myeloblastic cell line 32d cl3  (ATCC)

Journal: bioRxiv

Article Title: Targeting HMGA1-driven leukemic transformation in myeloproliferative neoplasms with pacritinib

doi: 10.1101/2025.06.01.657170

Figure Lengend Snippet: (a) Efficient shRNA-mediated knockdown of HMGA1 (sh1, sh2 vs. sh-NC control) in HEL and UKE-1 cells. Left: Relative HMGA1 mRNA levels by qRT-PCR (mean ± SD, n = 3). Right: Western blot analysis of HMGA1 protein; ACTB served as loading control. (b) Lentiviral-mediated overexpression of HMGA1 (OE vs. CMV-NC control) in HEL and UKE-1 cells. Left: Relative HMGA1 mRNA levels by qRT-PCR (mean ± SD, n = 3). Right: Western blot analysis of HMGA1 protein; Tubulin served as loading control. (c) Lentiviral-mediated overexpression of Hmga1 (J/OE vs. J/NC control) in murine Ba/F3 ( Jak2 wild type, or Jak2 V617F ) and 32D-cl3 ( Jak2 wild type, or Jak2 V617F ) cells. Left: Relative Hmga1 mRNA levels by qRT-PCR (mean ± SD, n = 3). Right: Western blot analysis of Hmga1 protein; Tubulin served as loading control. Statistical analyses for (a-c) by two-sample t-test or one-way ANOVA, as appropriate. (d) HMGA1 overexpression exacerbates disease phenotype in a HEL xenograft model. Hematological parameters (WBC, white blood cell count; HGB, hemoglobin; HCT, hematocrit; PLT, platelet count) in NSG mice engrafted with HEL cells stably expressing control vector (CMV-NC, n = 6) or HMGA1 (OE, n = 6) at 35 days post-transplantation. Data are presented as mean ± SD. Two-sample t -test. (e) HMGA1 knockdown alters chromatin accessibility and HMGA1 binding at key cell cycle regulatory gene loci. Integrative Genomics Viewer (IGV) snapshots displaying ATAC-seq and HMGA1 CUT&Tag signals at representative E2F target genes ( E2F1 , CCNE1 , CCNE2 , CDK2 , RB1 ), G2M checkpoint genes ( CCNB1 , CCNB2 , CDC2 , WEE1 , CDC25C , PLK1 , AURKA , AURKB ), and common cell cycle regulators ( CCNA2 , CDKN1A / p21 , CDKN1B / p27 ) in HEL cells following control (NC) versus HMGA1 knockdown (KD). (f) Enhanced E2F target and G2M checkpoint gene signatures in sAML patient cells. UMAP projections of single-cell CITE-seq data (GSE185381) from control and sAML patients, with cells colored by enrichment scores for E2F target and G2M checkpoint gene sets. Corresponding density plots illustrate score distributions.

Article Snippet: Murine IL-3-dependent pro-B cell line Ba/F3 (DSMZ ACC 300) and myeloblastic cell line 32D-cl3 (ATCC CRL-11346) were also used.

Techniques: shRNA, Knockdown, Control, Quantitative RT-PCR, Western Blot, Over Expression, Cell Counting, Stable Transfection, Expressing, Plasmid Preparation, Transplantation Assay, Binding Assay

Journal: bioRxiv

Article Title: Targeting HMGA1-driven leukemic transformation in myeloproliferative neoplasms with pacritinib

