Mass cytometry reveals blunting of TNFα responses by pevonedistat in MF and sAML. (A-C) viSNE plots of healthy, normal BM donors (NBM 43, 44) and MF and sAML patient samples after signaling mass cytometry. (A) CD34⁺ HSPC population identified through extracellular surface staining with anti-CD34 antibody. Intracellular staining of phospho-p65/RELA (S529) (B) and IκBα (C) in patient samples treated with 1 µM pevonedistat for 1 hour, 20 ng/mL TNFα for 15 minutes, or in combination. (D) Heatmaps of signaling markers from CD34⁺ cells from 3 normal BM donors, 5 primary MF patients, and 2 sAML patients analyzed by mass cytometry. Cells from patient samples were treated with 1 µM pevonedistat for 1 hour, 20 ng/mL TNFα for 15 minutes, or in combination. Signals from each patient sample per treatment condition were normalized to its basal signal. (E) Dual counts of intracellular phospho-p65/RELA and IκBα from CD34⁺ cells from the 10 patient samples in panel D. *P < .05 by Mann-Whitney U test. NS, not significant.