Poster Presentation 27th Annual Lorne Proteomics Symposium 2022

Assessment of redox post-translational modifications (OxPTMs) reveals the mechanisms underpinning oncogenic signaling in FLT3-mutant Acute Myeloid Leukaemia   (#117)

Zacary P Germon 1 2 , Jonathan R Sillar 1 2 3 , Abdul Mannan 1 2 , Ryan J Duchatel 1 2 , Heather C Murray 1 2 , Dilana Staudt 1 2 , Izac J Findlay 1 2 , Alicia Douglas 1 2 , Honggang Huang 4 , Frank Alvaro 2 5 , Janis Chamberlain 2 5 , Andrew Wei 6 , Geoffry N De luliis 7 , John Aitken 7 , Anoop K Enjeti 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
  5. John Hunter Children's Hospital, New Lambton Heights, NSW, Australia
  6. Australian Centre for Blood Diseases, Monash University, Melbourne, VIC, Australia
  7. Priority Research Centre for Reproductive Sciences, Faculty of Science, University of Newcastle, Callaghan, NSW, Australia

Reactive oxygen species (ROS) are crucial regulators of cell signalling via their ability to oxidise enzymes at cysteine residues. ROS driven oxidative stress is a hallmark of acute myeloid leukaemia (AML), driving genomic instability, clonal evolution, and treatment resistance. However, proteins influenced by ROS-induced redox post-translational modifications (oxPTMs) remain unknown. Here we aimed to characterise the oxPTMs in primary AML patient samples to identify novel treatment targets both alone and in combination with clinically relevant therapies. First, oxPTMs of AML patient samples were characterised using a Cysteine specific Phosphonate Adaptable Tag (CysPAT) to simultaneously enrich for peptides containing reversibly oxidised cysteine residues and phosphorylated peptides. In doing so, 2946 proteins were identified containing OxPTMs, associated with patients harbouring mutations in the receptor tyrosine kinase FLT3 (FLT3-ITD). FLT3-ITD+ patient samples showed increased oxPTMs in proteins of the Src-family kinases and the NADPH oxidase-2 complex (NOX2), directly responsible for ROS production. Combined NOX2 and FLT3 inhibition was synergistically cytotoxic to FLT3-mutant AML patient samples ex vivo and in vivo, significantly increasing the survival of FLT3-ITD+ AML patient derived xenograft (PDX) mouse models as a monotherapy. Analysis of global oxPTMs in AML cells harvested from the bone marrow of PDX models following NOX2 inhibition, revealed decreased total NOX2 expression and significantly decreased oxPTMs in FLT3 at Cys828, thereby inhibiting downstream signalling. Analysis of oncogenic signalling pathways downstream of FLT3 following NOX2 inhibition, revealed decreased activity of the AML transcription factor pSTAT5, and proliferation pathway pERK. Here, we highlight the importance of analysis of oxPTMs and their contribution to oncogenic signalling in kinase active AML patient samples and cell lines. In doing so, we reveal the clinical relevance of targeting ROS production using NOX-inhibitors which may improve the clinical utility and durability of precision therapies currently used in the clinic.