Statins markedly potentiate aminopeptidase inhibitor activity against (drug-resistant) human acute myeloid leukemia cells

Abstract

Aim: This study aimed to decipher the molecular mechanism underlying the synergistic effect of inhibitors of the mevalonate-cholesterol pathway (i.e., statins) and aminopeptidase inhibitors (APis) on APi-sensitive and -resistant acute myeloid leukemia (AML) cells. Methods: U937 cells and their sublines with low and high levels of acquired resistance to (6S)-[(R)-2-((S)-Hydroxy-hydroxycarbamoyl-methoxy-methyl)-4-methyl-pentanoylamino]-3,3 dimethyl-butyric acid cyclopentyl ester (CHR2863), an APi prodrug, served as main AML cell line models. Drug combination effects were assessed with CHR2863 and in vitro non-toxic concentrations of various statins upon cell growth inhibition, cell cycle effects, and apoptosis induction. Mechanistic studies involved analysis of Rheb prenylation required for mTOR activation. Results: A strong synergy of CHR2863 with the statins simvastatin, fluvastatin, lovastatin, and pravastatin was demonstrated in U937 cells and two CHR2863-resistant sublines. This potent synergy between simvastatin and CHR2863 was also observed with a series of other human AML cell lines (e.g., THP1, MV4-11, and KG1), but not with acute lymphocytic leukemia or multiple solid tumor cell lines. This synergistic activity was: (i) specific for APis (e.g., CHR2863 and Bestatin), rather than for other cytotoxic agents; and (ii) corroborated by enhanced induction of apoptosis and cell cycle arrest which increased the sub-G1 fraction. Consistently, statin potentiation of CHR2863 activity was abrogated by co-administration of mevalonate and/or farnesyl pyrophosphate, suggesting the involvement of protein prenylation; this was experimentally confirmed by impaired Rheb prenylation by simvastatin. Conclusion: These novel findings suggest that the combined inhibitory effect of impaired Rheb prenylation and CHR2863-dependent mTOR inhibition instigates a potent synergistic inhibition of statins and APis on human AML cells.

Keywords: Aminopeptidase; Rheb; carboxyl esterase; mTOR; mevalonate pathway; statins.

Figure 1

Growth inhibitory effects of CHR2863 for (A) U937/WT, (B) U937/CHR2863^R0.2 and (C) U937/CHR2863^R5 cells in the absence and presence of maximal non-toxic concentrations of simvastatin (2 µM, 2.5 µM, and 2.5 µM, respectively). Cell growth inhibition was determined after 72 h of drug exposure. The results depicted are the mean ± SE of 6-10 separate experiments; (D) Combination index - fraction affected plot from (A-C) of the combination simvastatin (fixed concentration) and CHR2863 for U937/WT, U937/CHR2863^R0.2 and U937/CHR2863^R5 cells. CHR2863: (6S)-[(R)-2-((S)-Hydroxy-hydroxycarbamoyl-methoxy-methyl)-4-methyl-pentanoylamino]-3,3 dimethyl-butyric acid cyclopentyl ester.

Figure 2

The potentiating effect of maximal non-toxic concentrations of various statins on the CHR2863 activity in U937/WT, U937/CHR2863^R0.2 and U937/CHR2863^R5 cells. The concentrations of simvastatin, lovastatin, pravastatin and fluvastatin were 2 µM, 2.5 µM, 100 µM and 0.5 µM, respectively, for U937/WT cells, and 2.5 µM, 5 µM, 200 µM and 1 µM, respectively, for U937/CHR2863^R0.2 and U937/CHR2863^R5 cells. CHR2863 dose response curves in combination with statins were generated over a CHR2863 concentration range of 0-1 μM for U937/WT cells, 0-5 μM for U937/CHR2863^R0.2 cells and 0-50 μM CHR2863 for U937/CHR2863^R5 cells, essentially as shown in Figure 1. Statin potentiating factor is defined as the ratio of IC₅₀ (50% growth inhibition) of cell culture without statins vs. IC₅₀ of cell cultures in the presence of statins. Cell growth inhibition was determined after 72 h of drug exposure. The results depicted are the mean ± SD of 3-4 independent experiments. CHR2863: (6S)-[(R)-2-((S)-Hydroxy-hydroxycarbamoyl-methoxy-methyl)-4-methyl-pentanoylamino]-3,3 dimethyl-butyric acid cyclopentyl ester.

