Extended Data Fig. 10: MCL-1 rescue of Ybx1-targeted cells and phosphoproteome analysis of primary Jak2-mutated cells upon JAK inhibitor treatment.
From: Splicing factor YBX1 mediates persistence of JAK2-mutated neoplasms
a, Proteome analysis of mouse Jak2-mutated cells following inactivation of Ybx1 by two shRNAs compared to non-targeting control. Heatmap representation of significantly enriched GO term biological processes in YBX1-depleted JAK2VF cells assessed by Fisher’s exact test (P (–log10) shown). b, Quantification of MCL-1 phosphosite pT144 in sh1Ybx1, sh2Ybx1 and shNT control in mouse JAK2VF cells. The y axis is the log2 intensity of the phosphopeptide (n = 4 biological replicates, two-tailed Student t-test). shNT: min = 23.8; max = 24.7; whisker = [23.8–24.7]; box = [24–24.2], sh1Ybx1: min = 19.8; max = 22; whisker = [19.8–22]; box = [21.3–21.6], sh2Ybx1: min = 19.2; max = 22.4; whisker = [19.2–22.4]; box = [21.8–22.4]. c, Scatter dot plot of MCL-1 phosphosite pT144 after respective kinase inhibitor treatment (n = 3, biological replicates, P values using two-tailed Student t-test, error bars represent ± s.d.). The y axis is the z-scored, log2 phosphopeptide intensity. DMSO: min = –0.09; max = 1.02; whisker = [–0.09 to 1.02], RUX: min = -0.554; max = 0.135; whisker = [–0.55 to 0.135], AKT inhibitor: min = 1.071; max = 1.263; whisker = [1.07–1.2634], PI3Ki: min = –1.008; max = –0.89; whisker = [–1.008 to 0.89], MEK inhibitor: min = –1.266; max = –0.699; whisker = [–1.266 to 0.699]. d, Western blot analysis of MCL-1, YBX1, BIM and BCL-XL following genetic inactivation of Ybx1 with four different shRNA constructs, compared to non-targeting control. n = 3 independent experiments. e, Measurement of apoptosis (annexin V- and Sytox-positive cells) after genetic inactivation of Ybx1 and concomitant JAK inhibitor treatment (RUX, 100 nM, 500 nM). Rescue by ectopic overexpression of MCL-1 (n = 4 independent experiments, two-tailed t-test). f, Western blot analysis of Ba/F3 JAK2VF cells showing downregulation of MKNK1 protein abundance following overnight combination treatment with RUX and Tram. n = 3 biological independent experiments. g, Peripheral blood cell analysis of human cell chimerism in NSGW41 humanized mice at week 4 and 20 (n = 5 per cohort). h, Heatmap shows unsupervised hierarchical clustering of significantly regulated (t-test with permutation-based FDR < 0.01) phosphosites with (n = 24) and without (n = 24) JAK inhibitor treatment in Jak2-mutated primary patient samples. Phosphoproteome analysis of Jak2-mutated primary patient samples (total n = 48) samples following in vitro (n = 18, JAK inhibitor treatment for 2 h) or in vivo (n = 6, 2 h post dosing of RUX samples) exposure to ruxolitinib. i, Network map of significantly enriched GO terms (P value <0.01) of dephosphorylated proteins upon JAK inhibitor treatment. Two-sided hypergeometric test, P value correction–Bonferroni step down. j, Box plot shows no significant changes in the MAPK1 and MAPK3 phosphorylation in control (n = 24) vs JAK inhibitor treated (n = 24) patient samples. MAPK1pT185 (DMSO): min = 19.61; max = 24.82; whisker = [19.61–24.82]; box = [21.34–23.11], MAPK1pT185 (RUX): min = 19.5; max = 24.81; whisker = [19.5–24.81]; box = [21.64–23.72], MAPK1pY187 (DMSO): min = 21.04; max = 26.45; whisker = [21.04–26.45];box = [23.22-25.46], MAPK1pY187(RUX): min = 21.09; max = 25.89; whisker = [21.09–25.89]; box = [22.63–24.77], MAPK3pT202(DMSO): min = 19.72; max = 26.06; whisker = [19.72–26.06]; box = [21.46–24.78], MAPK3pT202 (RUX): min = 20.41; max = 26.93; whisker = [20.41–26.93]; box = [22.05–23.88], MAPK3pY204 (DMSO): min = 20.70; max = 26.51; whisker = [20.70–26.51]; box = [22.02–24.99], MAPK3pY204 (RUX): min = 20.31; max = 25.76; whisker = [20.31–25.76]; box = [22.72–24.42]. k, Box plot shows significant changes in IKBKB, STAT3 and STAT5 phosphorylation in control (n = 24) vs JAK inhibitor treated (n = 24) patient samples. P values as determined by Mann–Whitney test. IKBKBpS697 (DMSO): min = 20.66; max = 25.79; whisker = [20.66–25.79]; box = [22.89–24.40], IKBKBpS697 (RUX): min = 20.66; max = 25.04; whisker = [20.66–25.04]; box = [21.88–23.88], IKBKBpS672 (DMSO): min = 20.71; max = 26.30; whisker = [20.71–26.30]; box = [22.5–23.86], IKBKBpS672 (RUX): min = 18.85; max = 24.73; whisker = [18.85–24.73]; box = [21.44–23.24], STAT5pY699 (DMSO): min = 22.02; max = 25.33; whisker = [22.02–25.33]; box = [22.73–23.63], STAT5pY699 (RUX): min = 19.90; max = 24.83; whisker = [19.90–24.83]; box = [21.8–23.2], STAT3pY705 (DMSO): min = 21.87; max = 26.66; whisker = [21.87–26.66]; box = [22.41–25.62], STAT3pY705 (RUX): min = 20.07; max = 26.17; whisker = [20.07–26.17]; box = [21.22–23.26]. l, Schematic depicting the mechanism of YBX1-mediated JAK inhibitor persistence.
