Fig. 4

Pharmacologic inhibition of LMP7/PSMB8 impairs KMT2A-r leukemia stem cell function without affecting normal hematopoietic stem cells. A Serial re-plating to assess for colony formation in methylcellulose using murine LSK cells transformed with KMT2A::MLLT3/KRAS, KMT2A::MLLT4 or AML1-ETO/KRAS. Cells were treated with DMSO as diluent control or PR-957 (100 nM, 200 nM). n = 3–4 independent experiments; mean with SD; paired Student t test. B-D 3 × 10⁵ KMT2A::MLLT3 murine BM cells transplanted into sublethally irradiated recipient mice. Mice were treated for 5 days/week for 3 cycles with 10 mg/kg PR-957 (n = 10) or NaCl 0.9% as diluent control (n = 11). Relative abundance of leukemic cells after 3 weeks of treatment in primary recipient mice in (B) peripheral blood, (C) spleen and (D) bone marrow. Two independent cohorts; mean with SD; Mann–Whitney U test. E Relative abundance of L-GMP (Lin-Kit + Sca1-CD34 + FcgR + GFP +) in diluent or PR-957 treated mice. Two independent cohorts; mean with SD; Mann–Whitney U test. F 2 × 10⁶ whole bone marrow cells from the PR-957 or NaCl in vivo treated KMT2A::MLLT3 mice were transplanted into secondary recipients (n = 14 recipients of PR-957 treated mice; n = 15 recipients of NaCl treated mice). Abundance of leukemic cells in the peripheral blood, 2 weeks after transplantation. Two independent cohorts; Mann–Whitney U test. G Survival of secondary recipient mice. Mantel-Cox test. H-I Limiting dilution (LD) assay using murine KMT2A::MLLT3 BM cells treated for 48 h with diluent or 200 nM PR-957. H Reduction of leukemia initiating cells and (I) Cell dose, animal numbers, LSC-frequency and confidence intervals (CI) following diluent or PR-957 exposure. n = 4 mice per dilution and treatment, analysis performed using ELDA (Extreme Limiting Dilution Assay) software [28]. J Schematic depicting competitive repopulation assay to investigate effects of PR-957 treatment on normal HSPCs. K-M Relative abundance of hematopoietic cells in CD45.1 mice after 3 weeks of treatment with 10 mg/kg PR-957 (n = 6) or NaCl 0.9% (n = 6), specifically in (K) Peripheral blood, (L-M) Bone marrow. L HSC abundance (HSC: Lin⁻ Sca1⁺ cKit⁺ CD48⁻ CD150⁺); (M) Progenitor cell abundance; (N) Peripheral blood chimerism of recipient animals using BM cells from in vivo treated mice with PR-957 or diluent control. O-Q Abundance of hematopoietic cells in the BM of recipient mice at week 16. O mature cell compartments; (P) progenitor cells; (Q) HSCs (HSC: Lin⁻ Sca1⁺ cKit⁺ CD34⁻)