Our research complies with all relevant ethical regulations. The mouse studies were approved by the National Cancer Institute (NCI) Animal Care and Use Committee and were conducted in compliance with the approved study protocols, and the human lung cancer samples were acquired under an Emory University institutional review board-approved protocol.

Plasmids

HA-tag vector (plasmid 38189) and HA-tagged KRASG12C (plasmid 58901) and KRASG12D (plasmid 58902) were obtained from Addgene. GFP-tagged HRAS-WT, NRAS-WT, KRAS-WT, KRAS-G12C, KRAS-G12D, KRAS-C185S, KRAS-C185S,G12C, KRAS-C185S,G12D, DDK tag vector, DDK-KRAS-WT and DDK-KRAS-K12D were provided by D. Esposito (Protein Expression Laboratory at the Frederick National Laboratory for Cancer Research).

Antibodies and fluorescent probes

Antibody information, including the catalog numbers and dilutions, is available in the Reporting Summary linked to this article. Rabbit anti-DLC1 was generated in our laboratory (clone 428, 1:500), as described previously39. ‘To make the anti-DLC1 specific antibody (clone 428), the cDNA encoding a DLC1 polypeptide (amino acids 82–251) was subcloned into the bacterial expression vector PGEX-4T-1 (Pharmacia) using EcoRI and XhoI, and its encoded GST fusion protein was induced by isopropyl β-d-1-thiogalactopyranoside (IPTG) in bacteria, purified by a Glutathione Sepharose 4B column, and used to immunize rabbits’. Alexa Fluor 568 anti-rabbit IgG (1:250), Alexa Fluor 488 anti-mouse IgG (1:250) and DAPI (1:2,500) were purchased from Thermo Fisher Scientific.

Cell lines, culture conditions and DNA transfection

NSCLC lines H1703, H157, A549, H358 and NCI-H23 were purchased from ATCC. All cancer cell lines were cultured in RPMI-1640 supplemented with 10% fetal bovine serum. HEK-293T cells and human fibroblastic WI-38 cells were purchased from ATCC and were cultured in DMEM and EMEM supplemented with 10% fetal bovine serum, respectively. HBECs were purchased from ATCC and were cultured in Airway Epithelial Cell Basal Medium with cell growth kit components. Where indicated, transient transfections were performed with Lipofectamine 3000 (Thermo Fisher Scientific) according to the manufacturer’s instructions. Stable clones expressing GFP, GFP–DLC1-WT, GFP–DLC1-K678A and GFP–DLC1-K678D were made by transfection of A549 cells with Lipofectamine 3000, followed by selection with G418 (0.9 µg ml–1).

siRNA transfection and treatment of cells with serum, EGF and inhibitors

To suppress expression of specific mRNAs, cells were transfected with 160 nM siRNAs targeting DLC1, XPO1, EZH2, KRAS, NTF2 or RANGAP1 or with scrambled siRNAs and collected 48 h later. Suppression of protein expression, at least with two different siRNAs, was confirmed by immunoblotting. Validated siRNAs for human DLC1 (Hs_DLC1 siRNA_5, SI03219909; Hs_DLC1 siRNA_11, SI04952213) were from Qiagen, as were scrambled siRNAs (control siRNA 1, 1027280; control siRNA 2, 1027310). The following siRNAs were purchased from Dharmacon: ON-TARGETplus Human KRAS (3845) Smart pool (L-005069-00-0005) siRNA, ON-TARGETplus Human EZH2 (2146) Smart pool L-004218-00-0005) siRNA, ON-TARGETplus Human XPO1 (7514) SMART pool (L-003030-00-0005) siRNA, ON-TARGETplus Human NUTF2 (nuclear transport factor 2) SMART pool (L-012132-00-0005) siRNA and ON-TARGETplus Human RANGAP1 SMART pool (L-006846-00-0005) siRNA. The sequences for each siRNA are described in Supplementary Table 1.

