Targeting ABCD1 inhibits peroxisomal fatty acid oxidation to selectively eliminate Acute Myeloid Leukemia cells Free

Acute myeloid leukemia (AML) is a devastating hematological malignancy driven by a strong reliance on fatty acid oxidation (FAO) for disease maintenance and chemotherapy resistance. FAO occurs in both mitochondria and peroxisomes, with peroxisomes specializing in the metabolism of very-long-chain fatty acids (VLCFAs, ≥20 carbons). VLCFAs are imported into peroxisomes via ATP-binding cassette subfamily D transporters, primarily ABCD1. Inside the peroxisomal matrix, VLCFAs are shortened into long- and medium-chain acyl-CoAs, generating acetyl-CoA that supports mitochondrial FAO (mFAO) and lipid biosynthesis. Although mFAO inhibition is well-studied as an anti-cancer strategy, pharmacological targeting of peroxisomal FAO (pFAO) remains unexplored, largely due to the scarcity of specific inhibitors (i.e., no existing agent target ABCD1). Importantly, direct pFAO inhibition in AML has never been investigated.

To examine the role of peroxisomes in AML, transcriptomic analyses of publicly available databases (GSE42519, GSE13159) showed significant dysregulation of peroxisomal genes in AML blasts versus healthy hematopoietic stem cells. Notably, ABCD1 was consistently upregulated, and this pattern was validated at the mRNA and protein levels in patient-derived AML (n=4) and normal cells (n=3). Functional assays confirmed that endogenous pFAO activity was significantly higher in AML-derived peroxisomes compared to normal peripheral blood mononuclear cells (n=10, p<0.05), demonstrating increased peroxisomal activity in leukemia over normal cells.

As a genetic approach to assess the functional role of ABCD1 on AML growth in vitro and in vivo, lentivirus-mediated shRNA generated three different AML cell lines lacking ABCD1. ABCD1 knockdown suppressed pFAO, reduced cell proliferation, and decreased clonogenic growth in vitro. Moreover, immunocompromised mice injected with ABCD1 knockdown cells lived significantly longer compared to those injected with isotype control cells (31 vs. 25 days, p<0.05). To recapitulate these effects biochemically, a novel ABCD1 inhibitor was discovered. A systematic screening approach identified eicosenol, a 20-carbon fatty alcohol, as a pFAO inhibitor that selectively reduced AML cell viability in vitro and in vivo. Eicosenol specifically accumulated in peroxisomes and bound selectively to ABCD1 leading to reduced ABCD1 protein expression, decreased pFAO and death of patient-derived AML but not normal cells; ABCD1 knockdown cells were resistant to eicosenol. In mouse leukemia models, eicosenol (300mg/kg/week) extended survival (Log-rank Mantel-Cox test, p<0.05) and decreased engraftment of patient-derived AML cells (300-400mg/kg/week, two different patient IDs, p<0.05) with no effect on engraftment of normal hematopoietic cells. To confirm in vivo targeting, cellular thermal shift assays demonstrated a shift in ABCD1 protein melting temperature only in mice treated with eicosenol.

To further determine the mechanisms of selective toxicity, lipidomic analysis following genetic and pharmacologic inhibition of ABCD1 was performed. ABCD1 inhibition caused accumulation of toxic VLCFAs alongside depletion of essential long-chain fatty acids such as palmitate, disrupting lipid homeostasis and inducing lipotoxicity. Remarkably, supplementation with palmitate rescued AML cell viability by replenishing the pool of critical lipids depleted following ABCD1 inhibition. By contrast, normal hematopoietic cells maintained their viability upon pFAO inhibition by compensating through upregulated glycolysis, as confirmed by increased glucose uptake measurements.

In summary, ABCD1 and pFAO are critical metabolic vulnerabilities in AML. Genetic and pharmacological inhibition of ABCD1 inhibits pFAO, disrupts VLCFA metabolism, induces toxic lipid accumulation, and selectively impairs AML, but not normal, cell survival. These findings establish, for the first time, ABCD1 as a peroxisome-specific metabolic vulnerability in AML and pFAO inhibition as a viable anti-AML strategy.