lentivector expression backbone plasmid plenti- cmv- blast- empty- (w263- 1)  (Addgene inc)

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) Colony-forming units (CFU) assays in human (HEL, UKE-1) and murine (Ba/F3- JAK2 V617F , 32D- JAK2 V617F ) cell lines following HMGA1 knockdown (shHMGA1) or overexpression (OE-HMGA1) relative to respective controls (NC). Data are mean ± SD. (n=5 per group). Representative colony images are shown (scale bar 300 μm).Two-sample t -test and one-way ANOVA with Tukey’s post-hoc test. (b) Schematic of the HEL cell xenograft experiment in NSG mice, comparing HMGA1-OE cells to vector control cells. (c) Representative bioluminescence images (left panel) and quantification of total photon flux (right panel) from NSG mice at day 35 post-engraftment with luciferase-tagged HEL cells transduced with vector control (CMV-NC, n=6) or HMGA1 overexpression constructs (OE, n=6). Data are mean ± SD. Two-sample t -test. (d) Correlation between HMGA1 mRNA expression (snRNA-seq) and the number of open chromatin peaks (scATAC-seq) in CD34 + cells from a patient during primary MF and after sAML transformation (GSE221946). Pearson correlation coefficient ( R ) and P -value from Wilcoxon ranksum test with BH correction. (e) Genomic distribution of HMGA1 binding sites lost upon HMGA1 knockdown in HEL cells, as determined by CUT&Tag (i), and chromatin regions with significantly reduced accessibility upon HMGA1 knockdown, as determined by ATAC-seq (ii). Stacked bar chats show the percentage of peaks located in promoter (±3kb of TSS), intronic, intergenic, and other genomic regions. (f) GSEA enrichment plots for E2F targets and G2M checkpoint Hallmark gene sets. Comparisons shown in figure. Normalized Enrichment Score (NES) and False Discovery Rate (FDR) q-value are indicated. (g) Cell cycle distribution (Propidium Iodide staining followed by flow cytometry) of HEL and UKE-1 cells with HMGA1 knockdown (sh1, sh2) or non-targeting control (NC). Data are mean ± SD. (n = 5 per group). One-way ANOVA with Tukey’s post-hoc test. (h) Western blots analysis of key E2F pathway, G2M checkpoint, and common cell cycle regulatory protein levels in HEL and UKE-1 cells following overexpression (OE vs. CMV-NC vector control; left panels) or HMGA1 knockdown (shHMGA1 vs. shNC control; right panels). GAPDH served as loading controls. Blots are representative of at least three independent experiments.

Article Snippet: HEL92.1.7 cells stably transduced with a luciferase reporter gene (pLenti-CMV-Luc2-Blast, Addgene plasmid # 21474, a gift from Eric Campeau) and either control vector (CMV-NC) or HMGA1 overexpression vector (HMGA1-OE) were prepared.

Techniques: Knockdown, Over Expression, Plasmid Preparation, Control, Luciferase, Transduction, Construct, Expressing, Transformation Assay, Binding Assay, Staining, Flow Cytometry, Western Blot

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) Prognostic significance of HMGA1 expression in the OHSU BeatAML MPN-sAML cohort (n = 31). Genes are ranked by their hazard ratio (HR) for overall survival (OS). Points are colored based on FDR significance: grey ( FDR > 0.05), blue ( FDR < 0.05 & HR < 1, good prognosis), red ( FDR < 0.05 & HR ≥ 1, poor prognosis) (b) Gene Set Enrichment Analysis (GSEA) showing Hallmark pathways enriched among genes associated with poor prognosis (ranked by HR) in the OHSU BeatAML MPN-sAML cohort. Heatmap displays NES for selected pathways. Asterisks (*) indicate FDR < 0.05. (c) Kaplan-Meier OS curves for MPN-sAML patients from the OHSU BeatAML cohort (n = 31, top panel) and an independent in-house cohort (n = 21, bottom pnel), stratified by high versus low HMGA1 expression (HMGA1 expression levels for BeatAML in-house cohort using median cut-off). Log-rank (Mantel-Cox) test. (d) Representative immunohistochemical (IHC) staining for HMGA1 in bone marrow biopsies from in-house MPN-sAML cohort patients, illustrating HMGA1 expression changes with therapy and clinical outcome. (i) HMGA1-low patient achieving complete remission (CR) post-ruxolitinib. (ii) HMGA1-low patient achieving CR post-decitabine + venetoclax. (iii) HMGA1-high patient with progressive disease (PD) despite 5-azacytidine + venetoclax, showing increased HMGA1 at relapse. (iv) HMGA1-high patient achieving durable remission with decreased HMGA1 staining post-allogeneic hematopoietic stem cell transplantation (allo-HCT). Scale bars: 80µm (overview), 20µm (insets). (e) Comparison of HMGA1 expression levels between MPN-sAML patients achieving complete remission (CR, includes CRh, CRi) and those not achieving CR (Non-CR). Top panel: HMGA1 transcript levels (Log2 normalized counts) in the OHSU BeatAML cohort (n=31). Bottom panel: Percentage of HMGA1-positive cells (IHC score) in the in-house cohort (n=21). Data are mean ± SD. Two-sample t -test. (f) Heatmap illustrating Hallmark GSEA of differentially expressed genes in HEL cells treated with DMSO (vehicle), ruxolitinib (Rux), fedratinib (Fed), pacritinib (Pac), or momelotinib (Mmb) for 4 hours or 48 hours (GSE229712) and in HEL cells with acquired resistance to ruxolitinib (Rux-Persistent, GSE190517) compared to DMSO control. Color intensity represents NES. * indicate FDR < 0.05. (g) Dose-response curves depicting cell viability of parental (NC) control versus ruxolitinib-persistent (Rux-P) HEL (left) and UKE-1 (right) cells treated with indicated concentrations of ruxolitinib for 72 hours. IC 50 values (mean± SD) are shown. Two-way ANOVA comparing IC 50 values. (h) HMGA1 mRNA expression (RNA-seq, normalized counts) in HEL cells: non-targeting control (NC), ruxolitinib-persistent (Rux-P), and fedratinib-persistent (Fed-P). (I) Immunoblot analysis of HMGA1 protein levels in parental (NC) and and ruxolitinib-persistent (Rux-P) HEL and UKE-1 cells. Tublin serves as a loading control. (J) Dose-response curve showing cell viability of HEL cells transduced with control vector (NC), HMGA1 overexpression (OE), or HMGA1 shRNA (Sh1) constructs, treated with indicated concentrations of ruxolitinib (Rux), fedratinib (Fed), pacritinib (Pac), and momelotinib (Mmb) for 72 hours. IC50 values (mean ± SD) are shown. Two-way ANOVA comparing IC50 values between OE/Sh1 and respective NC. (k) Schematic representation of the in vivo pacritinib treatment study in NSG mice engrafted with luciferase-expressing HEL cells (transduced with CMV-NC vector or HMGA1-OE construct). Following leukemia engraftment (Day 0-21), mice received pacritinib (100 mg/kg, BID) or vehicle orally for 14 days (Day 21-35). Endpoint analyses included bioluminescence imaging, spleen weight, flow cytometry, Wright-Giemsa staining, H&E, and IHC, alongside survival monitoring. (l) Representative bioluminescence image (left) and quatification of total tumor bioluminescence (total flux, right) at day 35 in NSG mice engrafted with CMV-NC or HMGA1-OE HEL cells and treated with vehicle or pacritinib (n=6 mice/group). Data are shown in mean ± SD. One-way ANOVA with Tukey’s post-hoc test.

