S1. In vitro effects of TSLP on target cells as assessed by Alamar Blue assay. S2. Demographics of phosphosites identified from MHH-CALL-4 and MUTZ-5 cells in global P-Tyr profiling. S3. In vitro single agent efficacy of BMS-754807 and ponatinib against CRLF2r cell lines and PDXs. S4. siRNA knockdown of IGF1R and FGFR1 in MHH-CALL-4 and MUTZ-5 cell lines. S5. Tolerability testing of ponatinib in combination with BMS754807 in naïve NSG mice as assessed by percentage of body weight change. S6. Ex vivo combination effect of BMS-754807 and ponatinib over time against CRLF2r Ph-like ALL PDXs. S7. Plasma concentrations of BMS-754807 and ponatinib at specific timepoints.
ARTICLE ABSTRACTPhiladelphia (Ph)-like acute lymphoblastic leukemia (ALL) is characterized by aberrant activation of signaling pathways and high risk of relapse. Approximately 50% of Ph-like ALL cases overexpress cytokine receptor-like factor 2 (CRLF2) associated with gene rearrangement. Activated by its ligand thymic stromal lymphopoietin (TSLP), CRLF2 signaling is critical for the development, proliferation, and survival of normal lymphocytes. To examine activation of tyrosine kinases regulated by TSLP/CRLF2, phosphotyrosine (P-Tyr) profiling coupled with stable isotope labeling of amino acids in cell culture (SILAC) was conducted using two CRLF2-rearranged (CRLF2r) Ph-like ALL cell lines stimulated with TSLP. As a result, increased P-Tyr was detected in previously reported TSLP-activated tyrosine kinases and substrates, including JAK1, JAK2, STAT5, and ERK1/2. Interestingly, TSLP also increased P-Tyr of insulin growth factor 1 receptor (IGF1R) and fibroblast growth factor receptor 1 (FGFR1), both of which can be targeted with small-molecule inhibitors. Fixed-ratio combination cytotoxicity assays using the tyrosine kinase inhibitors BMS-754807 and ponatinib that target IGF1R and FGFR1, respectively, revealed strong synergy against both cell line and patient-derived xenograft (PDX) models of CRLF2r Ph-like ALL. Further analyses also indicated off-target effects of ponatinib in the synergy, and novel association of the Ras-associated protein-1 (Rap1) signaling pathway with TSLP signaling in CRLF2r Ph-like ALL. When tested in vivo, the BMS-754807/ponatinib combination exerted minimal efficacy against 2 Ph-like ALL PDXs, associated with low achievable plasma drug concentrations. Although this study identified potential new targets in CRLF2r Ph-like ALL, it also highlights that in vivo validation of synergistic drug interactions is essential.
Quantitative phosphotyrosine profiling identified potential therapeutic targets for high-risk CRLF2-rearranged Ph-like ALL.