Multi-omics unveils BCAA metabolism markers L-leucine and HMGCS1 as prognostic marker for immunotherapy efficacy in non-small cell lung cancer

Abstract

Background: This study aims to identify branched-chain amino acid (BCAA) plasma metabolites and gene signatures that enhance prognostic assessments in non-small cell lung cancer (NSCLC) patients receiving immunotherapy.

Methods: Plasma metabolites were measured using untargeted UPLC-MS/MS (n = 94 and 40), with lymphocyte subset tests on 72 patients. BCAA-related subtypes were identified in NSCLC datasets (n = 274, 176, and 196). A prognostic risk model was developed and validated in NSCLC (n = 16, 27, 24, and 339), melanoma (n = 25), and pan-cancer ICIs cohorts (n = 330 and 81). Immune cell infiltration and prognostic signatures were validated using mIF (n = 21 in CHCAMS), scRNA-seq (n = 8 and 21), and spatial transcriptomics (n = 2 and 6). Cell and animal experiments involving HMGCS1 were conducted in a lung cancer model. Additionally, based on our previous findings that B cells with higher malignancy exhibited enhanced cholesterol homeostasis pathways in diffuse large B-cell lymphoma (DLBCL), we further analyzed the prognostic value of HMGCS1 using our spatial transcriptomics (n = 10) and immunohistochemistry (IHC, n = 39) in DLBCL.

Results: Our plasma metabolite analysis showed higher L-leucine levels were associated with better prognosis and had higher T cell counts and CD4⁺ T cell counts (P < 0.05). In GEO datasets, four NSCLC subtypes were identified, showing distinct prognostic outcomes and tumor microenvironment. Five BCAA-related genes (ACAT2, ALDH2, HMGCS1, MLYCD, and PPM1 K) formed a prognostic risk model for NSCLC, validated through Kaplan-Meier and ROC curve analyses in ICI cohorts (P < 0.05). HMGCS1 was an independent prognostic value in ICI cohorts and was negatively correlated with CD8⁺ T cell infiltration, while positively correlating with tumor severity, cholesterol homeostasis, and BCAA degradation across multiple platforms, including GEO datasets, our mIF cohort, public scRNA-seq, and spatial transcriptomics (P < 0.05). And our cell and animal function experiments found HMGCS1 overexpression promotes metabolic pathways and accelerates tumor growth, whereas HMGCS1 knockdown suppresses tumor progression in a mouse model treated with PD-1 monoclonal antibody (P < 0.05). In DLBCL, high HMGCS1 expression was associated with shorter overall survival, enriched in B cells and relapsed patients, correlated with cholesterol homeostasis and amino acid degradation pathways, and its prognostic value was further validated at the protein level by our IHC cohort (P < 0.05).

Conclusions: This study identifies a BCAA-related plasma metabolites and gene signature as effective prognostic markers for NSCLC patients receiving immunotherapy, with HMGCS1 as a key prognostic factor influencing tumor progression and immune response.

Keywords: Branched chain amino acids; Cholesterol homeostasis; Immunotherapy; Non-small cell lung cancer; Prognostic biomarker.

Fig. 1

Differential analysis of plasma metabolites receiving immunotherapy and distinct BCAA metabolism patterns in NSCLC. A Heatmap of differentially expressed metabolites between the DCB and NDB groups in the validation cohort. B Functional enrichment analysis of the differentially expressed metabolites. C Comparison of L-leucine levels between the DCB and NDB groups, along with the AUC curve and Kaplan–Meier survival analysis in both the discovery and validation cohorts. D Consensus clustering matrix for k = 4 in GSE41271, principal component analysis of the four clusters, and Heatmap displaying the distribution of 27 prognosis-related BCAA metabolism regulators across the four clusters. E Kaplan–Meier curves illustrating overall survival according to the four clusters in GSE41271 and GSE42127. F Differences in tumor-infiltrating immune cells among the four clusters. (Abbreviation: BCAA: branched chain amino acid; NSCLC: non-small cell lung cancer; NDB: non-durable clinical benefit; DCB: durable clinical benefit. Mann-Whitney test was performed between clusters.)

