Figure 1.
HMGA1 is upregulated during MPN progression and required for leukemia engraftment. (Ai) Relative HMGA1 expression (mean ± standard deviation [SD]) performed in triplicate (quantitative polymerase chain reaction) in CD34+ cells from patients with MPN with acquired JAK2V617F mutations (PV, n = 8; MF, n = 5; AML, n = 6) or control subjects (Normal; n = 2). RPLP0 was used to control for loading. *P < .05, 1-way ANOVA, followed by Tukey’s multiple-comparison test. (Aii) HMGA1 expression in CD34+ cells from BM of patients with MPN with JAK2V617F ET (n = 17), JAK2V617F PV (n = 26), or CALR-mutant ET (n = 7) or from unaffected individuals (n = 15) by microarray. Each point represents a single patient. *P < .05, **P <. 01, ***P < .001, 1-way ANOVA, followed by Tukey’s multiple-comparison test. (Aiii) HMGA1 expression in JAK2-mutant CD34+ PB cells from patients with MF compared with unmutated CD34+ cells from the same patient with MF (n = 8) and unaffected individuals (n = 2) by scRNAseq; each point represents the expression level for a single cell. **P < .01, ***P < .001, 1-way ANOVA, followed by Tukey’s multiple-comparison test. (Aiv) HMGA1 expression in lineage− CD34+ PB from patients with MF and age-matched healthy donors. HMGA1 was detected in 81.1% of cells (30 786/37 941) from healthy donors and in 89.7% of cells (34 038/37 941) from patients with MF. White dots on the violin plot indicate the mean level of expression; black points represent expression values for each single cell. ***P ≤ .001, Wilcoxon rank-sum test. (B) Western blot analysis of HMGA1, β-actin, and GAPDH (loading control) protein levels from DAMI, SET-2, and UKE-1 cells, with or without HMGA1 silencing. Western blots were performed 3 times; a representative blot is shown. Size markers (kDa) are indicated. (C) Proliferation (mean ± SD) estimated by MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; Promega)] in DAMI, SET-2, and UKE-1 cells, with or without HMGA1 silencing (control vs HMGA1-sh1 or HMGA1-sh2; mean ± SD, at the indicated time points. ***P < .001, 2-tailed Student t test. (D) Colony-forming units (CFU; mean ± SD) in DAMI, SET-2, and UKE-1 cells, with or without HMGA1 silencing (control vs HMGA1-sh1 or HMGA1-sh2), performed in triplicate from 2 independent experiments (upper panels). Representative images of colonies are shown (lower panels). Scale bars, 200 μm. ***P < .001; 2-tailed Student t test. (E) Edu (5-ethynyl-2′-deoxyuridine) cell cycle analysis (mean ± SD) by flow cytometry assessed in DAMI, SET-2 and UKE-1 cells, with or without HMGA1 silencing (control vs HMGA1-sh1 or HMGA1-sh2), performed in triplicate from 2 independent experiments; *P < .05, **P < .01, ***P < .001; 2-tailed Student t test. (F) Annexin V apoptosis by flow cytometry assessed in DAMI, SET-2, and UKE-1 cells, with or without HMGA1 silencing (control vs HMGA1-sh1 or HMGA1-sh2), performed in triplicate from 2 independent experiments, ***P < .001; 2-tailed Student t test. Representative density plots from each group are also shown. (G) Representative images of spleens and graphical comparisons of relative spleen weight (spleen/body weight %; mean ± SD); leukemia cell engraftment by flow cytometry (mean ± SD) in spleen from NSG mice injected with DAMI cells and SET-2 cells, with or without HMGA1 silencing (control vs HMGA1-sh1; n = 4 or 5 per group). *P < .05, **P < .01, ***P < .001; 2-tailed Student t test. (H) Leukemia cell engraftment (mean ± SD) assessed by flow cytometry in BM from NSG mice injected with DAMI cells (left panel) or SET-2 cells (middle panel), with or without HMGA1 silencing (control vs HMGA1-sh1; n = 4 or 5 per group). Representative flow cytometry plots from each group and hematoxylin and eosin-stained BM from DAMI cells (right panels). Scale bar, 200 μm. ***P < .001; 2-tailed Student t test. (I) Survival analysis by Kaplan-Meier estimate in NSG mice injected with DAMI cells, with or without HMGA1 silencing (Control: median survival, 24 days vs HMGA1-sh1: median survival, 38 days; n = 6 per group). ***P < .001, log-rank (Mantel-Cox) test. mRNA, messenger RNA; ns, not significant.