Figure 3

Selectivity of simvastatin-potentiating effect for APis. Effect of non-toxic concentrations of simvastatin (2-2.5 µM) on the growth inhibitory activity of the APis CHR2863 and bestatin, HDAC inhibitor prodrug CHR2875, and daunorubicin in U937/WT, U937/CHR2863^R0.2 and U937/CHR2863^R5 cells. Simvastatin potentiation factor is defined as the ratio of IC₅₀ (50% growth inhibition) of cell culture without statins vs. IC₅₀ of cell cultures in the presence of statins. Cell growth inhibition was determined after 72 h of drug exposure. Results depicted are the mean of two separate experiments (for bestatin) and the mean ± SD of 3-4 separate experiments for CHR2863, CHR2875 and daunorubicin. IC50 values of U937/WT, U937/CHR2863R0.2 and U937/CHR2863R5 cells for CHR2863 are: 52 ± 16 nM, 713 ± 212 nM, and 14,047 ± 5,521 nM, respectively; for Bestatin: 158 ± 15 μM, 169 ± 32 μM, and 177 ± 14 μM, respectively; for CHR2875: 158 ± 9 nM, 86 ± 13 nM, and 147 ± 36 nM, respectively; and for daunorubicin: 16 ± 1 nM, 16 ± 2 nM, and 15 ± 3 nM, respectively. APis: aminopeptidase inhibitors; CHR2863: (6S)-[(R)-2-((S)-Hydroxy-hydroxycarbamoyl-methoxy-methyl)-4-methyl-pentanoylamino]-3,3 dimethyl-butyric acid cyclopentyl ester.

Figure 4

Simvastatin potentiation of CHR2863 activity in human AML cell lines vs. human lymphoid and solid tumor cell lines. Cell growth inhibition was determined after 72 h of drug exposure in the absence or presence of maximal non-toxic concentrations of simvastatin, being (between brackets) for: U937 (2 µM), THP1 (2.5 µM), MV4-11 (2.5 µM), KG1 (10 µM), CCRF-CEM (2.5 µM), CEM/Vbl (2.5 µM), SW1573 (0.2 µM), 2008 (0.75 µM), 2008/MRP1 (2.5 µM), MCF7 (1 µM), MCF7/MR (2.5 µM) and KB (1 µM). Simvastatin potentiation factor is defined as the ratio of IC₅₀ (50% growth inhibition) of cell culture without statins vs. IC₅₀ of cell cultures in the presence of statins. IC₅₀ values (between brackets) for CHR2863 for the various cell lines (in the absence of simvastatin) were: U937 (61 ± 16 nM), THP1 (1172 ± 807 nM), MV4-11 (282 ± 51 nM), KG1 (394 ± 144 nM), CCRF-CEM (11,170 ± 5,100 nM), CEM/Vbl (29,100 ± 5,900 nM), SW1573 (6,625 ± 3,020 nM), 2008 (2,020 ± 1,080 nM), 2008/MRP1 (6,700 ± 2,560 nM), MCF7 (453 ± 400 nM), MCF7/MR (386 ± 64 nM), and KB (132 ± 50 nM). The results depicted are the mean ± SD of 3-5 independent experiments. CHR2863: (6S)-[(R)-2-((S)-Hydroxy-hydroxycarbamoyl-methoxy-methyl)-4-methyl-pentanoylamino]-3,3 dimethyl-butyric acid cyclopentyl ester.

