Mutant KRAS tumors are associated with poor outcomes, at least in part, due to decreased therapeutic sensitivity. Here, we show that KRAS mutations are associated with resistance to monotherapy and combination therapy with PARP inhibitors (PARPi) and immune checkpoint blockade with anti–PD-L1 antibodies. In mutant KRAS tumors, inhibition of KRAS signaling with MEK inhibitors (MEKi) triggered and amplified PARPi-induced DNA damage, cytosolic double-stranded DNA accumulation, STING pathway activation, and CD8+ T-cell recruitment. Moreover, MEKi decreased myeloid-derived suppressor cell infiltration, in part, by inhibiting IL6 and GMCSF production. Importantly, addition of MEKi to PARPi and anti–PD-L1 resulted in marked tumor inhibition in immunocompetent mutant KRAS tumor models. This study provides the underlying mechanistic data to support evaluation of PARPi, MEKi, and anti–PD-L1 combination in clinical trials of mutant KRAS tumors.

Significance:

This study provides key insights into the potential for using MEKi combined with PARPi and anti–PD-L1 for the treatment of all mutant KRAS tumors.

PARP inhibitors (PARPi) entered the clinic as a result of studies showing synthetic lethality with defects in homologous recombination (HR) DNA damage repair (DDR), such as those caused by BRCA mutations (1). PARPi have been approved for treatment of HR defective (HRD) tumors, including ovarian, breast, pancreatic, and prostate cancers (2). Unfortunately, in the majority of patients, resistance to PARP inhibition develops, leading to treatment failure (3). In addition, a proportion of patients exhibit primary resistance to PARPi, despite harboring genomic features of HR deficiency (4). Therefore, efforts are underway to develop new therapeutic strategies with novel drug combinations to extend the population of patients who would benefit from PARPi beyond HR deficiency and to increase the depth and duration of response to PARPi.

Recent studies have provided a compelling rationale for combining PARPi with immunotherapy with immune checkpoint blockade (ICB) to broaden responding populations, as well as to delay and overcome emerging resistance, leading to improved response duration and overall survival. Many potential mechanisms by which PARPi could increase the activity of immunotherapy have been proposed: (i) PARPi triggers cGAS/STING signaling through an accumulation of cytosolic double-stranded DNA (dsDNA), favoring lymphoid-attractant chemokine secretion and increasing immune cell infiltration and cytotoxic T-cell activation (5); (ii) in the context of defective DDR, PARPi triggers cancer cell death through replication stress and a mitotic catastrophe that is proposed to expose tumor-specific antigens to immune surveillance (6); (iii) PARPi, by inducing DNA damage, as well as decreasing DDR, is proposed to increase the mutational burden and promote immunogenicity and immune priming by increasing neoantigen expression (7); and (iv) PARPi upregulates PD-L1 through IFN-dependent pathways (8), or tumor cell intrinsic mechanisms (9). On the basis of these observations, a number of clinical trials are currently ongoing (ClinicalTrials.gov) with four different PARPi/anti–PD-1/L1 combinations being tested: olaparib/durvalumab (10, 11), niraparib/pembrolizumab (12), talazoparib/avelumab (13, 14), and BGB-A317/BGB-290 (15). Overall, clinical studies conducted to date suggest combinations of PARPi and anti–PD-1/L1 agents are well tolerated and demonstrate encouraging antitumor activity in a wide range of solid malignancies independent of DDR status, including mutations in BRCA1 or BRCA2. However, several key questions remain unanswered. Most critically: what is the magnitude and nature of benefit from combination treatment versus monotherapy? Does benefit varies across different tumor types? What is the contribution of HRD status? What approaches can be used to overcome resistance? Answers to these questions will be necessary to identify patient populations most likely to benefit from PARPi plus ICB and to increase the depth and duration of responses.

Increasing evidence suggests that activation of oncogenic pathways is associated with the generation of an immune desert or non–T-cell–inflamed tumor microenvironment (TME) with consequent immunotherapy resistance (16). KRAS represents one of the most frequently mutated oncogenes in cancer with alterations most common in pancreatic carcinoma (72%), colorectal cancer (69%), lung adenocarcinoma (33%), endometrial cancer (10%–31%), and low-grade serous ovarian cancer (35%; ref. 17). KRAS-mutant cancers are refractory to targeted therapies and anti–PD-1/PD-L1 therapies (18). The limited response to therapy could be attributed to an immunosuppressive TME, decreased cytotoxic T cells (19), increased regulatory T cells (Treg; ref. 20), and myeloid-derived suppressor cells (MDSC; ref. 19). This may be explained, in part, by KRAS mutations being associated with increased IL6 production, which recruits neutrophils, decreases T-cell infiltration, increases T-cell exhaustion markers (PD-1, CTLA-4, and TIM3), and increases expression of PD-L1 on tumor cells (21). Interestingly, MAPK pathway gene signatures are enriched in anti–PD-1 nonresponding patients (16).

We demonstrate that KRAS-mutant tumor models are resistant to PARPi, anti–PD-L1, and PARPi plus anti–PD-L1 combinations. MEK inhibitors (MEKi) both trigger and amplify PARPi-induced DNA damage, cytosolic dsDNA accumulation, STING pathway activation, and CD8+ T-cell recruitment. Moreover, MEKi decreases MDSC infiltration, at least, in part, by decreasing IL6 and GMCSF. Importantly, our results demonstrate remarkable efficacy of the triplet combination of PARPi, MEKi, and anti–PD-L1 blockade in immunocompetent KRAS-mutant tumor models.

Cell lines and cell culture

CT26, MC38, OVCAR3, A2780, HOC7, TOV-21G, LOVO, and A549 were obtained from the ATCC. NCI-H441 and NCI-H1793 were obtained from China Center for Type Culture Collection. MCAS and HeyA8 were from MD Anderson Cancer Center (MDACC, Houston, TX) Characterized Cell Line Core. Pa02c, Pa16c, and iKRAS murine cells (primary murine pancreatic cancer cells established from p48 Cre_tetO_LKrasG12D ROSA_rtTAL+p53L+ mice) were gifted from professor Anirban Maitra (Division of Pathology/Lab Medicine, The University of Texas MDACC, Houston, TX). iKRAS cells were induced in the medium with 2 μg/mL doxycycline (Sigma, D9891; doxycycline-on) for 48 hours or without doxycycline. All cell lines were authenticated through short tandem repeat profiling and tested monthly for Mycoplasma by PCR. Cell lines were not passaged more than 30 times. All cells were maintained in humidified incubators under 37°C, 5% CO2. KRAS mutation status for each cell line is listed in Supplementary Table S1.

Antibodies and compounds

GAPDH (AC033), STING (A3575), phosphorylated MEK1/2 (pMEK1/2) (AP0209), TBK1 (A14641), IRF3 (A11373) antibodies were from ABclonal. CD4 (ab183685), CD8 (ab209775), pERK1/2 (ab 214362), pSTAT1 (ab109461), and phosphorylated TBK1 (pTBK1) (ab 109272) antibodies were from Abcam. Phosphorylated ERK1/2 (pERK1/2) (4370), pIRF3 (29047), STAT1 (14994), and phosphorylation of histone H2AX at Ser139 (γH2AX) (2577) antibodies were from Cell Signaling Technology.

Olaparib, BMN673, and trametinib were from Selleck. Cobimetinib, H151, and C176 were from MCE. Compounds were dissolved in DMSO and stored as 10 mmol/L aliquots at −80°C. Anti–PD-L1 antibody (BE0101, clone B7-H1) and IgG isotype control (BE0090) were from BioXCell. CD45 (BV605 30-F11), CD3e (APC-Cy7 145-2C11), mouse CD4 (FITC RM4-5), CD25 (PE-Cy7 PC61), Foxp3 (BV421 MF23), CD8a (PerCP-Cy5.5 53-6.7), granzyme B (D2H2F), IFNγ (PE XMG1.2), CD11c (PE-Cy HL3), I-A/I-E (BV421 M5/114.15.2), F4/80 (PE T45-2342), CD11b (FITC M1/70), CD49b (APC DX5), CD3e (APC-Cy7 145-2C11), ly-6G (PE 1A8), ly-6C (APC AL-21), Gr-1 (PerCP-Cy5.5), Fixable Viability Stain (BV510), and Transcription Factor Buffer Set were from BD Biosciences.

Reverse phase protein array

Protein lysates were analyzed by reverse phase protein array (RPPA), performed as described previously (22) by the MDACC (Houston, TX) Cancer Center Support Grant–supported RPPA Core. Antibodies and approaches are described at the RPPA website: https://www.mdanderson.org/research/research-resources/core-facilities/functional-proteomics-rppa-core.html.

IHC staining

Briefly, formalin-fixed, paraffin-embedded tissues were subsequently sectioned at 4 μm and mounted on coated glass slides. Tissues were subjected to deparaffinization and antigen retrieval prior to antibody staining, and then stained with primary antibody overnight at 4°C. Primary antibodies used included pERK1/2 (1:500), CD4 (1:800), and CD8 (1:800). Negative controls were treated identically, but with normal serum. After mounting, slides were observed under microscope and pictures were taken. The expression of the CD4+ and CD8+ T cells was evaluated using an Optical Microscope (BX53F; Olympus). Quantitation of the positive cells in the tumor sections was performed using methods described by Ma and colleagues (23). Briefly, the number of positive cells was counted in five areas that included both cancer nest and stromal areas at a high-power field (200×), and the average number of positive cells per high-power field was calculated. pERK staining was assigned a score using a semiquantitative six-category grading system: 0, no staining; 1, 1% to 10% staining; 2, 11% to 25% staining; 3, 26% to 50% staining; 4, 51% to 75% staining; and 5, >75% staining. Staining intensity was assigned a score using a semiquantitative four-category grading system: 0, no staining; 1, weak staining; 2, moderate staining; and 3, strong staining. Every core was assessed individually and the mean of five readings was calculated for every slide. Staining score was determined separately by two experts under the same conditions. In rare cases, discordant scores were reevaluated and scored by another expert.

