BCKDK modification enhances the anticancer efficacy of CAR-T cells by reprogramming branched chain amino acid metabolism

Abstract

Altered branched chain amino acids (BCAAs), including leucine, isoleucine, and valine, are frequently observed in patients with advanced cancer. We evaluated the efficacy of chimeric antigen receptor (CAR) T cell-mediated cancer cell lysis potential in the immune microenvironment of BCAA supplementation and deletion. BCAA supplementation increased cancer cell killing percentage, while accelerating BCAA catabolism and decreasing BCAA transporter decreased cancer cell lysis efficacy. We thus designed BCKDK engineering CAR T cells for the reprogramming of BCAA metabolism in the tumor microenvironment based on the genotype and phenotype modification. BCKDK overexpression (OE) in CAR-T cells significantly improved cancer cell lysis, while BCKDK knockout (KO) resulted in inferior lysis potential. In an in vivo experiment, BCKDK-OE CAR-T cell treatment significantly prolonged the survival of mice bearing NALM6-GL cancer cells, with the differentiation of central memory cells and an increasing proportion of CAR-T cells in the peripheral circulation. BCKDK-KO CAR-T cell treatment resulted in shorter survival and a decreasing percentage of CAR-T cells in the peripheral circulation. In conclusion, BCKDK-engineered CAR-T cells exert a distinct phenotype for superior anticancer efficiency.

Keywords: BCKDK; branched chain amino acids; cancer metabolism; cancer microenvironment; chimeric antigen receptor; immunotherapy.

Graphical abstract

Figure 1

BCAA supplementation improved CAR-T cell-mediated cancer cell lysis effectiveness, while disruption of BCAA metabolism and transport through 4 inhibitors impaired CAR-T cell-mediated antitumor effectiveness (A) The strategy of evaluating BCAA metabolism on the cancer cell killing potential of CAR T cells. (B) The pseudocolor images of flow cytometry showed CAR percentages that were indicated by the FLAG tag of anti-DYKDDDDK. (C) The grouped scatterplots showed cancer cell lysis percentage from different killing assay at different E:T ratios after co-culture for 48 h with the addition of BCAA, inhibitors of BCAA metabolism, and transport. (D) The pseudocolor images of flow cytometry showed CAR-T cells and cancer cells of NALM6-GL percentages after co-culture for 48 h. (E) The histograms showed the NALM6-GL cancer cells percentages after co-culture for 48 h. (F) The histograms showed the CAR-T cells percentages after co-culture for 48 h. One-way or two-way ANOVA followed by post hoc Tukey’s multiple comparison tests were used for comparing multiple groups. Statistical significance is indicated in all figures by the following annotations: ∗p < 0.05; ∗∗p < 0.01, ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001. The original statistical data are included in Tables S4–S6.

Figure 2

Reprogramming of BCAA metabolism regulated CAR-T cell function, proliferation, and glucose uptake (A) The pseudocolor images of flow cytometry showed intracellular staining of IFN-γ and TNF-α after co-culture for 48 h at Effector CAR-T cells: Target cancer cells ratio of 1:1 with the addition of BCAA, inhibitors of BCAA metabolism and transport after co-culture for 2 days. (B) The histograms showed the IFN-γ⁺ and TNF-α⁺ CAR-T cell percentages after co-culture for 48 h. (C) The pseudocolor images of flow cytometry showed CFSE⁺ CAR-T with the addition of BCAA, inhibitors of BCAA metabolism, and transport after culture for 5 days. (D) The histograms showed CAR-T cell proliferation through the analysis of CFSE⁺ cell percentages. (E) The pseudocolor images of flow cytometry showed 2-NBDG⁺ CAR-T with the addition of BCAA, inhibitors of BCAA metabolism, and transports after culture for 60 min. (F) The histograms showed CAR-T cell glucose uptake through the analysis of 2-NBDG⁺ cell percentages. One-way ANOVA followed by post hoc Tukey’s multiple comparison test were used for comparing multiple groups. Statistical significance were indicated in all figures by the following annotations: ∗p < 0.05; ∗∗p < 0.01, ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001. The original statistical data are included in Tables S7–S9.

Figure 3

BCKDK-modified CAR-T cell function, proliferation, and metabolism (A) The metabolic pathways of BCAA catabolism and their key enzyme. (B) The strategy of production BCKDK modified CAR-T cells. (C) The pseudocolor images of flow cytometry showed CAR percentages that were indicated by the FLAG tag of anti-DYKDDDDK from BCKDK modified CAR-T cells. (D) The histograms showed BCKDK gene expression at different BCKDK-modified CAR-T cells. (E) The protein expression levels of BCKDK t different BCKDK modified CAR-T cells through Western blot gels (Figure S1). (F) The gel image of endonuclease T7E1 mismatch detection assay showed a significant KO of BCKDK in BCKDK-KO CAR-T cells (Figure S2). (G) The pseudocolor images of flow cytometry and the histograms showed CFSE⁺ cell percentages from BCKDK modified CAR-T cells after culture for 5 days. (H) The pseudocolor images of flow cytometry and the histograms showed 2-NBDG⁺ cell percentages from BCKDK modified CAR-T cells after cultured for 60 min. (I) The grouped histograms showed cancer cell lysis percentage from killing assay at different E:T ratios after co-culture for 48 h based on BCKDK modified CAR-T cells. (J) The pseudocolor images of flow cytometry and the histograms showed intracellular staining of IFN-γ and TNF-α after co-culture for 48 h at Effector CAR-T cells: target cancer cells ratio of 1:1 from BCKDK modified CAR-T cells. One-way or two-way ANOVA followed by post hoc Tukey’s multiple comparison tests were used for comparing multiple groups. Statistical significance was indicated in all figures by the following annotations: ∗p < 0.05; ∗∗p < 0.01, ∗∗∗p < 0.001; ∗∗∗p < 0.0001. The original statistical data are included in Tables S10–S15.