Figure 5

Effect of simvastatin and CHR2863 combinations on cell viability, apoptosis induction and cell cycle distribution in U937/WT, U937/CHR2863^R0.2 and U937/CHR2863^R5 cells. Simvastatin concentrations used for U937/WT cells, U937/CHR2863R0.2 and U937/CHR2863R5 cells were maximal in vitro non-toxic concentrations: 2 µM, 2.5 µM and 2.5 µM, respectively. For CHR2863, minimally cytotoxic (≈ IC10) were selected (from Figure 1), i.e., 25 nM, 250 nM and 5 μM for U937/WT cells, U937/CHR2863^R0.2 and U937/CHR2863^R5 cells, respectively. Cells (3 × 10⁵/mL in 10 mL medium) were incubated for 48 h with the indicated concentrations of simvastatin, CHR2863 and their combination and assessed for the impact on (A) cell viability, (B) apoptosis induction, (C) sub-G1 fraction and (D) cell cycle distribution. Cells incubated for 24 h with bortezomib or 48 h with 6 µM CHR2863 served as a control for cell growth inhibition and apoptosis induction. Percentages of apoptotic cells in control untreated U937/WT, U937/CHR2863^R0.2 and U937/CHR2863^R5 cells were 4.6% ± 1.9%, 5.2% ± 1.2% and 6.1% ± 0.9%, respectively. Sub-G1 fractions in control untreated U937/WT, U937/CHR2863^R0.2 and U937/CHR2863^R5 cells were 4.2% ± 2.0%, 9.1% ± 6.5% and 8.2% ± 2.2%, respectively. The results depicted are the mean ± SD of 4-5 independent experiments. *Combination statistically significant (P < 0.05) different compared to single drugs control cells. CHR2863: (6S)-[(R)-2-((S)-Hydroxy-hydroxycarbamoyl-methoxy-methyl)-4-methyl-pentanoylamino]-3,3 dimethyl-butyric acid cyclopentyl ester.

Figure 6

Effects of mevalonic acid, farnesyl pyrophosphate and geranylgeranyl pyrophosphate on simvastatin potentiation of CHR2863 activity. U937/WT, U937/CHR2863^R0.2 and U937/CHR2863^R5 cells were incubated for 72 h with the indicated concentrations of CHR2863 and non-toxic concentration of simvastatin in the presence of increasing concentrations of (A) mevalonic acid, (B) farnesyl pyrophosphate (FPP) and (C) geranylgeranyl pyrophosphate (GGPP). Results, presented as cell growth relative to control, are the mean ± SD of 4 independent experiments. The dashed line indicates the mean growth inhibition by CHR2863 alone. CHR2863: (6S)-[(R)-2-((S)-Hydroxy-hydroxycarbamoyl-methoxy-methyl)-4-methyl-pentanoylamino]-3,3 dimethyl-butyric acid cyclopentyl ester.

Figure 7

Effect of simvastatin and CHR2863 combinations on Rheb prenylation. U937/WT, U937/CHR2863^R0.2 and U937/CHR2863^R5 cells were incubated for 48 h with simvastatin, CHR2863, and their combination (as described in Figure 5), with or without the addition of MVA (100 µM), FPP (2 µM), GGPP (1 µM) or FTI-277 (10 µM). The slower (upper) migrating band represents unprenylated Rheb, and the faster (lower) migrating band represents prenylated Rheb. CHR2863: (6S)-[(R)-2-((S)-Hydroxy-hydroxycarbamoyl-methoxy-methyl)-4-methyl-pentanoylamino]-3,3 dimethyl-butyric acid cyclopentyl ester.

Figure 8

Proposed model for synergistic action of statins and APi CHR2863. (A) Peptide breakdown by aminopeptidases provides amino acids for re-utilization in protein synthesis. According to previously described models^{[49-51,56,62,63,66]}, an increased intralysomal amino acid content triggers dissociation of V-ATPase and Ragulator-Rag-mTORC1 complex. Binding of the latter complex to (prenylated) Rheb (in the lysosomal membrane) and membrane association of Ragulator will then induce mTOR activation and initiation of protein synthesis; (B) Inhibition of aminopeptidases by CHR2863 (or bestatin) will reduce the intralysomal amino acid content and dissociation of the Ragulator-Rag complex from mTORC1. By a different mechanism, statins may block Rheb prenylation and abolish its lysosomal membrane localization. The combined effect of CHR2863 and statins may then synergize in impairing mTOR activation, protein synthesis and inhibiting cell growth. The figure was created via BioRender. APi: Aminopeptidase inhibitor; CHR2863: (6S)-[(R)-2-((S)-Hydroxy-hydroxycarbamoyl-methoxy-methyl)-4-methyl-pentanoylamino]-3,3 dimethyl-butyric acid cyclopentyl ester.