Alkaline single-cell agarose gel electrophoresis (Comet) assays

Alkaline single-cell agarose gel electrophoresis assays were performed according to the manufacturer's instructions of Trevigin Comet Assays Kit. Briefly, cells were suspended in LMA agarose gel and then mounted on comet slides, followed by incubation in lysis solution for 1 hour at 4°C and freshly prepared alkaline unwinding solution (pH > 13) for 20 minutes at 25°C in the dark. Electrophoresis was performed at 21 V for 30 minutes in electrophoresis solution (pH > 13). Tail DNA content was analyzed with Comet score 1.6 software after staining with SYBR Green I. The tail moment, which is stand for DNA strand breakage, was measured for at least 100 cells per sample, and average damage from three independent experiments was calculated.

RNAi treatment

siRNAs (100 μmol/L) for human TMEM173 (SASI_Hs02_00371843, SASI_Hs01_00031033) and mouse TMEM173 (SASI_Mm02_00424289, SASI_Mm02_00424293) were obtained from Sigma-Aldrich and were transfected separately according to the manufacturer's instructions with Lipofectamine 3000 (Invitrogen). Proteins were assessed 48 hours after transfection.

Western blot analysis

Cells were lysed with RIPA Buffer (Servicebio, G2002-100) supplemented with Protease and Phosphatase Inhibitor Cocktail (Servicebio, G2006). After thorough mixing and incubation at 4°C for 45 minutes, lysates were centrifuged at 12,000 × g at 4°C for 25 minutes, and supernatants collected. Protein content was determined, separated by 10% SDS-PAGE, and electrotransferred onto 0.45 μm polyvinylidene difluoride membranes. After blocking with 5% BSA in TBST at room temperature for 1 hour, membranes were incubated with primary antibodies at 4°C overnight, followed by 1:3,000 horseradish peroxidase–conjugated secondary antibody (Antgene) incubation for 1 hour at room temperature. Bands were visualized using WesternBright ECL Kit (Advansta, 190113-13).

dsDNA90 stimulation

dsDNA90 was prepared as follows: a single-stranded DNA (ssDNA) sense strand (5′-TACAGATCTACTAGTGATCTATGACTGATCTGTACATGATCTACATACAGATCTACTAGTGATCTATGACTGATCTGTACATGATCTACA-3′) was annealed to an ssDNA90 antisense sequence. Cells were seeded onto a 6-well culture plate for 24 hours and then transfected with dsDNA90 (0.5 μg/mL) according to the manufacturer's instructions of Lipofectamine 3000 (Invitrogen). After transfection for 24 hours, cells were collected and used for further experiments.

Immunofluorescence staining and microscopy

Briefly, cells were fixed with 4% paraformaldehyde and then blocked with 5% normal blocking serum for 25 minutes and incubated with primary antibody of γH2AX (Ser139, #2577, 1:400 from Cell Signaling Technology) for 2 hours, followed by secondary antibody incubation for 1 hour at 25°C. Mounting was done in the medium containing DAPI (Sigma-Aldrich, D9542) for further image acquisition. OLYMPUS U-HGLGPS imaging acquisition system equipped with a 40× objective lens was used to capture images. At least 100 cells per slide were analyzed to count percentages for γH2AX foci–positive cells.

PicoGreen staining

PicoGreen staining was performed according to the manufacturer's instructions of QuantiT Pico-Green dsDNA Reagent (Thermo Fisher Scientific). For confocal microscopy, cells were incubated with 3 μL/mL PicoGreen for 1 hour at 37°C. Then cells were washed with PBS for 5 minutes × 5 times and fixed for confocal microscopy with DAPI counterstaining.

RT-qPCR assays

Total RNA (1 μg) was reverse transcribed into cDNA with the Hiscript Q-RT SuperMix for qPCR (Vazyme Biotech) according to the manufacturer's instructions. The SYBR Green Real-Time PCR Master Mixes Kit (Life Technologies) was used for the thermocycling reaction in a Bio-Rad CFX96 Real-Time System. The mRNA level analysis was carried out in triplicate and normalized by GAPDH. Primers sequences are listed in Supplementary Table S2.

ELISA

The cell culture supernatants were collected and processed according to the manufacturer's instructions. The IL6 [EHC007(H).96] and GMCSF (EHC105.96) levels were determined using ELISA Kit for human from Neobioscience. Human CXCL10 (EHC157.96) ELISA Kit was from Neobioscience, human CCL5 (DRN00B) ELISA Kit was from R&D Systems, while the mouse CXCL10 ELISA Kit was from Thermo Fisher Science (BMS6018).

Flow cytometry and intercellular cytokine staining

Tumor tissues were washed in PBS, minced into small fragments, and incubated in collagenase solution (1 mg/mL collagenase IV in RPMI1640) and DNase I (Sigma-Aldrich) at 37°C, 120 rpm for 2.5 hours. Dissociated cells were passed through a 40-μm strainer to achieve single-cell suspensions followed by erythrocyte lysis. The following mAbs were used in flow cytometry: fixable viability stain 510, anti-CD45 (BV605; 30-F11), anti-CD3e (APC-Cy7; 145-2C11), anti-CD4 (FITC; RM4-5), anti-CD25 (PE-Cy7; PC61), anti-CD8a (PerCP-Cy5.5; 53-6.7), anti-CD11c (PE-Cy7; HL3), anti–I-A/I-E (BV421; M5/114.15.2), anti-F4/80 (PE; T45-2342), anti-CD11b (FITC; M1/70), anti-CD49b (APC; DX), anti–Gr-1 (BV510), anti–Ly-6G (PE; 1A8), and anti–Ly-6C (APC; AL-21; all from BD Biosciences). Single-cell suspensions were stained with 1 μg/sample fluorochrome-labeled antibodies for specific surface marker at 4°C for 30 minutes in 100 μL PBS. Then, the cells were fixed, permeabilized, and stained for intracellular cytokines, including anti-Foxp3 (BV421; MF23) and anti-IFNγ (PE; XMG1.2) by using a Fixation/Permeabilization Kit (BD Biosciences). Stained single-cell suspensions of tumor tissue were processed to flow cytometry using CytoFlex. The data were analyzed by using FlowJo v10 software. Gating strategies are displayed in Supplementary Fig. S6.

Mass cytometry (cytometry by time of flight)

Single-cell suspensions were achieved as above. Tumor-infiltrating lymphocytes (TIL) were enriched on a gradient Ficoll (Sigma-Aldrich) solution. TILs were incubated with mouse anti-CD16/32 antibody (eBioscience) to block FcγR binding for 10 minutes and then applied to cytometry by time of flight (CyTOF). For CyTOF analysis, TILs were incubated with a mixture of metal-labeled antibodies (listed below) for 30 minutes at 25°C, washed twice by PBS, and incubated with Cell-ID Intercalator-103Rh (Fluidigm) overnight at 4°C. The samples were analyzed with the CyTOF2 Instrument (Fluidigm) in the Flow Cytometry and Cellular Imaging Core Facility at MDACC (Houston, TX). Detailed antibodies used for CyTOF analysis are presented in Supplementary Table S3 (provided by Fluidigm).

Cytokine protein arrays

HOC7 cells were treated with vehicle, olaparib (5 μmol/L), trametinib (500 nmol/L), and combined therapy for 24 hours and supernatant was collected for cytokine analysis. A semiquantitative membrane-based RayBio Human Inflammation Antibody Array Kit (Human inflammation Array C3) was used to analyze a panel of 40 inflammatory mediators according to the manufacturer's instructions. After immunoblotting, the reaction intensity was quantified using NIH ImageJ software. The data were normalized and expressed as Z-score of the mean fold changes as a ratio of vehicle cohort.

Animal models

All experiments were performed in accordance with the guidelines for the Care and Use of Laboratory Animals of Tongji Hospital (Wuhan, P.R. China). Female, 8-week-old NOD/SCID mice, FVB mice, nude mice, C57BL-6J mice, and Balb/c mice were purchased from Beijing HFK Bioscience and housed in laminar flow cabinets under specific pathogen–free conditions. Tumor size and mouse weight were monitored every 3 days. Tumor volumes were calculated using the formula: tumor volume = 1/2 (length × width2).

LPA1-T22 and LPA1-T127 syngeneic breast cancer model

LPA1-T22 tissues were obtained from a transgenic mouse model that expressed LPA1 receptor in mammary epithelium, and LPA1-T127 tissues were obtained from a transgenic mouse model that expressed LPA1 receptor in mammary epithelium and spontaneous KRASQ61H mutation. Minced fresh tumor tissue (0.1 cm3/mouse) was transplanted into the mammary fat pads of FVB mice. After the tumors were palpable, the mice were randomly assigned to treatment cohorts. In Fig. 1A and B, mice were randomized into treatment cohorts as: vehicle (0.5% hydroxypropylmethylcellulose and 0.2% Tween 80 and IgG isotype control), BMN673 (0.333 mg/kg/day), anti–PD-L1 antibody (αPD-L1, 200 μg/mouse every 3 days for six times), and doublet (n = 8 for each group). In Fig. 6A, LPA1-T127 mice were randomized into treatment cohorts as: vehicle [0.5% methylcellulose or 10% DMSO in (2-hydroxypropyl)-b-cyclodextrin/PBS solution], olaparib (50 mg/kg/every day), trametinib (1 mg/kg/every day), and doublet (n = 6 for each group). In Fig. 7A, LPA1-T127 mice were randomized into treatment cohorts as: vehicle [0.5% methylcellulose or 10% DMSO in (2-hydroxypropyl)-b-cyclodextrin/PBS solution and IgG isotype control], olaparib (50 mg/kg/every day), trametinib (1 mg/kg/every day), αPD-L1 (200 μg/mouse every 3 days for six times), the combination of olaparib and trametinib, the combination of olaparib and anti–PD-L1, the combination of trametinib and anti–PD-L1, and triplet treatment (n = 6 for each group).

