC-1 cells treated with the combination of RMC-7977 and the sanglifehrin A competitive CYPA inhibitor (3 mM), luminescence signal was normalized to that of the CYPA inhibitor treatment-only control (normalized signal (%) = (luminescence (treated)/mean luminescence (CYPA inhibitor only) × 100%)). Data were plotted as log [inhibitor (M)], and a four-parameter sigmoidal concentration response model was fitted to the data to calculate EC₅₀. Data were fit with top plateau constrained to 100% and lower plateau constrained depending on the cell line (Capan-1, AsPC-1 and Hs 766T, 25% ≥ lower plateau ≥ 0%; HCT 116, SKMEL30 and KU1919, 10% ≥ lower plateau ≥ 0%; NCI-H358, A375 PSN1 and HUPT3, lower plateau = 0%). Pa16C MEK1 mutant cells were evaluated by live-cell counting using Calcein AM and a SpectraMax i3X multi-mode detection platform (Molecular Devices). Growth percentages were calculated by normalizing the treated cell counts to their respective untreated cell counts.

Cellular RAS–RAF and RAS–CYPA assays

U2OS cells or U2OS cells with PPIA gene knockout were seeded at 500,000 cells per well in a 6-well plate and incubated overnight. KRAS4B, or other small GTPases, containing the indicated mutations were cloned in pNLF-N or pHTN plasmids for expression with an N-terminal nanoluciferase or HaloTag fusion, respectively. Full-length CYPA was cloned into pHTN, the RBD of RAF1 (residues 51–149) was cloned into pHTC, full-length RALGDS was cloned into pHTC, PIK3CA was cloned into pNLF-N, and the catalytic domain of SOS1 (residues 558–1049) was cloned into pNLF-N. U2OS cells were transfected with KRAS and effector plasmids, and U2OS PPIA-KO cells were transfected with small GTPase and CYPA plasmids, both using Fugene HD reagent according to manufacturer protocols. The following day, the cells were collected by Trypsin and reseeded in a white tissue culture-treated 96-well plate in OptiMem phenol red-free medium (Gibco) containing 4% FBS and a 1:1,000 dilution of NanoBRET 618 HaloTag ligand (Promega). For endpoint concentration response curves, vivazine nanoluciferase substrate was added to 1× concentration in OptiMem phenol red-free medium with 4% FBS. Varying concentrations of inhibitor were added and incubated for 1 or 4 h before the nano-BRET signal was measured on a Perkin Elmer Envision plate reader. For kinetic assays, endurazine nanoluciferase substrate was used in place of vivazine, and the plate was placed in a Cytation5 multi-mode reader pre-equilibrated to 37 °C and 5% CO₂. After 1 h of equilibration, RMC-7977 (50 nM) was added and the nano-BRET signal measured.

Generation of NCI-H358 expressing low and high CYPA

NCI-H358 cells were transduced by lentivirus encoding Cas9, a guide RNA targeting PPIA (which encodes CYPA), and the puromycin resistance gene at WarpDrive Bio. Following puromycin selection, Flag–CYPA was introduced under the control of a tet-inducible promoter by lentivirus. Clones with high and low expression levels of Flag–CYPA were isolated at Revolution Medicines. Flag–CYPA expression was induced by adding doxycycline (0.1 µg ml⁻¹) for at least 24 h.

Generation of cell lines with acquired resistance to adagrasib

NCI-H358 cells resistant to adagrasib were generated by continuously culturing in growth medium containing 1 µM adagrasib for approximately 2 months. Resistant cells were subsequently maintained in culture medium containing 1 µM adagrasib, which was removed during assays.

Generation of inducible full-length and fusion RTK overexpression cell lines

Plasmids encoding the tet-controlled transcriptional silencer (tTS), reverse tet-controlled transcriptional activator (rtTA), and tet-inducible receptor tyrosine kinases (RTKs) and fusion RTKs were synthesized and packaged into lentivirus at Vector Builder. Lentivirus transductions were performed with addition of polybrene (4 µg ml⁻¹). NCI-H358 and MiaPaCa2 cells were transduced with lentivirus encoding tTS or rtTA for 48 h prior to selection with blasticidin (5 µg ml⁻¹) for 12 days. The concentration of blasticidin was subsequently lowered to 2 µg ml⁻¹. NCI-H358 tTS/rtTA cells were then transduced with lentivirus encoding tet-inducible GFP, EGFR, EGFR(A289V), HER2, FGFR2 and RET(M918T). MiaPaCa2 tTS/rtTA cells were transduced with lentivirus encoding GFP or tet-inducible EML4–ALK, FGFR3–TACC3 and CCDC6–RET. Cells were cultured in growth medium containing puromycin (2 µg  ml⁻¹) and blasticidin (2 µg ml⁻¹) starting 48 h after transduction to maintain selective pressure for both plasmids. Expression of the transgene was induced by adding doxycycline (0.1–1 µg ml⁻¹) for at least 24 h.

