Figure S2 from Protein Kinase C Epsilon Is a Key Regulator of Mitochondrial Redox Homeostasis in Acute Myeloid Leukemia

https://doi.org/10.1158/1078-0432.22464702

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posted on 2023-03-31, 19:45 authored by Daniela Di Marcantonio, Esteban Martinez, Simone Sidoli, Jessica Vadaketh, Margaret Nieborowska-Skorska, Anushk Gupta, Jake M. Meadows, Francesca Ferraro, Elena Masselli, Grant A. Challen, Michael D. Milsom, Claudia Scholl, Stefan Fröhling, Siddharth Balachandran, Tomasz Skorski, Benjamin A. Garcia, Prisco Mirandola, Giuliana Gobbi, Ramiro Garzon, Marco Vitale, Stephen M. Sykes

<p>Supplemental figure 2. (A) Schematic representation of the in vivo experimental conditions carried out in Figures 1A-C. (B) FACS gating strategy for sorting 7AAD-/GFP+ mouse MLL-AF9 leukemia cells transduced with CTRL shRNA or PkcÎµ shRNA. Cell sorted 48 hours post-transduction and FACS isolated cells were analyzed for purity. (C) Western blot analysis of PKCÎµ expression in mouse MLL-AF9 leukemia cells transduced with CTRL shRNA or PkcÎµ shRNA GFP and sorted 24, 48 and 72 hours after transduction. The original exposure is shown in the upper panel. Brightness was modified in the lowest panel to clarify differences between conditions. (D & E) In vitro competitive growth curve of mouse MLL-AF9 leukemia cells transduced with CTRL shRNA or PkcÎµ shRNA were analyzed at the indicated time points for (D) percentage of GFP-positive (% GFP+) cells plotted versus (E) fold change in the percentage of GFP-positive cells relative to Day 2 post-transduction. The data is representative on one experiment.</p>

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NIH

Rotary Foundation

CURE supplement

Fox Chase Cancer Center

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DOI - Is supplement to Protein Kinase C Epsilon Is a Key Regulator of Mitochondrial Redox Homeostasis in Acute Myeloid Leukemia

ARTICLE ABSTRACT

Purpose: The intracellular redox environment of acute myeloid leukemia (AML) cells is often highly oxidized compared to healthy hematopoietic progenitors and this is purported to contribute to disease pathogenesis. However, the redox regulators that allow AML cell survival in this oxidized environment remain largely unknown.Experimental Design: Utilizing several chemical and genetically-encoded redox sensing probes across multiple human and mouse models of AML, we evaluated the role of the serine/threonine kinase PKC-epsilon (PKCϵ) in intracellular redox biology, cell survival and disease progression.Results: We show that RNA interference-mediated inhibition of PKCϵ significantly reduces patient-derived AML cell survival as well as disease onset in a genetically engineered mouse model (GEMM) of AML driven by MLL-AF9. We also show that PKCϵ inhibition induces multiple reactive oxygen species (ROS) and that neutralization of mitochondrial ROS with chemical antioxidants or co-expression of the mitochondrial ROS-buffering enzymes SOD2 and CAT, mitigates the anti-leukemia effects of PKCϵ inhibition. Moreover, direct inhibition of SOD2 increases mitochondrial ROS and significantly impedes AML progression in vivo. Furthermore, we report that PKCϵ over-expression protects AML cells from otherwise-lethal doses of mitochondrial ROS-inducing agents. Proteomic analysis reveals that PKCϵ may control mitochondrial ROS by controlling the expression of regulatory proteins of redox homeostasis, electron transport chain flux, as well as outer mitochondrial membrane potential and transport.Conclusions: This study uncovers a previously unrecognized role for PKCϵ in supporting AML cell survival and disease progression by regulating mitochondrial ROS biology and positions mitochondrial redox regulators as potential therapeutic targets in AML. Clin Cancer Res; 24(3); 608–18. ©2017 AACR.