Figure 1.

Mutant KRAS is associated with PARPi, anti–PD-L1, and doublet combination therapy resistance in vivo. A and B,KRASWT (LPA1-T22) and KRASQ61H mutation (LPA1-T127) tumors were transplanted into the mammary fat pads of FVB mice. Eight days later, mice were randomized into treatment cohorts: vehicle (0.5% hydroxypropylmethylcellulose and 0.2% Tween 80), BMN673 (0.333 mg/kg/day), anti–PD-L1 antibody (200 μg/mouse every 3 days), or the combination of BMN673 and anti–PD-L1 (n = 8 for each group). Tumor measurements were performed every 3 days by calipers, and average tumor volume ± SEM for each cohort is displayed. P values were determined by one-way ANOVA test. C, PCA was performed on each protein from RPPA data. Ellipses indicate 95% confidence interval of group membership. Axis percentages indicate variance contribution. D, Volcano plot shows differentially expressed proteins from RPPA data of LPA1-T22 and LPA1-T127. Proteins above the horizontal line and to the left and right of the vertical line exhibited over- (red circle) and underexpression (green circle), respectively. Black circles, nondifferentially expressed proteins. E, Heatmap of RPPA data represents differentially expressed proteins [log2 (fold change) ≥ 1; P < 0.001]. Different passages are shown on x-axis. Statistically significant changes (z-scores) are indicated in boxes. F, Box plot of differentially expressed pMAPK and pMEK1 from RPPA data (Student t test). G, Murine MC38 colorectal cancer cells (5 × 105) were subcutaneously injected into the right flank of C57BL-6J mice (6–8 weeks old). When tumors were palpable, mice were randomized into treatment cohorts accordingly (n = 8). H, Murine CT26 colorectal cancer cells (2 × 105) were subcutaneously injected into the right flank of Balb/c mice (6 to 8 weeks old). When tumors were palpable, mice were randomized into treatment cohorts accordingly (n = 5). A, B, G, and H, Data represent mean ± SEM. *, P < 0.05; ***, P < 0.001, nonparametric pairwise comparisons (Mann–Whitney). n.s., not significant.

Figure 1.

Mutant KRAS is associated with PARPi, anti–PD-L1, and doublet combination therapy resistance in vivo. A and B,KRASWT (LPA1-T22) and KRASQ61H mutation (LPA1-T127) tumors were transplanted into the mammary fat pads of FVB mice. Eight days later, mice were randomized into treatment cohorts: vehicle (0.5% hydroxypropylmethylcellulose and 0.2% Tween 80), BMN673 (0.333 mg/kg/day), anti–PD-L1 antibody (200 μg/mouse every 3 days), or the combination of BMN673 and anti–PD-L1 (n = 8 for each group). Tumor measurements were performed every 3 days by calipers, and average tumor volume ± SEM for each cohort is displayed. P values were determined by one-way ANOVA test. C, PCA was performed on each protein from RPPA data. Ellipses indicate 95% confidence interval of group membership. Axis percentages indicate variance contribution. D, Volcano plot shows differentially expressed proteins from RPPA data of LPA1-T22 and LPA1-T127. Proteins above the horizontal line and to the left and right of the vertical line exhibited over- (red circle) and underexpression (green circle), respectively. Black circles, nondifferentially expressed proteins. E, Heatmap of RPPA data represents differentially expressed proteins [log2 (fold change) ≥ 1; P < 0.001]. Different passages are shown on x-axis. Statistically significant changes (z-scores) are indicated in boxes. F, Box plot of differentially expressed pMAPK and pMEK1 from RPPA data (Student t test). G, Murine MC38 colorectal cancer cells (5 × 105) were subcutaneously injected into the right flank of C57BL-6J mice (6–8 weeks old). When tumors were palpable, mice were randomized into treatment cohorts accordingly (n = 8). H, Murine CT26 colorectal cancer cells (2 × 105) were subcutaneously injected into the right flank of Balb/c mice (6 to 8 weeks old). When tumors were palpable, mice were randomized into treatment cohorts accordingly (n = 5). A, B, G, and H, Data represent mean ± SEM. *, P < 0.05; ***, P < 0.001, nonparametric pairwise comparisons (Mann–Whitney). n.s., not significant.

Close modal

CT26 subcutaneous model

A total of 2 × 105 CT26 cells were injected subcutaneously into right flank of the Balb/c mice (female, 6–8 weeks old) in a 1:1 mixture of PBS and Matrigel. After the tumors were palpable, the mice were randomly assigned to treatment cohorts. In Fig. 1H, mice were randomized into treatment cohorts as: vehicle (0.5% hydroxypropylmethylcellulose and 0.2% Tween 80 and IgG isotype control), olaparib (50 mg/kg/every day), αPD-L1 (200 μg/mouse every 3 days for six times), and doublet (n = 5 each group). In Fig. 7A, CT26 mice were randomized into treatment cohorts as: vehicle [0.5% methylcellulose or 10% DMSO in (2-hydroxypropyl)-b-cyclodextrin/PBS solution and IgG isotype control], olaparib (50 mg/kg/every day), trametinib (1 mg/kg/every day), αPD-L1 (200 μg/mouse every 3 days for six times), the combination of olaparib and trametinib, the combination of olaparib and anti–PD-L1, the combination of trametinib and anti–PD-L1, and triplet treatment (n = 5 for each group). Treatments were terminated at day 15, and tumor growth was observed for another 2 weeks. In Fig. 7B, 2 × 105 CT26 cells were injected subcutaneously into left flank of the Balb/c mice (female, 6 to 8 weeks old). After the tumor volume reached 200 to 500 mm3, CT26 mice were randomized into treatment cohorts as accordingly (n = 6 for each group).

MC38 subcutaneous model

A total of 5 × 105 MC38 cells were injected subcutaneously into right flank of C57BL/6J mice (female, 6–8 weeks old). After tumors were palpable, mice were randomly assigned to treatment cohorts. In Fig. 1G, mice were randomized into treatment cohorts as: vehicle (0.5% hydroxypropylmethylcellulose and 0.2% Tween 80 and IgG isotype control), olaparib (50 mg/kg/every day), αPD-L1 (200 μg/mouse every 3 days for six times), and doublet (n = 8 each group). Treatment was terminated at day 21.

Nude mouse model

Minced LPA1-T127 tumor tissue (0.1 cm3/mouse) was transplanted into the mammary fat pads of nude mice. In Fig. 6A, after the tumors were palpable, the mice were randomly assigned to four groups, which were vehicle [0.5% methylcellulose or 10% DMSO in (2-hydroxypropyl)-b-cyclodextrin/PBS solution], olaparib (50 mg/kg/every day), trametinib (1 mg/kg/every day), and doublet (n = 6 for each group).

Computational analysis

The gene expression datasets of patient-derived xenografts (PDX) from patients with pancreatic adenocarcinoma, an inducible mouse model of NRAS-mutant melanoma, human pancreatic ductal epithelial cells transduced with KRASG12D, and bronchial epithelial cells 16HBE [Gene Expression Omnibus (GEO) access nos. GSE98399, GSE58055, GSE39984, and GSE63229] were downloaded from the NCBI GEO database (http://www.ncbi.nlm.nih.gov/geo/). Samples included were innately or inducible to be RAS mutant. The following information was also extracted from each identified study: GEO accession number, sample type, number of cases and controls, and gene expression data.

For gene set enrichment analysis (GSEA), the expression value of each gene was regarded as the average value corresponding to the same gene and was normalized using the LIMMA package of the R platform (version 3.5.1.; www.r-project.org). The biological pathways were further explored with GSEA using data derived from the Molecular Signatures Database of c2. P values were calculated by the clusterProfiler package, and P < 0.05 was determined to confer statistical significance.

Gene list of IFN responsive genes for heatmap analysis was obtained from Qiagen RT2 profiler PCR arrays (https://www.qiagen.com).

The analysis of correlation between activities of KRAS signaling and expression of MDSC signature was performed as described previously (24).

Quantification and statistical analysis

Two-sided Student t test was used to compare differences between two groups of cells in vitro. If the multiple groups data followed a normal distribution, we used ANOVA test for multiple comparisons. Nonparametric pairwise comparisons (Mann–Whitney) were conducted where xenograft size did not follow a Gaussian distribution in vivo. Data are presented as means ± SEM and P < 0.05 was considered significant. Correlation between groups was determined by Pearson correlation test. ANOVA was used to compare differences among multiple groups. Data were analyzed and plotted using GraphPad Prism 7 software. Statistical parameters, including sample size and statistical significance, are reported in the figures and corresponding figure legends.

Mutant KRAS is associated with PARPi, anti–PD-L1, and combination therapy resistance in vivo

To identify biomarkers for sensitivity to combined PARPi and anti–PD-L1 blockade, we assessed the effects of PARPi and anti–PD-L1 combinations in immunocompetent syngeneic MMTV-LPA receptor transgene-induced transplantable tumor models, which are a heterogeneous group of genetically engineered breast cancer mouse models (25). Like human PDX, the tumors have never been cultured on plastic, and, thus, may be more representative of human tumors (25). Strikingly, while combination therapy induced complete tumor regression and durable response in LPA1-T22 tumors (Fig. 1A), the LPA1-T127 model was not only highly resistant to PARPi or anti–PD-L1 monotherapy, but also to combination therapy (Fig. 1B). No significant changes in body weight were observed, indicating the overall safety of the therapy (Supplementary Fig. S1A). To reveal mechanisms underlying resistance of the LPA1-T127 tumors, we performed whole-exome sequencing and identified a spontaneous KRASQ61H mutation (25). Principal component analysis (PCA) of RPPAs with 170 total and phosphoproteins (Supplementary Fig. S1B; ref. 26) showed distinctive functional protein profiles between LPA1-T127 and LPA1-T22 tumors (Fig. 1C). Consistent with the acquired functional KRAS mutation, pMEK1/2 and pMAPK were remarkably upregulated in LPA1-T127 tumors (Fig. 1D–F). Fibronectin (a mesenchymal marker gene) and VEGFR, both of which are induced by KRAS mutation to promote tumor growth and migration (26), were elevated in LPA1-T127 tumors (Fig. 1E). Moreover, phospho-NF-κB p65 (pNF-κB) and STAT5a, two key inflammatory cytokine regulators, were decreased in LPA1-T127, consistent with MAPK pathway activation inducing an immunosuppressive TME (Fig. 1D; Supplementary Fig. S1C).

