Current chemotherapeutic regimens for acute myeloid leukemia (AML) often fail to eliminate leukemic stem cells (LSCs) which contribute to disease relapse. A key step towards the development of more effective therapies is the identification of vulnerabilities that are unique to LSCs. Here, we sought to identify LSC-specific metabolic dependencies by performing a flow cytometry-based screen of 110 metabolically-focused drugs against a primary human AML sample. This sample harbored distinct subsets defined by CD34 and CD38 expression, and LSC activity assayed by xenotransplantation was restricted to the CD34+CD38- fraction. Through this screen, we found that inhibitors of nicotinamide phosphoribosyltransferase (NAMPT), which catalyzes the rate-limiting step in the NAD+ salvage pathway, preferentially depleted CD34+CD38- cells, implicating NAMPT inhibitors as potential anti-LSC agents.
To evaluate the therapeutic potential of NAMPT inhibitors, we focused on KPT-9274, a small-molecule NAMPT inhibitor currently under clinical development for other cancer types. Treatment with KPT-9274 depleted the CD34+CD38- fraction across multiple primary human AML samples through induction of apoptosis. The preferential sensitivity of CD34+CD38- cells to NAMPT inhibition correlated with a lower basal level of intracellular NAD+ and greater dependency on NAMPT activity for NAD+ generation relative to the other fractions. In contrast, normal CD34+ HSPCs were largely resistant to the cytotoxic effects of KPT-9274 due to their capacity to utilize the Preiss-Handler pathway for NAD+ generation. Consistent with the in vitro findings, KPT-9274 treatment significantly reduced LSC activity as determined by secondary engraftment potential in 2 of 3 patient-derived xenograft (PDX) models of human AML and had minimal impact on normal HSC activity in mice engrafted cord blood cells.
To gain mechanistic insights into how NAMPT inhibition induces cell death, we performed transcriptomic analysis of sorted CD34+CD38- cells treated with KPT-9274. This analysis revealed a striking upregulation of genes involved in cholesterol and lipid synthesis including the stearoyl-CoA desaturase (SCD) gene. The upregulated genes were highly enriched for known targets of the sterol regulatory element binding protein (SREBP) transcription factors. Functional studies demonstrated that this transcriptional response was protective against the cytotoxic effect of NAMPT inhibition in AML cells. To uncover the metabolic basis of this protective effect, we performed global metabolomic profiling of AML cells treated with KPT-9274 and observed a decrease in the ratio of monounsaturated fatty acids (MUFAs) to saturated fatty acids (SFAs) upon drug treatment. This drop in MUFA:SFA ratio reflected a reduction in SCD activity which catalyzes the desaturation of SFAs to MUFAs in a NADPH-dependent reaction. Since depletion of intracellular MUFAs could trigger apoptosis, we hypothesized that the SREBP response might protect against cell death through upregulation of SCD activity and consequent increase in MUFA synthesis. In line with this hypothesis, we found that exogenous oleic acid, a MUFA, completely rescued cell death induced by KPT-9274, while treatment with SCD inhibitors sensitized AML cells to the cytotoxic effects of NAMPT inhibition. To explore the translational application of our findings, we tested whether dipyridamole (DP), a clinically approved anti-platelet agent with inhibitory activity against SREBP signaling, can be repurposed to enhance the anti-leukemic effects of KPT-9274. We showed that treatment with DP, at non-toxic concentrations, potentiated the cytotoxicity of KPT-9274 against AML cells in vitro. Importantly, in vivo combination treatment with KPT-9274 and DP effectively targeted LSC activity in a PDX model that was refractory to KPT-9274 as single agent.
In summary, our findings demonstrate that LSCs are preferentially dependent on NAMPT activity for survival over non-LSCs and normal HSCs. We further uncovered that NAMPT inhibition results in dysregulation of lipid homeostasis and induces a lipogenic response coordinated by SREBPs that protects AML cells against NAD+ depletion. These findings offer insights into drug combination strategies to enhance the efficacy of NAMPT inhibitors and provide the rationale for testing NAMPT inhibitors in the treatment of AML in clinical trials.
Disclosures
Dick: Bristol-Myers Squibb/Celgene: Research Funding. Wang:Trilium therapeutics: Patents & Royalties: There is an existing license agreement between TTI and University Health Network and J.C.Y.W. may be entitled to receive financial benefits further to this license and in accordance with UHN's intellectual property policies. .
Reorganization of Metabolism during Cardiomyogenesis Implies Time-Specific Signaling Pathway Regulation
