Potentiation of Kras peptide cancer vaccine by avasimibe, a cholesterol modulator

Abstract

Background: No effective approaches to target mutant Kras have yet been developed. Immunoprevention using KRAS-specific antigenic peptides to trigger T cells capable of targeting tumor cells relies heavily on lipid metabolism. To facilitate better TCR/peptide/MHC interactions that result in better cancer preventive efficacy, we combined KVax with avasimibe, a specific ACAT1 inhibitor, tested their anti-cancer efficacy in mouse lung cancer models, where Kras mutation was induced before vaccination.

Methods: Control of tumor growth utilizing a multi-peptide Kras vaccine was tested in combination with avasimibe in a syngeneic lung cancer mouse model and a genetically engineered mouse model (GEMM). Activation of immune responses after administration of Kras vaccine and avasimibe was also assessed by flow cytometry, ELISpot and IHC.

Findings: We found that Kras vaccine combined with avasimibe significantly decreased the presence of regulatory T cells in the tumor microenvironment and facilitated CD8+ T cell infiltration in tumor sites. Avasimibe also enhanced the efficacy of Kras vaccines target mutant Kras. Whereas the Kras vaccine significantly increased antigen-specific intracellular IFN-γ and granzyme B levels in CD8+ T cells, avasimibe significantly increased the number of tumor-infiltrating CD8+ T cells. Additionally, modulation of cholesterol metabolism was found to specifically impact in T cells, and not in cancer cells.

Interpretation: Avasimibe complements the efficacy of a multi-peptide Kras vaccine in controlling lung cancer development and growth. This treatment regimen represents a novel immunoprevention approach to prevent lung cancer.

Keywords: Avasimibe; Chemoimmunoprevention; Cholesterol metabolism; Kras; Peptide vaccine; T cell.

Fig. 1

Avasimibe shifts the balance from regulatory to effector T cells and increases CD8+ T cell infiltration in lung tumors. Kras-driven LKR13 mouse lung cancer cells were inoculated into sv129 mice, i.p. treatments with avasimibe (AVA) were started at different time points when tumors reached ∼200mm in size. At the experimental endpoint, tumors were processed into single cell suspension, enriched for TILs by Ficoll gradient separation, and flow cytometric analysis was conducted on the tumor-infiltrating leukocytes isolated from control or AVA-treated LKR13 lung tumors. (a, b) Representative flow cytometric histograms for CD4 and CD8 T cells and Tregs in the TME. (c) Percentages of CD8+, CD4+ and Tregs in the TME. (d) Representative flow cytometric histograms and results for monocytic cells n TME. (e) Representative flow cytometric histograms and combined results for staining of cells with CD44 and CD62L to assess naïve T cells, T_CM and T_EM cells. (f-g) Flow cytometric histograms and combined results for staining of cells for intracellular cytokines granzyme B, IFN-γ and TNF-α after stimulation with PMA and Ionomycin. Data are shown as the mean ± SE of three replicate samples per group, n = 3, *P ≤ 0.05 vs Ctrl, two-tailed Student's t-test.

Fig. 2

AVA plus the Kras peptide vaccine enhances the inhibition of lung tumor progression in a syngraft model. (a) Experimental design outlining the timing of vaccine and AVA administration, inoculation of tumor cells, and the experimental endpoint (Eut, euthanasia). Sv129 mice were given 4 weekly Kras multipeptide vaccinations, the Kras-driven mouse lung cancer cell LKR13 was then injected, and avasimibe treatments were given every other day until the experimental endpoint. Tumor sizes were measured weekly. The experiment was repeated three more times. (b) Tumor volume quantitation over time. (c) Representative ELISPOT assay figures (insets) and combined results. Upper left panel, splenocytes controls or Kras-vaccinated animals re-stimulated with a combination of KRAS peptides (p5–21, p5–21 G12D, p17–31, and p78–92). Upper right and lower panels, splenocytes re-stimulated with LKR13 cancer cells. Combined results are the mean ± SE, n = 10, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 vs Ctrl, two-tailed Student's t-test.

Fig. 3

Immunophenotyping of T cells from syngraft tumors of animals treated with AVA and/or KVax. Tumors and spleens were collected at the efficacy study experimental endpoint from LKR13 bearing mice as shown in Fig. 2. (a) Representative flow cytometry graphs showing CD8+ and IFN-γ staining cells. (b, c) Combined treatment with AVA and KVax promotes increased frequencies of tumor-infiltrating CD8+ T cells. (d, e) Combined treatment with AVA and KVax leads to increased cytokine-producing CD8+ T cells. Data are shown as the mean ± SE of three replicate samples per group, n = 5, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 vs Ctrl, one-way ANOVA.

Fig. 4

AVA modulates the cholesterol levels in CD8+ T cells. (a) Free cholesterol and cholesterol ester levels were measured in tumor cells and T cells isolated from LKR13 tumors using Amplex Ultra Red. (b) Comparison of cholesterol esterification gene transcription levels in CD8+ TILs as compared to cancer cells. (c) Upregulation of cholesterol synthesis and cholesterol transport gene transcription levels by AVA in cancer cells and CD8+ TILs when compared to non-treated controls. Data are shown as the mean ± SE of three replicate samples per group, n = 3, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 vs Ctrl, one-way ANOVA.

Fig. 5

The combination of AVA with KVax enhances the inhibition of Kras-driven lung tumors in the Kras^LA1 GEMM model. (a) Experimental design outlining the timing of vaccine, AVA administration, and experimental endpoint (Eut, euthanasia). (b) Tumor multiplicity and tumor volume quantitation at the experimental endpoint. (c) Representative IHC staining and quantitative data for Ki-67 from the Kras^LA1 model lung tumor. (d) Representative IHC staining and quantitative data for tumor-infiltrating CD8+ T cells from the Kras^LA1 model lung tumor. (e) Cytokine analysis by Mouse Cytokine Array/ Chemokine Array 31-Plex (MD31) using supernatant collected from splenocytes stimulated with peptides for 72hr. Data are shown as the mean ± SE, n = 8, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 vs Ctrl, two-tailed Student's t-test.