To confirm that the resistance of KRAS-mutant tumors to PARPi and anti–PD-L1 is not model specific, we investigated the effect of the PARPi and anti–PD-L1 combination in two additional murine colon cancer models with different KRAS-mutant status [CT26: (KRASG12D) and MC38 (KRASWT)]. Consistent with the observations in LPA1-T22 tumors, combination therapy with PARPi and anti–PD-L1 induced significant tumor regression in MC38 (KRASWT) tumors. However, CT26 tumors did not respond to olaparib or anti–PD-L1 alone or in combination (Fig. 1G and H). No significant weight changes were observed consistent with a lack of toxicity (Supplementary Fig. S1D).

PARPi-induced STING signaling and IFN responses are decreased in KRAS-mutant cancer cells

PARPi induces DNA double-strand breaks, causes cell-cycle arrest in S-phase, induces accumulation of cytosolic dsDNA, and activates the STING signaling pathway (27). STING, in turn, induces phosphorylation and nuclear translocation of the IFN transcriptional regulatory factors, TANK-binding kinase 1 (TBK1) and IFN regulatory factor 3 (IRF3; refs. 28, 29). To verify causal effects of KRAS mutation on PARPi-induced STING activation, we took advantage of an iKRASG12D cell (primary murine pancreatic cancer cells established from p48 Cre_tetO_LKrasG12D ROSA_rtTAL+p53L+ mice; ref. 30), in which oncogenic KRAS allele is inducible with doxycycline (30). Remarkably, while PARPi increased activation of TBK1, IRF3, and STAT1 in the absence of doxycycline, induction of KRASG12D with doxycycline abrogated the effects of PARPi (Fig. 2A). Consistent with this observation, CCL5 and CXCL10, the two major target genes downstream of STING activation that are involved in T-cell chemotaxis (31), as well as ISG15 and IFI44 mRNA, two IFN-stimulated genes (ISG; ref. 32), were upregulated in the absence of doxycycline, and induction of KRASG12D with doxycycline abrogated the effects of PARPi (Fig. 2B). Furthermore, PARPi did not increase CXCL10 secretion as detected by ELISA in doxycycline-on cells (Fig. 2C). This potent and specific effect supports a causal role for KRAS in blocking PARPi-induced STING activation.

Figure 2.

PARPi-induced STING signaling and IFN responses were alleviated in KRAS-mutant cancer cells. A, Doxycycline (Dox)-inducible iKRAS cells were incubated with doxycycline (2 μg/mL) for 48 hours and then treated with vehicle or olaparib (Olap; 5 μmol/L) for another 24 hours with doxycycline or withdrawal of doxycycline. Then cells were collected for Western blotting with indicated antibodies. B and C, Quantification of CCL5, CXCL10, ISG15, and IFI44 expression by RT-qPCR, and CXCL10 secretion levels by ELISA in iKRAS cells in the presence or absence of doxycycline (2 μg/mL) after treatment with vehicle or 5 μmol/L olaparib for 24 hours. D and E, Expression of pERK and STING pathway signaling (STING, pTBK1, pIRF3, and pSTAT1) in both KRASWT (MC38) and KRASG12D (CT26) cell lines after vehicle or 5 μmol/L olaparib treatment for 24 hours by Western blotting (left). CCL5, CXCL10, ISG15, and IFI44 gene expressions were quantified by RT-qPCR (middle), and CXCL10 secretion in culture media was quantified by ELISA (right). Data are shown as mean ± SEM from each of three independent experiments. n = 3. F, Heatmap shows z-score of relative expression changes of CCL5, CXCL10, ISG15, and IFI44 expression by RT-qPCR in KRASWT (A2780, MC38, NCI-H1793, and OVCAR3) and KRASMut (TOV-21G, HOC7, LOVO, A549, CT26, Pa02C, Pa16C, MACS, HEYA8, and NCI-H441) cell lines treated with 5 μmol/L olaparib for 24 hours (olaparib/vehicle). G, Western blotting of pERK and STING pathway signaling (STING, pTBK1, pIRF3, and pSTAT1) activation levels in CT26, LOVO, HOC7, and NCI-H441 after treatment with vehicle or trametinib (Tram; 500 nmol/L) for 24 hours. H, Quantification of CXCL10 secretion by ELISA in iKRAS cells induced by doxycycline for 48 hours and subsequently treated with or without 500 nmol/L trametinib for 24 hours. I, Heatmap shows relative expression changes of CCL5, CXCL10, ISG15, and IFI44 by RT-qPCR in KRASWT (A2780, MC38, NCI-H1793, and OVCAR3) and KRASMut (HOC7, LOVO, CT26, Pa02C, Pa16C, MACS, A549 HEYA8, and NCI-H441) cell lines treated with 500 nmol/L trametinib for 24 hours. J, GSEA identified the ISGs significantly elevated after treatment with MEK and ERK inhibitors and iKRAS depletion with doxycycline withdrawal in indicated GEO datasets. The bar graph shows enrichment score in indicated datasets. The top symbol indicates the suppression methods of the MAPK pathway. The P values were calculated by the clusterProfiler package of the R platform (version 3.5.1.; www.r-project.org). B and C, Data represent mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001, calculated by one-way ANOVA. D, E, and H, Analysis by Student t test. n.s., not significant.

Figure 2.

PARPi-induced STING signaling and IFN responses were alleviated in KRAS-mutant cancer cells. A, Doxycycline (Dox)-inducible iKRAS cells were incubated with doxycycline (2 μg/mL) for 48 hours and then treated with vehicle or olaparib (Olap; 5 μmol/L) for another 24 hours with doxycycline or withdrawal of doxycycline. Then cells were collected for Western blotting with indicated antibodies. B and C, Quantification of CCL5, CXCL10, ISG15, and IFI44 expression by RT-qPCR, and CXCL10 secretion levels by ELISA in iKRAS cells in the presence or absence of doxycycline (2 μg/mL) after treatment with vehicle or 5 μmol/L olaparib for 24 hours. D and E, Expression of pERK and STING pathway signaling (STING, pTBK1, pIRF3, and pSTAT1) in both KRASWT (MC38) and KRASG12D (CT26) cell lines after vehicle or 5 μmol/L olaparib treatment for 24 hours by Western blotting (left). CCL5, CXCL10, ISG15, and IFI44 gene expressions were quantified by RT-qPCR (middle), and CXCL10 secretion in culture media was quantified by ELISA (right). Data are shown as mean ± SEM from each of three independent experiments. n = 3. F, Heatmap shows z-score of relative expression changes of CCL5, CXCL10, ISG15, and IFI44 expression by RT-qPCR in KRASWT (A2780, MC38, NCI-H1793, and OVCAR3) and KRASMut (TOV-21G, HOC7, LOVO, A549, CT26, Pa02C, Pa16C, MACS, HEYA8, and NCI-H441) cell lines treated with 5 μmol/L olaparib for 24 hours (olaparib/vehicle). G, Western blotting of pERK and STING pathway signaling (STING, pTBK1, pIRF3, and pSTAT1) activation levels in CT26, LOVO, HOC7, and NCI-H441 after treatment with vehicle or trametinib (Tram; 500 nmol/L) for 24 hours. H, Quantification of CXCL10 secretion by ELISA in iKRAS cells induced by doxycycline for 48 hours and subsequently treated with or without 500 nmol/L trametinib for 24 hours. I, Heatmap shows relative expression changes of CCL5, CXCL10, ISG15, and IFI44 by RT-qPCR in KRASWT (A2780, MC38, NCI-H1793, and OVCAR3) and KRASMut (HOC7, LOVO, CT26, Pa02C, Pa16C, MACS, A549 HEYA8, and NCI-H441) cell lines treated with 500 nmol/L trametinib for 24 hours. J, GSEA identified the ISGs significantly elevated after treatment with MEK and ERK inhibitors and iKRAS depletion with doxycycline withdrawal in indicated GEO datasets. The bar graph shows enrichment score in indicated datasets. The top symbol indicates the suppression methods of the MAPK pathway. The P values were calculated by the clusterProfiler package of the R platform (version 3.5.1.; www.r-project.org). B and C, Data represent mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001, calculated by one-way ANOVA. D, E, and H, Analysis by Student t test. n.s., not significant.

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To explore whether mutant KRAS would block PARPi-induced STING activation, we assessed the effects of olaparib on STING signaling in MC38 and CT26 cells in vitro by Western blotting. While pIRF3, pTBK1, and pSTAT1 were markedly elevated by olaparib in MC38, STING activation by olaparib was suppressed in CT26 (Fig. 2D and E, left). CCL5, CXCL10, ISG15, and IFI44 mRNA and CXCL10 secretion were elevated in MC38 cells, but not in CT26 cells (Fig. 2D and E).

To determine the generalizability of the effects of KRAS on PARPi-induced STING signaling, we assessed CCL5, CXCL10, ISG15, and IFI44 mRNA levels with or without PARPi in 12 well-characterized cancer cell lines, including nine KRAS-mutant cell lines (four ovarian, two colon, two lung, and two pancreatic) and four KRASWT cancer cell lines (two ovarian, one lung, and one colon; Fig. 2F; Supplementary Fig. S2A–S2C). As expected, olaparib markedly elevated CCL5, CXCL10, ISG15, and IFI44 mRNA and CXCL10 secretion in KRASWT cells. In contrast, olaparib failed to induce STING activation in a broad array of KRAS-mutant cells demonstrating generalizability (Fig. 2F).