doi: 10.1101/2025.06.01.657170

Figure Lengend Snippet: (a) Relative proliferation curves of human (HEL, UKE-1) and murine (Ba/F3, 32D-cl3 transduced with Jak2 wild-type or Jak2 V617F ) cell lines following HMGA1/Hmga1 overexpression (OE) or shRNA-meidated knockdown (sh1, sh2) compared to respective controls (CMV-NC or sh-NC)NC.) 32D-cl3 cells were cultured with IL-3. Data are mean ± SD. (n = 5 per group). Two-way ANOVA. (b) Flow cytometric analysis of CD11b expression on 32D-cl3 cells transduced with Jak2 wild-type (J WT ) or Jak2 V617F (J VF ), and co-transduced with control vector (NC) or HMGA1 overexpression (OE), following G-CSF (100 ng/mL) induced differentiation. (i) Representative histograms of CD11b-FITC fluorescence. (ii) Quantification of HMGA1-PE mean fluorescence intensity (MFI). (iii) Quantification of CD11b-FITC MFI (n = 5 per group). Data are mean ± SD. Two-sample t -test. (c) Quantification of human CD45 + CD117 + HEL cells in peripheral blood of NSG mice at day 35 post-transplant, comparing HMGA1-OE versus vector control (CMV-NC) groups (n=6 per group). Data are mean ± SD. Two-sample t -test. (d) Wright-Giemsa stained peripheral blood smears from NSG mice engrafted with HMGA1-OE or CMV-NC HEL cells at day 35. Quantification of HEL cells (% of total nucleated cells) is shown (n = 6 per group). Data are mean ± SD. Two-sample t -test. (e-f) Representative H&E and HMGA1 IHC staining (left panels of e and f, respectively) and quantification of HMGA1-positive cells (%) (right panels fo e and f, respectively) in (e) femur bone marrow and (f) spleen sections from NSG mice engrafted with HMGA1-OE or CMV-NC HEL cells. Scale bars: 50 µm. Data are mean ± SD. Two-sample t -test. (g) Representative images of spleens (left) and relative spleen weights (spleen weight/body weight %, right) from NSG mice at day 35 post-engraftment with HMGA1-OE or CMV-NC HEL cells (n = 6 per group). Data are mean ± SD. Two-sample t -test. (h) Kaplan-Meier survival curves for NSG mice injected with HMGA1-OE ro CMV-NC HEL cells (n = 6 per group). Median survival times are indicated. Log-rank (Mantel-Cox) test. (i) Heatmaps showing HMGA1 binding intensity (CUT&Tag, left) and chromatin accessibility (ATAC-seq, right) centered on transcription start site (TSS ± 3kb) for genes in HEL cells transduced with shNC or shHMGA1. Color scale indicates normalized read counts (Max/Min normalized). (j) Top de novo motifs identified by HOMER analysis within ATAC-seq peak regions that either lose accessibility (left) or gain accessibility (right) upon HMGA1 knockdown in HEL cells. P -value for motif enrichment are indicated. (k) Quantification of apoptosis by Annexin V-APC/7-AAD staining and flow cytometry in HEL and UKE-1 cells after transduction with shNC or HMGA1 shRNAs (sh1, sh2). Representative flow cytometry plots are shown. Data are mean ± SD. (n = 5 per group). One-way ANOVA with Tukey’s post-hoc test.

Article Snippet: Murine IL-3-dependent pro-B cell line Ba/F3 (DSMZ ACC 300) and myeloblastic cell line 32D-cl3 (ATCC CRL-11346) were also used.

Techniques: Transduction, Over Expression, shRNA, Knockdown, Cell Culture, Expressing, Control, Plasmid Preparation, Fluorescence, Staining, Immunohistochemistry, Injection, Binding Assay, Flow Cytometry

Journal: bioRxiv

Article Title: Targeting HMGA1-driven leukemic transformation in myeloproliferative neoplasms with pacritinib

doi: 10.1101/2025.06.01.657170

Figure Lengend Snippet: (a-f) HMGA1 expression levels modulate sensitivity to diverse therapeutic agents. Dose-response curves showing viability of HEL, UKE-1, Ba/F3 ( Jak2 wild type, or Jak2 V617F ), and 32D-cl3 ( Jak2 wild type, or Jak2 V617F ) cells with engineered HMGA1/Hmga1 expression (OE vs. NC; sh1/sh2 vs. sh-NC) following 72-hour treatment with (a) IFNα, (b) 5-Azacytidine, (c) Decitabine, (d) Cytarabine, (e) Venetoclax, and (f) Hydroxyurea. Calculated IC50 values are shown. Data represent mean ± SD from n = 3 independent experiments. Two-way ANOVA. (g) Ruxolitinib treatment, particularly long-term exposure, alters key signaling and cell cycle protein expression. Western blot analysis of indicated JAK-STAT, E2F pathway, G2M checkpoint, and cell cycle regulatory proteins in HEL and UKE-1 cells treated with vehicle, short-term ruxolitinib (4 hours), or in ruxolitinib-persistent (Rux-P) lines. GAPDH served as loading control. (h-j) HMGA1/Hmga1 expression status influences sensitivity to JAK inhibitors. Dose-response curves assessing viability of (h) UKE-1 cells, (i) Ba/F3 cells (J VF /NC: Jak2 V617F /control vector; J VF /OE: Jak2 V617F /Hmga1 OE; J WT /NC: Jak2 wild-type/control vector; J WT /OE: Jak2 wild-type /Hmga1 OE), and (j) 32D-cl3 cells (similarly engineered) with engineered HMGA1/Hmga1 expression, following treatment with ruxolitinib, fedratinib, pacritinib, or momelotinib. Calculated IC50 values are shown. Data represent mean ± SD from n = 3 independent experiments. Two-way ANOVA. (k) Pacritinib mitigates weight loss in mice bearing HMGA1-overexpressing HEL xenografts. Percent body weight change in NSG mice engrafted with HEL-Luc cells (CMV-NC or HMGA1-OE) and treated with vehicle or pacritinib (100 mg/kg, BID, 14 days). Data are mean ± SD (n = 6 per group). One-way ANOVA. (l) Pacritinib treatment improves hematological parameters in the HMGA1-overexpressing HEL xenograft model. Peripheral blood counts (WBC, HGB, HCT, PLT) in xenografted mice at day 35 endpoint. Data are mean ± SD (n = 6 per group). One-way ANOVA.

Article Snippet: Murine IL-3-dependent pro-B cell line Ba/F3 (DSMZ ACC 300) and myeloblastic cell line 32D-cl3 (ATCC CRL-11346) were also used.

Techniques: Expressing, Western Blot, Control, Plasmid Preparation