Chromatin accessibility governs the differential response of cancer and T cells to arginine starvation

Abstract

Depleting the microenvironment of important nutrients such as arginine is a key strategy for immune evasion by cancer cells. Many tumors overexpress arginase, but it is unclear how these cancers, but not T cells, tolerate arginine depletion. In this study, we show that tumor cells synthesize arginine from citrulline by upregulating argininosuccinate synthetase 1 (ASS1). Under arginine starvation, ASS1 transcription is induced by ATF4 and CEBPβ binding to an enhancer within ASS1. T cells cannot induce ASS1, despite the presence of active ATF4 and CEBPβ, as the gene is repressed. Arginine starvation drives global chromatin compaction and repressive histone methylation, which disrupts ATF4/CEBPβ binding and target gene transcription. We find that T cell activation is impaired in arginine-depleted conditions, with significant metabolic perturbation linked to incomplete chromatin remodeling and misregulation of key genes. Our results highlight a T cell behavior mediated by nutritional stress, exploited by cancer cells to enable pathological immune evasion.

Keywords: ASS1; ATF4; H3K27me3; T cell chromatin; arginine; cancer metabolism; immunometabolism; immunosuppression; metabolic regulation; nutritional stress.

Graphical abstract

Figure 1

T cells and THP1 cells show differential responses to arginine starvation (A) Left: growth of stimulated CD4⁺ human T cells in complete (+Arg) or arginine-free medium with (−Arg +Citr) or without (−Arg) citrulline. Data are represented as mean ± SD; n = 4. Right: naive, central memory (Tcm), and effector memory (Tem) T cells (see Figure S1D for sort strategy) were stimulated and then incubated in the indicated media for 96 h and counted. Data are fold increase over cell number at 0 h; mean ± SD; n = 2. (B) Growth of THP1 cells in the indicated media. Data are represented as mean ± SD; n = 4. (C) Concentration of citrulline in the blood plasma of healthy (control) and plasma or bone marrow of AML patients. Center bar shows mean ± SD. ^∗∗∗∗p < 0.0001 (unpaired t test). (D) Microarray analysis of mRNA in THP1 or stimulated T cells incubated in +Arg or −Arg medium for 72 h. Each column represents a replicate. Class assignments (I–VI) for genes are indicated. (E) Overlap of differentially expressed genes in T cells and THP1 cells, with class assignments (I–VI) indicated. (F) Analysis of KEGG pathway enrichment within each class of differentially expressed genes following arginine starvation, shown in (D). Dot size is proportional to significance (Wallenius method). See also Figure S1 and Tables S1 and S2.

Figure 2

ATF4-induced ASS1 upregulation facilitates citrulline-dependent growth of THP1 cells (A) Key proteins in arginine uptake and biosynthesis. (B) Forest plot showing changes in gene expression, based on microarray analysis (see Figure 1D). Horizontal bars show interquartile range. (C) ASS1 and ATF4 expression in primary AML blasts or non-transformed monocytic and myelocytic cells from healthy donors (Quek et al., 2016). Samples are colored by donor. Bars show mean ± SD. ^∗∗p < 0.01, ^∗∗∗p < 0.001 (Mann-Whitney test). (D) Western blot for ASS1 and ATF4 in stimulated T cells and THP1 cells incubated for 72 h in complete medium (+), medium containing 20 μM arginine (low), or lacking arginine (−). Two exposures of the ASS1 blot are shown for clarity. Representative of five replicates. (E) Quantification of (D), normalized to GAPDH, relative to +Arg T cells. Expression in T cells is shown on a smaller scale for clarity. Data are represented as mean ± SEM; n = 5. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001 (Dunnett’s multiple comparison test). ns, not significant. (F) Growth of control (NT) THP1 cells or following KD of ASS1 or ATF4 in the indicated media. Data are represented as mean ± SD; n = 4. (G) Representative western blot for ASS1 and ATF4 in control (NT) THP1 cells or following KD of ASS1 or ATF4 in the presence (+) and absence (−) of arginine for 72 h. Right: quantification, normalized to GAPDH, relative to +Arg NT cells. Data are represented as mean ± SEM; n = 3. ^∗p < 0.05 (Dunnett’s multiple comparison test). (H) CTV-labeled CD8⁺ T cells were transduced with a GFP-ASS1 coexpression plasmid. Cells were analyzed for GFP and CTV levels after 96 h in the indicated media. Three technical replicates are shown. (I) Proportion of GFP-positive cells from the analysis in (H), normalized to the −Arg ratio. Data are represented as mean ± SD; n = 5 from two donors. ^∗∗p < 0.01 (paired t test). See also Figure S2.