After overnight incubation in serum-free medium, cells were treated with 15% serum or EGF (purchased from Sigma-Aldrich) for 20 min. The final concentration of EGF was 100 ng ml–1. AKT inhibitor (mk-2206) and SRC inhibitor (saracatinib; used at 10 µM each) were purchased from Selleck Chemicals. Inhibitors for KRAS-G12C (sotorasib), KRAS-G12D (mrtx-1133), EZH2 (tazemetostat), XPO1 (selinexor), MEK (selumetinib), PI3K (copanlisib) and other pharmacological compounds (used at 1–10 µM each) were provided by the Developmental Therapeutics Program Chemicals Repository, Division of Cancer Treatment and Diagnosis, NCI.

Coimmunoprecipitation and immunoblotting

Coimmunoprecipitation and immunoblotting were performed according to the previously described protocol40. ‘For co-IP experiments, equal amounts of protein from each cell lysate were precleared with Protein G Agarose (Thermo Fisher Scientific) and then incubated with the indicated antibodies or control IgG for 1 h at room temperature. After incubation, 30 µl of Protein G Agarose was added to each immune reaction and rotated overnight at 4 °C. The immunopellets were washed three times with RIPA buffer. Co-IP proteins were eluted by boiling for 5 min in 50 µl of Laemmli sample buffer containing 5% (vol/vol) 2-mercaptoethanol. Eluted proteins were resolved on a NuPage 4–12% BisTris gel and detected by IB using specific antibodies. Immunoreactive bands were detected by enhanced chemiluminescence (ECL Plus; GE Healthcare) using horseradish peroxidase-linked anti-rabbit or anti-mouse secondary antibodies’.

Immunofluorescence staining

Immunostaining was performed according to the previously described protocol40. ‘Tumor tissue sections or cells were seeded onto glass chambers, incubated for 24 h, and fixed with 4% paraformaldehyde for 20 min. Fixed cells or deparaffinized tissues sections were permeabilized with 0.25% Triton X-100 in PBS and then blocked with 3% BSA in PBS for 2 h. The cells or tissue sections were incubated with the indicated primary antibodies at 4 °C overnight. After being thoroughly washed with PBS, cells were incubated with the appropriate 1:250 Alexa Fluor-conjugated secondary antibodies for 1 h. To visualize nuclei, cells were incubated with DAPI (1:2,500) for 1 h. After staining, cells were thoroughly washed with PBS and mounted with gel mounting solution (Biomeda).’

PLA

PLA was used to visualize proximity colocalization (<40 nm) of KRAS and RanGAP1 in NSCLC lines or PDX tissue sections using a Duolink Detection kit (Olink Proteomics), as per the manufacturer’s instructions. The cells were fixed with 4% paraformaldehyde for 20 min at room temperature, and fixed cells or deparaffinized tissues sections were permeabilized with 0.25% Triton X-100 for 5 min. After incubating with Duolink blocking solution, cells were incubated overnight at 4 °C with mouse anti-KRAS (WH0003845M1; 1:200) and rabbit anti-RanGAP1 (ab92360, 1:500) or the indicated primary antibody in Duolink antibody diluent. After washing, cells were incubated with secondary antibodies conjugated to PLA probes (MINUS probe-conjugated anti-rabbit IgG and PLUS probe-conjugated anti-mouse IgG, Olink Proteomics). Circularization and ligation of the oligonucleotides in the probes were followed by an amplification step. A complementary fluorescence-labeled probe was used to detect the product of the rolling circle amplification. Slides were mounted with Duolink II mounting medium containing DAPI. Images were obtained with an LSM 780 confocal microscope (ZEISS) using ZEN software (ZEISS).

Fluorescence confocal microscopy

Confocal microscopy was performed according to a previously described protocol40. ‘Confocal microscopy of fluorescent-labeled cells was performed using a confocal microscope (LSM 780; Carl Zeiss). Alexa Fluor probes were viewed with excitation wavelengths of 488 nm (Alexa Fluor 488) and 568 nm (Alexa Fluor 568). Images were made at room temperature using photomultiplier tubes with a Plan-Apochromat ×63/1.4-NA oil differential interference contrast objective lens with a 2× magnifier to produce a 125× magnification. The images were minimally processed for levels/contrast adjustment in DAPI panels, and the adjustment was done for all images using Adobe Photoshop 2024 (25.0.0) software. The colocalization of two proteins was analyzed by ZEN microscopy software (version ZEN 2.3 SP1). The adjustments do not enhance, erase or misrepresent any information present in the original images’.

