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. 2016 Oct 14;291(42):21984-22000.
doi: 10.1074/jbc.M116.750653. Epub 2016 Aug 29.

Rational Design of a Parthenolide-based Drug Regimen That Selectively Eradicates Acute Myelogenous Leukemia Stem Cells

Shanshan Pei  1 , Mohammad Minhajuddin  1 , Angelo D'Alessandro  2 , Travis Nemkov  2 , Brett M Stevens  1 , Biniam Adane  1 , Nabilah Khan  1 , Fred K Hagen  3 , Vinod K Yadav  4 , Subhajyoti De  4 , John M Ashton  5 , Kirk C Hansen  2 , Jonathan A Gutman  1 , Daniel A Pollyea  1 , Peter A Crooks  6 , Clayton Smith  1 , Craig T Jordan  7
Affiliations

Rational Design of a Parthenolide-based Drug Regimen That Selectively Eradicates Acute Myelogenous Leukemia Stem Cells

Shanshan Pei et al. J Biol Chem. .

Erratum in

Abstract

Although multidrug approaches to cancer therapy are common, few strategies are based on rigorous scientific principles. Rather, drug combinations are largely dictated by empirical or clinical parameters. In the present study we developed a strategy for rational design of a regimen that selectively targets human acute myelogenous leukemia (AML) stem cells. As a starting point, we used parthenolide, an agent shown to target critical mechanisms of redox balance in primary AML cells. Next, using proteomic, genomic, and metabolomic methods, we determined that treatment with parthenolide leads to induction of compensatory mechanisms that include up-regulated NADPH production via the pentose phosphate pathway as well as activation of the Nrf2-mediated oxidative stress response pathway. Using this knowledge we identified 2-deoxyglucose and temsirolimus as agents that can be added to a parthenolide regimen as a means to inhibit such compensatory events and thereby further enhance eradication of AML cells. We demonstrate that the parthenolide, 2-deoxyglucose, temsirolimus (termed PDT) regimen is a potent means of targeting AML stem cells but has little to no effect on normal stem cells. Taken together our findings illustrate a comprehensive approach to designing combination anticancer drug regimens.

Keywords: 2-deoxyglucose; anticancer drug; drug combination; drug development; drug resistance; endoplasmic reticulum stress (ER stress); leukemia; oxidative stress; parthenolide; temsirolimus.

