2035 – LIPOYLATION PATHWAY DEPENDENCY IN LEUKEMIC STEM CELLS CONFERS VULNERABILITY TO CUPROPTOSIS

Abstract

Acute myeloid leukemia (AML) remains a clinically challenging disease due to therapy resistance and relapse driven by leukemic stem cells (LSCs). Identifying LSC-specific vulnerabilities is essential to improve therapeutic outcomes.

We performed a high-throughput screen of 3,247 anticancer compounds using a patient-derived xenograft (PDX) sample enriched for CD34⁺CD38^– cells. We identified two copper ionophores, disulfiram and 8-hydroxyquinoline (8-HQ), as top hits, suggesting marked sensitivity of LSCs to copper overload. Validation in primary AML samples (n = 8) confirmed that disulfiram, 8-HQ, and elesclomol preferentially induced cell death in the CD34⁺CD38^– compartment.

Single-cell RNA sequencing (scRNA-seq) of CD34⁺CD38^– cells revealed that LSC-enriched clusters exhibited elevated cuproptosis-related gene signatures. Consistently, treatment with copper ionophores induced aggregation of dihydrolipoamide S-acetyltransferase (DLAT), hallmarks of cuproptosis.

We found that LSCs displayed lower intracellular copper levels compared with progenitor cells. Transcriptomic analyses revealed increased expression of copper efflux transporters ATP7A and ATP7B in LSCs. Their silencing impaired clonogenicity and induced mitochondrial dysfunction and cell death. It also reduced leukemic burden in PDX models (n = 3) in both primary and secondary transplants, highlighting a critical role for copper homeostasis in LSC self-renewal.

Finally, scRNA-seq and western blot analyses demonstrated elevated levels of lipoylated DLAT in the LSC compartment. Inhibition of DLAT or the lipoylation enzyme LIAS reduced clonogenic potential and promoted myeloid differentiation in primary AML samples.

These findings identify LSC dependence on the lipoylation pathway, creating vulnerability to cuproptosis. Targeting copper homeostasis and lipoylation represents a promising therapeutic strategy in AML.

Acute myeloid leukemia (AML) remains a clinically challenging disease due to therapy resistance and relapse driven by leukemic stem cells (LSCs). Identifying LSC-specific vulnerabilities is essential to improve therapeutic outcomes.

We performed a high-throughput screen of 3,247 anticancer compounds using a patient-derived xenograft (PDX) sample enriched for CD34⁺CD38^– cells. We identified two copper ionophores, disulfiram and 8-hydroxyquinoline (8-HQ), as top hits, suggesting marked sensitivity of LSCs to copper overload. Validation in primary AML samples (n = 8) confirmed that disulfiram, 8-HQ, and elesclomol preferentially induced cell death in the CD34⁺CD38^– compartment.

Single-cell RNA sequencing (scRNA-seq) of CD34⁺CD38^– cells revealed that LSC-enriched clusters exhibited elevated cuproptosis-related gene signatures. Consistently, treatment with copper ionophores induced aggregation of dihydrolipoamide S-acetyltransferase (DLAT), hallmarks of cuproptosis.

We found that LSCs displayed lower intracellular copper levels compared with progenitor cells. Transcriptomic analyses revealed increased expression of copper efflux transporters ATP7A and ATP7B in LSCs. Their silencing impaired clonogenicity and induced mitochondrial dysfunction and cell death. It also reduced leukemic burden in PDX models (n = 3) in both primary and secondary transplants, highlighting a critical role for copper homeostasis in LSC self-renewal.

Finally, scRNA-seq and western blot analyses demonstrated elevated levels of lipoylated DLAT in the LSC compartment. Inhibition of DLAT or the lipoylation enzyme LIAS reduced clonogenic potential and promoted myeloid differentiation in primary AML samples.

These findings identify LSC dependence on the lipoylation pathway, creating vulnerability to cuproptosis. Targeting copper homeostasis and lipoylation represents a promising therapeutic strategy in AML.