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. 2022 Dec;9(34):e2202104.
doi: 10.1002/advs.202202104. Epub 2022 Oct 30.

Genome-Wide CRISPR/Cas9 Library Screening Revealed Dietary Restriction of Glutamine in Combination with Inhibition of Pyruvate Metabolism as Effective Liver Cancer Treatment

Chunxue Yang  1   2   3 , Derek Lee  1   2   4 , Misty Shuo Zhang  1   2   4 , Aki Pui-Wah Tse  1   2   4 , Lai Wei  1   2 , Macus Hao-Ran Bao  1   2   4 , Bowie Po-Yee Wong  1   2   4 , Cerise Yuen-Ki Chan  1   2   4 , Vincent Wai-Hin Yuen  1   2   4 , Yiling Chen  1   2   4 , Carmen Chak-Lui Wong  1   2   4   5
Affiliations

Genome-Wide CRISPR/Cas9 Library Screening Revealed Dietary Restriction of Glutamine in Combination with Inhibition of Pyruvate Metabolism as Effective Liver Cancer Treatment

Chunxue Yang et al. Adv Sci (Weinh). 2022 Dec.

Abstract

Hepatocellular carcinoma (HCC) is the second most lethal cancer worldwide. Glutamine is an essential, extracellular nutrient which supports HCC growth. Dietary glutamine deficiency may be a potential therapeutic approach for HCC. HCC cells overcome metabolic challenges by rewiring their metabolic pathways for rapid adaptations. The efficiency of dietary glutamine deficiency as HCC treatment is examined and the adaptation machinery under glutamine depletion in HCC cells is unraveled. Using genome-wide CRISPR/Cas9 knockout library screening, this study identifies that pyruvate dehydrogenase α (PDHA), pyruvate dehydrogenase β (PDHB), and pyruvate carboxylase (PC) in pyruvate metabolism are crucial to the adaptation of glutamine depletion in HCC cells. Knockout of either PDHA, PDHB or PC induced metabolic reprogramming of the tricarboxylic acid (TCA) cycle, disrupts mitochondrial function, leading to the suppression of HCC cell proliferation under glutamine depletion. Surprisingly, dietary glutamine restriction improves therapeutic responses of HCC to PDH or PC inhibitor in mouse HCC models. Stable isotope carbon tracing confirms that PDH or PC inhibitors further disrupt the metabolic rewiring of the TCA cycle induced by dietary glutamine depletion in HCC. In summary, the results demonstrate that pyruvate metabolism acts as novel targetable metabolic vulnerabilities for HCC treatment in combination with a glutamine-deficient diet.