Interestingly, inhibition of the MAPK pathway in KRAS-mutant cells with trametinib was sufficient to increase IRF3, STAT1, and TBK1 activation in murine CT26 cells (KRAS mutant) and in human HOC7 (KRASG12A), LOVO (KRASG13D), and NCI-H441 (KRASG12V; Fig. 2G), and induce CXCL10 protein secretion in iKRAS cells with doxycycline-on (Fig. 2H). To determine the generalizability of the effects of trametinib-induced STING signaling in KRAS-mutant cells, we assessed CCL5, CXCL10, ISG15, and IFI44 mRNA levels with or without MEKi in 12 well-characterized cancer cell lines (Fig. 2I). As expected, trametinib markedly elevated CCL5, CXCL10, ISG15, and IFI44 mRNA in all KRAS-mutant cells, but A549 cells (Supplementary Fig. S2D). Considering A549 cells (KRAS mutant and LKB1 mutant) do not have STING pathway (33), this result further supports the important role STING plays in activating ISGs after MEKi treatment. In contrast, MEKi failed to induce STING activation in KRASWT cells (Fig. 2I). Moreover, cobimetinib, another MEKi, recapitulated the effects of trametinib on STING and ISG activation (Supplementary Fig. S2E and S2F).

We subsequently assessed the effects of MAPK inhibition on ISG levels in KRAS-mutant tumors by GSEA (Supplementary Fig. S2G–S2J). Four GEO datasets (GSE98399, GSE63229, GSE39984, and GSE58055) revealed that molecular or genetic inhibition of depletion of MAPK induced IFN signatures and ISG expression (Fig. 2J). Thus, inhibition of the RAS/MAPK pathway is sufficient to induce STING signaling and IFN pathway activation in KRAS-mutant cells.

MEKi triggers and amplifies PARPi-induced STING signaling and IFN responses in vitro

We subsequently explored the effects of combination MEK and PARP inhibitors in activating STING signaling. PARPi did not activate TBK1, IRF3, and STAT1 in iKRAS cells with doxycycline-induced KRAS expression (Fig. 3A and B). In contrast, the MEKi and PAPRi combination remarkably enhanced CCL5, CXCL10, ISG15, and IFI44 levels in doxycycline-on iKRAS cells (Fig. 3A and B).

Figure 3.

MEKi triggers and amplifies PARPi-induced STING signaling and IFN responses in vitro. A and B, iKRAS cells were pretreated with doxycycline (Dox; 2 μg/mL) for 48 hours and then treated with vehicle, olaparib (Olap; 5 μmol/L), trametinib (Tram; 500 nmol/L), as well as combined therapy for 24 hours. Then cells were collected for Western blotting with indicated antibodies (A) and RT-qPCR analysis of CCL5, CXCL10, ISG15, and IFI44 expression (B). C, NCI-H441, human lung cancer cells, were treated with vehicle, olaparib (5 μmol/L), trametinib (500 nmol/L), as well as combined therapy for 24 hours. Then cells were assessed with indicated antibodies by Western blotting. Human LOVO (D), HOC7 (E), and murine CT26 (F) cells were treated with vehicle, olaparib (5 μmol/L), trametinib (500 nmol/L), as well as combined therapy for 24 hours. Then cells were assessed with indicated antibodies by Western blotting and expression of CCL5, CXCL10, ISG15, and IFI44 by RT-qPCR, and CXCL10 secretion by ELISA. B and D–F, Data represent mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001. P values were determined by ANOVA.

Figure 3.

MEKi triggers and amplifies PARPi-induced STING signaling and IFN responses in vitro. A and B, iKRAS cells were pretreated with doxycycline (Dox; 2 μg/mL) for 48 hours and then treated with vehicle, olaparib (Olap; 5 μmol/L), trametinib (Tram; 500 nmol/L), as well as combined therapy for 24 hours. Then cells were collected for Western blotting with indicated antibodies (A) and RT-qPCR analysis of CCL5, CXCL10, ISG15, and IFI44 expression (B). C, NCI-H441, human lung cancer cells, were treated with vehicle, olaparib (5 μmol/L), trametinib (500 nmol/L), as well as combined therapy for 24 hours. Then cells were assessed with indicated antibodies by Western blotting. Human LOVO (D), HOC7 (E), and murine CT26 (F) cells were treated with vehicle, olaparib (5 μmol/L), trametinib (500 nmol/L), as well as combined therapy for 24 hours. Then cells were assessed with indicated antibodies by Western blotting and expression of CCL5, CXCL10, ISG15, and IFI44 by RT-qPCR, and CXCL10 secretion by ELISA. B and D–F, Data represent mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001. P values were determined by ANOVA.

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To demonstrate generalizability, we assessed STING signaling and IFN in seven additional KRAS-mutant cancer cell lines. Olaparib alone had limited effects on STING signaling (pTBK1, pIRF3, and pSTAT1) or IFN responses as detected by RT-qPCR and ELISA (CXCL10 and CCL5). Trametinib monotherapy modestly activated STING signaling and IFN responses. Once again, combination therapy with olaparib and trametinib markedly activated STING signaling and downstream IFN responses in NCI-H441, LOVO, HOC7, CT26, HeyA8, Pa02c, and Pa16c cells (Fig. 3C–F; Supplementary Fig. S3A–S3E). Furthermore, olaparib and cobimetinib induced similar effects in HOC7 (Supplementary Fig. S3F). Together, these data indicate that combined MAPK pathway inhibition and PARPi activate STING and IFN signaling in KRAS-mutant cells.

MEKi and combination therapy induce DNA damage and cytosolic dsDNA accumulation in KRAS-mutant cells

The presence of cytosolic dsDNA triggers cGAS/STING-mediated innate immune responses (34). Given that MEKi decrease HR capacity in RAS-mutant cells (35), we reasoned that MEKi itself might activate and exacerbate PARPi-induced STING signaling by generating cytosolic dsDNA. Combination therapy with PARPi and MEKi indeed markedly induced DNA damage (γH2AX) as detected by immunofluorescence and Western blotting in iKRAS with doxycycline-on, LOVO, CT26, HOC7, and NCI-H441 cells (Fig. 4A; Supplementary Fig. S4A–S4D). It is noteworthy that PARPi alone triggered DNA damage in iKRAS cells without doxycycline, and induction of KRAS with doxycycline abrogated the effect (Fig. 4B). However, trametinib augmented PARPi-induced DNA damage as evidenced by an increased amount of DNA in tails in Comet assays in iKRAS cells with doxycycline-on, CT26, and HOC7 cells (Fig. 4B). We further hypothesized that MEKi or combination therapy might specifically favor the formation of cytosolic DNA, so we detected dsDNA with PicoGreen in cells after various treatments. Trametinib, but not olaparib monotherapy, led to cytosolic dsDNA accumulation in iKRAS cells with doxycycline-on, CT26, and LOVO cells (Fig. 4C and D; Supplementary Fig. S4E and S4F). Importantly, dsDNA accumulation was exacerbated by combined PARPi and MEKi treatment (Fig. 4C and D; Supplementary Fig. S4E and S4F).

Figure 4.

MEKi and combination therapy induce DNA damage and cytosolic dsDNA accumulation in KRAS-mutant cells. A, CT26, HOC7, iKRAS with doxycycline-on (Dox-on), and NCI-H441 cells were treated with vehicle, olaparib (Olap; 5 μmol/L), trametinib (Tram; 500 nmol/L), and combined therapy for 24 hours and subjected to Western blotting with pERK1/2 and γH2AX. B, iKRAS cells were pretreated with doxycycline (2 μg/mL) for 24 hours and then treated with vehicle, olaparib (5 μmol/L), trametinib (500 nmol/L), as well as combined therapy for 24 hours, and iKRAS cells were directly treated with vehicle and olaparib (5 μmol/L) for 24 hours and subjected to Comet analysis. HOC7 and CT26 were treated as in A and subjected to Comet analysis. DNA damage was quantified as percentage DNA in tails from three independent experiments. C and D, Representative images and quantification of PicoGreen staining after treatment as in B in iKRAS cells. White arrows in C, cytosolic DNA. E and F, RT-qPCR analysis of CCL5, CXCL10, ISG15, and IFI44 expression in HOC7 with depletion of STING by STING inhibition (1 μmol/L H151) for 24 hours (E) or si-STING (100 μmol/L) for 48 hours (F). G, iKRAS cells with or without doxycycline were treated with dsDNA90 (the well-known cGAS/STING activator), and CCL5, CXCL10, ISG15, and IFI44 expression was quantified by RT-qPCR. B and E–G, Data represent mean ± SEM of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; P values were determined by one-way ANOVA. n.s, not significant. D, Data represent mean ± SEM of three independent experiments, and P values were determined by Student t test when comparing two groups of iKRAS cells, while one-way ANOVA was performed when comparing four groups of iKRAS doxycycline-on.

Figure 4.

MEKi and combination therapy induce DNA damage and cytosolic dsDNA accumulation in KRAS-mutant cells. A, CT26, HOC7, iKRAS with doxycycline-on (Dox-on), and NCI-H441 cells were treated with vehicle, olaparib (Olap; 5 μmol/L), trametinib (Tram; 500 nmol/L), and combined therapy for 24 hours and subjected to Western blotting with pERK1/2 and γH2AX. B, iKRAS cells were pretreated with doxycycline (2 μg/mL) for 24 hours and then treated with vehicle, olaparib (5 μmol/L), trametinib (500 nmol/L), as well as combined therapy for 24 hours, and iKRAS cells were directly treated with vehicle and olaparib (5 μmol/L) for 24 hours and subjected to Comet analysis. HOC7 and CT26 were treated as in A and subjected to Comet analysis. DNA damage was quantified as percentage DNA in tails from three independent experiments. C and D, Representative images and quantification of PicoGreen staining after treatment as in B in iKRAS cells. White arrows in C, cytosolic DNA. E and F, RT-qPCR analysis of CCL5, CXCL10, ISG15, and IFI44 expression in HOC7 with depletion of STING by STING inhibition (1 μmol/L H151) for 24 hours (E) or si-STING (100 μmol/L) for 48 hours (F). G, iKRAS cells with or without doxycycline were treated with dsDNA90 (the well-known cGAS/STING activator), and CCL5, CXCL10, ISG15, and IFI44 expression was quantified by RT-qPCR. B and E–G, Data represent mean ± SEM of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; P values were determined by one-way ANOVA. n.s, not significant. D, Data represent mean ± SEM of three independent experiments, and P values were determined by Student t test when comparing two groups of iKRAS cells, while one-way ANOVA was performed when comparing four groups of iKRAS doxycycline-on.

