Glioblastoma (GBM) presents an immunosuppressive tumor microenvironment (inhibition of T-cell infiltration, activation, and action) that also limits the efficacy of immunotherapy. In GBM, self-renewal of glioblastoma stem cells (GCS) promotes cell differentiation and drug resistance. Amino acid metabolism affects T cell activation and function, and limiting amino acid intake can effectively inhibit tumor growth in vivo. Lysine metabolism produces a variety of bioactive substances, such as crotonyl coenzyme A. Crotonyl coenzyme A, a precursor of histone crotonylation modification (Kcr), affects the local chromosomal microenvironment and thus regulates stem cell differentiation, but its role in tumors is unclear.
On May 17, 2023, Professor Jeremy N Rich and Professor Nathaniel W. Snyder of the University of Pittsburgh Medical Center, in collaboration with Professor Yingming Zhao of the University of Chicago, published a paper entitled "Lysine catabolism reprograms tumour immunity through histone crotonylation" in Nature. Lysine catabolism reprograms tumour immunity through histone crotonylation". The article found that the reprogramming of lysine metabolism in glioblastoma stem cells leads to the production of crotonyl coenzyme A, which increases the level of overall crotonylation modification of the cells, especially the level of histone H4 crotonylation modification. Histone crotonylation modification affects H3K27ac and H3K9me3, influencing interferon signaling as well as CD8+ T-cell infiltration, ultimately promoting tumor growth.
Case 1 Lysine Metabolism Mediates Histone Crotonylation to Remodel Tumor Immunity (1)
Glioblastoma stem cells (GSC) specifically increase lysine uptake and promote tumor growth
Through metabolomics analysis, researchers found an enrichment of lysine, folate, pyrimidine, purine, and arginine metabolism-related pathways in GSC compared to matched differentiated glioblastoma cells (DGC). Folate, pyrimidine and purine metabolism have been found to maintain GSC and promote glioblastoma growth, but the role of lysine metabolism in this has not been reported. Using mass spectrometry to examine free amino acid levels in patient-derived GSCs, matched DGCs, and neural stem cells (NSCs), the investigators found a sustained increase in lysine in GSCs. Further, the investigators examined changes in the levels of SLC7A1, SLC7A2, and SLC7A3, which are responsible for the transport of lysine, and found that promoter activation and expression of SLC7A2 was increased in GSC. Finally, the researchers found that knockdown of SLC7A2 in GSC cells and mouse models significantly affected GSC growth as well as tumorigenesis. The above findings demonstrate the important role of lysine in tumors and revealed that upregulation of SLC7A2 expression in GSC specifically increased lysine uptake and promoted tumor growth.
GSC up-regulates lysine catabolism by SLC7A2
GSC reprograms lysine catabolism to promote Kcr and tumorigenesis.
Lysine tracing analysis reveals an increase in the conversion of lysine to acetyl-CoA in GSCs. This is accompanied by elevated expression of glutaryl-CoA dehydrogenase (GCDH), involved in metabolizing to produce glutaryl-CoA. In contrast, the key rate-limiting enzyme involved in breaking down glutaryl-CoA, ECHS1, is downregulated in typical GBM samples. This suggests a reprogramming of lysine metabolism in GSCs, leading to the accumulation of glutaryl-CoA through upregulation of GCDH and downregulation of ECHS1. Glutaryl-CoA serves as a precursor for protein lysine crotonylation (Kcr), which participates in the regulation of protein structure and function. Researchers compared the levels of histone Kcr, histone succinylation (Kglu), and histone acetylation (Kac) between GSCs and matched DGCs, as well as the expression of enzymes associated with lysine catabolism. Analysis revealed a specific enhancement of Kcr in GSCs, without an increase in glutaryl-CoA or short-chain fatty acids, and Kcr was primarily enriched on histone H4. Transplanting GSC cells with reduced GCDH expression into mice significantly improved mouse survival rates, while overexpression of ECHS1 weakened histone Kcr and cell proliferation in GSCs. Overall, the researchers confirmed the presence of lysine catabolism reprogramming in GSCs, promoting histone Kcr and tumor growth through enhanced GCDH and reduced ECHS1 expression.
GSC reprograms lysine catabolism to promote Kcr and tumorigenesis
Kcr influences interferon signaling to regulate GSC fate.
