Most AML patients who receive intensive chemotherapy achieve a significant clinical response; however, the majority will relapse and succumb to their disease, indicating that leukemia stem cells (LSCs) are not effectively targeted. Further, it has recently been shown that LSC frequency and phenotypic diversity are increased at relapse (Ho et al. Blood, 2016), thereby creating an even more challenging clinical scenario. Thus, novel therapies specifically designed to target LSCs in relapsed AML patients are urgently needed. Previously, we have shown that LSCs can be targeted by perturbing energy metabolism (Lagadinou et al. Cell Stem Cell, 2013). Therefore, the goal of the current study was to identify and target metabolic dependencies of relapsed LSCs, with the hope that this would allow improved efficacy for AML patients with relapsed disease.
To achieve this objective we first measured metabolic differences in LSCs isolated from de novo and relapsed patients. This analysis revealed that relapsed LSCs have significantly increased levels of nicotinamide compared to de novo LSCs (Figure 1A). Nicotinamide is a precursor of NAD+, an essential coenzyme in energy metabolism. We hypothesized that relapsed LSCs are dependent on nicotinamide metabolism to maintain energy metabolism. To test this hypothesis, we targeted nicotinamide metabolism with the small molecule APO866, an inhibitor of Nampt, the rate-limiting enzyme for conversion of nicotinamide to NAD+. This resulted in a significant decrease in NAD+ in LSCs isolated from both de novo and relapsed AML specimens (data not shown). However, strikingly, inhibition of nicotinamide metabolism only decreased viability and colony-forming ability of LSCs isolated from relapsed AML patients, not LSCs from untreated patients (Figure 1B). To verify that inhibition of Nampt was targeting functional LSCs, we treated a relapsed AML patient specimen with APO866 for 24 hours and measured the ability of the leukemia cells to engraft into immune deficient mice. We observed a significant reduction in leukemia engraftment upon APO866 treatment (data not shown). Importantly, inhibition of nicotinamide metabolism did not affect normal hematopoietic stem cell frequency or colony forming ability (data not shown). Altogether, these data suggest that inhibition of nicotinamide metabolism specifically targets relapsed LSCs.
We next sought to understand the mechanism by which inhibiting nicotinamide metabolism targets relapsed LSCs. To this end we measured changes in the major energy metabolism pathways (oxidative phosphorylation [OXPHOS] and glycolysis) in LSCs isolated de novo and relapsed AML patient specimens. Upon APO866 treatment, we observed a significant decrease in OXPHOS and OXPHOS capacity in relapsed LSCs but not de novo LSCs (Figure 1C). Furthermore, no change in glycolysis was observed (data not shown). These data demonstrate that inhibition of nicotinamide metabolism targets OXPHOS specifically in relapsed LSCs. To determine how APO866 reduced OXPHOS, we measured stable isotope metabolic flux of amino acids, the fatty acid palmitate, and glucose into the TCA cycle after APO866 treatment. We observed an increased accumulation of citrate, malate, and α-ketoglutarate from amino acids and palmitate, consistent with decreased activity of the NAD+ dependent enzymes isocitrate dehydrogenase, α-ketoglutarate dehydrogenase and malate dehydrogenase (data not shown). Through direct measurement of enzyme activity, we confirmed that isocitrate dehydrogenase, α-ketoglutarate dehydrogenase and malate dehydrogenase activity were each significantly decreased upon APO866 treatment (Figure 1D). Consistent with our previous findings we did not observe any changes in glycolysis or glucose contribution to the TCA cycle (data not shown). Overall, these data suggest that inhibition of nicotinamide metabolism through Nampt inhibition results in decreased OXPHOS through decreased TCA cycle activity.
In conclusion, we have shown that relapsed LSCs have distinct metabolic properties including increased levels of nicotinamide, which can be selectively targeted to eradicate relapsed LSCs. We propose that therapeutic strategies designed to target nicotinamide metabolism may be useful for relapsed AML patients and may allow for broad efficacy such as that observed when LSCs are targeted in the up-front treatment setting.
Nemkov:Omix Technologies inc: Equity Ownership. Pollyea:Curis: Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Consultancy, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Celyad: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Argenx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy; Karyopharm: Membership on an entity's Board of Directors or advisory committees.
Asterisk with author names denotes non-ASH members.