Anchorage-independent growth assay

The anchorage-independent growth assay was performed according to our previously described protocol4. ‘For soft agar assays, a 0.6% agar (BD) base in RPMI-1640 medium was placed in 60-mm dishes for 1 h at room temperature. 1.0 × 105 cells were mixed with complete medium containing 0.4% agar and placed over 0.6% basal agar in 60-mm dishes.’ Cells were grown for 3 weeks and were continuously treated without or with selinexor, tazemetostat, sotorasib, selumetinib, copanlisib, mk-2206 and saracatinib, as indicated, and colonies were photographed microscopically and quantified with a colony counter.

Generation of DLC1-KO NCI-H23 cells

CRISPR–Cas9-mediated knockout of DLC1 in the NCI-H23 lung cancer cell line was performed by targeting exon 5 of DLC1 transcript variant 2 (ref. 4; NM_006094). The targeted region of DLC1 was amplified from NCI-H23 clones that were negative for DLC1 protein expression by western blotting, and PCR products were sequenced to confirm the presence of insertion/deletion mutations that would cause premature translation termination of the DLC1 polypeptide. Cells were transfected with two different constructs (pAG0266 and pAG0267) with single-guide RNA (sgRNA) for DLC1. Lenti-SpCas9-2A-GFP-DLC1-IVT vector was used to deliver individual sgRNAs. The sequences of sgRNA primers for DLC1 and nontargeted control sgRNA are described in Supplementary Table 2. Lipofectamine 3000 (Life Technologies) was used to transfect plasmid DNA according to the manufacturer’s instructions. GFP+ single cells were sorted using a FACSAria UV into a sterile 96-well culture plate, yielding single-cell DLC1-KO clones.

Purification of recombinant proteins and preparation of exclusively GDP-bound and GTP analog GppNHp-bound KRAS proteins

DDK–RanGAP1 (Origene) and DDK–CDCP1 (a gift from the laboratory of B. Mock at the NCI) were transfected into HEK-293T cells for 48 h and lysed, as described previously41. ‘Two days after transfection, cells were lysed with Golden Lysis Buffer (GLB: 20 mM Tris (pH 7.9), 137 mM NaCl, 10% glycerol, 1% Triton, 5 mM EDTA, 1 mM EGTA, 1 mM Na3VO4, 10 mM NaF, 1 mM sodium pyrophosphate, 0.5 mM β-glycerophosphate and protease inhibitor cocktail tablet (Roche)). The cleared supernatants were collected, and the amount of protein estimated by BCA kit (Pierce).’ The cell extracts were immunoprecipitated by DDK Flag beads (Sigma-Aldrich) and washed thoroughly with HNTG (20 mM HEPES buffer (pH 7.5) containing 150 mM NaCl, 0.1% (wt/vol) Triton X-100 and 10% (wt/vol) glycerol) buffer. The purified GST-tagged RAF-RBD protein was purchased from EMD Millipore. KRAS4b (1–169) expression clones of both wild-type and KRAS-G12D mutant for Escherichia coli production were generated using a His×6–maltose-binding protein fusion. All proteins were purified as outlined for G-Hs.KRAS4b (1–169), as described previously42. ‘Cell pellets were resuspended in 20 mM HEPES, pH 7.3, 300 mM NaCl, 1 mM TCEP and 1:200 (vol/vol) protease inhibitor cocktail. Homogenized cells were lysed by passing twice through a Microfluidizer at 9,000 psi. Lysates were clarified by centrifugation at 7,900g for 90 min at 4 °C. Clarified lysates were filtered through 0.45-μm Whatman PES syringe filters, and proteins were purified using NGC medium-pressure chromatography systems. Clarified lysates were thawed, adjusted to 35 mM imidazole and loaded at 3 ml min–1 onto IMAC columns equilibrated in IMAC equilibration buffer (EB) of 20 mM HEPES, pH 7.3, 300 mM NaCl, 1 mM TCEP, 35 mM imidazole and 1:1,000 protease inhibitor cocktail. The columns were washed to baseline with EB and proteins eluted with a 20 column-volume gradient from 35 mM to 500 mM imidazole in EB. Elution fractions were analyzed by SDS–PAGE.’ The quantity of all purified proteins was estimated by Coomassie blue stained gel compared to known concentrations of the albumin standard. KRAS nucleotide exchange efficiency was determined using high-performance liquid chromatography. Exchanged proteins were diluted into 0.1 M K2HPO4 and 1 mM tetrabutyl ammonium hydrogen sulfate (buffer A) and injected onto an Ultrasphere 5 ODS, 250 ×4.6 mm column (Hichrom). Bound nucleotides were eluted using a linear gradient of buffer A containing 30% acetonitrile at a flow rate of 0.6 ml min–1. Standards of GDP and GMPPNP (GTP) were included to validate the identity of the bound nucleotide. GMPPNP exchange efficiency was routinely >95% pure as measured by this assay.

