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.

Activation of the Wnt/b-catenin pathway has recently been shown to be crucial to the establishment of leukemic stem cells in chronic myeloid leukemia. We sought to determine whether b-catenin was correlated to clonogenic capacity also in the acute myeloid leukemia (AML) setting. Eighty-two patients were retrospectively evaluated for b-catenin expression by Western blot. b-Catenin was expressed (although at various protein levels) in 61% of patients, and was undetectable in the remaining cases. In our cohort, b-catenin expression was correlated with the clonogenic proliferation of AML-colony forming cells (AML-CFC or CFU-L) in methylcellulose in the presence of 5637-conditioned medium, and more strikingly with self-renewing of leukemic cells, as assessed in vitro by a replating assay. In survival analyses, b-catenin appeared as a new independent prognostic factor predicting poor event-free survival and shortened overall survival (both with Po0.05). Furthermore, variations in b-catenin protein levels were dependent on post-transcriptional mechanisms involving the Wnt/ b-catenin pathway only in leukemic cells. Indeed, b-catenin negative leukemic cells were found to increase b-catenin in response to Wnt3a agonist in contrast to normal counterparts. Altogether, our data pave the way to the evaluation of Wnt pathway inhibition as a new rationale for eradicating the clonogenic pool of AML cells.