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Pharmacologic or siRNA-mediated STING inhibition resulted in significantly reduced expression of CCL5, CXCL10, and ISG post-MEKi or combination treatment in human HOC7 (Fig. 4E and F; Supplementary Fig. S4G–S4I) and murine CT26 (Supplementary Fig. S4G and S4J) cells, indicating the involvement of cGAS/STING in this process. To exclude the possibility of dsDNA sensor defects caused by KRAS, iKRAS cells were treated with dsDNA90 to directly activate cGAS/STING (36) with or without doxycycline. Interestingly, doxycycline-induced KRAS expression did not alleviate dsDNA90-induced CCL5, CXCL10, and ISG expression, indicating that the STING pathway was intact (Fig. 4G). Moreover, iKRAS depletion did not further enhance dsDNA90-induced ISG expression (Supplementary Fig. S4K). Collectively, these results support the notion that MEKi was sufficient to trigger and exacerbate PARPi-induced innate immune activation in KRAS-mutant cells by inducing DNA damage and cytosolic dsDNA accumulation in a STING-dependent manner.

Induction of MAPK activity in KRASWT cells alleviates PARPi-induced IFN activation

FBS contains growth factors, such as EGF (37) and lysophosphatidic acid (38), that are sufficient to induce activation of the MAPK pathway in KRASWT Hela S3 cells. Remarkably, while PARPi increased CCL5, CXCL10, ISG15, and IFI44 mRNA under serum-starvation conditions, in the presence of FBS, the effects of PARPi on chemokine production were abrogated (Fig. 5A). Inhibition of MEKi reversed the effects of serum on PARPi-induced production of chemokines (Fig. 5B). The effects of serum were recapitulated by EGF, including the inhibition of the effect of EGF by MEKi (Fig. 5B). Furthermore, MEKi was sufficient to increase STING signaling (pTBK1, pIRF3, and pSTAT1; Fig. 5C), CCL5, CXCL10, ISG15, and IFI44 mRNA (Fig. 5D), and CXCL10 secretion in Hela S3 cells cultured with FBS (Fig. 5E). Together, these data indicate that MAPK pathway activation in KRASWT cells can alleviate PARPi-induced STING and IFN signaling.

Figure 5.

Induction of MAPK activity in KRASWT cells alleviates PARPi-induced IFN activation. A, First, Hela-S3 cells were precultured in serum-free or complete medium for 48 hours. DMSO (vehicle) or olaparib (Olap; 5 μmol/L) was then added to cells, and the cells were further incubated for 24 hours in the same medium. Cell lysates were subjected to RT-qPCR with expression of CCL5, CXCL10, ISG15, and IFI44 genes. B, Serum-starved Hela-S3 cells were treated as indicated: DMSO (vehicle), olaparib (5 μmol/L), EGF (50 ng/mL), and trametinib (Tram; 500 nmol/L) for further 24 hours. Expression of CCL5, CXCL10, ISG15, and IFI44 genes was assessed by RT-qPCR. Hela-S3 cells treated with DMSO (vehicle), olaparib (5 μmol/L), and trametinib (500 nmol/L) for 24 hours, and combinational therapy and subjected to Western blotting with indicated proteins (C) and RT-qPCR with expression of CCL5, CXCL10, ISG15, and IFI44 genes (D). E, Hela-S3 cells treated with DMSO (vehicle), olaparib (5 μmol/L), and trametinib (500 nmol/L) for 24 hours and combinational therapy, and subjected to evaluation of CXCL10 secretion in different treatment cohorts and different time points (6, 24, and 48 hours) by ELISA. F, Hela-S3 cells were treated as in C and subjected to Western blotting with γH2AX. G, Representative images and quantification (left) for γH2AX foci and dsDNA by immunofluorescence staining and PicoGreen staining in Hela-S3 cells treated as in A. DAPI (blue) was used to visualize the nuclei. White arrows, cytosolic DNA. Data represent mean ± SEM of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001, one-way ANOVA. H, Hela-S3 cells were treated as in C and subjected to Comet analysis. DNA damage was quantified as percentage DNA in tails from three independent experiments. ***, P < 0.001, one-way ANOVA. n.s., not significant.

Figure 5.

Induction of MAPK activity in KRASWT cells alleviates PARPi-induced IFN activation. A, First, Hela-S3 cells were precultured in serum-free or complete medium for 48 hours. DMSO (vehicle) or olaparib (Olap; 5 μmol/L) was then added to cells, and the cells were further incubated for 24 hours in the same medium. Cell lysates were subjected to RT-qPCR with expression of CCL5, CXCL10, ISG15, and IFI44 genes. B, Serum-starved Hela-S3 cells were treated as indicated: DMSO (vehicle), olaparib (5 μmol/L), EGF (50 ng/mL), and trametinib (Tram; 500 nmol/L) for further 24 hours. Expression of CCL5, CXCL10, ISG15, and IFI44 genes was assessed by RT-qPCR. Hela-S3 cells treated with DMSO (vehicle), olaparib (5 μmol/L), and trametinib (500 nmol/L) for 24 hours, and combinational therapy and subjected to Western blotting with indicated proteins (C) and RT-qPCR with expression of CCL5, CXCL10, ISG15, and IFI44 genes (D). E, Hela-S3 cells treated with DMSO (vehicle), olaparib (5 μmol/L), and trametinib (500 nmol/L) for 24 hours and combinational therapy, and subjected to evaluation of CXCL10 secretion in different treatment cohorts and different time points (6, 24, and 48 hours) by ELISA. F, Hela-S3 cells were treated as in C and subjected to Western blotting with γH2AX. G, Representative images and quantification (left) for γH2AX foci and dsDNA by immunofluorescence staining and PicoGreen staining in Hela-S3 cells treated as in A. DAPI (blue) was used to visualize the nuclei. White arrows, cytosolic DNA. Data represent mean ± SEM of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001, one-way ANOVA. H, Hela-S3 cells were treated as in C and subjected to Comet analysis. DNA damage was quantified as percentage DNA in tails from three independent experiments. ***, P < 0.001, one-way ANOVA. n.s., not significant.

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Furthermore, in Hela cells cultured with FBS, where the MAPK pathway was activated (Fig. 5A), combination therapy with PARPi and MEKi markedly induced DNA damage as assessed by γH2AX protein (Fig. 5F), γH2AX foci (Fig. 5G), and Comet assay (Fig. 5H). Combination therapy also increased cytosolic dsDNA (Fig. 5G), mimicking the effects observed in KRAS-mutant cells.

MEKi reshapes the TME by decreasing myeloid cells in KRAS-mutant tumors

Interestingly, the effects of MEKi and PARPi on growth of KRAS-mutant LPA1-T127 tumors in immunocompetent FVB mice were mitigated in immunocompromised nude mice (Fig. 6A), consistent with the adaptive immune system participating in the antitumor activity of olaparib and trametinib. We thus determined whether MEKi and a combination of MEKi and PARPi would induce changes in tumor-infiltrating immune cells. Using mass cytometry (CyTOF) immunophenotyping with 11 lineage markers, we profiled the TME of LPA1-T127 tumors in FVB mice (Fig. 6A; Supplementary Fig. S5A). Cytobank-based viSNE analysis revealed a complex cellular landscape of dendritic cells, T/B cells, and MDSCs/macrophage cells with a high level of myeloid cells in LPA1-T127 tumors (Fig. 6B). MEKi treatment slightly decreased myeloid cells, and in combination with PARPi dramatically diminished myeloid cells (Fig. 6B). In contrast, PARPi alone had only modest effects on the TME. CyTOF analysis revealed that MEKi significantly increased the percentage of T cells, including CD4+ and CD8+ cells, which was further enhanced by combination treatment with PARPi (Fig. 6B). These data suggest that KRAS-mutant tumors are characterized by an immunosuppressive TME with high myeloid cell and low T-cell infiltration. MEKi remodels immunity by decreasing myeloid cells and elevating T-cell infiltration to activate antitumor immune effects, while the addition of PARPi to the MEKi reinforces these effects.

Figure 6.

MEKi reshapes the TME by decreasing myeloid cells in KRAS-mutant tumors. A, LPA1-T127 tumors were transplanted into the mammary fat pads of FVB immunocompetent mice (top) or nude mice (bottom). Eight days later, mice were randomized into treatment cohorts: vehicle (0.5% hydroxypropylmethylcellulose and 0.2% Tween 80), olaparib (Olap; 50 mg/kg per day), trametinib (Tram; 1 mg/kg per day), or the combination of olaparib and trametinib (n = 6 for each group). Average tumor volumes ± SEM for each cohort are displayed. P values were determined by nonparametric pairwise comparisons (Mann–Whitney). *, P < 0.05; **, P < 0.01. B, LPA1-T127 tumors in FVB mice treated as in A were harvested on day 21 for CyTOF analysis. t-SNE analysis of immune cells by relative expression of CyTOF markers, with population indicated. Quantification of MDSCs/macrophages (CD45+CD11b+), CD8+ T (CD45+ CD3+ CD8+) cells, and CD4+ T (CD45+ CD3+ CD4+) cells in different therapy cohorts. Data across panels are mean ± SEM. P values were determined by ANOVA. **, P < 0.01; ***, P < 0.001. DC, dendritic cell. C, HOC7 cells were treated with vehicle, olaparib (5 μmol/L), trametinib (500 nmol/L), and combined therapy for 24 hours, and then culture supernatants were collected for assessment of cytokines and chemokines by inflammatory cytokine arrays. Heatmap of statistically different (z-score) cytokines is shown. D, Heatmap of relative expression of IL6, GMCSF, CXCL1, CXCL2, TGFα, TGFβ, and IL10 in KRASMUT (HOC7, LOVO, and CT26) cell lines after treatment with vehicle or trametinib (500 nmol/L) for 24 hours. E, HOC7 and LOVO were treated as in C, and IL6 and GMCSF secretion was quantified in culture media by ELISA. Data are mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001, one-way ANOVA.

