Elevated mitochondrial protein import in acute myeloid leukemia increases reliance on mitochondrial protease LONP1
Matthew Tcheng, Veronique Voisin, Geethu Emily Thomas, Anastasija A. Piric, Marcela Gronda, Rose Hurren, Dakai Ling, Yongran Yan, Lan Xin Zhang, Yue Feng, Ali Chegini, Nathan Duong, Ross S. Mancini, Stefan Quinn W. Currie, Zaynab Mamai, Brady Stock, Shahbaz Khan, Yulia Jitkova, Chaitra Sarathy, Edward Ayoub, Po Yee Mak, Andrea Arruda, Thomas Kislinger, Mark A. Reed, Bing Z. Carter, Michael Andreeff, Steven M. Kornblau, Mark D. Minden, Siavash Vahidi, Aaron D. Schimmer
Matthew Tcheng, Veronique Voisin, Geethu Emily Thomas, Anastasija A. Piric, Marcela Gronda, Rose Hurren, Dakai Ling, Yongran Yan, Lan Xin Zhang, Yue Feng, Ali Chegini, Nathan Duong, Ross S. Mancini, Stefan Quinn W. Currie, Zaynab Mamai, Brady Stock, Shahbaz Khan, Yulia Jitkova, Chaitra Sarathy, Edward Ayoub, Po Yee Mak, Andrea Arruda, Thomas Kislinger, Mark A. Reed, Bing Z. Carter, Michael Andreeff, Steven M. Kornblau, Mark D. Minden, Siavash Vahidi, Aaron D. Schimmer
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Research Article Cell biology Metabolism Oncology

Elevated mitochondrial protein import in acute myeloid leukemia increases reliance on mitochondrial protease LONP1

Abstract

Most mitochondrial proteins are nucleus-encoded, translated in the cytosol, and imported into the mitochondria. Through gene expression analysis and functional assays, we demonstrated that mitochondrial protein import was increased in acute myeloid leukemia (AML) cells compared with normal hematopoietic cells. Increased mitochondrial protein import was positively correlated with an increase in the mitochondrial unfolded protein response (UPRmt), a stress-activated pathway of mitochondrial proteases and chaperones that maintains protein solubility and prevents the formation of toxic aggregates. The UPRmt protease LONP1 (Lon peptidase 1) was upregulated in AML and positively correlated with increased mitochondrial protein import and UPRmt. Genetic or chemical inhibition of the LONP1 ATPase domain induced mitochondrial protein aggregation and selectively killed AML cells with high LONP1 expression, while sparing AML cells with low LONP1 expression and normal hematopoietic cells in vitro and in vivo. Thus, we uncovered a critical role of the UPRmt protease LONP1 in buffering stress from mitochondrial protein import in AML.

Authors

Matthew Tcheng, Veronique Voisin, Geethu Emily Thomas, Anastasija A. Piric, Marcela Gronda, Rose Hurren, Dakai Ling, Yongran Yan, Lan Xin Zhang, Yue Feng, Ali Chegini, Nathan Duong, Ross S. Mancini, Stefan Quinn W. Currie, Zaynab Mamai, Brady Stock, Shahbaz Khan, Yulia Jitkova, Chaitra Sarathy, Edward Ayoub, Po Yee Mak, Andrea Arruda, Thomas Kislinger, Mark A. Reed, Bing Z. Carter, Michael Andreeff, Steven M. Kornblau, Mark D. Minden, Siavash Vahidi, Aaron D. Schimmer

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Figure 4

Inhibition of LONP1 leads to the accumulation of aggregated mitochondrial proteins in AML cells with high LONP1 expression.

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Inhibition of LONP1 leads to the accumulation of aggregated mitochondria...
(A) OCI-AML2 or OCI-M2 cells were transduced with shRNA or sgRNA targeting LONP1 or control sequences or treated with 250 nM omaveloxolone or bardoxolone methyl. After 7 days (shRNA) or 14 days (gRNA) or 30 hours (chemical inhibition), cells were stained with proteostat (aggregated proteins) and FITC anti-TOMM20 (mitochondria). Cells were imaged by confocal microscopy, and colocalization of proteostat with TOMM20 was quantified by HALO software. ****P < 0.0001, by 1-way ANOVA with Dunnett’s multiple-comparison test. (B) Primary AML samples (n = 8) were treated with 250 nM omaveloxolone or bardoxolone methyl for 30 hours. Mitochondrial protein aggregation was measured as in A, and LONP1 expression relative to OCI-AML2 cells was measured by immunoblotting (omaveloxolone: r2 = 0.64; bardoxolone methyl: r2 = 0.65). (C) Normal mononuclear hematopoietic cells were treated with 250 nM omaveloxolone for 30 hours. Mitochondrial protein aggregation was measured as in A. NS, P > 0.05, by unpaired, 2-tailed Student’s t test. (D) OCI-AML2 cells were transduced with an shRNA targeting LONP1 or control sequences. After 7 days, LONP1 substrates (CLPX, TUFM, NDUFA9) were measured in detergent-insoluble fractions of mitochondrial lysates (Supplemental Figure 18) and quantified by densitometry. Data represent the mean ± SD of 3 biological replicates. CLPX: shLONP1 1755 (*P = 0.0152), shLONP1 3′-UTR (**P = 0.034); TUFM: shLONP1 1755 (**P = 0.0015), shLONP1 3′-UTR (***P = 0.0004), NDUFA9: shLONP1 1755 (***P = 0.0005), shLONP1 3′-UTR (***P = 0.0004) by 1-way ANOVA with Dunnett’s multiple-comparison test. (E) OCI-AML2 cells were transduced with an shRNA targeting LONP1 or control sequences. After 7 days, the oxygen consumption rate was measured (n = 14–18 per group). (F) OCI-AML2 cells were transduced with an shRNA targeting LONP1 or control sequences. After 7 days, mitochondrial superoxide levels were measured by flow cytometry using MitoSOX reagent. ***P = 0.0001, by 1-way ANOVA with Dunnett’s multiple-comparison test. Data are presented as mean ± SD.