Global phosphotyrosine profiling identifies new therapeutic targets in CRLF2-rearranged Ph-like acute lymphoblastic leukaemia

KCS Sia1, L Zhong2, MJ Raftery2 and RB Lock1

  1. Children's Cancer Institute, Lowy Cancer Research Centre, UNSW Australia, Sydney, Australia
  2. Bioanalytical Mass Spectrometry Facility, Mark Wainwright Analytical Centre, UNSW Australia, Sydney, Australia

A recently identified high-risk subtype, termed “Ph-like”, comprises up to 15% of childhood B-cell precursor ALL and is associated with very poor clinical outcome. Approximately 50% of paediatric Ph-like cases harbour rearrangements and overexpression of the receptor tyrosine kinase cytokine receptor-like factor 2 (CRLF2). While clinically approved agents targeting CRLF2 do not yet exist, tyrosine kinase inhibitors (TKIs) can be used to target kinases downstream of CRLF2. This study aimed to identify targetable tyrosine kinases deregulated by CRLF2 that are critical for the survival of Ph-like ALL cells. We used an immunoaffinity enrichment phosphotyrosine profiling technique coupled with stable isotope labelling of amino acids in cell culture (SILAC) and mass spectrometry to identify TKs regulated by CRLF2 in Ph-like ALL cell lines (MHH-CALL-4 and MUTZ-5) stimulated with the CRLF2 ligand thymic stromal lymphopoietin (TSLP). TSLP caused increased tyrosine phosphorylation of several well-known TKs and substrates, including JAK1, JAK2, STAT5, and ERK1/2. TSLP also increased tyrosine phosphorylation of the insulin receptor (INSR) and fibroblast growth factor receptor 1 (FGFR1). Based on these findings, fixed-ratio cytotoxicity assays were carried out to investigate the possible synergistic effects of combining specific pathway inhibitors. In both MHH-CALL-4 and MUTZ-5, dual inhibition of INSR and FGFR1 using the TKIs BMS-754807 and ponatinib was strongly synergistic, and was replicated in patient-derived xenografts. In conclusion, this study has demonstrated the feasibility of rationally designing combination drug treatments using unbiased phosphotyrosine profiling. The dual inhibition of INSR and FGFR1 represents a novel strategy to target CRLF2-rearranged Ph-like ALL.