Figure 7.
Combined inhibition of MEK and BET downregulates inhibitory immune checkpoint pathways and AML transformation gene signature and prolongs the survival of NrasG12D/+;Asxl1−/− AML mice. Sublethally irradiated mice (CD45.1+) were transplanted with 2.5 × 105 bone marrow cells from moribund primary NA-AML recipients. Two weeks after transplantation, mice were randomly separated into (A-G) 2 groups and treated with vehicle (Veh) or a combination (Combo) of trametinib and GSK525762 once per day or (H) 4 groups treated with vehicle, trametinib (Tra), and/or GSK525762 (GSK) once per day. (A-F) Recipients were euthanized at ∼5 weeks posttransplant. Quantification of (A) spleen and liver weights; (B) bone marrow leukemia cells (CD45.2+) expressing PD-L1, CD86, CD80, PD-L2, or CD155 using flow cytometry; (C) bone marrow CD4 and CD8 T cells expressing PD-1, TIGIT, or CTLA-4; and (D) exhausted T cells (PD-1+TIGIT+LAG3+) in spleen CD4 and CD8 T cells. (E) T-cell cytotoxicity assay. CD45.2+ leukemia cells (Target) were sorted from moribund NA-AML recipients treated with vehicle. The same number of Target cells were cocultured with splenic CD8 T cells (Effector), which were sorted from recipients treated with vehicle (Veh) or combination (Com) of drugs at different ratios. The live leukemia cells were quantified 48 hours after coculture. (F) Quantification of AP-1 complex genes (Fos, Fosb, Jun, Junb, Jund, and Atf3) and Flt3 mRNA levels using qRT-PCR in sorted leukemia cells (CD45.2+). (G-H) Kaplan-Meier survival curves were plotted against days after transplantation using NA-AML cells from different donors. (I) 1.78 million bone marrow mononuclear cells from a RAS and ASXL1 double-mutant CMML patient were transplanted into irradiated NRGS mice (n = 4 mice per group). Five days after transplantation, mice were randomly separated into 2 groups and treated with vehicle or trametinib plus GSK525762 until moribund stage as described in “Materials and methods.” Kaplan-Meier survival curves were plotted against days after transplantation. (G-I) Data are presented as mean + SD. P values were determined using the log-rank test. *P < .05; **P < .01; ***P < .001.

Combined inhibition of MEK and BET downregulates inhibitory immune checkpoint pathways and AML transformation gene signature and prolongs the survival of NrasG12D/+;Asxl1−/− AML mice. Sublethally irradiated mice (CD45.1+) were transplanted with 2.5 × 105 bone marrow cells from moribund primary NA-AML recipients. Two weeks after transplantation, mice were randomly separated into (A-G) 2 groups and treated with vehicle (Veh) or a combination (Combo) of trametinib and GSK525762 once per day or (H) 4 groups treated with vehicle, trametinib (Tra), and/or GSK525762 (GSK) once per day. (A-F) Recipients were euthanized at ∼5 weeks posttransplant. Quantification of (A) spleen and liver weights; (B) bone marrow leukemia cells (CD45.2+) expressing PD-L1, CD86, CD80, PD-L2, or CD155 using flow cytometry; (C) bone marrow CD4 and CD8 T cells expressing PD-1, TIGIT, or CTLA-4; and (D) exhausted T cells (PD-1+TIGIT+LAG3+) in spleen CD4 and CD8 T cells. (E) T-cell cytotoxicity assay. CD45.2+ leukemia cells (Target) were sorted from moribund NA-AML recipients treated with vehicle. The same number of Target cells were cocultured with splenic CD8 T cells (Effector), which were sorted from recipients treated with vehicle (Veh) or combination (Com) of drugs at different ratios. The live leukemia cells were quantified 48 hours after coculture. (F) Quantification of AP-1 complex genes (Fos, Fosb, Jun, Junb, Jund, and Atf3) and Flt3 mRNA levels using qRT-PCR in sorted leukemia cells (CD45.2+). (G-H) Kaplan-Meier survival curves were plotted against days after transplantation using NA-AML cells from different donors. (I) 1.78 million bone marrow mononuclear cells from a RAS and ASXL1 double-mutant CMML patient were transplanted into irradiated NRGS mice (n = 4 mice per group). Five days after transplantation, mice were randomly separated into 2 groups and treated with vehicle or trametinib plus GSK525762 until moribund stage as described in “Materials and methods.” Kaplan-Meier survival curves were plotted against days after transplantation. (G-I) Data are presented as mean + SD. P values were determined using the log-rank test. *P < .05; **P < .01; ***P < .001.

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