沉默ACACA使急性髓系白血病细胞对venetoclax敏感

Introduction: Acute myeloid leukemia (AML) is an aggressive blood cancer marked by malignant myeloid stem/progenitor cell proliferation in the bone marrow, with a long-term survival rate below 30%. The FDA-approved BCL-2 inhibitor venetoclax (VEN), combined with Azacitidine, is a standard-of-care therapy in older, unfit for chemotherapy AML patients, but this treatment achieves only about 50% clearance of minimal residual disease. AML cells rely on oxidative phosphorylation and fatty acid oxidation, along with other amino acids metabolism for survival. Acetyl-CoA-carboxylases are known to regulate fatty acid metabolism. While ACACA is involved in fatty acid synthesis, ACACB governs fatty acid oxidation. Thus, we hypothesize that fatty acid oxidation (FAO) inhibition by ACACA knockdown might sensitize AML cells to BCL-2 inhibitors (Venetoclax). Methods: Knockout of ACACA was performed using CRISPR Cas9 system in AML cell lines and patient samples. Cell viability was assessed by CTG assay and colony assay was performed using methylcellulose-enriched media. Medium and long-chain fatty acid profiling was conducted via a gas chromatography mass spectrometry approach. Seahorse assay was performed to measure fatty acid oxidation. Results: Genome-wide CRISPR screen in human AML cells treated with BH3 mimetics combinations (Glytsou et al., Cancer Discovery, 2023), demonstrated enrichment of fatty acid metabolism. We found that genes involved in both FAO, such as CPT1b, and fatty acid synthesis, such as Acetyl-CoA Carboxylase Alpha (ACACA), are top significant synthetic lethal targets with VEN/AZA. We first tested the efficacy of ND-630, known to inhibit ACC carboxylases (ACACA and ACACB) in combination with VEN. The combination was found to be effective in VEN-resistant THP-1, OCI-3 and SKM-1 cell lines in 1% FBS media. In THP-1 cell line, ND-630 reduces colony formation by 35±8%, VEN by 29±6% and the combination yielded 62±3% reduction. VEN-resistant patient samples (n=2) responded well to the combination as compared to VEN-sensitive primary AML samples (n=1). Ablating ACACA through CRISPR Cas9 system led to a reduction in colony formation in THP-1 and SKM-1 cells (36±8% and 28±4%, respectively). There was 26±4% reduction in cell viability in ACACA KO VEN-resistant primary AML cells treated with lower doses of Ven (100nM) as compared to the control. Additional primary samples are being tested and will be reported. Knockdown of ACACA resulted in a reduction of fatty acid synthesis in primary AML sample. Seahorse fatty acid oxidation assay was performed in THP-1 cell line after ACACA knockdown. We found that maximal respiration in ACACA knockdown cells was reduced compared to control cells, suggesting limited fatty acid oxidation. Conclusion: These findings suggest that VEN-resistant AML rewire metabolism through ACACA-driven fatty acid synthesis and that inhibition of Acetyl-CoA-carboxylase restores VEN sensitivity in AML.