Figure 6.

MEKi reshapes the TME by decreasing myeloid cells in KRAS-mutant tumors. A, LPA1-T127 tumors were transplanted into the mammary fat pads of FVB immunocompetent mice (top) or nude mice (bottom). Eight days later, mice were randomized into treatment cohorts: vehicle (0.5% hydroxypropylmethylcellulose and 0.2% Tween 80), olaparib (Olap; 50 mg/kg per day), trametinib (Tram; 1 mg/kg per day), or the combination of olaparib and trametinib (n = 6 for each group). Average tumor volumes ± SEM for each cohort are displayed. P values were determined by nonparametric pairwise comparisons (Mann–Whitney). *, P < 0.05; **, P < 0.01. B, LPA1-T127 tumors in FVB mice treated as in A were harvested on day 21 for CyTOF analysis. t-SNE analysis of immune cells by relative expression of CyTOF markers, with population indicated. Quantification of MDSCs/macrophages (CD45+CD11b+), CD8+ T (CD45+ CD3+ CD8+) cells, and CD4+ T (CD45+ CD3+ CD4+) cells in different therapy cohorts. Data across panels are mean ± SEM. P values were determined by ANOVA. **, P < 0.01; ***, P < 0.001. DC, dendritic cell. C, HOC7 cells were treated with vehicle, olaparib (5 μmol/L), trametinib (500 nmol/L), and combined therapy for 24 hours, and then culture supernatants were collected for assessment of cytokines and chemokines by inflammatory cytokine arrays. Heatmap of statistically different (z-score) cytokines is shown. D, Heatmap of relative expression of IL6, GMCSF, CXCL1, CXCL2, TGFα, TGFβ, and IL10 in KRASMUT (HOC7, LOVO, and CT26) cell lines after treatment with vehicle or trametinib (500 nmol/L) for 24 hours. E, HOC7 and LOVO were treated as in C, and IL6 and GMCSF secretion was quantified in culture media by ELISA. Data are mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001, one-way ANOVA.

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Cytokines secreted from tumor cells are critical determinants of immune cell recruitment. We thus audited 40 cytokines and chemokines in culture supernatants of HOC7 cells treated with vehicle, olaparib, trametinib, or combined therapy (Supplementary Fig. S5B). Densitometry analysis of these protein arrays revealed that several proinflammatory cytokines, including CXCL10, CCL3, CCL5, and TNFα/β, were modestly increased after trametinib monotherapy and markedly enhanced by combination with PARPi (Fig. 6C). Similar to the modest effects on the immune TME, PARPi had limited effects on the production of proinflammatory cytokines. Interestingly, immunosuppressive factors, including IL6, GMCSF, and TNFRSF1A, were decreased by trametinib monotherapy or combination treatment with PARPi, but in contrast, were increased by olaparib monotherapy (Fig. 6C). GMCSF and IL6 contribute to MDSC recruitment (39, 40). Consistent with the protein arrays, MEKi inhibited mRNA levels for IL6 and GMCSF, and other immunosuppressive chemokines, including CXCL1 and CXCL2, and IL10 and TGFβ expression in CT26, HOC7, and LOVO cells (Fig. 6D). Furthermore, trametinib alone or combined with olaparib substantially downregulated the secretion of IL6 and GMCSF as detected by ELISA in HOC7 and LOVO cells (Fig. 6E). GSEA of the GSE58055 dataset also revealed that IL6 and GMCSF pathway activity was suppressed when KRAS was decreased by removal of doxycycline (Supplementary Fig. S5C). In conclusion, these data point to a potential effect of MEKi on modulating the immune repressive environment in KRAS-mutant tumors through decreasing IL6 and GMCSF, and, thus, MDSCs' recruitment resulting in a shift of the balance from a protumorigenic to an antitumorigenic immune TME.

To assess a potential link between KRAS mutations and MDSCs in the TME in human tumors, we analyzed The Cancer Genome Atlas pan-cancer data using an established MDSC signature (41) in 10 tumor types in which RAS family mutational frequency was more than 10%. These tumor types showed a strong positive correlation between high MAPK signaling and enrichment of the MDSC signature, consistent with the effects of KRAS mutations on the immune microenvironment being manifest in human tumors (Supplementary Fig. S5D).

MEKi, PARPi, and anti–PD-L1 combination is highly effective in KRAS-mutant tumors

We next investigated the efficacy of triplet therapy with PARPi, MEKi, and anti–PD-L1 in LPA1-T127 tumors. Neither PARPi or anti–PD-L1 alone nor the combination had a significant impact on tumor growth (Fig. 7A, left). While MEKi alone modestly decreased tumor growth, combination treatment with anti–PD-L1 augmented the decreased tumor burden. MEKi plus PARPi, and triplet therapy showed similar antitumor activity at day 20, when therapy was stopped because of control tumors reaching a tumor volume endpoint requiring termination. Importantly, following therapy termination, triplet therapy with MEKi, PARPi, and anti–PD-L1 induced a statistically significant more durable response (Fig. 7A, left).

Figure 7.

MEKi, PARPi, and anti–PD-L1 combination is highly effective in KRAS-mutant tumors. A, LPA1-T127 tumor tissues were transplanted into the mammary fat pads of FVB immunocompetent mice. Eight days later, mice were randomized into treatment cohorts: vehicle (0.5% hydroxypropylmethylcellulose, 0.2% Tween 80 and IgG isotype control), olaparib (Olap; 50 mg/kg/day), trametinib (Tram; 1 mg/kg/day), or the combination of olaparib and trametinib and/or αPD-L1 antibody (200 μg/mouse every 3 days, six times; n = 6 for each group). Treatment was terminated on day 20, while trametinib plus olaparib and triplet therapy groups continued to be observed for the change of tumor volume (left). CT26 cells (2 × 105) were subcutaneously injected into the right flank of Balb/c mice (6 to 8 weeks old). After the tumors were palpable, the mice were randomly assigned to treatment cohorts accordingly (n = 6 for each group). Treatment was terminated on day 15, and then tumor growth was observed for another 2 weeks (right). P values were determined by nonparametric pairwise comparisons (Mann–Whitney). *, P < 0.05; **, P < 0.01; ***, P < 0.001. Red line, vehicle group; green line, olaparib group; blue, trametinib group; carmine, αPD-L1 group; purple, combination of trametinib and olaparib group; orange, combination of olaparib and αPD-L1 group; blue green, combination of trametinib and αPD-L1; army green, triplet therapy group. B, CT26 cells (2 × 105) were subcutaneously injected into the left flank of Balb/c mice (6–8 weeks old). After the tumor volume reached 200 to 500 mm3, CT26 mice were randomized into treatment cohorts accordingly (n = 6 for each group). Tumor size and mouse weight were monitored every 3 days. We stopped treatment on day 15, and then tumor growth was observed for another 16 days. P values were determined by nonparametric pairwise comparisons (Mann–Whitney). *, P < 0.05; **, P < 0.01; ***, P < 0.001. C, Quantification of MDSC, polymorphonuclear MDSC (PMN-MDSC), monocytic MDSC (M-MDSC) proportions in tumors from each group, respectively. n = 3. P values were determined by ANOVA. *, P < 0.05; **, P < 0.01; ***, P < 0.001. G-MDSC, granulocytic MDSC. D, Representative images of IHC with indicated antibodies in tumor tissues from CT26 models (A, top). Quantification of number of CD4+ and CD8+ cells, and pERK scores after treatments (bottom). P values were determined by ANOVA (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

Figure 7.

MEKi, PARPi, and anti–PD-L1 combination is highly effective in KRAS-mutant tumors. A, LPA1-T127 tumor tissues were transplanted into the mammary fat pads of FVB immunocompetent mice. Eight days later, mice were randomized into treatment cohorts: vehicle (0.5% hydroxypropylmethylcellulose, 0.2% Tween 80 and IgG isotype control), olaparib (Olap; 50 mg/kg/day), trametinib (Tram; 1 mg/kg/day), or the combination of olaparib and trametinib and/or αPD-L1 antibody (200 μg/mouse every 3 days, six times; n = 6 for each group). Treatment was terminated on day 20, while trametinib plus olaparib and triplet therapy groups continued to be observed for the change of tumor volume (left). CT26 cells (2 × 105) were subcutaneously injected into the right flank of Balb/c mice (6 to 8 weeks old). After the tumors were palpable, the mice were randomly assigned to treatment cohorts accordingly (n = 6 for each group). Treatment was terminated on day 15, and then tumor growth was observed for another 2 weeks (right). P values were determined by nonparametric pairwise comparisons (Mann–Whitney). *, P < 0.05; **, P < 0.01; ***, P < 0.001. Red line, vehicle group; green line, olaparib group; blue, trametinib group; carmine, αPD-L1 group; purple, combination of trametinib and olaparib group; orange, combination of olaparib and αPD-L1 group; blue green, combination of trametinib and αPD-L1; army green, triplet therapy group. B, CT26 cells (2 × 105) were subcutaneously injected into the left flank of Balb/c mice (6–8 weeks old). After the tumor volume reached 200 to 500 mm3, CT26 mice were randomized into treatment cohorts accordingly (n = 6 for each group). Tumor size and mouse weight were monitored every 3 days. We stopped treatment on day 15, and then tumor growth was observed for another 16 days. P values were determined by nonparametric pairwise comparisons (Mann–Whitney). *, P < 0.05; **, P < 0.01; ***, P < 0.001. C, Quantification of MDSC, polymorphonuclear MDSC (PMN-MDSC), monocytic MDSC (M-MDSC) proportions in tumors from each group, respectively. n = 3. P values were determined by ANOVA. *, P < 0.05; **, P < 0.01; ***, P < 0.001. G-MDSC, granulocytic MDSC. D, Representative images of IHC with indicated antibodies in tumor tissues from CT26 models (A, top). Quantification of number of CD4+ and CD8+ cells, and pERK scores after treatments (bottom). P values were determined by ANOVA (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

