Pkc Epsilon Regulates Mitochondrial Redox Biology to Support the Differentiation Blockade in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a genetically heterogeneous disease that is characterized by the clonal expansion of myeloid progenitors that have impaired differentiation capacity. Determining the molecular machinery that regulate the survival and differentiation blockade of AML cells could serve as a foundation for designing novel therapies. PKCε is a serine-threonine kinase belonging to the subgroup of the Protein Kinase C family called Novel PKCs. Aberrant PKCε expression and activation is associated with the pathogenesis and chemotherapy resistance of many solid cancers. However, the contribution of PKCε in blood malignances such as AML is not well defined.

To evaluate the role of PKCe in AML biology, we employed short hairpin RNA (shRNA)-mediated approaches to down-regulate PKCε expression in human and murine AML cell lines. We found that shRNA-mediated knockdown of PKCε significantly reduces the in vitro expansion of several human AML cell lines (MOLM-14, NOMO-1, OCI-AML3, THP-1 and U937). We also observed that blocking PKCε induces caspase-3 cleavage and increases the number of annexin V-positive cells (P<0.05), suggesting that PKCε antagonizes AML cell apoptosis. Additionally, we have also found that prior to cell death, AML cells expressing PKCε-targeting shRNAs display characteristics of myeloid differentiation. Specifically, down-regulation of PKCε results in altered expression of the myeloid differentiation transcription factors C/EBPa and PU.1 and increased expression of the mature myeloid marker CD11b (P<0.001). Moreover, upon PKCε inhibition, AML cells acquire morphological changes associated with differentiation, such as increased cytoplasmic volume, granule formation and nuclear segmentation.

Interestingly, we observed similar phenotypic changes when we inhibited PKCε expression in murine AML cell lines driven by the leukemogenic fusion protein MLL-AF9 alone (MLL-AF9) or in combination with the internal tandem duplication mutation of murine Flt3 (MLL-AF9;Flt3-ITD). Specifically, we observed that PKCε down-modulation significantly reduces murine AML cell survival (P<0.001) and colony formation in methylcellulose (P<0.001) of both MLL-AF9 and MLL-AF9;Flt3-ITD cells compared to non-targeting shRNA-expressing cells. We are currently investigating how PKCε inhibition impacts AML progression in vivo using mouse models of AML driven by MLL-AF9 or MLL-AF9;Flt3-ITD.

At the molecular level, we have found that PKCε is a key regulator of reactive oxygen species (ROS) biology in AML cells. Specifically, using a fluorogenic probe (CellRox) that indiscriminately detects most types of ROS, we have observed that PKCε knockdown in human AML cell lines results in increased steady-state levels of intracellular ROS compared to shRNA control cells (P<0.002). Total intracellular ROS levels are influenced by the production and clearance of distinct ROS types in various cellular compartments. To further characterize the localization and specific type(s) of ROS regulated by PKCε, we utilized four redox-sensitive GFP (roGFP) probes, which allow for direct measurement of cytoplasmic and mitochrondrial glutathione redox potential (Grx1-roGFP-Cyto and Grx1-roGFP-Mito, respectively) and hydrogen peroxide (H₂O₂) levels (Orp1-roGFP-Cyto and Orp1-roGFP-Mito, respectively) in live cells. Down-modulation of PKCε in NOMO-1 and OCI-AML-3 cells expressing each of these roGFP constructs resulted in a significant increase in the oxidation of Grx1-roGFP (P<.0007) and Orp1-roGFP-Mito (P<0.02) but not either of cytoplasmic constructs, suggesting that PKCε regulates the production of ROS in the mitochondria of AML cells. Since increased H₂O₂ production and glutathione oxidation results from increased superoxide (O₂^-) production in mitochondria, we next evaluated the impact of PKCε on O₂^- production. Using a fluorogenic probe (MitoSOX) that specifically detects O₂^- in live cells, we found that PKCε down-modulation increases the production of O₂^- in AML cells (P<0.05). Our future studies are focused on determining the precise molecular events that connect alterations in redox biology with the survival and differentiation of AML cells.

Collectively, these results uncover the previously unrecognized role of PKCε as a critical regulator of mitochondrial redox biology and supporter of cell survival and impaired differentiation in AML.