Figure 2.

Identification of effective single agents and drug combinations using a ZMYM2:FGFR1 Ba/F3 cell line model. A. The wild type ZMYM2:FGFR1 fusion (ZMYM2:FGFR1^WT) transformed murine Ba/F3 pro-B cell lines and supported interleukin 3 (IL-3)-independent growth. No growth was observed in the Ba/F3 cells expressing the empty vector (MSCV puro). The average of total viable cells was recorded and plotted over days post-IL-3 withdrawal. B-C. Evaluation of sensitivity of Ba/F3 ZMYM2:FGFR1^WT cells to tyrosine kinase inhibitors (TKIs; (B)) and FGFR inhibitors (FGFRi; (C)) that have been FDA-approved or are in advanced stages of approval. Sensitivities are represented as IC₅₀ values derived for a 12-point concentration series ranging from 1 μM to 0.005 nM. Cells transduced with MSCV puro empty vector were used as a control. Cells were treated in triplicate and viabilities were measured at 72 hours by MTS assay. D-E. FGFR inhibitors pemigatinib, ponatinib, and infigratinib were used in combination with either midostaurin or trametinib and plotted to display areas of synergy. Pemigatinib and infigratinib were used at concentration ranges of 0.041 to 10 nM, and ponatinib was used at a concentration range of 0.205 to 50 nM in combination with either (D) midostaurin (2.057 to 500 nM) or (E) trametinib (0.411 to 100 nM). Combination dose response curves compare ZMYM2:FGFR1^WT sensitivities (bottom) with empty vector control (MSCV; top) at increasing concentrations. Heatmaps of synergy scores were calculated using the highest single agent (HSA) model (¹⁰; top right plot) across the full matrix of combined doses tested for each drug pair. Dose-response curves for the ZMYM2:FGFR1^WT cells using only constant ratio dose combinations of FGFRi with either midostaurin or trametinib (indicated as blue squares on synergy plots) are plotted in comparison to sensitivities for single-agent pemigatinib, ponatinib, or infigratinib (bottom right graphs of each panel). F. Ponatinib combined with midostaurin inhibits activation of downstream signaling pathways. Cells were serum starved overnight and treated with either a DMSO vehicle control, each single agent (midostaurin or ponatinib), or a combination of ponatinib and midostaurin at the concentrations indicated for 4 hours at 37°C. Western blots were performed to evaluate downstream signaling proteins: ph-AKT, ph-PLCy1, ph-Stat3, and ph-FLT3. V5 antibodies were used to detect the ZMYM2:FGFR1^WT protein. GAPDH was used as a loading control