Fig. 2

Performance of HMGCS1 in predicting survival in immunotherapy cohorts. A, E Kaplan–Meier curves for PFS based on HMGCS1 in NSCLC and melanoma immunotherapy cohorts. B Comparison of HMGCS1 expression between NDB and DCB groups. C, F Correlations between HMGCS1 and cholesterol biosynthesis in NSCLC and melanoma immunotherapy cohorts. D Correlations between HMGCS1 and CD8⁺ T cell infiltration in NSCLC immunotherapy cohort. G Comparison of HMGCS1 in MPR and NMPR groups in neoadjuvant immunotherapy cohort (GSE207422). H Correlations between HMGCS1 and residual tumor (%) in neoadjuvant immunotherapy cohort. I, J Kaplan–Meier curves for OS based on HMGCS1 in pan-cancer immunotherapy cohorts pre-treatment and on-treatment (KM Plot database), and GSE218989 cohort. K Correlation between HMGCS1 and cholesterol biosynthesis pathway score in GSE41271, GSE42127, and GSE37745. L Kaplan–Meier analysis for PFS based on HMGCS1 intensity, comparison of CD8⁺ T cells intensity in HMGCS1_high and HMGCS1_low groups, and representative mIF staining of DAPI, CD8⁺ T cells, HMGCS1, and pan Cytokeratin in patient #1 (PFS = 40 days) and patient #2 (PFS = 748 days) in the mIF cohort (n = 21, 5X). (Abbreviation: PFS: progression free survival; NSCLC: non-small cell lung cancer; NDB: non-durable clinical benefit; DCB: durable clinical benefit; MPR: major pathological response; NMPR: non-major pathological response; OS: overall survival. Mann–Whitney test was performed between groups; LUSC: lung squamous cell carcinoma; LUAD: lung adenocarcinoma; mIF: multiple immunofluorescence. Mann–Whitney test was performed between groups.)

Fig. 3

Distribution of HMGCS1 in NSCLC by single-cell RNA sequencing and spatial transcriptome. A UMAP of cell distribution and HMGCS1 expression in a LUSC sample (LUNG_FFPE_CytAssist_10x). B, C Spatial plots of HMGCS1 expression, tumor cells, CD8⁺ T cells, hallmark cholesterol homeostasis score, and valine leucine and isoleucine degradation score in LUSC (LUNG_FFPE_CytAssist_10x) and LUAD (GSM5420751) samples. D UMAP and Vlnplot plots displaying HMGCS1 expression in NSCLC samples (SCAR database). E Comparison of HMGCS1 expression among AIS, MIA, and IAC samples in GSE189487. F Comparison of HMGCS1 protein expression in grade 2 and grade 3 LUAD in CPTAC database. G Comparison of CNV score in HMGCS1.⁺ malignant cells and HMGCS1- malignant cells in GSE131907. H GSEA of hallmark cholesterol homeostasis and valine leucine and isoleucine degradation pathways between HMGCS1 positive and HMGCS1 negative tumor cells in GSE131907. (Abbreviation: NSCLC: non-small cell lung cancer; UMAP: uniform manifold approximation and projection; LUSC: lung squamous cell carcinoma; LUAD: lung adenocarcinoma; AIS: adenocarcinoma in situ; MIA: minimally invasive adenocarcinoma; IAC: invasive adenocarcinoma. **** p < 0.001.)

Fig. 4

RNA-seq and animal experiments validation of HMGCS1 knockdown and overexpression in LLC1 cell line. A RNA-seq and KEGG functional enrichment of HMGCS1 KD cells, WT_KD cells, HMGCS1 OE cells, and WT_OE cells. B qPCR analysis of HMGCS1 KD cells, WT_KD cells, HMGCS1 OE cells, and WT_OE cells. C, D Tumor volume and body weight measurements of four animal groups treated with anti-PD-1 therapy. (Abbreviation: NSCLC: non-small cell lung cancer; KD: knock down, OE: over express.* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.)

Fig. 5

Survival and distribution of HMGCS1 in DLBCL by spatial transcriptomics (n = 10) and immunohistochemistry (20X) (n = 39). A Kaplan–Meier curves of OS in HMGCS1_high and HMGCS1_low groups in GSE31212. B, C Spatial plot and vlnplot illustrating the cell distribution of HMGCS1 in non_relapsed and relapsed groups. D Correlation between HMGCS1 expression and hallmark cholesterol homeostasis score, and valine leucine and isoleucine degradation score. E Representative IHC staining images showing HMGCS1 expression levels from patient 1 (PFS = 90 months) and patient 2 (PFS = 10.4 months) are depicted, along with kaplan–meier survival curves for PFS grouped by HMGCS1 expression levels. (Abbreviation: DLBCL: diffuse large B-cell lymphoma; IHC: immunohistochemistry; PFS: progression-free survival.)