INCA033989 antagonizes mutCALR-induced oncogenic signaling and megakaryocyte differentiation in primary CD34⁺ patient cells. (A-C) A subpopulation of CD34⁺ cells isolated from mutCALR-positive patients with MF express mutCALR. (A) Representative flow cytometric analysis of the Lin^– peripheral blood mononuclear cells from a healthy individual (control) or a CALR^del52 patient with MF (mutCALR). (B) Flow cytometric analysis of Lin^–CD34⁺CD38^– cells from a healthy individual (control) and a patient with MF (mutCALR). (C) Normalized mutCALR signal intensity in Lin^–/CD34⁺/CD38^–/CD45Ra^–/CD90⁺ HSCs, Lin⁻/CD34⁺/CD38⁻ HSPCs, and Lin⁻/CD34⁺/CD41⁺ MkPs from 7 patients with MF (mutCALR). (D) Western blot analysis of CD34⁺ cells isolated from a patient with MF (mutCALR) or from human cord blood from a healthy individual (wtCD34⁺) treated with different concentrations of INCA033989 or an IgG control (10 μg/mL). pSTAT3/5 expression was induced by TPO (50 ng/mL) in wtCD34⁺ cells. (E-G) CD34⁺ progenitor cells from a healthy individual (wt) or patients with MF carrying the JAK2^V617F or CALR mutations were cultured in stem cell factor alone or the presence of the indicated concentrations of INCA033989 or control IgG (10 μg/mL) for 6 days. The impact of INCA033989 on CD34⁺-derived cell populations was assessed by flow cytometry. Significance was calculated using analysis of variance (ANOVA). ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .000001. Percentages of wt and mutCALR Lin^–/CD34⁺/CD38^– HSPCs. (E) Representative data from cells from a single patient. (F) JAK2^V617F (n = 5), CALR^del52 (n = 15), and CALR^ins5 (n = 9) megakaryocytes after treatment with 10 or 25 μg/mL of isotype or INCA033989. (G) Percentages of mutCALR Lin^–/CD34⁺/CD38^– HSPCs after 6 and 12 days of INCA033989 treatment. (H) INCA033989 is internalized upon the binding of mutCALR to the surface of primary CD34⁺ cells. CD34⁺ cells from a mutCALR-positive patient with MF were treated with 2 μg/mL of AF647-labeled INCA033989 for 18 hours, prepared for microscopy, and evaluated using the ZEN software suite. Green: cell membrane; blue: nucleus; magenta: INCA033989. Lin, lineage; MkP, megakaryocyte progenitor; ns, not significant.