Oral Presentation 35th Lorne Cancer Conference 2023

Second messenger signalling influences the kinome and methylome of kinase-activated paediatric acute myeloid leukemia. (#24)

Zacary P Germon 1 2 , Abdul Mannan 1 2 , Jonathan R Sillar 1 2 3 , Mika Persson 1 2 , Alicia Douglas 1 2 , Ryan Duchatel 1 2 , Andrew H Wei 4 , Martin R. R Larsen 5 , Frank Alvaro 6 , Janis Chamberlain 2 6 , Anoop K Enjeti 2 , Geoffry De Iuliis 7 , Nicole M Verrills 1 2 , Myron Evans 8 , Heather Lee 1 2 , Matthew D Dun 1 2
  1. Cancer Signalling Research Group, School of Biomedical Sciences and Pharmacy, College of Health, Medicine and Wellbeing, Callaghan, NSW, Australia
  2. Precision Medicine Program, Hunter Medical Research Institute, New Lambton Heights, NSW, Australia
  3. Haematology Department, Calvary Mater Hospital, Newcastle, NSW, Australia
  4. Australian Centre for Blood Diseases, Monash University, Melbourne, VIC, Australia
  5. Department of Molecular Biology and Biochemistry, Protein Research Group, University of Southern Denmark, Odense, Denmark
  6. John Hunter Children's Hospital, New Lambton Heights, NSW, Australia
  7. Priority Research Centre for Reproductive Sciences, Faculty of Science, University of Newcastle, Callaghan, NSW, Australia
  8. Ben Towne Center for Childhood Cancer Research, Seattle Children's Hospital, Seattle, Washington, USA

Acute myeloid leukaemia (AML) is the second most common paediatric leukaemia (25%) with the FMS-like tyrosine kinase 3 (FLT3) gene frequently mutated (20% of cases). FLT3-targeted therapies have entered the clinic; however, relapse rates remain high. Notably, FLT3-mutant patients harbour increased abundance of reactive oxygen species (ROS), that cause DNA damage, driving genomic instability and potentiating leukaemic cell proliferation. Using primary AML patient samples, AML cell lines and CD34+ normal bone marrow cells (NBM), we have simultaneously characterised proteins harbouring redox modifications (cysteine oxidation) and phosphorylation to determine how elevated ROS in AML contributes to oncogenic signalling. Redox-proteomics identified for the first time, that patients harbouring FLT3-mutations carry site specific reversible oxidation of cysteines residues within the catalytic domain of the DNA methyltransferase 1 (DNMT1), altering its methyltransferase activity. We identified that the NADPH oxidase 2 (NOX2) was the primary source of ROS/redox second messenger signalling in FLT3-mutant patients. Targeting NOX2 using specific inhibitors, and/or CRISPR-Cas9 gene knockout (CYBB-/-), reduced levels of oxidative stress, and when combined with FLT3-inhibitors, induced synergistic cell death in vitro and in vivo using AML patient-derived xenograft (PDX) mouse models. Importantly, either molecular or pharmacological inhibition of NOX2 decreased oxidation of DNMT1, resulting in a reciprocal increase in methylation of CpG islands in the oncogene Protein kinase B (AKT1) among others. NOX2 inhibition, also reduced cysteine oxidation of FLT3, in AML cells isolated from the bone marrow of AML PDX models, reducing phosphorylation and hence activity of the oncogenes STAT5 and MAPK/ERK. Here, we provide evidence that the oncogenic activity of FLT3-NOX2 promotes global second messenger signalling in a feed-forward loop. FLT3-ITD driven NOX2 activity influences methyltransferase activity and promotes epigenetic reprograming, driving expression of the oncogene AKT; an important novel link between oxidative stress and the regulation of the epigenome. These discoveries may have important clinical implications in the resistance setting for patients receiving standard of care pro-oxidant cytotoxic chemotherapies that promote ROS production.