Figure 6.
GATA2 restoration partially rescues antileukemogenic effects of HMGA1 silencing; GATA2 and HMGA1 are coexpressed in primary MPN and HMGA1 networks and are activated in leukemic blasts from patients with MPN. (A) Relative HMGA1 and GATA2 expression (mean ± standard deviation [SD]) from 2 experiments performed in triplicate (quantitative polymerase chain reaction) in DAMI, SET-2, and UKE-1 cells (control vs HMGA1-sh1 vs HMGA1-sh1 + GATA2 overexpression [OE]). GAPDH was used to control for loading. (B) Western blots of HMGA1, GATA2, β-actin, and GAPDH (loading control) protein levels from DAMI, SET-2, and UKE-1 cells transduced with lentivirus (control vs HMGA1-sh1 vs HMGA1-sh1 + GATA2 OE). Western blots were performed 3 times with similar results; a representative blot is shown. Size markers (kDa) are indicated. (C) Colony-forming units (CFU; mean ± SD) from DAMI, SET-2, and UKE-1 cells (control vs HMGA1-sh1 vs HMGA1-sh1 + GATA2 OE) in 2 experiments. Representative images of colonies are shown. Scale bars, 200 μm. (D) Annexin V apoptosis by flow cytometry in DAMI, SET-2, and UKE-1 cells (control vs HMGA1-sh1 vs HMGA1-sh1 + GATA2 OE) in 2 experiments. (E) Survival curve (Kaplan-Meier plots) in NSG mice injected with DAMI cells (control vs HMGA1-sh1 vs HMGA1-sh1 + GATA2 OE; n = 6). (F) Leukemia cell engraftment assessed by flow cytometry (mean ± SD) in BM from NSG mice injected with DAMI cells transduced with lentivirus (control vs HMGA1-sh1 vs HMGA1-sh1 + GATA2 OE; n = 4 per group). Representative BM histology (hematoxylin and eosin stain) from each group are shown. Scale bar, 200 μm. (Gi) HMGA1 and GATA2 expression (RNAseq) from PBMCs from a cohort of matched patient samples with MF that progressed to AML (n = 11 per group). (Gii) HMGA1 and GATA2 are positively correlated (P < .001); Pearson correlation coefficient. (Giii) HMGA1 and GATA2 are positively correlated (P < .01) in JAK2-mutant CD34+ BM cells from patients with ET or PV and unaffected individuals (GSE103237); Pearson correlation coefficient. (Giv) HMGA1 and GATA2 in JAK2-mutant CD34+ PB cells from patients with MF compared with unmutated CD34+ cells from the same patients (n = 8) or unaffected individuals (n = 2) by scRNAseq (GSE122198). Each dot represents the expression value for each single cell (P < .0010); Pearson correlation coefficient. (H) Transcriptional networks activated in MPN PBMCs after leukemic transformation correlate positively with HMGA1 expression levels (P < .05; Spearman correlation > 0.6) and overlap with the top HMGA1 networks (red) identified in JAKV617F MPN cell lines (DAMI, SET-2). *P < .05, **P < .01, ***P <. 001; 1-way ANOVA, followed by Tukey’s multiple-comparison test (A-D,F), log-rank (Mantel-Cox) test, followed by Bonferroni correction (E).

GATA2 restoration partially rescues antileukemogenic effects of HMGA1 silencing; GATA2 and HMGA1 are coexpressed in primary MPN and HMGA1 networks and are activated in leukemic blasts from patients with MPN. (A) Relative HMGA1 and GATA2 expression (mean ± standard deviation [SD]) from 2 experiments performed in triplicate (quantitative polymerase chain reaction) in DAMI, SET-2, and UKE-1 cells (control vs HMGA1-sh1 vs HMGA1-sh1 + GATA2 overexpression [OE]). GAPDH was used to control for loading. (B) Western blots of HMGA1, GATA2, β-actin, and GAPDH (loading control) protein levels from DAMI, SET-2, and UKE-1 cells transduced with lentivirus (control vs HMGA1-sh1 vs HMGA1-sh1 + GATA2 OE). Western blots were performed 3 times with similar results; a representative blot is shown. Size markers (kDa) are indicated. (C) Colony-forming units (CFU; mean ± SD) from DAMI, SET-2, and UKE-1 cells (control vs HMGA1-sh1 vs HMGA1-sh1 + GATA2 OE) in 2 experiments. Representative images of colonies are shown. Scale bars, 200 μm. (D) Annexin V apoptosis by flow cytometry in DAMI, SET-2, and UKE-1 cells (control vs HMGA1-sh1 vs HMGA1-sh1 + GATA2 OE) in 2 experiments. (E) Survival curve (Kaplan-Meier plots) in NSG mice injected with DAMI cells (control vs HMGA1-sh1 vs HMGA1-sh1 + GATA2 OE; n = 6). (F) Leukemia cell engraftment assessed by flow cytometry (mean ± SD) in BM from NSG mice injected with DAMI cells transduced with lentivirus (control vs HMGA1-sh1 vs HMGA1-sh1 + GATA2 OE; n = 4 per group). Representative BM histology (hematoxylin and eosin stain) from each group are shown. Scale bar, 200 μm. (Gi) HMGA1 and GATA2 expression (RNAseq) from PBMCs from a cohort of matched patient samples with MF that progressed to AML (n = 11 per group). (Gii) HMGA1 and GATA2 are positively correlated (P < .001); Pearson correlation coefficient. (Giii) HMGA1 and GATA2 are positively correlated (P < .01) in JAK2-mutant CD34+ BM cells from patients with ET or PV and unaffected individuals (GSE103237); Pearson correlation coefficient. (Giv) HMGA1 and GATA2 in JAK2-mutant CD34+ PB cells from patients with MF compared with unmutated CD34+ cells from the same patients (n = 8) or unaffected individuals (n = 2) by scRNAseq (GSE122198). Each dot represents the expression value for each single cell (P < .0010); Pearson correlation coefficient. (H) Transcriptional networks activated in MPN PBMCs after leukemic transformation correlate positively with HMGA1 expression levels (P < .05; Spearman correlation > 0.6) and overlap with the top HMGA1 networks (red) identified in JAKV617F MPN cell lines (DAMI, SET-2). *P < .05, **P < .01, ***P <. 001; 1-way ANOVA, followed by Tukey’s multiple-comparison test (A-D,F), log-rank (Mantel-Cox) test, followed by Bonferroni correction (E).

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal