BET Bromodomain Inhibition Cooperates with PD-1 Blockade to Facilitate Antitumor Response in Kras-Mutant Non-Small Cell Lung Cancer

Abstract

KRAS mutation is present in approximately 30% of human lung adenocarcinomas. Although recent advances in targeted therapy have shown great promise, effective targeting of KRAS remains elusive, and concurrent alterations in tumor suppressors render KRAS-mutant tumors even more resistant to existing therapies. Contributing to the refractoriness of KRAS-mutant tumors are immunosuppressive mechanisms, such as increased presence of suppressive regulatory T cells (Treg) in tumors and elevated expression of the inhibitory receptor PD-1 on tumor-infiltrating T cells. Treatment with BET bromodomain inhibitors is beneficial for hematologic malignancies, and they have Treg-disruptive effects in a non-small cell lung cancer (NSCLC) model. Targeting PD-1-inhibitory signals through PD-1 antibody blockade also has substantial therapeutic impact in lung cancer, although these outcomes are limited to a minority of patients. We hypothesized that the BET bromodomain inhibitor JQ1 would synergize with PD-1 blockade to promote a robust antitumor response in lung cancer. In the present study, using Kras^+/LSL-G12D ; Trp53^L/L (KP) mouse models of NSCLC, we identified cooperative effects between JQ1 and PD-1 antibody. The numbers of tumor-infiltrating Tregs were reduced and activation of tumor-infiltrating T cells, which had a T-helper type 1 (Th1) cytokine profile, was enhanced, underlying their improved effector function. Furthermore, lung tumor-bearing mice treated with this combination showed robust and long-lasting antitumor responses compared with either agent alone, culminating in substantial improvement in the overall survival of treated mice. Thus, combining BET bromodomain inhibition with immune checkpoint blockade offers a promising therapeutic approach for solid malignancies such as lung adenocarcinoma. Cancer Immunol Res; 6(10); 1234-45. ©2018 AACR.

Fig 1.

PD-1 blockade and BET bromodomain inhibition promotes reduced Treg proportions and expression of inhibitory receptors on tumor-infiltrating T cells in GEMM of NSCLC. (A) Schematics of treatment study and immune analysis in KP GEM. KP mice were induced with adeno-Cre intranasally, and treatment was started upon tumor establishment as confirmed by MRI. After two weeks of treatment, tumor nodules were excised from the lungs of JQ1 and/or α-PD-1-treated mice, and multiparameter flow cytometric analysis was conducted on single cell suspensions to assess the frequencies and phenotype of tumor-infiltrating T cell subsets. (B) Percent CD4⁺Foxp3⁺ Tregs within CD45⁺ leukocytes (left) and absolute Treg counts within the tumors of KP mice treated as indicated. (C, D) Representative histograms (left) and summary (right) of expression levels for (C) PD-1 and (D) CTLA-4 on tumor-infiltrating CD8⁺ T cells. Data are mean ± SEM for 4–5 mice per group. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig 2.

PD-1 blockade and BET bromodomain inhibition promote increased activation and effector capacity of tumor-infiltrating T cells in GEMM of NSCLC. Phenotypic assessments were conducted by flow cytometry of tumor cell suspensions to evaluate activation status of T cells. (A, B) Representative histograms (left) and summary (right) of CD69 expression on tumor-infiltrating (A) CD8⁺ and (B) CD4⁺ T cells. (C, D) Immune cells from KP tumors treated with vehicle or indicated agents were isolated by Ficoll gradient and stimulated for 6 hours with leukocyte activation cocktail (PMA and Ionomycin); golgi plug and CD107a antibody were added in the last 5 hours of culture. (C) Representative histograms with corresponding percentages and (D) summary of CD8⁺ T cells secreting IFNγ after intracellular cytokine staining. Data are mean ± SEM for 3–4 independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig 3.

PD-1 blockade and BET bromodomain inhibition is associated with a Th1 cytokine skewing in the BAL fluid of treated GEMM of NSCLC. BAL fluid obtained from the lungs of KP-tumor-bearing mice treated with α-PD-1, JQ1 or the combination were subjected to multiplex cytokine evaluation. (A) Summary of listed cytokines detected in the BAL fluid of mice that were treated as indicated shown as log2 fold-change relative to samples from vehicle-treated mice. (B) Mean fold change for Th1 (IL2, IFNγ, TNFα) and Th2 (IL4, IL5, IL6, IL10, IL13) cytokines relative to the vehicle controls in BAL fluids. (C) Ratio of the mean values for Th1 versus Th2 cytokines concentrations. Data in panel A are the average value for 4 replicates expressed as Log2 fold change over vehicle for each cytokine. Data shown in panels B and C represent mean ± SEM for 4 replicates/group.

Fig 4.

Combination of BET bromodomain inhibitor and anti-PD-1 promotes long-lasting therapeutic outcomes in KP-driven lung adenocarcinomas. (A) Waterfall plots showing tumor volume change (%) under indicated treatments after two weeks compared to pre-treatment tumor burden. Each column represents one individual mouse under each treatment (B) Representative MRI images of lung tumors in KP mice on day zero and day 14 after vehicle (control), JQ1, and/or α-PD-1 treatments. (C) Overall survival curve of KP mice under each indicated treatment with corresponding median survival time in days. Data are from 5–7 mice/treatment group.

Fig 5.

Reduced proportions of KLRG1+ Tregs is associated with low tumor burden and improved survival and response to treatment in KP mice. Following identification of a subset of tumor-infiltrating KLRG1⁺ CD4⁺Foxp3⁺ Tregs , their proportions were further assessed. (A) Representative histograms for the expression of KLRG1 on splenic versus tumor-Tregs. (B) Percent of KLRG1⁺ Tregs in the tumors of KP mice as a function of tumor volume. (C) Representative histograms with corresponding percentages and (D) summary of KLRG1⁺ Tregs in the tumors of vehicle and JQ1/anti-PD-1-treated KP mice. (E) Percent of KLRG1⁺ Tregs in the tumors of KP mice as a function of survival time in days. Data in D are mean ± SEM for 4–5 mice/group. *P < 0.05; **P < 0.01.

Fig. 6.

Proposed model for JQ1/α-PD-1 therapeutic effects in the KP model of NSCLC. In the absence of therapeutic intervention, inhibitory mechanisms including increased Treg presence and PD-1/CTLA-4 expression on tumor-infiltrating T cells outweigh stimulatory mechanisms leading to impairment in antitumor T cell function. Treatment with JQ1 and α-PD-1 cooperatively reduce these inhibitory mechanisms in distinct ways to allow stimulatory signals to prevail and tip the scale in favor of enhanced T cell function.