Generation of Pa16C cells expressing MEK1 mutants

MEK1 mutants were generated using quick change mutagenesis in MEK1-GFP (Addgene plasmid #14746). MEK1 was PCR amplified with flanking NheI and AgeI sites and digested. Luciferase-PCW107-V5 (Addgene plasmid #64649) was also digested with NheI and AgeI, removing luciferase, and ligated with the MEK1 insert in front of the V5 tag. Lentivirus was made from the control construct (Luciferase-PCW107-V5) and each of the MEK1 constructs by co-transfection with psPax packaging plasmid into HEK293T cells using Fugene 6 Transfection Reagent (Promega). Viral supernatant was collected, combined with polybrene (8 µg ml⁻¹), and used to transduce Pa16C (PDAC, KRAS(G12D)) cells in DMEM supplemented with 10% FBS. Cells were infected for 12 h and then selected using puromycin.

PRISM assay

RMC-7977 was screened in 931 PRISM DNA-barcoded cell lines established by the Broad Institute. In brief, 20–25 cell lines per pool were plated in 384 well plates and treated with RMC-7977 at 8 doses in threefold dilutions starting at 10 µM for 5 days. Cells were then lysed in TCL mRNA lysis buffer, and then PCR with reverse transcription was performed. Detection of the barcodes and univariate and multivariate analysis was then performed as previously described⁴³. Data analysis is described in the Supplementary Methods. Up-to-date code for our analysis is at the github link: https://github.com/cmap/dockerized_mts.

Cell panel

A panel of 183 cancer cell lines harbouring mutant and wild-type RAS was screened for response to RMC-7977 by cell proliferation and viability inhibition at Crown Bioscience. The panel consisted of cell lines with any substitution at position 12 of KRAS, NRAS or HRAS (KRAS(G12X), NRAS(G12X), HRAS(G12X)); substitutions in KRAS, NRAS or HRAS at any position other than 12, 13 and 61 (KRAS(other/VUS), NRAS(other/VUS), HRAS(other/VUS)); other oncogenic mutations in the RAS pathway (ABL1, ALK, ARAF, BRAF, CBL, EGFR, ERBB2, ERBB3, ERBB4, ERRFI1, FGFR1, FGFR2, FGFR3, FGFR4, FLT3, HRAS, IGF1R, JAK2, KIT, MAP2K1, MAP2K2, MAPK1, MET, NF1, NRAS, NTRK1, NTRK2, NTRK3, PDGFRA, PTPN11, RAC1, RAF1, RASA1, RET, RIT1, ROS1 and SOS1); and no oncogenic mutations in the RAS pathway. Cells were cultured in methylcellulose and treated in triplicate with serial dilutions of RMC-7977 or DMSO. Cells were incubated for 120 h, and cell viability was determined using the CellTiter-Glo Luminescent Cell Viability Assay (CTG) (Promega, G7572) according to the manufacturer’s instructions. Data were plotted as a function of log [inhibitor (M)] and a four-parameter sigmoidal concentration response model was fitted to the data to estimate the inhibitor EC₅₀ using Genedata Screener.

Western blot analysis

Antibodies and protocols are described in the Supplementary Methods.

Quantification of CYPA protein level in cell and tumour samples

Cells were seeded at 1 × 10⁶ cells per well in triplicate in 6-well plates and incubated overnight. The following day, cells were collected by Trypsin, washed in PBS, pelleted by centrifugation, and snap frozen in a slurry of dry ice and ethanol. Tumour samples were collected and flash frozen (see Supplementary Methods). Samples were transferred to IQ Proteomics for analysis. The samples were lysed by bead beating in 8 M urea + 200 mM EPPS pH 8.0 + HALT protease inhibitor cocktail. Following bead beating, SDS was added to the lysate, 1% final (w/v). Following quantification by BCA assay, lysate corresponding to 16 μg of total protein was aliquoted for downstream processing. Samples were reduced and alkylated via DTT/Iodoacetamide, and protein was isolated via ethanol precipitation. Protein was digested in 100 mM EPPS pH 8.1, using LysC (overnight, room temperature) and Trypsin (6 h, 37 °C). 5 stable isotope labelled standard peptides spanning the CYPA protein sequence (sequences VSFELFADK, ALSTGEK, FEDENFILK, TEWLDGK and EGMNIVEAMER) were spiked into each sample at a ratio of 25 fmol μg⁻¹ total protein digested. Endogenous (light) and internal standard (heavy) peptides were quantified via custom targeted assay on an Orbitrap Lumos instrument (Thermo).

