RIPK3 is significantly increased in MF and correlates with disease burden. (A) Intracellular RIPK3 protein expression of PBMCs from 9 healthy control subjects, 11 patients with ET, 10 patients with PV, 8 patients with pre-MF, 16 patients with MF, and 3 patients with sAML after a history of MPN were quantified via flow cytometry. RIPK3 protein expression was calculated as the ratio of stained antibody MFI divided by isotype control MFI. Shown are the mean and error bars denoting standard deviation. The presence of a JAK2 V16F mutation is indicated by the coloration of the individual patient samples. One-way ANOVA P < .0001; P values from post hoc analysis with Tukey test as indicated in the figure. (B) RIPK3 gene expression (mRNA) was analyzed in primary human CD34⁺ PBMCs from 39 patients with PMF (including 18 JAK2 V16F mutated and 21 JAK2 V617F wild-type patients) and 16 healthy control subjects using the Human Genome U219 Array (GEO: GSE53482). Shown are the mean and error bars denoting standard deviation. P values from Student t test as indicated in the figure. (C-D) Analysis of single-cell RNA sequencing data of primary human CD34⁺ HSPCs from a public database (GEO: GSE144568). (C) Dimensionality reduction by t-SNE summarizing all MF and control cells analyzed. Each dot represents a single cell. Left panel: t-SNE colored by RIPK3 expression (low [gray] or high [blue]). Middle panel: t-SNE colored by disease status (healthy donors [blue] or MF [red]). Right panel: t-SNE colored by hematopoietic cell types according to the expression of lineage signature genes as indicated in the figure. (D) Boxplots indicating RIPK3 expression within positive cells in MF and control cells, normalized by the number of positive cells. (E-G) RIPK3 protein expression in PBMCs was analyzed and calculated as described in Figure 1A. (E) RIPK3 protein expression in PBMCs from 17 patients with MF (including PMF, post-PV MF, and post-ET MF) including 6 patients with DIPSS-based classification as intermediate-risk I, 8 intermediate-risk II patients, and 3 high-risk patients. Shown are the mean and error bars denoting standard deviation. One-way ANOVA P = .0133; P values from post hoc Tukey test as indicated in the figure. (F) Linear regression analysis of RIPK3 protein levels and leucocyte count (G/L) in patients with MF (including PMF, post-ET MF, and post-PV MF) (n = 16). Pearson r = 0.562, P = .0235, y = 2.552x – 0.5553. (G) Linear regression analysis of RIPK3 protein levels with spleen length (cm) in patients with MF (including PMF, post-ET M, F, and, post-PV MF; n = 8). Pearson r = 0.7705, P = .0252, y = 3.095x + 5.833. All panels: level of significance .05: ∗P < .05, ∗∗P < .005, ∗∗∗P < .0005 and ∗∗∗∗P <.0001. ANOVA, analysis of variance; DIPSS, Dynamic International Prognostic Scoring System; HSPC, hematopoietic stem and progenitor cell; MFI, mean fluorescence intensity; mRNA, messenger RNA; PMF, primary myelofibrosis; sAML, secondary acute myeloid leukemia; t-SNE, t-distributed stochastic neighbor embedding.