HMGA1 is upregulated during MPN progression and required for leukemia engraftment. (Ai) Relative HMGA1 expression (mean ± standard deviation [SD]) performed in triplicate (quantitative polymerase chain reaction) in CD34+ cells from patients with MPN with acquired JAK2V617F mutations (PV, n = 8; MF, n = 5; AML, n = 6) or control subjects (Normal; n = 2). RPLP0 was used to control for loading. *P < .05, 1-way ANOVA, followed by Tukey’s multiple-comparison test. (Aii) HMGA1 expression in CD34+ cells from BM of patients with MPN with JAK2V617F ET (n = 17), JAK2V617F PV (n = 26), or CALR-mutant ET (n = 7) or from unaffected individuals (n = 15) by microarray. Each point represents a single patient. *P < .05, **P <. 01, ***P < .001, 1-way ANOVA, followed by Tukey’s multiple-comparison test. (Aiii) HMGA1 expression in JAK2-mutant CD34+ PB cells from patients with MF compared with unmutated CD34+ cells from the same patient with MF (n = 8) and unaffected individuals (n = 2) by scRNAseq; each point represents the expression level for a single cell. **P < .01, ***P < .001, 1-way ANOVA, followed by Tukey’s multiple-comparison test. (Aiv) HMGA1 expression in lineage− CD34+ PB from patients with MF and age-matched healthy donors. HMGA1 was detected in 81.1% of cells (30 786/37 941) from healthy donors and in 89.7% of cells (34 038/37 941) from patients with MF. White dots on the violin plot indicate the mean level of expression; black points represent expression values for each single cell. ***P ≤ .001, Wilcoxon rank-sum test. (B) Western blot analysis of HMGA1, β-actin, and GAPDH (loading control) protein levels from DAMI, SET-2, and UKE-1 cells, with or without HMGA1 silencing. Western blots were performed 3 times; a representative blot is shown. Size markers (kDa) are indicated. (C) Proliferation (mean ± SD) estimated by MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; Promega)] in DAMI, SET-2, and UKE-1 cells, with or without HMGA1 silencing (control vs HMGA1-sh1 or HMGA1-sh2; mean ± SD, at the indicated time points. ***P < .001, 2-tailed Student t test. (D) Colony-forming units (CFU; mean ± SD) in DAMI, SET-2, and UKE-1 cells, with or without HMGA1 silencing (control vs HMGA1-sh1 or HMGA1-sh2), performed in triplicate from 2 independent experiments (upper panels). Representative images of colonies are shown (lower panels). Scale bars, 200 μm. ***P < .001; 2-tailed Student t test. (E) Edu (5-ethynyl-2′-deoxyuridine) cell cycle analysis (mean ± SD) by flow cytometry assessed in DAMI, SET-2 and UKE-1 cells, with or without HMGA1 silencing (control vs HMGA1-sh1 or HMGA1-sh2), performed in triplicate from 2 independent experiments; *P < .05, **P < .01, ***P < .001; 2-tailed Student t test. (F) Annexin V apoptosis by flow cytometry assessed in DAMI, SET-2, and UKE-1 cells, with or without HMGA1 silencing (control vs HMGA1-sh1 or HMGA1-sh2), performed in triplicate from 2 independent experiments, ***P < .001; 2-tailed Student t test. Representative density plots from each group are also shown. (G) Representative images of spleens and graphical comparisons of relative spleen weight (spleen/body weight %; mean ± SD); leukemia cell engraftment by flow cytometry (mean ± SD) in spleen from NSG mice injected with DAMI cells and SET-2 cells, with or without HMGA1 silencing (control vs HMGA1-sh1; n = 4 or 5 per group). *P < .05, **P < .01, ***P < .001; 2-tailed Student t test. (H) Leukemia cell engraftment (mean ± SD) assessed by flow cytometry in BM from NSG mice injected with DAMI cells (left panel) or SET-2 cells (middle panel), with or without HMGA1 silencing (control vs HMGA1-sh1; n = 4 or 5 per group). Representative flow cytometry plots from each group and hematoxylin and eosin-stained BM from DAMI cells (right panels). Scale bar, 200 μm. ***P < .001; 2-tailed Student t test. (I) Survival analysis by Kaplan-Meier estimate in NSG mice injected with DAMI cells, with or without HMGA1 silencing (Control: median survival, 24 days vs HMGA1-sh1: median survival, 38 days; n = 6 per group). ***P < .001, log-rank (Mantel-Cox) test. mRNA, messenger RNA; ns, not significant.

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