Bioanalysis of cells and supernatant

Ten-million cells were exposed to RMC-7977 (10, 100 or 1,000 nM) in suspension at 1 × 10⁶ cells ml⁻¹ for 1 h at 37 °C. Cells were pelleted by centrifugation, and 1 ml of supernatant was reserved and frozen at −80 °C. Cell pellets were washed twice in cold PBS, and pre-weighed tubes containing the cell pellets were weighed prior to snap freezing in a slurry of dry ice and ethanol. Concentrations of RMC-7977 in cell pellets and supernatant were determined by liquid chromatography–tandem mass spectrometry (LC–MS/MS) methods. Cell pellet samples were resuspended in cell medium (diluted as needed), then treated as supernatant. An aliquot of supernatant or resuspended cells (50 µL) was quenched with a 3× volume of acetonitrile containing the internal standard terfenadine (2.5 ng ml⁻¹). Samples were vortexed, centrifuged, and analysed on a Sciex 6500+ triple quadrupole mass spectrometer equipped with a Shimadzu AD LC system. A Waters ACQUITY UPLC BEH C4 1.7 µm (2.1 × 50 mm) column was used with gradient elution for compound separation. RMC-7977 and internal standard were detected by positive electrospray ionization using multiple reaction monitoring (RMC-7977: m/z 865.273/833.500; terfenadine: m/z 471.939/436.300). The lower limit of quantification was 0.25 ng ml⁻¹, and the calibration range was 0.25 to 400 ng ml⁻¹. The intracellular concentration of RMC-7977 was calculated using the mass of each cell pellet (mass of empty tube subtracted) and the known cell number, with the assumptions that the volume of a cell is ~2,000 µm³, that the density of a cell is approximately the density of water (thus, cell volume = cell mass); and that any compound in CYPA-KO cells in excess of the medium concentration is probably membrane-bound. The ratio of compound concentration in the cell pellet to compound in medium was determined for each concentration of RMC-7977 tested.

Animal studies

Xenograft studies were conducted at GenenDesign, Pharmaron, Wuxi AppTec, the laboratory of P. Lito, and the laboratory of C. Ambrogio. Animals were assigned to study groups using stratified randomization based upon their tumour volumes. All procedures related to animal handling, care and treatment were conducted in compliance with all applicable regulations and guidelines of the relevant Institutional Animal Care and Use Committee (IACUC). For the sotorasib-resistance xenograft study, all procedures and animal housing conformed to the regulatory standards and were approved by the Italian Health Minister (authorization no. 1227/2020-PR); all experiments were performed in accordance with the guideline for Ethical Conduct in the Care and Use of Animals as stated in The International Guiding Principles for Biomedical Research Involving Animals, developed by the Council for International Organizations of Medical Sciences. Experimental details are supplied in the Supplementary Methods.

Mouse blood and tumour sample bioanalysis

Whole-blood and tumour concentrations of RMC-7977 were determined using LC–MS/MS methods performed at WuXi AppTec. Tumour tissue samples were homogenized with a 10× volume of methanol/15 mM PBS (1:2, v:v). Sample preparation and analysis on a Sciex 6500+ triple quadrupole mass spectrometer equipped with an ACQUITY UPLC system were performed as previously described¹². RMC-7977 and internal standard verapamil were detected by positive electrospray ionization using multiple reaction monitoring (RMC-7977: m/z 865.4/706.4; verapamil: m/z 455.2/164.9).

In vivo pharmacodynamic analysis by DUSP6 qPCR

RNA extraction and analysis of DUSP6 levels by in tumour tissue were performed as previously described¹².

OVA peptide vaccination

Experimental details are described in Supplementary Methods.

Immune cell response in vivo

Experimental details are described in Supplementary Methods.

Ethics statement

All CDX and PDX mouse efficacy and pharmacodynamics and pharmacokinetics studies and procedures related to animal handling, care and treatment were conducted in compliance with all applicable regulations and guidelines of the relevant Institutional Animal Care and Use Committee (IACUC). For the sotorasib-resistance PDX studies, all experiments were performed in accordance with the guideline for Ethical Conduct in the Care and Use of Animals as stated in The International Guiding Principles for Biomedical Research Involving Animals, developed by the Council for International Organizations of Medical Sciences.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.