Figure 3

ATF4 activates ASS1 transcription via an intronic enhancer (A) Reference-normalized ChIP-seq for ATF4 and CEBPβ at ASS1 in stimulated T cells and THP1 cells incubated in the indicated media for 72 h, and ChIP-seq for H3K4me3, H3K27ac, and H3K4me1 in THP1 cells in +Arg medium (Godfrey et al., 2019). Gray bars show qPCR primer locations. (B) Reference-normalized ChIP-seq for ATF4 and CEBPβ at SLC7A1, as in (A). (C) Sequences of the enhancer region in parental (wild type [WT]) and mutant THP1 cells. PAM sequences are underlined. (D) ChIP-qPCR for ATF4 and H3K27ac in WT and mutant THP1 cells, incubated in +Arg or −Arg medium for 72 h. Data are represented as mean ± SEM; n = 3. (E) Western blot for ASS1 and ATF4 in WT and mutant THP1 cells, incubated in +Arg or −Arg medium for 72 h. Representative of three replicates. (F) Growth of WT and mutant THP1 cells, incubated in the indicated media. Data are represented as mean ± SD; n = 3. See also Figure S3.

Figure 4

ASS1 is repressed in T cells (A) ATAC-seq at ASS1 in THP1 cells incubated in the indicated media for 72 h. ATF4 ChIP-seq from −Arg cells is shown for comparison. Bottom: overlay of ATAC-seq traces at the highlighted region of ASS1, mean of three replicates. (B) ATAC-seq at ASS1 in stimulated T cells, as in (A). (C) ChIP-qPCR for H3K9me3, H3K27me3, and H3K4me3 in stimulated T cells and THP1 cells incubated in the indicated media for 72 h. Data are represented as mean ± SEM; n = 4. (D) ChIP-qPCR for H3K9me3, H3K27me3, and ATF4 in stimulated T cells incubated for 72 h in complete medium (+Arg), medium containing 20 μM arginine, without (low Arg) or with (low Arg + 2HG) addition of 500 μM 2HG, or lacking arginine (−Arg). Data are represented as mean ± SEM; n = 4. (E) Representative western blot for ASS1 and ATF4 in stimulated T cells incubated in the indicated media for 72 h. Non-specific bands are indicated by an asterisk. Right: quantification, normalized to GAPDH, relative to +Arg. Data are represented as mean ± SEM; n = 5. ^∗∗p < 0.01, ^∗∗∗∗p < 0.0001 (Dunnett’s multiple comparison test). (F) Model for ASS1 regulation in T cells and THP1 cells in response to arginine depletion. In THP1 cells accessibility at ASS1 allows ATF4 binding under low and −Arg conditions, inducing ASS1 expression. In T cells, ATF4 binding and ASS1 expression are regulated by two competing processes: ATF4 is active under low or −Arg conditions, but the ASS1 promoter is repressed. Under −Arg, elevated H3K9me3/H3K27me3 and reduced accessibility at ASS1 block ATF4 binding. See also Figure S4.

