Skip to main content
. 2023 Nov 22;30(3):586–599. doi: 10.1158/1078-0432.CCR-23-0163

Figure 6.

Figure 6. Targeting MAPK pathway abrogates acquired resistance to type II JAK2 inhibition in vivo. A, Schema of in vivo model with subcutaneous engraftment. JAK2 inhibitor resistant SET2 cells (JAK2i-R) were injected into the flank of NSG mice. Animals were treated orally with type II JAK2 inhibitor CHZ868 15 mg/kg every day, MAPK inhibitor trametinib (tram) 0.3 mg/kg every day, combined CHZ868 15 mg/kg every day/MAPK inhibitor trametinib 0.3 mg/kg every day, or vehicle control. Treatment was initiated at 100 mm3 tumor size and continued until maximal tumor size was reached in vehicle-treated mice. B, Tumor size over time showed tumor growth in vehicle-treated mice similarly to CHZ868-treated mice. Combined JAK2/MAPK inhibition effectively reduced tumor size (n = 13/group). C, Analysis of tumor weight at end of treatment confirmed significant reduction of tumor growth in vivo by combined JAK2/MAPK inhibition with CHZ868 15 mg/kg every day/MAPK inhibitor trametinib 0.3 mg/kg every day (n = 13/group). D, Expression of MAPK pathway target DUSP6 was significantly reduced by combined JAK2/MAPK inhibition with CHZ868 15 mg/kg every day/MAPK inhibitor trametinib 0.3 mg/kg every day (n = 4–5/group). E, Expression of MAPK pathway target ETV5 was significantly reduced by combined JAK2/MAPK inhibition with CHZ868 15 mg/kg every day/MAPK inhibitor trametinib 0.3 mg/kg every day (n = 4–5/group). F, Schema of in vivo model with intravenous engraftment. JAK2 inhibitor resistant SET2 cells (JAK2i-R) stably expressing luciferase were injected intravenously into NSG mice and engraftment documented by bioluminescent imaging. Animals were treated orally with type II JAK2 inhibitor CHZ868 15 mg/kg every day, MAPK inhibitor trametinib 0.3 mg/kg every day, combined CHZ868 15 mg/kg every day/MAPK inhibitor trametinib 0.3 mg/kg every day, or vehicle control. G, Bioluminescent signal was significantly reduced in mice treated with combined CHZ868/trametinib at 7 days of treatment (n = 7–9/group). H, Representative bioluminescence images are shown. I, BM infiltration of JAK2i-R cells was determined as human CD45 positivity by IHC on BM sections. A significant reduction of BM infiltration was observed with combined CHZ868 15 mg/kg every day/MAPK inhibitor trametinib 0.3 mg/kg every day (n = 5–6/group). J, Representative images of BM sections stained with H&E (top) and IHC for hCD45 (lower panel) showed reduced infiltration with combined CHZ868 15 mg/kg every day/MAPK inhibitor trametinib 0.3 mg/kg every day. Original magnification x400. Data are presented as mean ± SD and analyzed by one-way ANOVA. ns, not significant; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.

Targeting MAPK pathway abrogates acquired resistance to type II JAK2 inhibition in vivo. A, Schema of in vivo model with subcutaneous engraftment. JAK2 inhibitor resistant SET2 cells (JAK2i-R) were injected into the flank of NSG mice. Animals were treated orally with type II JAK2 inhibitor CHZ868 15 mg/kg every day, MAPK inhibitor trametinib (tram) 0.3 mg/kg every day, combined CHZ868 15 mg/kg every day/MAPK inhibitor trametinib 0.3 mg/kg every day, or vehicle control. Treatment was initiated at 100 mm3 tumor size and continued until maximal tumor size was reached in vehicle-treated mice. B, Tumor size over time showed tumor growth in vehicle-treated mice similarly to CHZ868-treated mice. Combined JAK2/MAPK inhibition effectively reduced tumor size (n = 13/group). C, Analysis of tumor weight at end of treatment confirmed significant reduction of tumor growth in vivo by combined JAK2/MAPK inhibition with CHZ868 15 mg/kg every day/MAPK inhibitor trametinib 0.3 mg/kg every day (n = 13/group). D, Expression of MAPK pathway target DUSP6 was significantly reduced by combined JAK2/MAPK inhibition with CHZ868 15 mg/kg every day/MAPK inhibitor trametinib 0.3 mg/kg every day (n = 4–5/group). E, Expression of MAPK pathway target ETV5 was significantly reduced by combined JAK2/MAPK inhibition with CHZ868 15 mg/kg every day/MAPK inhibitor trametinib 0.3 mg/kg every day (n = 4–5/group). F, Schema of in vivo model with intravenous engraftment. JAK2 inhibitor resistant SET2 cells (JAK2i-R) stably expressing luciferase were injected intravenously into NSG mice and engraftment documented by bioluminescent imaging. Animals were treated orally with type II JAK2 inhibitor CHZ868 15 mg/kg every day, MAPK inhibitor trametinib 0.3 mg/kg every day, combined CHZ868 15 mg/kg every day/MAPK inhibitor trametinib 0.3 mg/kg every day, or vehicle control. G, Bioluminescent signal was significantly reduced in mice treated with combined CHZ868/trametinib at 7 days of treatment (n = 7–9/group). H, Representative bioluminescence images are shown. I, BM infiltration of JAK2i-R cells was determined as human CD45 positivity by IHC on BM sections. A significant reduction of BM infiltration was observed with combined CHZ868 15 mg/kg every day/MAPK inhibitor trametinib 0.3 mg/kg every day (n = 5–6/group). J, Representative images of BM sections stained with H&E (top) and IHC for hCD45 (lower panel) showed reduced infiltration with combined CHZ868 15 mg/kg every day/MAPK inhibitor trametinib 0.3 mg/kg every day. Original magnification x400. Data are presented as mean ± SD and analyzed by one-way ANOVA. ns, not significant; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.