Figure 4

BCKDK-OE CAR-T cells increase cancer cell apoptosis and inhibit CAR-T cell apoptosis (A) The pseudocolor images of flow cytometry showed BCKDK-modified CAR-T cells and NALM6-GL cancer cells percentages after co-culture for 48 h. (B) The histograms showed the CAR-T cell and the NALM6-GL cancer cell percentages after co-culture for 48 h from BCKDK-modified CAR-T cells. (C) The pseudocolor images of flow cytometry showed apoptosis cell percentages of BCKDK-modified CAR-T cells and NALM6-GL cancer cells. (D) The histograms showed apoptosis cell percentages of the NALM6-GL cancer cells and the BCKDK-modified CAR-T cells. (E) The pseudocolor images of flow cytometry showed markers PD-1 and LAG-3 expression of BCKDK modified CAR-T cells after co-culture with NALM6-GL cancer cells at an E:T ratio of 1:1 for 48 h with the addition of 10 ng/mL IFN-γ. (F) The histograms showed expression of PD-1 and LAG-3 in BCKDK modified CAR-T cells after co-culture with NALM6-GL cancer cells at an E:T ratio of 1:1 for 48 h. One-way ANOVA or two-way ANOVA followed by post hoc Tukey’s multiple comparison tests were used for comparing multiple groups. Statistical significance is indicated in all figures by the following annotations: ∗p < 0.05; ∗∗p < 0.01, ∗∗∗p < 0.001; ∗∗∗p < 0.0001. The original statistical data are included Tables S16–S18.

Figure 5

BCKDK modified CAR-T cells showed distinct metabolic features and differentiation phenotypes (A) The heatmap of metabolomics showed metabolic distinction of BCKDK-modified CAR-T cells based on 19 metabolites. (B) The biplot showed that two principal components discriminated BCKDK-modified CAR-T cells and their relevant metabolites. (C) The pathway plot showed the main metabolic pathway resulted from BCKDK modification. (D) The protein expression levels of mTOR, p70S6K, 4EBP1, and their phosphorylation expression. The histograms showed expression phosphorylation levels of mTOR, p70S6K, and 4EBP1 at BCKDK-modified CAR-T cells (Figure S3). (E) The pseudocolor images of flow cytometry showed memory differentiation markers CD45RA and CD62L from BCKDK modified CAR-T cells after co-culture with NALM6-GL cancer cells at an E:T ratio of 1:1 for 2 days and 14 days. (F) The histograms showed percentages of Tscm (CD45RA⁺ CD62L⁺), Tcm (CD45RA⁻ CD62L⁺), Tcm (CD45RA⁻ CD62L⁻), and Temra (CD45RA⁺ CD62L⁻) from BCKDK-modified CAR-T cells after co-culture with NALM6-GL cancer cells at an E:T ratio of 1:1 for 2 days and 14 days. One-way or two-way ANOVA or two-way ANOVA followed by post hoc Tukey’s multiple comparison tests were used for comparing multiple groups. Statistical significance were indicated in all figures by the following annotations: ∗p < 0.05; ∗∗p < 0.01, ∗∗∗p < 0.001; ∗∗∗p < 0.0001. The original statistical data are included in Tables S19 and S20.

Figure 6

BCKDK-OE CAR-T cells exert superior cancer cell lysis ability, while BCKDK-KO CAR-T cell treatment resulted in shorter survival (A) The in vivo strategy of BCKDK modified CAR-T cells for the treatment cancer mouse bearing NALM6-GL cells and the disease progression following. (B) The survival plot of BCKDK modified CAR-T cells for the treatment cancer mouse bearing NALM6-GL cells. (C) The pseudocolor images of flow cytometry showed CAR-T cells percentages and NALM6-GL cancer cells percentages from BCKDK modified CAR-T cell-treated mice bearing NALM6-GL cancer cells at day 14. (D) The histograms showed NALM6-GL cancer cells percentage and CAR-T cell percentages from BCKDK modified CAR-T cell-treated mice bearing NALM6-GL cancer cells at day14. (E) The histograms showed sera IFN-γ and TNF-α levels from BCKDK-modified CAR-T cell-treated mice bearing NALM6-GL cancer cells at day 14. (F) The pseudocolor images of flow cytometry showed CD45RA and CD62L expression in CAR-T cells from BCKDK modified CAR-T cell-treated mice bearing NALM6-GL cancer cells at days 14 and 28. (G) The histograms showed percentages of Tscm (CD45RA⁺ CD62L⁺), Tcm (CD45RA⁻ CD62L⁺), Tcm (CD45RA⁻ CD62L⁻), and Temra (CD45RA⁺ CD62L⁻) from BCKDK modified CAR-T cell-treated mice bearing NALM6-GL cancer cells at days 14 and 28. Survival analysis was conducted using the Kaplan-Meier method, and significance was determined using the log rank test. One-way or two-way ANOVA or two-way ANOVA followed by post hoc Tukey’s multiple comparison tests were used for comparing multiple groups. Statistical significance were indicated in all figures by the following annotations: ∗p < 0.05; ∗∗p < 0.01, ∗∗∗p < 0.001; ∗∗∗p < 0.0001. The original statistical data are included in Tables S21–S23.