Chemotherapy-resistant human acute myeloid leukemia (AML) cells are thought to be enriched in quiescent immature leukemic stem cells (LSCs). To validate this hypothesis in vivo, we developed a clinically relevant chemotherapeutic approach treating patient-derived xenograft (PDX) with cytarabine. Cytarabine residual AML cells are enriched neither in immature, quiescent cells nor LSCs. Strikingly, cytarabine-resistant pre-existing and persisting cells displayed high levels of reactive oxygen species, showed increased mitochondrial mass, and retained active polarized mitochondria, consistent with a high oxidative phosphorylation (OXPHOS) status. Cytarabine residual cells exhibited increased fatty acid oxidation, upregulated CD36 expression and a HIGH OXPHOS gene signature predictive for treatment response in PDX and AML patients. HIGH OXPHOS but not LOW OXPHOS human AML cell lines were chemoresistant in vivo. Targeting mitochondrial protein synthesis, electron transfer, or fatty acid oxidation induced an energetic shift towards LOW OXPHOS and markedly enhanced anti-leukemic effects of cytarabine. Together, this study demonstrates that essential mitochondrial functions contribute to cytarabine resistance in AML and are a robust hallmark of cytarabine sensitivity and a promising therapeutic avenue to treat AML residual disease.

Autophagy has been associated with oncogenesis with one of its emerging key functions being its contribution to the metabolism of tumors. Therefore, deciphering the mechanisms of how autophagy supports tumor cell metabolism is essential. Here, we demonstrate that the inhibition of autophagy induces an accumulation of lipid droplets (LD) due to a decrease in fatty acid β-oxidation, that leads to a reduction of oxidative phosphorylation (OxPHOS) in acute myeloid leukemia (AML), but not in normal cells. Thus, the autophagic process participates in lipid catabolism that supports OxPHOS in AML cells. Interestingly, the inhibition of OxPHOS leads to LD accumulation with the concomitant inhibition of autophagy. Mechanistically, we show that the disruption of mitochondria–endoplasmic reticulum (ER) contact sites (MERCs) phenocopies OxPHOS inhibition. Altogether, our data establish that mitochondria, through the regulation of MERCs, controls autophagy that, in turn finely tunes lipid degradation to fuel OxPHOS supporting proliferation and growth in leukemia.