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. 2023 Sep 7;15(18):4467.
doi: 10.3390/cancers15184467.

Increased Response to Immune Checkpoint Inhibitors with Dietary Methionine Restriction in a Colorectal Cancer Model

Lauren C Morehead  1 , Sarita Garg  1 , Katherine F Wallis  1 , Camila C Simoes  2 , Eric R Siegel  3 , Alan J Tackett  1 , Isabelle R Miousse  1
Affiliations

Increased Response to Immune Checkpoint Inhibitors with Dietary Methionine Restriction in a Colorectal Cancer Model

Lauren C Morehead et al. Cancers (Basel). .

Abstract

Dietary methionine restriction (MR), defined as a reduction of methionine intake by around 80%, has been shown to reproducibly decrease tumor growth and synergize with cancer therapies. In this study, we combined DMR with immune checkpoint inhibitors (ICIs) in a model of colon adenocarcinoma. In vitro, we observed that MR increased the expression of MHC-I and PD-L1 in both mouse and human colorectal cancer cells. We also saw an increase in the gene expression of STING, a known inducer of type I interferon signaling. Inhibition of the cGAS-STING pathway, pharmacologically or with siRNA, blunted the increase in MHC-I and PD-L1 surface and gene expression following MR. This indicated that the cGAS-STING pathway, and interferon in general, played a role in the immune response to MR. We then combined dietary MR with ICIs targeting CTLA-4 and PD-1 in an MC38 colorectal cancer tumor model developed in immunocompetent C57BL/6 mice. The combination treatment was five times more effective at reducing the tumor size than ICIs alone in male mice. We noted sex differences in the response to dietary MR, with males showing a greater response than females. Finally, we observed an increase in membrane staining for the PD-L1 protein in MC38 tumors from animals who were fed an MR diet. MHC-I was highly expressed in all tumors and showed no expression difference when comparing tumors from control and MR-treated mice. These results indicated that MR increased PD-L1 expression both in vitro and in vivo and improved the response to ICIs in mice.

Keywords: PD-L1; STING; colorectal; immune checkpoint inhibitors; methionine restriction.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
MR increases MHC-I and PD-L1 expression through interferon signaling.
Figure 2
Figure 2
Methionine restriction increases MHC-I and PD-L1 expression. HT29 human colon cancer cells were grown for 24 h in media containing 200 µM (CTL) or 5 µM (MR) methionine. Gene expression for HLAA (A) and CD274 (B) increased with MR. Surface abundance measured by flow cytometry (C,D) also increased with MR. In B16-OVA cells, lowering methionine also increased MHC-I surface abundance and the presentation of the SIINFEKL peptide (E). t-test with mean and SEM is shown.
Figure 3
Figure 3
Inhibiting STING blunts the increase in MHC-I. STING was increased by MR at the gene and protein expression levels (A) and insert, t-test with mean and SEM). Inhibiting STING with C-176 led to a reduction in the increase in HLAA gene expression (B) and in its cell surface representation (C). CD274 gene expression was increased by C-176 and did not increase further with MR (D). Two-way ANOVA with mean and SEM.
Figure 4
Figure 4
MHC-I and PD-L1 are altered by methionine restriction in a model cell line. MR increased H2Kb gene expression in vitro in the cell line MC38 (A). The cell surface expression of MHC-I, however, decreased with MR (B). The gene expression for Cd274 also increased with MR (C), with a parallel increase in surface protein abundance (D). t-test with mean and SEM. Inhibiting JAK (interferon signaling) with 4 µM ruxolitinib led to a blunting of the effect of MR on Cd274 gene expression (E). Two-way ANOVA with mean and SEM.
Figure 5
Figure 5
Dietary methionine restriction synergizes with ICIs in males. After subjecting the mice to a combination treatment consisting of anti-CTLA-4 and anti-PD-1 with MRD, females showed no benefit from MRD (A). In males, there was a 5× reduction in tumor volume with ICIs and MRD compared with ICIs alone (B) and (C). There was no significant change in body weight measured after 10 days of MRD (C).
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