Keywords: CRISPR/Cas9 library screening; dietary intervention; glutamine depletion; hepatocellular carcinoma; pyruvate metabolism.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
CRISPR/Cas9 library screening identified PDH and PC as critical drivers for glutamine depletion. A) Survival curve of Tp53KOc‐MycOE HCC mice induced by hydrodynamic tail vein injection (HDTVi) of genome editing plasmids and fed with different diets (n = 10). B) Schematic diagram illustrates the workflow of genome‐wide CRISPR/Cas9 knockout library screening. C) Pyruvate dehydrogenase α (PDHA), pyruvate carboxylase (PC), and pyruvate dehydrogenase β (PDHB) were identified as the most significant genes in the library screen. D) The sgRNAs targeting PDHA, PC, and PDHB were negatively selected during glutamine depletion. E–G) Cell proliferation assays demonstrated that knockout (KO) of E) PDHA, F) PDHB, and G) PC significantly suppressed HCC proliferation under glutamine depletion (GLN−). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 versus EV as indicated. Student's t‐test. Error bars indicate mean ± SEM (n = 3).
Figure 2
Figure 2
Metabolic characteristics of HCC cells in response to glutamine depletion. A) TCA cycle flux under glutamine supplementation or glutamine depletion. B–D) Stable‐isotope tracing with [U‐13C3] pyruvate revealed that B) PDHA, C) PDHB, or D) PC KO slowed TCA flux by decreasing the production of α‐ketoglutarate (α‐KG) and succinate. E) Cell proliferation assays demonstrated that the addition of dimethyl ketoglutarate (DMKG) rescued the proliferation of PDHA, PDHB, and PC KO cells under glutamine depletion (GLN−). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 versus EV as indicated. Student's t‐test. Error bars indicate mean ± SEM (n = 3).
Figure 3
Figure 3
Mitochondrial function affected by knockout of PDH and PC in HCC cells under glutamine depletion. A,C,E) Seahorse XF Cell Mito Stress assay demonstrated that KO of A) PDHA, C) PDHB, and E) PC significantly suppressed oxygen consumption rate (OCR) of MHCC97L cells under glutamine depletion (GLN−) compared to normal conditions with glutamine (GLN+). B,D,F) JC‐1 staining in B) PDHA, D) PDHB, and F) PC KO cells revealed significantly impaired mitochondrial membrane potential of MHCC97L cells under glutamine depletion (GLN−) compared to normal conditions with glutamine (GLN+). GLSi (BPTES): 1 µm. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 versus EV as indicated. Student's t‐test. Error bars indicate mean ± SEM (n = 3).
Figure 4
Figure 4
The effects of PDH, PC, and GLS inhibitors on HCC cells. A,B) Higher GI50 for A) PDH and B) PC inhibitors were detected on MIHA compared to MHCC97L cells using XTT assay. C,D) Cell proliferation assay demonstrated C) PDH and D) PC inhibitor significantly suppressed MHCC97L cell proliferation in dose‐dependent manner under glutamine depletion (GLN−) or in combination with GLS inhibitor (GLSi) treatment. E) Proliferation of MHCC97L‐PDHA, ‐PDHB, and ‐PC KO cells in the presence of GLS inhibitor (GLSi) were significantly suppressed. GLSi (BPTES): 1 µm, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 versus Ctrl or EV as indicated. Student's t‐test. Error bars indicate mean ± SEM (n = 3).
Figure 5
Figure 5
Metabolic flux of HCC cells in response to glutaminase (GLS) inhibition (BPTES) tracing with [U‐13C3] pyruvate. A) TCA cycle flux under glutamine supplementation with or without GLS inhibitor (BPTES). BPTES: 1 µm. B–D) Stable isotopic carbon tracing using [U‐13C3] pyruvate revealed that B) PDHA, C) PDHB, or D) PC KO slowed down TCA flux by decreasing the production of α‐ketoglutarate (α‐KG) and succinate. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 versus EV as indicated. Student's t‐test. Error bars indicate mean ± SEM (n = 3).
Figure 6
Figure 6
PDH inhibitor (PDHi) or PC inhibitor (PCi) sensitized HCC to glutamine deficient diet. A) Tumor images and tumor volume growth curves after treated with PDH inhibitor (PDHi; CPI‐613, 4 mg kg−1) and normal, glutamine supplemented (GLN+) or glutamine deficient (GLN−) diets. Ctrl/GLN+: control with normal diet; PDHi/GLN+: CPI‐613 treatment with normal diet; Ctrl/GLN−: control with glutamine deficient diet; PDHi/GLN−: CPI‐613 treatment with glutamine deficient diet; tumor volumes and tumor weights of mice in treated groups. (n = 10). B) Tumor images and tumor volume growth curves after treated with PC inhibitor (PCi; CHCA, 40 mg kg−1) and normal, glutamine supplemented (GLN+) or glutamine deficient (GLN−) diets. Ctrl/GLN+: control with normal diet; PCi/GLN+: CHCA treatment with normal diet; Ctrl/GLN−: control with glutamine deficient diet; PCi/GLN−: CHCA treatment with glutamine deficient diet; tumor volumes and tumor weights of mice in treated groups (n = 8). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 versus Ctrl or as indicated. Student's t‐test. Error bars indicate mean ± SEM.
Figure 7
Figure 7
Efficacy of glutamine deficient diet combined with PDH or PC inhibitor in orthotopic HCC model. A) Bioluminescent image of orthotopic tumors implanted in mice treated with Ctrl or PDH inhibitor (PDHi, CPI‐613, 4 mg kg−1) and glutamine supplemented or deficient (GLN+/−) diets. B) Representative image of the orthotopic tumors and tumor volumes. C) Labelled metabolites detected by in vivo carbon tracing with U‐13C6‐glucose. D) Bioluminescent image of orthotopic tumors implanted in mice treated with Ctrl or PC inhibitor (PCi; CHCA, 40 mg kg−1) and glutamine supplemented or deficient (GLN+/−) diets. E) Representative image of the orthotopic tumors and tumor volumes. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 versus Ctrl or as indicated. Student's t‐test. Error bars indicate mean ± SEM.
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