Figure 4. Gli1⁺ cells are myofibroblast precursors in JAK2(V617F) induced myelofibrosis and can be targeted pharmacologically by GANT61.

(A) Bigenic Gli1CreER^t2;tdTomato mice received tamoxifen (3x10mg p.o.) and were lethally irradiated 10 days after the last tamoxifen dose and transplanted with 2x10⁵ c-kit purified bone marrow (BM) cells from wild type littermates that had been transduced with either control (n=10) or Jak2(V617F) (n=10) cDNA (both MCSV-IRES-GFP retroviral backbone vector). Mice were injected with GANT61 (50mg/kg body weight) or vehicle (corn oil / ethanol) every other day between 8 and 17 weeks after transplantation (control + vehicle n=3, 2 males; control+GANT61 n=5, 3 males; Jak2(V617F) + vehicle n=4, 3 males; Jak2(V617F) + GANT61 n=4, 3 males). (B) Grading of reticulin fibrosis. ***p<0.001 versus all other groups by one way ANOVA with posthoc Tukey; mean±SEM. (C) Spleen weight was determined as a percentage of the body weight. *p<0.05, **p<0.001 by one way ANOVA with posthoc Tukey; mean±SEM. (D) Representative images of sternal bones. Scale bars 50μm, inserts 25μm. (E) Quantification of all alpha smooth muscle actin expressing cells (α-SMA⁺), Gli1 cells (tdTomato⁺) and Gli1-derived myofibroblasts (α-SMA⁺/tdTomato⁺) in the BM of mice transplanted with Jak2(V617F) (Jak2) or control that have been treated with GANT61 or vehicle. ***p<0.001 by one way ANOVA with posthoc Tukey; mean±SEM. (F) Representative flow cytometric plots and quantification of Gli1⁺ cells and within the lineage depleted BM. *p<0.05 by one way ANOVA with posthoc Tukey; mean±SEM. (G) Analysis of long-term (LT; lin^lowSca1⁺ckit⁺CD48^-CD150⁺) and multipotent progenitor cells (MPP, lin^lowSca1⁺ckit⁺CD48⁺CD150^-) as well as erythroid cells (Gr1^-CD11b^-CD3^-CD19^-Ter119⁺) by flow cytometry. *p<0.05, **p<0.01 by one way ANOVA with posthoc Tukey; mean±SEM. (H) c-kit⁺ purified HSPCs from wild type mice were transduced with either control or Jak2(V617F) cDNA (both MCSV-IRES-GFP retroviral backbone vector). 48 hours after transduction, cells were treated with GANT61 or vehicle. The gene marking (GFP⁺) was quantified by flow cytometry 48 hours after treatment. ***p<0.001 versus all other groups by one way ANOVA with posthoc Tukey; mean±SEM. (I) Quantification of the mean fluorescence intensity (MFI) and representative flow cytometric analysis of levels of phospho-STAT5 (p-STAT5) in c-kit+ cells transduced with Jak2(V617F) or control (GFP⁺) 48 hours after treatment with GANT61 or vehicle. GFP⁺ cells are shown in the histogram. ***p<0.001 versus all other groups by one way ANOVA with posthoc Tukey; mean±SEM. (J) c-kit⁺ purified HSPCs were transduced with Jak2(V617F) or control cDNA, were sort-purified by GFP-expression 48 hours after transduction and treated with GANT61 or vehicle 24 hours later. 24 hours after treatment, cells were harvested for RNA isolation and qt-RT-PCRs. Relative mRNA expression for Phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K), Akt and Gli1 is shown. *p<0.05 ns=non-signifcant by one way ANOVA with posthoc Tukey; mean±SEM. See also Figure S4.