NK cell-mediated cytotoxicity contributes to tumor control by a cytostatic drug combination

Abstract

Molecularly targeted therapies aim to obstruct cell autonomous programs required for tumor growth. We show that mitogen-activated protein kinase (MAPK) and cyclin-dependent kinase 4/6 inhibitors act in combination to suppress the proliferation of KRAS-mutant lung cancer cells while simultaneously provoking a natural killer (NK) cell surveillance program leading to tumor cell death. The drug combination, but neither agent alone, promotes retinoblastoma (RB) protein-mediated cellular senescence and activation of the immunomodulatory senescence-associated secretory phenotype (SASP). SASP components tumor necrosis factor-α and intercellular adhesion molecule-1 are required for NK cell surveillance of drug-treated tumor cells, which contributes to tumor regressions and prolonged survival in a KRAS-mutant lung cancer mouse model. Therefore, molecularly targeted agents capable of inducing senescence can produce tumor control through non-cell autonomous mechanisms involving NK cell surveillance.

Fig. 1.. NK cell immunity is required for the efficacy of combination MEK and CDK4/6 inhibitor therapy.

(A) Clonogenic assay of A549 lung cancer cells treated with MEK (trametinib) and/or various CDK4/6 inhibitors (palbociclib, ribociclib, abemaciclib); representative of three biological replicates. (B) Tumor volumes of mice bearing KRAS-mutant MSK-LX27 PDX lung tumors treated with vehicle, trametinib (3 mg/kg body weight), palbociclib (150 mg/kg body weight), or both in combination (Combo) for indicated times (n = 5 mice per group). (C) Tumor volumes of mice bearing KRAS-mutant MSK-LX68 PDX lung tumors treated with vehicle, trametinib [1 mg/kg (lo) or 3 mg/kg (hi) body weight], palbociclib (150 mg/kg body weight), or both in combination for indicated times (n = 8 mice per group). n.s., not significant. (D) Syngeneic KP transplant lung cancer model. (E) Kaplan-Meier survival curve of KP transplant mice treated with vehicle, trametinib (1 mg/kg body weight), palbociclib (100 mg/kg body weight), or both in combination (n ≥ 8 per group) (log-rank test). (F) Representative flow cytometry plots of NK cell populations in lung tumors from KP transplant mice treated for 1 week as in (E). (G) Percentage of NK cells within the CD45⁺ population (left), total NK cells relative to tumor cell number (middle), and percentage of CD107a⁺ degranulating NK cells (right) (n ≥ 4 mice per group). Palbo, palbociclib; Tramet, trametinib. (H) Kaplan-Meier survival curve of KP transplant mice treated with vehicle or combined trametinib (1 mg/kg body weight) and palbociclib (100 mg/kg body weight) and either an isotype control antibody (C1.18.4) or NK1.1-depleting antibody (PK136) (n ≥ 8 per group) (log-rank test). (B and C) Two-way ANOVA. (G) One-way ANOVA. Error bars, mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 2.. Senescence and SASP induction after combination MEK and CDK4/6 inhibitor therapy induces NK cell immune surveillance.

(A) Quantification of SA-β-gal⁺ cells in human KRAS-mutant lung tumor cell lines after 8-day treatment with trametinib (25 nM) and/or palbociclib (500 nM). Mean of three biological replicates is plotted. (B) Clonogenic assay of human KRAS-mutant lung cancer cells replated in the absence of drugs after 8-day pretreatment as in (A); representative of three biological replicates. RB and p53 genomic status is indicated on right. (C) Heat map of senescence-associated cell cycle and SASP gene expression in human KRAS-mutant lung cancer cell lines after treatment as in (A), as assessed by RNA-seq.Two biological replicates per cell line are shown. C, combination; P, palbociclib; T, trametinib; V, vehicle. (D) Heat map of cytokine array results from human KRAS-mutant lung tumor cells treated with trametinib (25 nM) and/or palbociclib (500 nM) for 8 days. Data are presented as mean of three biological replicates. (E) Flow cytometry analysis of ICAM-1 expression and levels of other NK cell–activating ligands after 8-day treatment of A549 lung tumor cells as described in (A). Quantification of mean fluorescence intensity (MFI) from three biological replicates is shown on the right. (F) Quantification of NK cell cytotoxicity (by live cell imaging) after pretreatment of A549 and H2030 lung tumor cell lines with indicated drugs for 8 days and coculturing with the YT NK cell line at a 10:1 effector to target cell (E:T) ratio for 20 hours in the presence or absence of indicated drugs. Change in tumor cells is normalized to control wells lacking NK cells. Mean of three biological replicates is plotted. (G) Representative SA-β-gal staining of KP transplant lung tumors after 1-week treatment with combined trametinib (1 mg/kg body weight) and palbociclib (100 mg/kg body weight) and either an isotype control antibody (C1.18.4) or NK1.1 depleting antibody (PK136) (scale bar, 50 mm). Quantification shown on right (n = 3 mice per group). (H) Effect of 1-week treatment as in (G) on lung tumor burden (relative to vehicle) (n = 5 mice per group). (E, F, and H) One-way ANOVA. (G) Unpaired two-tailed t test. Error bars, mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 3.. NF-κB-mediated SASP and TNF-α secretion is required for treatment-induced NK cell activity.

(A) Quantitative reverse transcription polymerase chain reaction analysis of SASP gene expression in KP tumor cells transduced with indicated shRNAs after treatment with vehicle or trametinib (25 nM) and palbociclib (500 nM) for 8 days. Mean of two biological replicates associated with three technical replicates is plotted. AU, arbitrary units. (B) Representative images of KP tumor cells with indicated shRNAs (green) pretreated as in (A) and cocultured with primary murine splenic NK cells (red) at a 20:1 E:T ratio for 20 hours in the presence or absence of indicated drugs (scale bar, 25 μm). Quantification of NK cell cytotoxicity (by live cell imaging) is shown on the right. Change in tumor cells is normalized to control wells lacking NK cells. Mean of three biological replicates is plotted. (C) Flow cytometry analysis of total and CD107a⁺ degranulating NK cells within the CD45⁺ population in the lungs after 1-week treatment of mice transplanted with KP tumor cells containing control Renilla (Ren) or p65 shRNAs with vehicle or combined trametinib (1 mg/kg body weight) and palbociclib (100 mg/kg body weight) (n ≥ 3 mice per group). (D) Kaplan-Meier survival curve of KP transplant mice treated with vehicle or combined trametinib (1 mg/kg body weight) and palbociclib (100 mg/kg body weight) and either an isotype control (HRPN), NK1.1 (PK136), or TNF-α (XT3.11) targeting antibody (n ≥ 6 per group) (log-rank test). (E) Flow cytometry analysis as in (C) after 1-week treatment of KP transplant mice with vehicle or combined trametinib (1 mg/kg body weight) and palbociclib (100 mg/kg body weight) and either an isotype control (HRPN) or TNF-α (XT3.11) targeting antibody (n = 5 per group). (F) Quantification of NK cell cytotoxicity (by live cell imaging) toward KP tumor cells with indicated shRNAs pretreated as in (B) and cocultured with primary murine splenic NK cells at a 20:1 E:T ratio for 20 hours in the presence or absence of indicated drugs. (A to C and E and F) One-way ANOVA. Error bars, mean ± SEM. **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 4.. Combination trametinib and palbociclib treatment drives NK cell–mediated lung tumor regressions in genetically engineered mice.

(A) Immunohistochemical staining of KP GEMM tumors treated with vehicle or combined trametinib (1 mg/kg body weight) and palbociclib (100 mg/kg body weight) for 2 weeks (scale bar, 50 μm). CC3, cleaved caspase-3; pERK, phosphorylated extracellular signal–regulated kinase. (B) Representative μCT images of KP GEMM lung tumors prior to treatment and after 2 weeks of treatment with vehicle or combined trametinib (1 mg/kg body weight) and palbociclib (100 mg/kg body weight) and either an isotype control antibody (C1.18.4) or NK1.1 depleting antibody (PK136). Yellow boxes indicate lung tumors. (C) A waterfall representation of the response of each tumor after 2 weeks of treatment with vehicle, trametinib (1 mg/kg body weight), palbociclib (100 mg/kg body weight), or both, and either an isotype control (C1.18.4), NK1.1 (PK136), TNF-α (XT3.11), or ICAM-1 (YN1/1.7.4) blocking antibody (n ≥ 6 per group). (D) Kaplan-Meier survival curve of KP GEMM mice treated as in (C) (n ≥ 6 per group) (log-rank test). (C) One-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.