Figure 3.

Effects of Pharmacological Inhibition of the ERK5 Pathway In Vivo and on Primary CML and Normal CD34+ Cells

(A) Effects of MEK5/ERK5 inhibitors on the number of viable primary CML cells. CML BMMCs were incubated at 0.1% O₂ and treated with DMSO (Vehicle) or the indicated inhibitors (XMD, XMD8-92; BIX, BIX02189; IM, imatinib; DAS, dasatinib) and viable cells counted at day 3. Values are means ± SD. See Figure S4A for single patient data. The number of patients for each group is indicated (vehicle group: n = 10). ^∗p ≤ 0.05; ^∗∗p ≤ 0.01.

(B) Effects of MEK5/ERK5 inhibitors on the CFA of primary CML cells. CML BMMCs were treated with DMSO (Vehicle) or inhibitors from time 0 and colonies scored after 7 days. Colony formation efficiency (CFE) values are means ± SD of data from single experiments performed in duplicate; ^∗p ≤ 0.05; ^∗∗p ≤ 0.01.

(C) Effects of XMD8-92 in vivo. CML mice (mice/group: n = 6) were treated twice daily with XMD8-92 (50 mg/kg) or placebo and euthanized after 1 additional day. Number of GFP+ (leukemic) or GFP− (non-leukemic) myeloid (Gr-1+) BM cells; data are means ± SD (left graph). Total number of viable BM cells; data are means ± SD (right graph). ^∗p < 0.05; ns, not significant.

(D) Effects of MEK5/ERK5 inhibitors on the number of viable primary CML and normal CD34+ cells. CD34+-enriched CML BMMCs or healthy donor PBMCs were treated with DMSO (Vehicle) or inhibitors at the indicated concentrations and incubated in 0.1% O₂ and viable cells counted at day 2. Values are means ± SD of data normalized for the respective vehicle-treated control. The number of patients/healthy donors is indicated; ^∗∗p ≤ 0.01; ^∗∗∗p ≤ 0.001; ns, not significant.

(E) Effects of MEK5/ERK5 inhibitors on CFA of primary CML or normal CD34+ cells. CD34+-enriched CML BMMCs or healthy donor PBMCs were treated with DMSO (Vehicle) or inhibitors at the indicated concentrations from time 0 and colonies scored after 7 days. CFE values are means ± SD of data normalized for the respective vehicle-treated control. The number of patients/healthy donors is indicated (see Figures S4C and S4D for data of single experiments); ^∗p ≤ 0.05; ^∗∗∗p ≤ 0.001; ns, not significant.

(F and G) Effects of MEK5/ERK5 inhibitors on the differentiation potential of primary CML or normal CD34+ cells. In the same experiments shown in (E), differential CFA was scored for primary CML (F) or normal (G) CD34+ cells. CFE values are means ± SEM of data normalized for the respective vehicle-treated control. ^∗p ≤ 0.05; ^∗∗p ≤ 0.01; ^∗∗∗p ≤ 0.001; to reduce figure cluttering, non-significant differences are not indicated.

(H) Effects of MEK5/ERK5 inhibitors on primary CD11b+ CML cells. Patient-derived cells were incubated in normoxia and treated with DMSO (Vehicle), 10 μM XMD8-92 (XMD), 10 μM BIX02189 (BIX), or 1 μM imatinib (IM) from time 0 to day 3. The percentages of CD11b-expressing cells were measured by flow cytometry. Data are means ± SD of data from three patients; ^∗∗∗p < 0.001 versus time 0 (t0).