Pkcε Is a Central Regulator of Mitochondrial Function and Metabolism in Acute Myeloid Leukemia
Authors: Di Marcantonio Daniela , Masselli Elena , Martinez Esteban , Araiza-Olivera Daniela , Gobbi Giuliana , Mirandola Prisco , Vitale Marco , Sykes Stephen M. 
Published: October 2019

Mitochondria are multi-functional organelles and the epicenters of cellular metabolism. The mitochondria of AML cells from patients as well as those from genetically engineered mouse models (GEMMs) are frequently larger and display altered electron transport chain (ETC) activity and reactive oxygen species (ROS) production compared to healthy hematopoietic tissue. Notably, AML cells are more sensitive than their healthy counterparts to mitochondrial targeting agents indicating that regulators of mitochondrial biology in AML may represent novel therapeutic entry points.

We recently reported that the kinase PKCε supports AML cell survival and disease progression by maintaining mitochondrial ROS homeostasis. Briefly, we found that shRNA-mediated inhibition of PKCε leads to a lethal burst of mitochondrial ROS that can be mitigated by neutralizing ROS production. We also reported that shRNA-mediated inhibition of PKCε both significantly delays disease onset in a GEMM of AML driven by MLL-AF9 and reduces the survival of leukemia cells derived from patients with MLL-AF9-driven AML. Importantly, we have also found that inhibition of PKCε significantly impedes the colony forming capacity (CFC) of mouse AML cells expressing MLL-AF9 but not that of healthy hematopoietic stem and progenitor cells (HSPCs). In addition to AMLs bearing MLL-AF9, we also observed that PKCε supports the ex vivo survival of multiple normal karyotype AML (NK-AML) samples, particularly those expressing DNMT3A and/or FLT3-ITD mutations. Therefore, we evaluated the role of PKCε in a GEMM of AML driven by deletion of Dnmt3a and Tet2 in combination with FLT3-ITD expression, hereafter referred to as DTF. From this analysis, we observed that shRNA-mediated inhibition of PKCε also delays disease onset in this DTF-driven GEMM of AML (p=0.0135).

To determine the underlying mechanism by which PKCε supports mitochondrial ROS production and AML cell survival, we performed whole cell proteomics on OCI-AML3 cells, which carry a DNMT3A-R882C mutation, expressing control- or PKCε-targeting shRNAs. From this analysis, we observed that 34 mitochondrial proteins, many of which are related to ETC activity, the tricarboxylic acid cycle and mitochondrial membrane transport, were differently expressed in AML cells expressing PKCε-targeting shRNAs. Given that mitochondrial dysfunction is often related with an aberrant production of mitochondrial ROS, we next investigated how PKCε inhibition impacts mitochondrial function in human and mouse AML cell models bearing MLL-AF9, Dnmt3a and/or FLT3-ITD mutations.

To investigate the role of PKCε in mitochondrial function, we first examined how PKCε inhibition impacted outer mitochondrial membrane (OMM) potential and mitochondrial mass using mitotracker dyes. From this analysis, we found that PKCε inhibition diminished OMM potential (p<0.01) but did not impact mitochondrial mass as measured by both mitotracker green and levels of mitochondrial DNA. Since maintenance of the OMM potential is critical for mitochondrial ATP generation, we then assessed whether PKCε influenced ATP levels. We found that PKCε inhibition did reduce total cellular ATP content, suggesting that PKCε signaling is required for efficient energy production in AML cells. Defects in OMM potential and ATP production often stem from perturbations in mitochondrial respiration. Therefore, we evaluated the role of PKCε in AML cell aerobic respiration by performing a mitochondrial stress test using the Agilent XF Seahorse Bioanalyzer. As predicted, this analysis showed that PKCε inhibition significantly reduced both basal (p<0.01) and maximal (p<0.001) mitochondrial respiration. A more detailed analysis of individual ETC complex activity revealed that PKCε inhibition severely inhibits oxygen consumption in the presence of succinate and rotenone, which is indicative of a metabolic defect related to complex II (p<0.05). Additionally, we observed that PKCε over-expression protected AML cells from otherwise lethal doses of the complex II inhibitor, 2-Thenoyltrifluoroacetone (p<0.01) further suggesting that PKCε regulates ETC complex II function in managing mitochondrial energy production and ROS homeostasis.

Collectively, these results highlight a previously unrecognized role of PKCε regulating the activity of the ETC and cellular ATP production to support AML cell survival and disease progression.

Disclosures

No relevant conflicts of interest to declare.