In Vivo Response to Cytarabine Chemotherapy Uncovers the Role of the Oxidative and Energetic Metabolism in the Chemoresistance of Human Primary AML Stem Cells

Resistant cells to conventional chemotherapy - which targets DNA synthesis - are thought to be rare and enriched in quiescent leukemic stem cells (LSCs) in acute myeloid leukemia (AML). In order to develop an improved understanding of chemotherapy resistance in AML, we analyzed the response to cytarabine (Ara-C) in our xenograft model with 20 primary AML patient specimens from two clinical sites. After demonstrating AML engraftment, mice are treated with Ara-C given IP daily for 5 days as a single agent at 60 mg/kg daily, which correlates with human dosing. In all mice treated with this regimen, there was a 4- to 46-fold cytoreductive effect at 1 week post-treatment. While in vivo Ara-C treatment induces changes in CD34^pos CD38^pos/neg phenotypes of 50% of AML-engrafted mice, this treatment does not induce any significant changes in cell cycle distribution and does not enrich in G₀ quiescent cells in AML-engrafted mice. Surprisingly we observed that Ara-C induces a decrease or no change in the frequency of SL-ICs (control, 1:2,500-21,900 versus Ara-C, 1:4,500-34,200), suggesting that Ara-C would not spare LSCs in vivo and equally kills both cycling and quiescent cells. Furthermore, transcriptomic analysis of Ara-C residual leukemic cells (RLCs) from three different patient-derived xenografts confirmed that RLCs are not enriched in stem cell marker genes at 1 week post-treatment. However, our gene signature of the in vivo AML resistance is enriched in genes involved in inflammatory, immune and stress response. When tested in three independent AML cohorts (Verhaak et al. 2009; TGCA, 2011; Metzeler et al. 2011), this signature is associated with an unfavorable prognosis in patients treated with intensive chemotherapy. Finally we have shown that RLCs are PIMO^pos cells with both high ROS content and active mitochondrial mass and membrane potential in AraC-treated xenografted mice compared to control mice. Accordingly, AML cells exhibiting high OXPHOS energetic phenotype are resistant to Ara-C chemotherapy in vivo while low OXPHOS AML cells are sensitive to AraC in NSG mice. Altogether these results suggest a novel model of cytarabine chemotherapy resistance in AML based on the oxidative and mitochondrial energy metabolism in vivo.