A novel and highly effective mitochondrial uncoupling drug in T-cell leukemia

Abstract

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy. Despite recent advances in treatments with intensified chemotherapy regimens, relapse rates and associated morbidities remain high. In this context, metabolic dependencies have emerged as a druggable opportunity for the treatment of leukemia. Here, we tested the antileukemic effects of MB1-47, a newly developed mitochondrial uncoupling compound. MB1-47 treatment in T-ALL cells robustly inhibited cell proliferation via both cytostatic and cytotoxic effects as a result of compromised mitochondrial energy and metabolite depletion, which severely impaired nucleotide biosynthesis. Mechanistically, acute treatment with MB1-47 in primary leukemias promoted adenosine monophosphate-activated serine/threonine protein kinase (AMPK) activation and downregulation of mammalian target of rapamycin (mTOR) signaling, stalling anabolic pathways that support leukemic cell survival. Indeed, MB1-47 treatment in mice harboring either murine NOTCH1-induced primary leukemias or human T-ALL patient-derived xenografts (PDXs) led to potent antileukemic effects with a significant extension in survival without overlapping toxicities. Overall, our findings demonstrate a critical role for mitochondrial oxidative phosphorylation in T-ALL and uncover MB1-47-driven mitochondrial uncoupling as a novel therapeutic strategy for the treatment of this disease.

Graphical abstract

Figure 1.

Chemical structure and mitochondrial uncoupling properties of MB1-47. (A) Schematic illustration of the ETC and the effects of uncoupling depolarizing the mitochondrial inner membrane. (B) Chemical structure of NEN and MB1-47, niclosamide-based first- and second-generation mitochondrial uncouplers, respectively. (C) Pharmacokinetic properties of NEN and MB1-47 in mice in vivo. (D) Representative flow cytometry histograms showing mitochondrial membrane potential measured by tetramethylrhodamine ethyl ester (TMRE) staining in live DND41 and JURKAT cells after 72 hours of treatment with MB1-47 (4 μM and 2 μM, respectively). (E) Geometric mean ± standard deviation (SD) quantification of TMRE staining from triplicates in 2 PTEN⁺ (DND41 and HPB-ALL) and 2 PTEN⁻ (JURKAT and MOLT-3) cell lines upon 72 hours of MB-47 treatment. (F) Oxygen consumption rate (OCR) in DND41 cells, under basal conditions or in response to the indicated mitochondrial inhibitors, measured in real time using a Seahorse XF24 instrument. Data are presented as mean ± SD of n = 4 wells. (G) Metabolite levels in FCCP-treated vs MB1-47–treated DND41 cells for 24 hours (mean; n = 3). (H) Relative quantification of ATP levels from DND41 triplicates treated with DMSO, FCCP, or MB1-47 for 24 hours (mean ± SD). The goodness of fit (R²) was determined by using a simple linear regression model. Statistical significance (P) was determined by using an unpaired, 2-tailed Student t test. *P < .05; ***P < .005. AUC, area under the curve; MFI, mean fluorescence intensity.

Figure 2.

MB1-47 antileukemic effects in T-ALL cell lines in vitro. (A) Relative cell survival of 6 independent human T-ALL cell lines (including PTEN⁺ and PTEN⁻ cells) in the presence of MB1-47 at the indicated concentrations for 72 hours (mean ± SD; n = 3). (B) Representative flow cytometry histograms showing cell-size changes in G₁-gated DND41 and JURKAT cells treated with DMSO (control) or MB1-47 (4 or 2 µM, respectively) for 72 hours. (C) Representative flow cytometry plots of annexin V (apoptotic cells) and 7-aminoactinomycin D (7-AAD; dead cells) staining. Numbers in quadrants indicate percentage of cells. (D) Quantification of apoptosis from DND41 triplicates treated for 72 hours with DMSO (control) or 4 μM MB1-47 (mean ± SD). Statistical significance (P) was determined by using the 2-tailed Student t test; ***P < .005. (E-F) Flow cytometry representation (E) and quantification (F) of cell-cycle analysis of DND41 cells treated with DMSO (control) or MB1-47 (4 μM) for 72 hours (n = 3; mean ± SD). P values in panel F were calculated using 2-way analysis of variance (ANOVA) for multiple comparisons. ***P < .005. FSC-H, forward scatter height; PI, propidium iodide.

Figure 3.

MB1-47 depletes TCA intermediates and NTPs in T-ALL cells. (A) Relative glucose abundance in media from DND41 cells cultured in the presence or absence of MB1-47 (mean ± SD; n = 3). (B) Relative lactate abundance in media from DND41 cells cultured in the presence or absence of MB1-47 (mean ± SD; n = 3). (C) Significantly altered metabolites after MB1-47 exposure, ranked by P value (−log₁₀ transformed). (D) Intracellular ratio of NAD⁺/NADH (left) and pyruvate/lactate (right) in DND41 cells cultured in the presence or absence of MB1-47 (n = 6, from 2 independent replicates; bar graphs represent mean ± SD). (E) Relative abundance of indicated TCA intermediates in DND41 cells cultured in the presence or absence of MB1-47 (n = 9, from 3 independent experiments). (F) Heat map showing differential intracellular amino acid abundances (log₂) after MB-47 treatment, relative to DMSO-treated (control) cells. (G) Relative abundance of aspartate in DND41 cells cultured in the presence or absence of MB1-47 (n = 9, from 3 independent experiments; mean ± SD). (H) Heat map showing differential intracellular nucleotide abundance (log₂) after exposure to MB1-47, relative to DMSO-treated (control) cells. (I) Relative abundance of indicated UDP sugars in DND41 cells cultured in the presence or absence of MB1-47. All measurements were determined after MB1-47 (4 μM) treatment of 24 hours and are relative to DMSO-treated (control) cells. (J) Immunoblot analyses of AMPK, ACC, and 4E-BP1 in DND41 cells treated with DMSO or MB1-47 (4 μM) for either 2 or 4 hours. Statistical significance (P) was determined by using the 2-tailed Student t test. *P < .05; ***P < .005.