Generation of full-length GST–RanGAP1 and truncated (1–416) GST–RanGAP1 constructs by PCR cloning

DDK–RanGAP1 expressing wild-type RanGAP1 was used as a template. The designed PCR primers included 5′ KpnI and 3′ NotI restriction sites. All primer sequences are described in Supplementary Table 2. Twenty cycles of PCR were performed. The PEBG mammalian expression vector41 was used for GST-tagged proteins after subcloning the PCR products with KpnI and NotI restriction sites.

In vitro KRAS–RanGAP1 binding assay

Purified KRAS proteins (wild-type KRAS or KRAS-G12D) were mixed with purified DDK–RanGAP1, DDK–CDCP1 or RAF-RBD in Mg++ lysis buffer (EMD Millipore) and rotated for 3 h at 4 °C, followed by washing with HNTC buffer. The pulldown beads were resuspended in 50 μl of Laemmli sample buffer separated on a reducing SDS–PAGE gel and immunoblotted with antibodies to DDK and KRAS, followed by secondary anti-IgG conjugated to anti-horseradish peroxidase (1:5,000). The signals bound to the membranes were detected by an ECL plus kit (GE Healthcare).

Nuclear and cytoplasmic fractionation

Nuclear and cytoplasmic fractionation was performed according to our previously described protocol4. ‘Nuclear and cytoplasmic fractions of cells were purified using a Nuclear/Cytosolic Fractionation Kit (AKR-171, Cell Biolabs), as per the manufacturer’s instructions. Briefly, cells were pelleted by centrifugation for 5 min at 4 °C (600g) and washed with ice-cold PBS. The cell pellets were resuspended with 500 μl of ice-cold extraction buffer containing DTT and protease inhibitors. The cell suspension was transferred into a prechilled microcentrifuge tube and incubated on ice for 10 min, 25 μl of cell lysis reagent was added, vortexed for 10 s and centrifuged at 800g for 10 min at 4 °C. The resulting supernatant (cytoplasmic fraction) was transferred to a clean, chilled microcentrifuge tube and stored at –80 °C until use. For nuclear protein extraction, the pellet was gently resuspended in 100 μl of ice-cold nuclear extraction buffer containing DTT and protease inhibitors, incubated on ice for 30 min, vortexed for 10 s and centrifuged at 14,000g for 30 min at 4 °C. The supernatant (nuclear protein extract) was stored at –80 °C until use. All buffers were supplemented with protease cocktail and phosphatase inhibitors’.

