Fig. 4: MAPK activation confers fitness advantage and increased clonogenic potential to JAK2V617F and JAK2WT hematopoietic cells upon ruxolitinib.
From: JAK2 inhibition mediates clonal selection of RAS pathway mutations in myeloproliferative neoplasms
A Percentage of CD45.1/2 NrasG12D cells among CD45.1/2 NrasG12D and CD45.1 NrasWT Lin− murine cells (50/50 ratio) treated with DMSO or ruxolitinib (0.25 µM). B Proliferation curves of NrasG12D or NrasWT Lin− cells after DMSO or ruxolitinib (0.25 µM). A, B Mean of n = 2 biological replicates. C In vivo NrasG12D competition model. Created in BioRender59. D Percentage of CD45.1/2 NrasG12D cells in peripheral blood of mice transplanted with 10% CD45.1/2 Jak2WT NrasG12D and 90% CD45.2 Jak2V617F NrasWT Lin− cells (n = 4 per group). Ruxolitinib (90 mg/kg twice daily) was started 4 weeks post-transplantation. E In vivo NrasQ61K competition model. Created in BioRender59. F Percentage of GFP+ NrasQ61K Jak2V617F cells in the bone marrow of mice transplanted with Jak2V617F cells expressing a GFP+ NrasQ61K vector (n = 5 per group). Ruxolitinib (90 mg/kg twice daily) was started 3 weeks post-transplantation. G, H Colony formation assay for cKit+ bone marrow cells from Jak2WT (G) or Jak2V617F (H) mice expressing Empty or NrasQ61K vectors. Colony number after at least 6 days of DMSO or ruxolitinib (1 µM) (n = 4 biological replicates). I Western blot for the indicated proteins in HEL cells expressing GFP, Empty or NRASQ61K vectors, treated 24 h with ruxolitinib (3 µM). J In vitro competition model. Created in BioRender59. K Percentage of GFP+ and GFP−_Empty or GFP−_NRASQ61K HEL or UKE-1 cells. 24 days after ruxolitinib (3 µM and 15 µM for HEL and UKE-1, respectively), the indicated cells were treated 3 days with ruxolitinib or ruxolitinib (Ruxo.) and trametinib (Trame.) (10 µM and 0.1 µM for HEL and UKE-1, respectively) (n = 3 biological replicates). L Western blot for the indicated proteins in Ba/F3 MPL-CALRWT and MPL-CALRdel52 cells expressing Empty or NRASQ61K vectors, treated 24 h with ruxolitinib (75 nM). M Percentage of Crimson+ and Crimson−_Empty or Crimson−_NRASQ61K Ba/F3 MPL-CALRWT and MPL-CALRdel52 cells after ruxolitinib (n = 3 biological replicates). Statistical significance using two-tailed Mann–Whitney (D, F) or Welch’s t-test (H, K, M). Experiments(I, L) were performed twice with similar results. Error bars represent mean ± SEM. P-values in the figure. Source data provided as Source Data file. Schemas created using BioRender.
