The Mitochondrial Unfolded Protein Response (UPRmt) Is Upregulated in Acute Myeloid Leukemia (AML) and Inhibiting the UPRmt Protease, LONP1, Leads to Mitochondrial Protein Aggregation and Cell Death Selectively in AML

Compared to normal hematopoietic cells, AML are uniquely reliant on the mitochondria and its proteome for survival and proliferation. The mitochondrial proteome contains ~1100 unique proteins, of which all but 13 are encoded by nuclear DNA, translated in the cytoplasm and imported into the mitochondria. To measure mitochondrial protein import in AML and normal cells, we developed a novel assay to quantify the mitochondrial import of puromycin labeled proteins. Compared to normal cells, primary AML samples had a 10-fold increase in mitochondrial protein import. Likewise, expression of mitochondrial protein import genes were higher in AML compared to normal.

Failure to properly process newly imported proteins into their mature forms leads to protein aggregation resulting in mitochondrial dysfunction. The mitochondrial unfolded protein response (UPR^mt) pathway utilizes chaperones and proteases to cleave, fold and degrade newly imported and damaged proteins. Since there are no dedicated UPR^mt gene sets in GO, KEGG, and Reactome databases, we curated a 39 gene signature that encompasses the components of UPR^mt. UPR^mt gene expression was increased in primary AML, compared to normal, and positively correlated with mitochondrial protein import gene expression.

We next assessed the dependencies of UPR^mt genes in 24 AML cell lines (DepMap database). Across shRNA and CRISPR screens, the mitochondrial AAA+ protease LONP1 was a top dependency. LONP1 is a serine protease that unfolds and degrades damaged or misfolded protein through its ATPase and protease domains, respectively. Compared to normal cells, LONP1 protein was increased >2-fold in 16/30 primary AML samples by immunoblotting. LONP1 was equally expressed across the molecular and cytogenetic AML subgroups in 730 primary AML samples by Reverse Phase Protein Array. LONP1 mRNA expression positively correlated with UPR^mt and mitochondrial protein import gene expression.

Through its ATPase activity, LONP1 acts in concert with mitochondrial chaperones to fold and solubilize newly imported mitochondrial proteins. We developed a novel confocal-based assay to measure mitochondrial protein aggregation using Proteostat, a dye that fluoresces when bound to aggregated proteins. We visualized the co-localization of Proteostat and the mitochondrial marker TOM20 with confocal microscopy and quantified the co-localization with HALO image analysis. Genetic depletion and chemical inhibition (Omaveloxolone and Bardoxolone methyl) of LONP1 increased mitochondrial protein aggregation in AML cell lines and primary AML samples with high LONP1 but not normal cells or AML samples with low LONP1 and low mitochondrial protein import. We confirmed the increase in mitochondrial protein aggregation after LONP1 inhibition using immunoblotting of LONP1 substrates in detergent-soluble and -insoluble fractions of mitochondrial lysates. We also demonstrated that the ATPase domain but not the proteolytic domain of LONP1 was necessary for mitochondrial protein solubility.

Genetic depletion and chemical inhibition of LONP1 killed AML cell lines and primary AML samples. In primary AML, LONP1 expression positively correlated with sensitivity to LONP1 chemical inhibition or genetic depletion (Omaveloxolone (n= 16, R²=0.64); Bardoxolone methyl (n= 30, R²=0.65); LONP1 shRNA (n=9, R²=0.72)). We also discovered that the ATPase domain but not the proteolytic domain of LONP1 was necessary for AML viability.

LONP1 knockdown decreased engraftment of AML lines and primary AML samples with high LONP1 into the marrow of NSG mice. Likewise, systemic treatment with Omaveloxolone and Bardoxolone methyl reduced AML growth in mice xenografted with AML cells and primary samples with high LONP1. Daily treatment of mice with Omaveloxolone and Bardoxolone methyl for 6 days increased mitochondrial protein aggregation in xenografted AML cells but not in normal mouse tissues, despite its cross-reactivity with murine LONP1.

In summary, a subset of AML patients have increased mitochondrial protein import and upregulate UPR^mt as a protective response. Targeting UPR^mt and the processing of newly imported mitochondrial proteins at the level of LONP1 leads to increased mitochondrial protein aggregation and cell death in AML while sparing normal hematopoietic cells in vitro and in vivo.