PM isolation

The PM was isolated using a Minute Plasma Membrane Protein Isolation and Cell Fractionation kit (SM-005, Invent Biotechnologies), as per the manufacturer’s instructions. Cells were pelleted by centrifugation for 5 min at 4 °C (600g), washed with cold PBS, incubated with Buffer A for 10 min on ice, vortexed at high speed for 30 s, transferred to a prechilled filter cartridge assembly collection tube and centrifuged at 16,000g for 30 s at 4 °C. The pellet was resuspended and centrifuged at 700g for 1 min, and the supernatant was transferred to a new microcentrifuge tube and centrifuged at 16,000g for 30 min at 4 °C. For the PM, the pellet was resuspended in 200 μl of Buffer B by vortexing at moderate intensity for 30 s and centrifuging at 7,800g for 5 min at 4 °C. The supernatant was transferred to a new microcentrifuge tube, and 1.5 ml of chilled PBS was added, mixed vigorously for 30 s and centrifuged at 16,000g for 30 min at 4 °C. The pellet containing the PM was resuspended in RIPA buffer with protease and phosphatase inhibitors plus 1.0% Triton X-100.

NE isolation

The NE was isolated using a Minute Nuclear Envelope Protein Extraction kit (NE-013, Invent Biotechnologies), as per the manufacturer’s instructions. Cells were pelleted by centrifugation at 600g for 5 min at 4 °C, washed twice with PBS, resuspended in Buffer A, incubated for 10 min on ice, mixed vigorously, transferred into a prechilled filter cartridge assembly tube and centrifuged at 14,000g for 30 s at 4 °C. The pellet was washed with cold PBS, resuspended in Buffer B by vortexing, incubated on ice for 5 min and centrifuged at 5,000g for 5 min at 4 °C. The supernatant was transferred to a new tube, and 1.0 ml of cold PBS was added, mixed vigorously for 15 s and centrifuged at 16,000g for 15 min at 4 °C. The NE pellet was then resuspended in RIPA buffer with protease and phosphatase inhibitors plus 0.25% Triton X-100.

Ran•GTP assay

A Ran activation assay kit (81701, NewEast Biosciences) was used for measuring GTP-bound Ran, as per the manufacturer’s instructions. Briefly, equal amounts of each cytoplasmic fraction (1,000 µg of protein) was incubated with 2 µl of anti-Ran•GTP for 1 h. After incubation, 30 µl of Protein A/G Agarose was added to each immune reaction and rotated at 4 °C for 2 h. The beads were then washed three times with washing buffer. Washed samples were subjected to separation on 4–12% SDS–PAGE gels, transferred onto nitrocellulose membranes and detected by immunoblotting using antibody to Ran (Cell Signaling Technology).

Primary human lung adenocarcinoma samples

The primary human lung adenocarcinoma samples were provided by the lung SPORE from Winship Cancer Institute, Emory University. Snap-frozen remnant lung tumor tissues were obtained from deidentified individuals by assigning random IDs for the purpose of this study under an Emory University institutional review board-approved protocol.

PDX models

Flash-frozen tumor fragments from PDX models 941728-121-R (lung adenocarcinoma), 422866-222-R5 (pancreatic adenocarcinoma), 463931-005-R (pancreatic adenocarcinoma), 572918-348-R (colon adenocarcinoma), 144555-231-T (colon adenocarcinoma), 765993-094-R (nasopharyngeal carcinoma with mutant HRAS-G12D) and 782815-120-R (colon adenocarcinoma with mutant NRAS-Q61R) were obtained from the NCI’s Patient Derived Models Repository (NCI Frederick, Frederick National Laboratory for Cancer Research; https://pdmr.cancer.gov/). Flash-frozen tumor fragments from PDX models LG0703-F948 (lung adenocarcinoma) and K00052-001-T (lung adenocarcinoma) were developed by The Jackson Laboratory and are available from the NCI Patient Derived Models Repository.

