Abstract 5898: Inhibition of the LONP1 ATPase domain induces mitochondrial proteotoxic stress and is cytotoxic to acute myeloid leukemia cells

Abstract

Abstract Compared to normal hematopoietic cells, AML cells exhibit a heightened reliance on mitochondrial metabolism for survival and proliferation. To support this unique phenotype, AML cells increase import of nuclear-encoded mitochondrial proteins which must be properly folded by mitochondrial chaperones, proteases, and heat shock proteins. Failure to properly fold these precursors leads to protein aggregation, mitochondrial dysfunction, and cell death. To evaluate the reliance of AML cells on maintaining mitochondrial proteostasis, we assessed gene dependency datasets (eg: depmap.org) and identified the mitochondrial protease LONP1, as the top essential gene. LONP1 is a matrix-localized nuclear-encoded AAA+ protease. Proteins are unfolded by its ATPase domain and degraded by its serine-catalyzed proteolytic domain. Compared to hematopoietic cells, LONP1 mRNA was overexpressed in AML across three publicly available datasets. By immunoblotting, LONP1 protein was increased in 16/30 primary AML samples compared to bulk (n=8) and CD34+ blood cells (n=3). LONP1 genetic depletion reduced the growth and viability of OCI-AML2, OCI-M2, NB4, and TEX leukemia cells as well as primary AML cells. LONP1 depletion increased aggregated mitochondrial proteins and reactive oxygen species (ROS) production while reducing mitochondrial respiration and membrane potential. To determine the domain of LONP1 that was necessary for mitochondrial protein solubility, mitochondrial function, and AML survival, we over-expressed wild type, ATPase dead (E591A), or proteolytically dead (S855A) LONP1 cDNA in OCI-AML2 cells and knocked down endogenous LONP1 with shRNA targeting the 3’UTR of the endogenous gene. Wild type or proteolytically dead (S855A) but not the ATPase mutant (E591A) rescued mitochondrial protein solubility, mitochondrial respiration, ROS generation, membrane potential, and cell viability. Thus, the ATPase domain but not the proteolytic activity of LONP1 is necessary for mitochondrial proteostasis. Bardoxolone methyl (CDDO-Me) is a synthetic triterpenoid that allosterically inhibits the LONP1 ATPase site. CDDO-Me killed OCI-AML2 and NB4 cells with IC50 values of 178.5±29.7 and 156.5±39.7 nM, respectively. CDDO-Me (200 nM) killed 3 out of 4 primary AML samples with high LONP1 expression but none of the 10 primary AML samples with low LONP1. Likewise, CDDO-Me induced mitochondrial protein aggregation in AML cell lines and primary AML patient samples with high LONP1 expression but not AML cells or primary AML patient samples with low LONP1. In summary, LONP1 is over-expressed in a subset of AML cells and primary samples where it maintains mitochondrial protein solubility. Selective inhibition of the LONP1 ATPase domain leads to mitochondrial protein aggregation and AML cell death, representing a novel therapeutic strategy for AML. Citation Format: Matthew Tcheng, Marcela Gronda, Rose Hurren, Lan Xin Zhang, Chaitra Sarathy, Yongran Yan, Andrea Arruda, Mark D. Minden, Aaron D. Schimmer. Inhibition of the LONP1 ATPase domain induces mitochondrial proteotoxic stress and is cytotoxic to acute myeloid leukemia cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5898.