Targeting ACSS2 with a Transition-State Mimetic Inhibits Triple-Negative Breast Cancer Growth

Abstract

Acetyl-CoA is a vitally important and versatile metabolite used for many cellular processes including fatty acid synthesis, ATP production, and protein acetylation. Recent studies have shown that cancer cells upregulate acetyl-CoA synthetase 2 (ACSS2), an enzyme that converts acetate to acetyl-CoA, in response to stresses such as low nutrient availability and hypoxia. Stressed cancer cells use ACSS2 as a means to exploit acetate as an alternative nutrient source. Genetic depletion of ACSS2 in tumors inhibits the growth of a wide variety of cancers. However, there are no studies on the use of an ACSS2 inhibitor to block tumor growth. In this study, we synthesized a small-molecule inhibitor that acts as a transition-state mimetic to block ACSS2 activity in vitro and in vivo. Pharmacologic inhibition of ACSS2 as a single agent impaired breast tumor growth. Collectively, our findings suggest that targeting ACSS2 may be an effective therapeutic approach for the treatment of patients with breast cancer. SIGNIFICANCE: These findings suggest that targeting acetate metabolism through ACSS2 inhibitors has the potential to safely and effectively treat a wide range of patients with cancer.

Figure 1.. VY-3-135 is a potent, stable ACSS2 inhibitor with good bioavailability.

(A) Schematic of the forward reaction catalyzed by ACSS2. (B-C) Chemical structures of VY-3-249 and VY-3-135 and IC50 determinations for inhibitors against ACSS1 (blue squares) and ACSS2 (red circles). Data points represent mean (filled shapes) and replicates (empty shapes). (D-E) Mouse and human microsomal stability assay for VY-3-135 and VY-3-249. n = 1. (F) Pharmacokinetic analysis of VY-3-135 by oral gavage, intraperitoneal, and intravenous injection. Data represent mean ± S.D., n = 3 mice/group/time point. Table describes the calculations of pharmacokinetic analysis.

Figure 2.. Model of Salmonella enterica acetyl-CoA synthetase in complex with VY-3-135.

(A) Amino acid sequence alignment of yeast, salmonella, human and mouse ACSS2. Key residues that create the nucleotide binding pocket of ACSS2 are highlighted in yellow. (B) Docking of VY-3-135 into acetyl-CoA synthetase shows that the inhibitor has a preference for the acetyl-AMP binding site of the protein but also occupies a portion of the CoA site. (C) Superposition of acetyl-CoA synthetase in complex with VY-3-135 with the published structure of acetyl-CoA synthetase in complex with adenosine-5’-propylphosphate and CoA. The overlay shows how VY-3-135 interferes with both acetyl-AMP and CoA binding. (D) The surface (acetyl-CoA synthetase) and stick (VY-3-135) representation shows that the inhibitor fits into the site with no clashes with the protein.

Figure 3.. VY-3-135 is a potent inhibitor of ACSS2 in cells.

(A) Immunoblot for ACSS2, EGFR, HER2 expression in a panel of human breast cancer cell lines. GAPDH is the loading control for ACSS2 and EGFR. ACTB is the loading control for HER2. (B) Enrichment of 100 μM ¹³C₂-acetate in the intracellular palmitate pool in SKBr3 cells treated with vehicle or VY-3-135 and cultured in normoxia and SMEM+10% serum (N10) or hypoxia and SMEM+1% serum (H1) over a 24 hr period. Data represent mean ± S.D., n = 3. (C) Enrichment of 100 μM ¹³C₂-acetate into palmitate in BT474 cells cultured in H1 conditions over a 24 hr period using a 10-fold dilution series of VY-3-135. Data represent mean ± S.D., n = 3. (D) Growth of BT474 and SKBr3 cells in 10 μM VY-3-135 for 72 hours in H1 culture conditions supplemented with 200 μM sodium acetate. Data are mean ± S.D. n = 2 performed in triplicate. p values are student t tests. (E) Enrichment of ¹³C₂-acetate into the intracellular citrate pool. Experimental parameters were identical to panel C. For all metabolomic data adjusted p values are reported on the graphs. p values were generated by 2-way ANOVA with Tukey’s multiple comparisons testing of mole percent enrichment of carbon-13 was used.

Figure 4.. Knockout or VY-3-135 inhibition of ACSS2 inhibits tumor growth.

(A) Enrichment of 100 μM ¹³C₂-acetate into palmitate in A7C11 and Brpkp110 cells cultured in N10 and H1 conditions over a 24 hr period in the presence and absence of VY-3-135. Data represent mean ± S.D., n = 3. Adjusted p values are reported on the graph. 2-way ANOVA Tukey’s multiple comparisons testing of mole percent enrichment of carbon-13. (B) Immunoblot for ACSS2 in A7C11 and Brpkp110 pools after CRISPR-Cas9 targeting of Acss2. Lysates were prepared from cells grown in N10 or H1 over a 24 hr period. sgNTC = single guide RNA against non-targeting control. sgACSS2 = single guide RNA against Acss2. (C) CRISPR-Cas9 knockout of Acss2 in A7C11 cells has a modest effect on tumor growth. Data represent mean ± standard error of the means (S.E.M.) with ANOVA p value displayed, n = 5. (D) CRISPR-Cas9 knockout of Acss2 in Brpkp110 cells causes a significant decrease in tumor growth. Data represent mean ± S.E.M. with ANOVA p value displayed, n = 5. (E) VY-3-135 treatment (100 mpk daily IP) causes a significant decrease in Brpkp110 tumor growth. Data represent mean ± S.E.M. with ANOVA p value displayed, n = 5.

Figure 5.. VY-3-135 inhibits growth of ACSS2^high but not ACSS2^low human breast tumors.

(A) Immunoblots for ACSS2, ACSS1, and HER2 expression. (B) Immunoblot for EGFR expression. (C) WHIM12 tumor growth ± VY-3-135. Data represent mean ± S.E.M. with ANOVA p value displayed, n = 6 per group. Black arrow indicates start of treatment (100 mpk daily PO). (D) MDA-MB-468 tumor growth ± VY-3-135. Data represent mean ± S.E.M. with ANOVA p value displayed, n = 8 per group. Black arrow indicates start of treatment (100 mpk daily PO). (E) BT474 tumor growth ± VY-3-135. Black arrow indicates start of treatment (100 mpk daily IP). ANOVA p value is displayed, n = 5 mice per group. (F-G) D3-acetate dependent labeling of palmitate in WHIM12 tumors (panel F) and MDA-MB-468 tumors (panel G). N.D. = not detected. n ≥ 5 (H-I) D3-acetate dependent labeling of UDP-GlcNAc in WHIM12 tumors (panel H) and MDA-MB-468 tumors (panel I). (J-K) D3-acetate dependent labeling of citrate in WHIM12 tumors (panel H) and MDA-MB-468 tumors (panel I). Data represent mean ± S.D., n ≥ 5. For all metabolomic data adjusted p values are reported on the graphs. p values were generated from 2-way ANOVA Tukey’s multiple comparisons testing of mole percent enrichment of carbon-13.