Extended Data Fig. 10: Humanized PDX models of MF and sAML.
From: DUSP6 mediates resistance to JAK2 inhibition and drives leukemic progression
a) Percentage of myeloid cells from hCD45+ PB (left) and CD71 + CD235a + from hCD45- BM (right) from NSGS mice transplanted with CD34 + cells ectopically expressing control (n = 9 mice) and DUSP6 (n = 10 mice) at endpoint. Both statistics assessed by two-tailed non-parametric Mann-Whitney U test after testing for normality by Shapiro-Wilk test. b) DUSP6 overexpression PDX with CD34 + cells from a second MF patient (MF106). Plots show percentage of human CD45 (hCD45) in peripheral blood and bone marrow of transplanted mice ectopically expressing control (n = 5) or DUSP6 (n = 5) across multiple timepoints, and spleen and liver weights of mice at endpoint normalized by mouse weight. %hCD45 in PB statistics assessed by two-way ANOVA incorporating weeks 4-8 post transplant. %hCD45 in BM, and normalized spleen and liver weights statistics were assessed by two-tailed Student’s t test. Data are presented as mean values + /- s.d. c) Kaplan-Meier survival analysis of mice from control or DUSP6 cohorts assessed by log-rank test. d) Colony assay of CD34 + cells from sAML15 after transduction with shRNAs targeting DUSP6 or control vector. Sorted cells were grown in MethoCult H4034 Optimum for 12 days. Samples were plated in triplicate (n = 3 replicates). Statistics were assessed by Two-tailed Student’s t test. Data are presented as mean values + /- s.d. e) CD34 + healthy donor normal bone marrow (NBM) PDX model. Cells were transduced with 2 independent shRNAs targeting DUSP6 or control and transplanted into NSGS mice. Plots show percentage of human CD45 (hCD45) in peripheral blood and bone marrow of transplanted mice treated ectopically expressing control (n = 5), shDUSP6 #1 (n = 5), or shDUSP6 #2 (n = 5) across multiple timepoints, and spleen and liver weights of mice at endpoint normalized by mouse weight. %hCD45 in PB statistics assessed by two-way ANOVA with Dunnett’s multiple comparisons test with control. %hCD45 in BM, and normalized spleen and liver weights statistics were assessed by two-tailed Student’s t test with Dunnett’s multiple comparisons test with control. f) Normalized spleen and liver weights from mice at end-point from sAML14 CD34 + PDX. Mice were treated with vehicle (n = 7), 25 mg/kg BCI (n = 8), 90 mg/kg ruxolitinib (n = 7), or combination (n = 7). Data are presented as mean values + /- s.d. g) tSNE dimensional reduction clustering of mouse and human CD45 + cells from bone marrow of sAML14 PDX mice. h) sAML PDX14 mass cytometry analysis showing percentage of CD123 + CD33 + leukemic cells gated from hCD45+ cells from 3 mice in each treatment group. Statistics were assessed by one-way ANOVA with Dunnett’s multiple comparison test. i) Erythroblast progenitors gated from hCD45+ cells. Statistics were assessed by one-way ANOVA with Dunnett’s multiple comparison test. j) SPADE tree cluster algorithm showing CD123 + and CD71 + populations from vehicle and BCI treated groups. k) UMAP clustering showing CD123 + and CD71 + populations. l) Schematic of the CD34 + healthy donor PDX model. CD34 + cells were isolated from BMMCs from normal donors, pooled, and transplanted into NSGS mice. Mice were treated with vehicle (n = 6), 25 mg/kg BCI (n = 6), 90 mg/kg ruxolitinib (n = 6), or combination (n = 6) following weekly schedule of 5 days on, 2 days off treatment starting on day 38. m) Plots show % hCD45 in peripheral blood and bone marrow of transplanted mice treated with vehicle or BCI across multiple timepoints and spleen weights of mice at endpoint normalized by mouse weight %hCD45 in PB statistics were assessed by two-way ANOVA comparing vehicle vs each individual treatment group with Dunnett’s multiple comparison test. %hCD45 in BM, and spleen and liver weights statistics were assessed by one-way ANOVA with Dunnett’s multiple comparison test. Data are presented as mean values + /- s.d.