Development and treatment of the KRAS-G12D mouse lung cancer model

All mouse studies were approved by the NCI Animal Care and Use Committee and were conducted in compliance with the approved protocols. The animals were housed under standard laboratory conditions on a 12-h dark/12-h light cycle (0600 to 1800 h) at ambient temperature 20–24 °C with 30–70% humidity and were provided with continuous food and water. Mouse lung tumors were generated by conditional expression of oncogenic KRAS and inactivation of p53 (ref. 25). The KrasLSL-G12D/+ (B6.129S4-Krastm4Tyj/J) and Trp53fl/fl (B6.129P2-Trp53tm1Brn/J) mouse strains were purchased from The Jackson Laboratory and were bred to produce KrasLSL-G12D/+; Trp53fl/fl mice. Adenovirus expressing Cre recombinase (Ad5CMVCre) was provided by the University of Iowa Viral Vector Core Facility, and a dose of 2.5 × 107 plaque-forming units per mouse was delivered to the respiratory tract of mice anesthetized with isoflurane, using the modified intranasal method of Santry et al.43. Three months after adenovirus infection, mice were randomly divided into groups and were treated daily for 3 weeks via intraperitoneal injection of KRAS-G12D inhibitor mrtx-1133 (15 mg per kg (body weight)) alone, oral administration of the XPO1 inhibitor selinexor (30 mg per kg (body weight)) alone, the three-drug combination of mrtx-1133 (15 mg per kg (body weight)) + saracatinib (50 mg per kg (body weight)) + mk-2206 (50 mg per kg (body weight)) or selinexor (30 mg per kg (body weight)) + saracatinib (50 mg per kg (body weight)) + mk-2206 (50 mg per kg (body weight)), the two-drug combination of selumetinib (15 mg per kg (body weight)) + copanlisib (15 mg per kg (body weight)) or saracatinib (50 mg per kg (body weight)) + mk-2206 (50 mg per kg (body weight)) or vehicle (Captisol), and intact lungs with residual tumors were then excised and processed for biochemical assays after treatment.

In vivo tumorigenesis and treatment of mice with inhibitors

For the development and treatment of mice with xenograft tumors, A549 or NCI-H23 cells with DLC1-WT or DLC1-KO were trypsinized, washed with cold PBS, diluted to 107 cells per ml with serum-free medium/Matrigel basement membrane matrix (BD Biosciences) at a ratio of 3:1 and injected subcutaneously into NOD-scid mice (106 cells per injection). When tumors were approximately 0.5 cm in diameter, mice were randomly divided into groups and were treated daily with oral EZH2 inhibitor tazemetostat (25 mg per kg (body weight)), the XPO1 inhibitor selinexor (30 mg per kg (body weight)) or the KRAS-G12C inhibitor sotorasib (15 mg per kg (body weight)), the two-drug combination of selumetinib (15 mg per kg (body weight)) + copanlisib (15 mg per kg (body weight)) for 1 week, followed by treatment with the indicated combination of tazemetostat (25 mg per kg (body weight)) + saracatinib (50 mg per kg (body weight)) + mk-2206 (50 mg per kg (body weight)) or sotorasib (15 mg per kg (body weight)) + saracatinib (50 mg per kg (body weight)) + mk-2206 (50 mg per kg (body weight)), all three drugs in the indicated combination or vehicle (Captisol) for an additional 2 weeks, and the remaining tumor tissues were excised, weighed and processed for biochemical assays after treatment. The maximal tumor size was not exceeded as permitted by the ethics committee and approved protocols. Sex was not considered in the study design because sex-based analysis was not relevant to the study. Therefore, this information was not collected.

Data analysis, statistics and reproducibility

At least two independent experiments were performed for all experiments. Immunoblots were quantified by densitometric scanning using Fiji software. Results in bar graphs are displayed as mean values ± s.d. from two or three experiments. No statistical methods were used to predetermine sample sizes, but our sample sizes are similar to those reported in previous publications within this field of research4,28. All animal experiments were grouped randomly based on genetically related cohorts and tumor size. For all other experiments, the sample allocation was random. The investigators were blinded to group allocation during data collection and/or analysis. No animals and data points were excluded from the analyses. All experiments were designed with matched control conditions within each experiment. Data distribution was assumed to be normal, but this was not formally tested. For the statistical analyses, parametric unpaired one-tailed t-test with Welch’s correction was performed using Prism software (version 10.1.1 (270), GraphPad), and no adjustments were made for multiple comparisons. A P value of <0.05 was considered statistically significant.

Reporting summary

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