INCA033989 antagonizes mutCALR-induced oncogenic signaling. (A-B) INCA033989 cell-binding profile. Binding of INCA033989 or isotype to Ba/F3-TPOR or Ba/F3-TPOR/CALR^del52 (A) and UT-7-TPOR or UT-7-TPOR/CALR^del52 (B) cells was assessed by flow cytometry (see supplemental Methods for details). The data represent the GMFI of 3 independent experiments. (C) INCA033989 antagonizes mutCALR-induced oncogenic signaling. Ba/F3-TPOR/CALR^del52 cells treated with increasing concentrations of INCA033989, isotype, or vehicle for 4 hours were barcoded, stained with phospho-specific antibodies, and evaluated using flow cytometry. The heat maps show mean fluorescence intensity from n = 3 independent experiments. The color scale represents the fold change over untreated Ba/F3-TPOR cells (baseline). (D-E) INCA033989 antagonizes mutCALR-induced oncogenic cell proliferation. Ba/F3-TPOR/CALR^del52 (D) and UT-7-TPOR/CALR^del52 (E) cells were treated with INCA033989 or isotype for 72 hours and cell proliferation was assessed. Inhibition of cell proliferation was calculated after normalization to maximal (100%) inhibition achieved by cell treatment with 2 to 5 μM of ruxolitinib and no inhibition (0%) in isotype-treated cells. The data are representative of 3 independent experiments. (F) INCA033989 induces the death of mutCALR-positive cells. Ba/F3-EPOR/JAK2^V617F and Ba/F3-TPOR/CALR^del52 cells were treated with INCA033989 or isotype for 48-hours pulse labeled with 5-bromo-2ʹ-deoxyuridine and stained with 7-Aminoactinomycin for cell cycle evaluation by flow cytometry (see supplemental Methods for details). Data represent the mean of triplicates ± SD and are representative of 2 independent experiments. AKT, protein kinase B; CREB, cyclic adenosine monophosphate responsive element binding protein; ERK, extracellular signal–regulated kinase; GMFI, geometric mean of fluorescence intensity; mTOR, mammalian target of rapamycin; SD, standard deviation.