Poster Presentation HUPO 2019 - 18th Human Proteome Organization World Congress

Quantitative redox proteomics in FLT3-ITD acute myeloid leukaemia patient samples revealed reactive oxygen species regulated oncogenic signalling, a novel treatment target. (#536)

Zacary P Germon 1 2 , Jonathan R Sillar 1 2 3 , Honggang Huang 4 , Heather C Murray 1 2 , Ryan Duchatel 1 2 , Anoop K Enjeti 1 2 3 , Martin R Larsen 4 , Nicole M Verrills 1 2 , Matthew D Dun 1 2
  1. Faculty of Health and Medicine , University of Newcastle, Newcastle, NSW, Australia
  2. Cancer Research Program, Hunter Medical Research Institute, Newcastle, NSW, Australia
  3. Haematology Department, Calvary Mater Hospital, Newcastle, NSW, Australia
  4. Department of Molecular Biology and Biochemistry, Protein Research Group, University of Southern Denmark, Odense, Denmark

Reactive oxygen species (ROS) are a heterogeneous group of molecules including superoxide anion and hydrogen peroxide, once considered a benign by-product of cellular metabolism. It is now widely accepted that ROS play an intricate role in regulating cellular signalling, implicit in haematopoietic stem cell growth, differentiation and self-renewal. However, under situations of oxidative stress, ROS promote tumourigeneses in all cancers, particularly haematopoietic malignancies. The radical nature of ROS, promote rapid oxidative modifications to proteins via the oxidation of critical cysteines of signalling protein when cellular defences (antioxidants) become depleted. To determine the role ROS play in leukaemogenesis we assessed the oxidative status of cysteines known to be redox sensitive. Blast cells from 14 acute myeloid leukaemia (AML) patients, either harbouring FLT3-ITD mutations or wild-type FLT3 were subjected to high-resolution quantitative phosphoproteomic profiling and protein cysteine oxidation analysis using Cysteine-specific Phosphonate Adaptable Tags (CysPAT) to selectively label cysteine-containing peptides. These peptides were then enriched with titanium dioxide (TiO2) and subjected to mass spectrometry analysis. FLT3-ITD+ patients showed significantly increased expression of NADPH oxidase 2 (NOX2) and associated subunits, directly responsible for the production of ROS. Oxidation and inactivation of key FLT3 tumour suppressor proteins were seen in FLT3-ITD+ compared to wild-type patients. Proteins important in maintaining cellular homeostasis, such as antioxidants were differentially oxidised between patient subtypes supporting the notion of Redox dysfunction in FLT3-ITD+ AML. Targeting NOX2 in combination with FLT3 inhibitors currently in clinical use, significantly reduced cytoplasmic ROS and synergistically triggered cytotoxic cell death. Reducing oxidative stress switched off oncogenic signalling in key growth and survival signalling pathways, to activate apoptotic pathways. Our investigations shows, cooperation between oncogenic kinases, metabolism and oxidative stress, revealing a novel treatment paradigm currently under preclinical evaluation.