To further delineate the impact of targeting GCDH or ECHS1 on GSCs, researchers conducted RNA-seq analysis and found that GCDH knockdown induced the expression of 308 genes, with a significant enrichment of Type I interferon (IFN) signaling among upregulated genes. Furthermore, reintroduction of wild-type GCDH reduced activation of the IFN pathway, indicating the importance of GCDH catalytic activity in IFN signaling suppression. In contrast to the function of GCDH in GSCs, RNA-seq analysis revealed that knocking down ECHS1 in DGCs decreased activation of IFN signaling, reduced levels of senescence markers, and increased Kcr.
IFN signaling suppresses tumor growth by inducing senescence, inhibiting proliferation, and modulating immune responses. Researchers found that two days after GCDH loss, expression of cyclin-dependent kinase inhibitors did not change, but IFN signaling was activated, suggesting that enhanced IFN signaling might be the cause of cell cycle arrest. In intracranial tumors derived from GSCs, GSC proportions decreased after GCDH knockdown, and recovery was observed with treatment using human IFNAR-blocking antibodies. Thus, researchers confirmed that dysregulated lysine catabolism induces IFN signaling transduction to regulate GSC fate.
Reprogramming of lysine catabolism affects type I interferon signaling
Cellular GCDH binds with CBP to enhance Kcr.
Approximately 20% of GCDH is localized in the nucleus, and besides ECHS1, other lysine degradation enzymes also partially localize to the nucleus, suggesting a potential significant impact of lysine catabolism on the nucleus. Therefore, researchers further explored whether GCDH could directly interact with histone acetyltransferases. Based on mass spectrometry, researchers detected CBP in the Flag-GCDH protein complex and further validated the interaction between endogenous nuclear GCDH and CBP, indicating the crucial role of nuclear GCDH bound with CBP in Kcr regulation. As there are currently no specific GCDH inhibitors, researchers, based on computational biology framework - Lisa, identified MYC as the only predicted candidate GCDH inhibitor. Subsequently, through experimental validation, researchers found that combined use of lysine restriction and MYC inhibitor (MYCi) indeed inhibited cellular Kcr levels and GSC growth.
GCDH interacts with CBP in the nucleus to regulate Kcr
Previous studies before the enhancement of tumor immunity by restricting lysine intake suggested that CBP is also one of the acetyl-CoA transferases. Therefore, researchers hypothesized that glutaryl-CoA competes with acetyl-CoA as a substrate for CBP. Researchers found that knocking out GCDH in GSCs significantly decreased levels of histone Kcr but significantly increased levels of H3K27ac and decreased levels of H3K9me3. Additionally, the sites of decreased Kcr binding resulting from GCDH knockout were localized to transposons, and ChIP-seq analysis confirmed the attenuation of Kcr in transposons. This indicates that lysine catabolism reprogramming-induced Kcr restricts immunogenic transposons by affecting H3K27ac and H3K9me3. Previous studies have shown that transcription of transposons contributes to the generation of immunogenic cytoplasmic dsRNA and DNA and activation of IFN. Consistently, researchers also found that localized loss of histone Kcr promotes the generation of cellular dsRNA and DNA, thereby triggering IFN signaling dependent on MDA5 and cGAS in GSCs. From data of glioma patients, researchers also found that inhibition of lysine catabolism is positively correlated with characteristics of CD4+ T cells, CD8+ T cells, and natural killer cells, indicating that restricting lysine intake promotes IFN action and infiltration of CD8+ T cells, ultimately inhibiting tumor growth.
Inhibition of lysine catabolism promotes anti-tumor immunity
In summary, glioblastoma stem cells reprogram lysine metabolism to produce a large amount of glutaryl-CoA, thereby promoting overall crotonylation modification of cells. Histone H4 crotonylation modification restricts immunogenic transposons by affecting H3K27ac and H3K9me3, influencing interferon signaling, suppressing CD8+ T cell infiltration, and ultimately inhibiting tumor immune responses to promote tumor growth. Finally, the authors also propose that synergistic effects of lysine-restricted diet with MYC inhibitors or anti-PD-1 therapy can limit tumor growth.
Reference
- Yuan, Huairui, et al. "Lysine catabolism reprograms tumour immunity through histone crotonylation." Nature (2023): 1-9.