Mitochondrial ACSS1 Regulates Oncometabolite 2-Hydroxyglutarate and De Novo Pyrimidine Biosynthesis in Lymphoma Cells: Functional Insights and Translational Implications

Introduction:

The mitochondrial acetyl-CoA synthetase short-chain family member 1 (ACSS1) converts acetate to mitochondrial acetyl-CoA, an energy source in nutrient-deprived conditions. While it is known that the expression of ACSS1 is regulated in certain non-lymphoid cells in an estrogen receptor-dependent manner, and that estrogen plays a role in regulating the incorporation of ¹⁴C-acetate into DNA, the fundamental mechanisms involved in ACSS1 expression and function remain largely undetermined in both normal and malignant cells.

Methods:

To assess the levels of ACSS1 expression in a diverse group of cancers, we used the publicly available database, The Cancer Genome Atlas (TCGA). We then analyzed the expression of ACSS1 in mantle cell lymphoma (MCL) and diffuse large B-cell lymphoma (DLBCL) cell lines, which are sensitive and resistant to the Bruton Tyrosine kinase (BTK) inhibitor ibrutinib (IBR), by quantitative (q)PCR and immunoblotting. We then conducted immunohistochemistry (IHC) analysis with the anti-ACSS1 antibody on specimens from MCL, DLBCL, and chronic lymphocytic leukemia (CLL) using tissue microarrays (TMA). For a comprehensive study of the role of ACSS1 in acetate metabolism in the low-expressing MCL-RL cell line compared to the ACSS1 overexpressing JeKo-1 and Maver cell lines, we utilized ¹³C-isotope tracing with three carbon sources: ¹³C-glucose, ¹³C-glutamine, and ¹³C-acetate using mass-spectrometry-based LC-MS/MS metabolomics analysis. Next, we investigated whether ACSS1 is responsible for ¹³C-acetate-derived ¹³C-carbon incorporation in TCA cycle metabolites by generating shRNA-mediated ACSS1 depletion in JeKo-1 and Maver cells, followed by ¹³C-acetate tracing analysis. Additionally, we measured the oxygen consumption rate (OCR) (Seahorse XF) under different conditions in these cells. We also evaluated the impact of ACSS1 impairment on acetate metabolism in nutrient-deprived conditions on the pyrimidine synthesis pathway using the appropriate antibodies in immunoblotting. Finally, we assessed the effect of the ACSS1 depletion cell growth and death by cell counting and annexin V staining.

Results:

The TCGA database analysis revealed ACSS1 overexpression in many hematological malignancies and solid tumors compared to normal tissues. Next, our qPCR and immunoblotting studies showed ACSS1 overexpression in MCL and DLBCL IBR-resistant cell lines compared to IBR-sensitive cell lines. Immunohistochemistry (IHC) analysis of biopsy samples of MCL (N=27), DLBCL (N=44), and CLL(N=13) revealed intense positive staining for ACSS1 in 92.59%, 79.54%, and 53.84% of cases, respectively compared to normal lymphoid tissue. Using ¹³C-isotope tracing with three different carbon sources in three MCL cell lines, MCL-RL (sensitive to IBR), JeKo-1, and Maver (both resistant to IBR), the study revealed a significant correlation between ACSS1 expression and the incorporation of ¹³C-acetate-derived TCA cycle metabolites. In the genetic approach of shRNA-mediated ACSS1 depletion, JeKo-1 and Maver cells showed that the ACSS1 loss reduced the incorporation of ¹³C-acetate into various metabolites such as acetyl-CoA, acetylcarnitine, TCA cycle intermediates, glutamine, aspartate, dihydroorotate and orotate involved in pyrimidine synthesis. Furthermore, ¹³C-acetate tracing revealed ACSS1's role in converting acetate to the oncometabolite D-2-Hydroxyglutarate, which is involved in epigenetic gene regulation in many cancer types. The study also shows reduced oxygen consumption in the ACSS1-depleted cells during a mitochondrial stress test. Next, we showed ACSS1's interaction with mitochondrial Dihydroorotate dehydrogenase (DHODH), a key enzyme in the de novo pyrimidine biosynthesis pathway, fosters the conversion of dihydroorotate (substrate) to orotate (product). Notably, depletion of ACSS1 and inhibition of DHODH reduce in MCL cells expression of the key oncogene Cyclin D1 and inhibit cell growth.

Conclusions:

This comprehensive metabolomics study reveals the role played by acetate/ACSS1 in lymphoma and cancer in general. Our results show that ACSS1 profoundly affects the metabolism of MCL cells. The study also suggests that this metabolic pathway may become a novel therapeutic target in MCL and, possibly, other types of lymphoid and non-lymphoid malignancies.