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

Pathways identification by phosphoproteomic profiling identifies DNA-PK as a novel therapeutic target in Acute Myeloid Leukaemia (AML) (#740)

Heather Murray 1 , Anoop Enjeti 2 , Richard Kahl 1 , Hayley Flanagan 1 , Jonathan Sillar 1 2 , David Skerrett-Byrne 1 , Juhura (Trisha) Al-mazi 1 , Nathan Smith 3 , Gough Au 4 , Kathryn Evans 5 , Richard Lock 5 , Charles DeBock 6 , Martin Larsen 7 , Nicole Verrills 1 8 , Matthew Dun 1 8
  1. Precision Medicine Research Program, Hunter Medical Research Institute, and School of Biomedical Sciences and Pharmacy Faculty of Health and Medicine, University of Newcastle, Callaghan, NSW, Australia
  2. Calvary Mater Hospital, Newcastle, NSW, Australia
  3. Analytical and Biomolecular Research Facility Advanced Mass Spectrometry Unit, University of Newcastle, Newcastle, New South Wales, Australia
  4. Viralytics, Hunter Medical Research Institute, and School of Biomedical Sciences and Pharmacy Faculty of Health and Medicine, Newcastle, New South Wales, Australia
  5. Children’s Cancer Institute, Lowy Cancer Research Centre, University of New South Wales , Sydney, New South Wales, Australia
  6. VIB Center for the Biology of Disease, and KU Leuven Center for Human Genetics, Leuven, Belgium
  7. Department of Molecular Biology and Biochemistry, Protein Research Group, University of Southern Denmark, Odense, Denmark
  8. *these authors contributed equally to this work, ,

Acute Myeloid Leukaemia (AML) is the most aggressive form of acute leukaemia, with a 5-year survival rate of 24%. Activating mutations in the receptor tyrosine kinase FLT3 are the most common driver mutations in AML (25-30% of patients). Inhibiting the FLT3 receptor as a mono-therapeutic strategy in AML has proven difficult however, due to the development of treatment resistance and relapse. Characterisation of the oncogenic signalling pathways downstream of FLT3 is required to identify improved therapeutic approaches for AML. To this end, we have performed phosphoproteomic analysis of primary blasts from 7 AML patients (4 FLT3-mutant, 3 FLT3-wildtype). Tryptic peptides were labelled with isobaric tags prior to multistage phosphopeptide enrichment using titanium dioxide, sequential elution from IMAC, and offline HPLC fractionation, followed by analysis on an Orbitrap Velos. 6,303 phosphopeptides were identified across the 7 AML patient blast samples. Analysis of differentially expressed phosphoproteins in FLT3-mutant versus FLT3-wildtype AML patients revealed dysregulation of DNA double strand break repair pathways, with increased phosphorylation of Non-Homologous End joining (NHEJ) proteins; indicating NHEJ pathway activation. Kinase enrichment analysis predicted increased activity of the NHEJ core kinase, DNA-PK, in FLT3-mutant samples. Accordingly, cell viability assays revealed that FLT3-mutant cell lines were sensitive to pharmacological inhibition of DNA-PK. Inhibition of DNA-PK kinase activity combined with inhibition of the FLT3 receptor led to synergistic induction of cell death, selectively in FLT3-mutant cell lines and in FLT3-mutant primary AML patient samples ex vivo. Furthermore, DNA-PK inhibitor therapy combined with FLT3 inhibition significantly prolonged survival compared to either monotherapy in an orthotopic human xenograft mouse model of AML. In conclusion, phosphoproteomic analysis of primary AML samples has enabled identification of novel therapeutic avenues. Targeting DNA-PK in combination with standard therapeutic agents has the potential to improve outcomes for this poor prognosis cancer.