Figure 5

ASS1 upregulation is a common tumor response to arginine starvation (A) Growth of tumor cell lines in the indicated media. AML, acute myeloid leukemia; APL, acute promyelocytic leukemia; ALL, acute lymphoblastic leukemia. Data are represented as mean ± SD; n = 4. (B) qRT-PCR for ASS1 in the indicated cell lines, cultured in +Arg and −Arg media. Data are normalized to GAPDH, relative to +Arg in each cell line, represented as mean ± SD; n = 4 or 6 (HeLa). ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001 (Šidák’s multiple comparison test). (C) Representative western blot for ASS1 and ATF4 in control (NT) HeLa cells or following KD of ASS1 or ATF4 in the indicated media for 72 h. (Right) Quantification, normalized to GAPDH, relative to +Arg NT cells. Data are represented as mean ± SEM; n = 3. ^∗p < 0.05 (Dunnett’s multiple comparison test). (D) Growth of control (NT) HeLa cells or following KD of ASS1 or ATF4, incubated in the indicated media. Data are represented as mean ± SD; n = 3. (E) ChIP-qPCR for ATF4 and CEBPβ levels in THP1 and HeLa cells incubated in the indicated media for 72 h. Data are represented as mean ± SEM; n = 3. (F) ChIP-qPCR for H3K9me3 and H3K27me3 in HeLa cells incubated in the indicated media for 72 h. Data are represented as mean ± SEM; n = 3. See also Figure S5.

Figure 6

Arginine-starved T cells show reduced ATF4/CEBPβ binding and chromatin accessibility (A) Left: number of ATF4 and CEBPβ peaks identified in ChIP-seq from stimulated T cells and THP1 cells incubated in the indicated media for 72 h. Right: overlap of ATF4 ChIP-seq peaks identified in T cells in low Arg or −Arg conditions. (B) Reference-normalized ATF4 (left) and CEBPβ (right) ChIP-seq levels at ATF4 peaks from T cells and THP1 cells incubated in the indicated media (colored lines). Mean level is displayed for T cell ATF4 peaks found only under low Arg conditions, only under arginine starvation, or under both conditions (common), as in (A). (C) Differential chromatin accessibility between stimulated T cells incubated in +Arg and −Arg medium. Red and blue dots indicate significantly increased and decreased ATAC peaks under −Arg; false discovery rate (FDR) < 0.05. (D) Chromatin accessibility (ATAC-seq) at T cell ATF4 peaks, as in (B). (E) Reference-normalized ATF4 and CEBPβ ChIP-seq levels at ATAC peaks from T cells incubated in the indicated media. Mean level is displayed for peaks that show reduced accessibility (more closed), increased accessibility (more open), or no change (unaffected) in arginine-starved T cells, as in (C). (F) Reference-normalized H3K27me3 ChIP-seq levels at T cell ATAC peaks, as in (E). (G) Reference-normalized H3K27me3 ChIP-seq levels at T cell ATF4 peaks, as in (B). See also Figure S6.

Figure 7

Arginine starvation disrupts chromatin and metabolic reprogramming of T cells (A) Differential chromatin accessibility in T cells upon stimulation in the indicated media for 72 h. Red and blue dots indicate significantly increased and decreased ATAC peaks upon stimulation; FDR < 0.05. (B) K-means clustering (k = 5) of differential ATAC peaks following stimulation in +Arg medium (see A, left), using ATAC-seq from unstimulated T cells (US) and T cells stimulated in the indicated media for 72 h. Each column is a sample, with each row an ATAC peak. (C) Principal component analysis of ATAC-seq data in (B). Each dot represents a sample; n = 3. PC, principal component. (D) Representative western blot of S6 phosphorylation (Ser235/Ser236) in unstimulated T cells and T cells stimulated for 24 h in +Arg, low Arg, or −Arg medium, or +Arg medium in the presence of 20 nM rapamycin (rapa). Bottom: quantification, normalized to GAPDH, relative to unstimulated T cells. Data are represented as mean ± SEM; n = 4. ^∗∗p < 0.01, ^∗∗∗∗p < 0.0001 (Dunnett’s multiple comparison test). (E) Growth of T cells stimulated in the indicated media. Data are represented as mean ± SD; n = 5. (F) Hierarchical clustering analysis of metabolite levels from unstimulated T cells (US) and T cells stimulated in the indicated media for 72 h. Each column is a sample, and each row is a metabolite. (G) Principal component analysis of metabolite data in (F). Each dot represents one sample; n = 5. (H) Seahorse analysis of unstimulated T cells and T cells stimulated in the indicated media for 72 h. Data are the mean of four donors ± SEM. ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001, comparisons made with +Arg across all time points (Tukey’s multiple comparison test). See also Figure S7 and Table S3.