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Figures

FIGURE 1.
FIGURE 1.
Characterization of PTL's proteomic interactome in primary AML cells. A, chemical structure of parthenolide. B, chemical structure of MMB-biotin. C, silver stain of primary AML total cell lysates and subcellular fractions on a SDS-PAGE gel. D, Western blot (WB) showing the purity of subcellular fractions. E, Western blot showing the SA pulldown products from total lysates and all subcellular fractions, probed with streptavidin-HRP. F, a schematic diagram illustrating the methodology used to compare the proteomic interactome and transcriptomic signature of PTL in primary AML cells. G, number of PTL binding targets within the top five most perturbed signaling pathways induced by PTL in primary AML cells. p values were calculated by IPA analysis to test if a particular pathway is significantly represented by the proteomic interactome of PTL.
FIGURE 2.
FIGURE 2.
Metabolomic analyses reveal increased PPP activity for NADPH production in PTL-treated AML cells. A, a schematic diagram showing the workflow used to characterize PTL-induced metabolomic changes in primary AML cells. Results from the uniform labeled [U-13C6]glucose (B) and the dual-labeled [13C1,2]glucose (C) labeling experiments showing quantification of 13C-labeled metabolites in glycolysis, PPP, and TCA cycle in primary AML cells after DMSO or 7.5 μm PTL treatment. Data represent the mean ± S.D. (n = 3). For each metabolite, M+x indicates specific isotopologoues of a metabolite labeled with x numbers of the heavy 13C isotopic carbons. A circle diagram is also given to each metabolite to illustrate its carbon frame labeled with different numbers of 13C carbons (red solid circles represent heavy 13C, clear open circles represent 12C). D, quantification of NADPH and NADPH/NADP+ ratio in primary AML cells treated with DMSO control or 7.5 μm PTL using a bioluminescent assay. Data represent the mean ± S.D. (n = 3).
FIGURE 3.
FIGURE 3.
The PDT regimen depletes glutathione and induces ROS without activating Nrf2-mediated oxidative stress response and NADPH production. A, a simplified diagram illustrating the basic rationale of the triple drug regimen PDT. B, amount of total glutathione in primary AML cells after 4 h of treatment. C, intracellular ROS level in primary AML cells after each treatment, quantified by the CM-H2DCFDA dye. MFI, mean fluorescence intensity. D, QPCR data showing -fold changes of mRNA expression induced by 7.5 μm PTL or the PDT regimen. Each line represents an individual AML specimen (n = 4). ns, not significant. E, Western blot (WB) data showing protein expression of HMOX1 after various treatments for 6 h. F, QPCR data showing -fold changes of mRNA expression induced by 7.5 μm PTL or the PDT regimen. The bars represent the mean ± S.D. (n = 4). G, quantification of NADPH level in primary AML cells after various treatments. UNT, untreated; RFU, relative fluorescence units. Bars represent the mean ± S.D. (n = 3); * indicates p < 0.05; ** indicates p < 0.01.
FIGURE 4.
FIGURE 4.
The PDT regimen does not inhibit NF-κB but induces strong protein misfolding stress in primary AML cells. A, NF-κB EMSA of primary AML cells after various treatments for 6 h. Intensity of EMSA signal in each lane was quantified and normalized to the untreated (UNT) group and expressed as relative intensity values below each lane. B, Western blot (WB) showing level of phospho-p65 (Ser-536) in primary AML cells after various treatments for 6 h. C, QPCR results showing mRNA expression of NF-κB downstream gene IL6 in primary AML cells following various treatments for 6 h. D, GSEA enrichment plots showing up-regulation of both KEGG_PROTEASOME and REACTOME_UNFOLDED_PROTEIN_RESPONSE gene sets in AML cells treated with the PDT regimen. E, heat maps showing up-regulation of genes related to chaperone function, ER stress response, and proteasome activity. -Fold change (FC) and standard deviation (SD) are given for each gene (n = 4). Red indicates increase. Blue indicates decrease. F, QPCR data showing expression of CHOP mRNA in primary AML cells after various treatments for 6 h. In all sub panels, unless explicitly stated, the doses used in all drug combinations including the PDT regimen are 2.5 μm, 0.1125 mg/ml, and 2.5 μg/ml for PTL, 2DG, and TEM, respectively. In C and F, bars represent the mean ± S.D. (n = 3).
FIGURE 5.
FIGURE 5.
The PDT regimen effectively eradicates primary AML. A, viability of 10 primary AML specimens after being treated with various drugs alone or in combination for 24 h. Data are plotted as % of untreated. Bars represent the mean ± S.D. B, viability of 5 PTL-resistant primary AML specimens after treatment with 7.5 μm PTL or the PDT regimen. Data are plotted as % of untreated. Bars represent the mean ± S.D. C, number of colonies in methylcellulose culture per 100,000 primary AML cells treated with the PDT regimen or 7.5 μm PTL. Bars represent the mean ± S.D. In all drug combinations including the PDT regimen, PTL, 2DG, and TEM was used at 2.5 μm, 0.1125 mg/ml, and 2.5 μg/ml, respectively. ** indicates p < 0.01; **** indicates p < 0.0001.
FIGURE 6.
FIGURE 6.
The PDT regimen is not toxic to normal hematopoietic cells. A, viability of total normal MNC cells after treatment with various drugs alone or in combination for 24 h. Data are plotted as % of untreated. Bars represent the mean ± S.D. (three individual MNC specimens). B, representative flow cytometry plots showing the impact of PDT treatment to both total MNCs and CD34+ MNCs. Viable MNCs were determined by double negative staining of 7-AAD and annexin V. Percentage of CD34+ cells were quantified within the viable MNCs. C, viable CD34+ normal MNCs after being treated with various drugs alone or in combination for 24 h. Data are normalized to untreated control. Bars represent mean ± S.D. (three individual MNC specimens). D, number of colonies in methylcellulose culture per 1 × 106 normal MNCs treated with or without the PDT regimen. Bars represent the mean ± S.D. (n = 3). CFU, colony forming unit; UNT, untreated. In all different drug combinations including the PDT regimen, PTL, 2DG, and TEM were used at 2.5 μm, 0.1125 mg/ml, and 2.5 μg/ml, respectively.
FIGURE 7.
FIGURE 7.
The PDT regimen preferentially targets LSCs but spares HSCs. A, engraftment potential of AML cells treated with various drug combinations in NSG mice. Each dot represents an individual mouse. Lines represent mean ± S.D. (n = 10). UNT, untreated. B, engraftment potential of normal MNCs treated with or without the PDT regimen in NSG mice. Each dot represents an individual mouse. Lines represent mean ± S.D. (n = 10). In all sub panels, the PDT regimen is composed of 2.5 μm PTL, 0.1125 mg/ml 2DG, and 2.5 μg/ml TEM. ns, not significant. C, a working model describing the anti-leukemia mechanism of the PDT regimen. ** indicates p < 0.01; *** indicates p < 0.001.
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