Figure 4.

MB1-47 promotes increased glycolysis and glucose flux into the TCA cycle. (A) Schematic diagram of TCA cycle intermediates ¹³C-labeling patterns in cells cultured with [U-¹³C]glucose. (B) Schematic diagram of TCA cycle intermediates ¹³C-labeling patterns in cells cultured with [U-¹³C]glutamine. (C) Isotopologs for the indicated metabolites after 4 hours of [U-¹³C]glucose labeling in DND41 triplicates treated with MB1-47 or DMSO (control). (D) Isotopologs for the indicated metabolites after 4 hours of [U-¹³C]glutamine labeling in DND41 triplicates treated with MB1-47 or DMSO (control). (E) Relative flux activity of TCA cycle in untreated (DMSO) or MB1-47–treated DND41 cells. Statistical significance (P) was determined by using multiple Student t tests. ^#P < .1; *P < .05; **P < .01; ***P < .005. ac-CoA, acetyl coenzyme A; CS, citrate synthase; FH, fumarate hydratase; fwd, forward; GDH, glutamate dehydrogenase; GLS, glutaminase; GOT, glutamic oxaloacetic transaminase; IDH, isocitrate dehydrogenase; α-KG, α-ketoglutarate; MDH, malate dehydrogenase; OGDH, oxoglutarate dehydrogenase; PC, pyruvate carboxylase; PDH, pyruvate dehydrogenase; rv, reverse; SDH, succinate dehydrogenase.

Figure 5.

Metabolic and signaling effects upon acute MB1-47 treatment of leukemic mice in vivo. (A) Schematic illustration of acute exposure to MB1-47 in vivo via gavage (detailed in “Methods”). (B) Significantly altered metabolites after acute exposure to MB1-47, ranked by P value (−log₂ transformed). (C) Ratio of pyruvate/lactate abundances from leukemic spleens after acute treatment with MB1-47 in vivo (mean ± SD). (D) Relative abundance of the indicated metabolites in leukemic spleens after acute treatment with MB1-47 in vivo. (E) Immunoblot analyses of cleaved PARP in leukemic spleens after acute treatment with MB1-47 in vivo. (F) Immunoblot analyses of AMPK, ACC, and 4E-BP1 in leukemic spleens after acute treatment with MB1-47 in vivo. Statistical significance (P) was determined by using the 2-tailed Student t test. **P < .01; ***P < .005.

Figure 6.

MB1-47 shows AMPK-mediated antileukemic effects in mouse primary leukemias in vivo. (A) Schematic illustration of treatment with MB1-47–containing diet in leukemic mice in vivo for 3 consecutive days (detailed in “Methods”; n = 5). (B-D) Changes in leukemia burden after 3 days of MB1-47 treatment, as assessed by total spleen weight (B) or by flow cytometry detection of leukemic GFP⁺ cells in spleen (C) and bone marrow (D). Statistical significance (P) was determined by using the 1-tailed Student t test. *P < .05; ***P < .005. (E) Immunoblot analyses of cleaved PARP in leukemic spleens after 3 days of MB1-47 treatment. (F) Flow cytometry representation of cell-cycle analysis of T-ALL cells from leukemic spleens after 3 days of MB1-47 treatment. P values were calculated using 2-way ANOVA for multiple comparisons; ***P < .005. (G-H) Kaplan-Meier survival curves of mice harboring isogenic Pten⁺ (G) and Pten⁻ (H) T-ALLs treated with an MB1-47–containing diet or a control diet. (I) Schematic illustration of bone marrow progenitor-retroviral transduction protocol for the generation and analysis of NOTCH1-induced Ampk-conditional knockout T-ALLs. (J) Kaplan-Meier survival curves of mice harboring Ampk⁺ and Ampk⁻ isogenic leukemias treated with an MB1-47–containing diet or a control diet. P values in survival curves were calculated with the log-rank test; n = 10 mice per group. ***P < .005.

Figure 7.

MB1-47 shows antileukemic effects in human T-ALL PDXs in vivo. (A) Kaplan-Meier survival curve of mice harboring PDTALL19, a NOTCH1-mutated PTEN⁺ human T- ALL PDX, treated with a control diet or an MB1-47–containing diet (n = 10 mice per group). (B) Kaplan-Meier survival curve of mice harboring CU-L-157, a NOTCH1–wild-type PTEN⁻ human T- ALL PDX, treated with a control diet or an MB1-47–containing diet (n = 6 mice per group). P values were calculated with the log-rank test. **P < .01; ***P < .005.