Article Snippet: HEL92.1.7 cells stably transduced with a luciferase reporter gene (pLenti-CMV-Luc2-Blast, Addgene plasmid # 21474, a gift from Eric Campeau) and either control vector (CMV-NC) or HMGA1 overexpression vector (HMGA1-OE) were prepared.

Techniques: Expressing, Immunohistochemical staining, Immunohistochemistry, Staining, Transplantation Assay, Comparison, Control, RNA Sequencing, Western Blot, Transduction, Plasmid Preparation, Over Expression, shRNA, Construct, In Vivo, Luciferase, Imaging, 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) Forest plot illustrating multivariable Cox proportional hazards analysis of overall survival (OS) in the OHSU BeatAML cohort (n = 31) and the in-house MPN-sAML cohort (n = 21). Harzard ratio (HR) and 95% confidence intervals (CI) are shown for HMGA1 epxression (high vs. low), age, gender and ELN 2022 risk category. (b) Kaplan-Meier OS curves for MPN-sAML patients from the OHSU BeatAML cohort stratified by specific therapies received (ruxolitinib, 5-azacytidine, decitabine, cytarabine,and hydroxyurea). Log-rank (Mantel-Cox) test. (c) Western blot analysis key proteins in JAK-STAT, E2F, and G2/M pathways in HEL and UKE-1 cells with vector control (CMV-NC) or HMGA1 overexpression (OE), following 4-hour treatment with vehicle (DMSO) or pacritinib (500nM). (d) Quantification of human HEL cells in peripheral blood of NSG mice at day 35 post-transplantation by flow cytometric analysis (CD45 + CD117 + ) or Writght_Giemsa smear, from mice engrafted with vector control (CMV-NC) or HMGA1-OE HEL cells and treated with vehicle or pacritinib (n=6 mice/group). Data are mean ± SD. One-way ANOVA. (e-f) Pacritinib reduces leukemic infiltration and HMGA1 expression in bone marrow and spleen of HMGA1-OE xenografted mice. Representative IHC staining for HMGA1 and quantification of human HEL cell engraftment (% of HEL cells) and HMGA1-positive cells (%) in (e) bone marrow and (f) spleen sections. NSG mice (n=6 per group) were engrafted with luciferase-expressing HEL cells transduced with CMV-NC or HMGA1-OE constructs, and subsequently treated with vehicle or pacritinib (100 mg/kg, BID) for 14 days. Scale bars for IHC images, 50 µm. Bar graphs depict mean ± SD. One-way ANOVA with Tukey’s post-hoc test. (g) Representative images of spleens and relative spleen weights (spleen weight/body weight %) from NSG mice engrafted with CMV-NC or HMGA1-OE HEL cells, treated with vehicle or pacritinib. One-way ANOVA with Tukey’s post-hoc test. (h) Kaplan–Meier survival curves for NSG mice engrafted with vector control HEL cells (CMV-NC) or HMGA1-OE HEL cells, treated with vehicle or pacritinib (n=6 per group). Log-rank (Mantel-Cox) test.

Article Snippet: HEL92.1.7 cells stably transduced with a luciferase reporter gene (pLenti-CMV-Luc2-Blast, Addgene plasmid # 21474, a gift from Eric Campeau) and either control vector (CMV-NC) or HMGA1 overexpression vector (HMGA1-OE) were prepared.

Techniques: Western Blot, Plasmid Preparation, Control, Over Expression, Transplantation Assay, Expressing, Immunohistochemistry, Luciferase, Transduction, Construct