Close modal

We further assessed triplet combination therapy in the CT26 KRAS-mutant tumor model. Trametinib monotherapy was highly effective in CT26 models. We thus stopped treatment at day 15 and continued observation until tumors in the vehicle group reached the designated tumor volume endpoint at day 24. Combination of MEKi with PARPi or anti–PD-L1 was more active than trametinib alone. Importantly, combination therapy of PARPi and MEKi with anti–PD-L1 achieved optimal effects with prolonged tumor control after cessation of therapy (Fig. 7A, right). To further explore the antitumor effects of the triple combination of PARPi, MEKi, and anti–PD-L1, we initiated therapy after tumors reached a larger volume of approximately 200 to 500 mm3. Interestingly, while tumors grew gradually in PARPi and MEKi combination treatment after 14 days, 7 of 8 mice in the triplet cohort remained tumor free at day 31 (Fig. 7B), consistent with the potential that mice were “cured” by the combination. There was no evidence of toxicity as indicated by weight loss in mice with any therapy (Supplementary Fig. S6A and S6B).

CT26 tumors were analyzed by multicolor flow cytometry for changes in the immune TME (Fig. 7C; Supplementary Fig. S6C–S6E). Consistent with sensitivity to trametinib monotherapy, trametinib markedly diminished MDSC (both monocytic MDSCs and granulocytic MDSCs) subsets. In contrast, olaparib or olaparib plus anti–PD-L1, which was ineffective as therapy, had limited effects on MDSCs. Furthermore, olaparib or anti–PD-L1, or triple combinations did not reduce MDSC populations compared with MEKi alone (Fig. 7C). Trametinib monotherapy also increased CD3+ T-cell infiltration and CD4+ Th cells compared with vehicle-treated tumors. In contrast to the effects on MDSCs, addition of PARPi or PARPi and PD-L1 to MEKi increased CD3+ T cell and CD4+ Th-cell infiltration (Fig. 7D; Supplementary Fig. S6E). Interestingly, despite a substantial increase in total CD4+ T cells, we observed a significant decrease in the percentage of immunosuppressive CD4+ CD25+/FOXP3+ Tregs in response to trametinib, or in combinations including trametinib (Supplementary Fig. S6E). Furthermore, IFNγ+ cytotoxic CD8+ T cells were remarkably increased by trametinib alone and in combinations with PARPi and anti–PD-L1 (Supplementary Fig. S6E).

PARPi combined with PD-1/PD-L1 blockade has recently shown promising activity in preclinical models and clinical trials (42). Our previous study, which has been confirmed by others, showed that MEKi decreases the expression of a number of members of the HR pathway (BRCA1, MRE11, BRCA2, or RAD51), as well as DDR in KRAS-mutant cells (30, 35). These studies confirm a critical role of the RAS/MAPK pathway in protecting cells from DNA damage potentially induced by KRAS-mediated oncogenic stress. Multiple studies, although somewhat controversial, indicated that PARPi induces an effective STING response potentially contributing to the efficacy of PARPi and anti–PD-L1 (27, 43).

Here, we demonstrate that KRAS mutations mediate resistance to PARPi and anti–PD-L1 combinations, which could be effectively reversed by RAS/MAPK pathway inhibition. Mechanistically, KRAS-mutant tumors are resistant to PARPi-induced DNA damage, as well as subsequent STING-TBK1-IRF3 and IFN pathway activation. The important role of STING in ISGs activation after MEKi treatment was demonstrated by the remarkably diminished ISG activation following depletion of STING activity by siRNAs or inhibitors in HOC7 and CT26 cells. However, ISGs were not entirely reduced by knocking down STING (Fig. 4F; Supplementary Fig. S4H), indicating that additional STING-independent mechanisms could contribute to cytokine release after MEKi treatment. For example, MEKi augments IRF3-driven type I and III IFN response in primary human airway epithelial cells (44). Moreover, PARPi and MEKi have been reported to modulate dsRNA-related immune responses, with upregulation of the related cytokines (45).

Here, we found the intrinsic cytosolic DNA sensor pathway remains intact in KRAS-mutant tumors. Notably, MEKi itself generates a low level of cytoplasmic dsDNA that activates cGAS/STING and downstream IFN signaling. MEKi also facilitates CCL5 and CXCL10 secretion in KRAS-mutant tumors, consistent with STING pathway activation. Importantly, MEKi sensitizes KRAS-mutant cells to PARPi, amplifying PARPi-induced DNA damage and cytosolic dsDNA accumulation. In addition, MEKi reshapes the TME by depleting MDSCs likely through decreasing GMCSF and IL6 production, modifies recruitment of CD8+ T lymphocytes, and importantly alters the types of CD3+ and CD8+ present to a more tumor suppressive environment. Together, TME reprogramming results in an adaptive immune response–dependent decrease in tumor growth. Although the PARPi and MEKi combination engages the immune system, addition of PD-L1 blockade augments the therapeutic efficacy of the combination in KRAS-mutant tumors.

KRAS-mutant cancers are characterized by abundant myeloid cell infiltration (46) with the extensive immunosuppressive myeloid cell environment, including MDSCs being postulated as a key cause of immunotherapy resistance (47). KRAS-driven cancers secrete high levels of GMCSF and IL6 that contribute to the accumulation of immunosuppressive MDSCs (46). Here, we showed that MEKi decreased GMCSF and IL6 production and reshaped the tumor immune microenvironment to unleash the immune system by excluding MDSCs and increasing TIL infiltration in KRAS-mutant tumors. Consistent with these results, BRAF and/or MEK inhibitors reduce immunosuppressive cytokine levels and increase T-cell infiltration in melanoma (48). Importantly, when combined with PARPi, MEKi enhances DNA damage and cancer cell death and also provokes a more favorable immune microenvironment that sensitizes the tumor to the effects of immune checkpoint inhibitors.

Recently, AMG 510 (49) or MRTX849 (50), two selective KRASG12C inhibitors, have been shown to enhance immune cell infiltration and render tumors sensitive to immunotherapy. These data also support the role of MAPK pathway activation in inhibiting antitumor immune responses. However, the restriction of this class of inhibitors to KRASG12C-mutant tumors limits their application in tumors with other forms of mutant KRAS. The approaches described herein with trametinib may have applicability to tumors with all types of KRAS mutations.

We delivered PARPi, MEKi, and anti–PD-L1 simultaneously with a high antitumor activity. However, although it appears that there was minimal toxicity (in terms of body weight loss of mice) using the triplet combination, caution should be paid to utilizing the triplet combination in patients with KRASWT tumors, especially when the doublet combination of PARPi and anti–PD-L1 is already effective in controlling tumor growth. While the potential for added toxicity may be acceptable for patients with KRAS-mutant tumors, the potential added toxicity of the triplet combination may not be justified for patients with wild-type KRAS tumors. It remains to be determined whether concurrent therapy is needed or whether there are more optimal schedules. For example, pulsatile MEKi treatment may be more effective at controlling tumor progression and enhancing T-cell activation (51). We evaluated two alternative dosing schedules, which could potentially minimize toxicity. Importantly, 48 hours of MEK inhibition with specific inhibitors (trametinib or cobimetinib) at low concentrations, around their IC50 value for inhibiting downstream ERK1/ERK2 phosphorylation, was sufficient to activate and amplify PARPi-induced STING pathway activation in KRAS-mutant cells (Supplementary Fig. S7A and S7B). Moreover, sequential therapy with 24 hours of trametinib followed by a PARPi induced STING signaling in the HOC7 cell line more efficiently than either agent alone (Supplementary Fig. S7C). Thus, it will be important to establish a dose and schedule that are able to maintain efficacy while ameliorating toxicity in clinical trials.

G.B. Mills reports personal fees from AstraZeneca, Chrysalis, Ionis, PDX Pharma, Symphogen, and Myriad; personal fees and other support from ImmunoMet, Lilly, Signalchem Lifesciences, Tarveda, Zentalis, and Turbine; other support from Catena, Genentech, and GlaxoSmithKline; and grants from NanoString and Ionis during the conduct of the study. No disclosures were reported by the other authors.

B. Yang: Data curation, validation, investigation, methodology, writing–original draft. X. Li: Data curation, validation, investigation. Y. Fu: Data curation, investigation. E. Guo: Conceptualization, supervision. Y. Ye: Visualization, methodology. F. Li: Methodology. S. Liu: Investigation, methodology. R. Xiao: Software, methodology. C. Liu: Visualization, methodology. F. Lu: Visualization, methodology. J. Huang: Investigation, methodology. T. Qin: Visualization, methodology. L. Han: Software, methodology. G. Peng: Visualization, methodology. G.B. Mills: Formal analysis, supervision, funding acquisition, writing–review and editing. C. Sun: Conceptualization, supervision, funding acquisition, project administration, writing–review and editing. G. Chen: Conceptualization, resources, supervision, funding acquisition, project administration.

The authors thank A. Maitra (Division of Pathology/Lab Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX) for iKRAS cells. This study was supported by grants from Nature and Science Foundation of China (81572569 and 81874106 to G. Chen), National Key R&D Program of China (2016YFC1303100 to G. Chen), and Nature and Science Foundation of China (81402163 and 81974408 to C. Sun). G.B. Mills has support from NIH P50CA217685 and U01 CA217842, the Ovarian Cancer Research Foundation, and a kind gift from the Miriam and Sheldon Adelson Medical Research Fund. G.B. Mills is supported by the Breast Cancer Research Foundation.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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