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 5

The LONP1 AAA+ domain, but not the protease domain, is necessary for mitochondrial protein solubility.

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The LONP1 AAA+ domain, but not the protease domain, is necessary for mit...
(A) OCI-AML2 cells were transduced with empty vector, FLAG-tagged WT cDNA (LONP1WT), ATPase-deficient (LONP1E591A) cDNA, or protease-deficient LONP1 cDNA (LONP1S855A). Fourteen days later, cells were transduced with either an shRNA targeting the 3′-UTR of endogenous LONP1 or control sequences. After 7 days, levels of LONP1 and MnSOD protein were measured in mitochondrial lysates by immunoblotting. A representative immunoblot from 3 biological replicates is shown. (B) Mean ± SD growth and viability of cells from A were measured by trypan blue staining. Representative data from 3 biological replicates are shown. (C) Cells from A were stained as in Figure 4A to assess mitochondria-localized protein aggregation (n = 122–166 cells per group). ****P < 0.0001 (empty vector+shControl vs. empty vector+shLONP1 3′-UTR or LONP1E591A+shLONP1 3′-UTR); NS, P > 0.05 (empty vector+shControl vs. LONP1WT+shLONP1 3′-UTR or LONP1S855A+shLONP1 3′-UTR), by 1-way ANOVA with Dunnett’s multiple-comparison test. (D) Levels of CLPX, TUFM, and NDUFA9 protein in the detergent insoluble fraction of isolated mitochondria from cells in A were measured by immunoblotting (n = 3). CLPX: **P < 0.01 (empty vector+shControl vs. empty vector+shLONP1 3′-UTR or LONP1E591A+shLONP1 3′-UTR); NS, P > 0.05 (empty vector+shControl vs. LONP1WT+shLONP1 3′-UTR or LONP1S855A+shLONP1 3′-UTR). TUFM: ***P < 0.001 (empty vector+shControl vs. empty vector+shLONP1 3′-UTR or LONP1E591A+shLONP1 3′-UTR); NS, P > 0.05 (empty vector+shControl vs. LONP1WT+shLONP1 3′-UTR or LONP1S855A+shLONP1 3′-UTR). NDUFA9: ***P = 0.0004 (empty vector+shControl vs. empty vector+shLONP1 3′-UTR); *P = 0.0179 (empty vector+shControl vs. LONP1E591A+shLONP1 3′-UTR); NS, P > 0.05 (empty vector+shControl vs. LONP1WT+shLONP1 3′-UTR or LONP1S855A+shLONP1 3′-UTR). (E) Oxygen consumption of cells from A was measured with a Seahorse Metabolic Flux Bioanalyzer (n = 9–15 wells per group). (F) Mitochondrial superoxide in cells from A was assessed as in Figure 4F. ****P < 0.0001 (empty vector+shControl vs. empty vector+shLONP1 3′-UTR or LONP1E591A+shLONP1 3′-UTR); NS, P > 0.05 (empty vector+shControl vs. LONP1WT+shLONP1 3′-UTR or LONP1S855A+shLONP1 3′-UTR) by 1-way ANOVA with Dunnett’s multiple-comparison test. Data are presented as mean ± SD.