Pharmacologic suppression of Ca⁺⁺-signaling inhibits AML1-ETO LSC function in vitro and in vivo. (A) Unsupervised hierarchical clustering of significantly downregulated proteins following genetic inactivation of PLCG1 by a specific gRNA in SKNO-1_Cas9-Blast cells. (B) Network map displaying the significantly enriched signaling pathways upon genetic inactivation of PLCG1, annotation from Reactome. The node size and color represent number of proteins participating in each node. (C) Proliferation assayed by cell counting after trypan blue exclusion for Kasumi-1, SKNO-1, HL-60, and OCI-AML3 cells following treatment with the calcineurin inhibitor cyclosporin A (CsA, 5 μM) or diluent control (NaCl 0.9%). n = 4 independent experiments, 1-way ANOVA. (D) Analysis scheme of primary recipient mice following ciclosporin A (CsA) treatment vs diluent control (NaCl 0.9%). (E) Spleen weight of AE/K primary recipient mice; Mann-Whitney U test. (F) Histologic analysis of liver, lung, and spleen morphology after onset of AML in AE/K primary recipient mice treated with CsA or diluent control (NaCl 0.9%). Scale bars, 100 μm. (G) Immunophenotypic analysis of AE/K GFP⁺ BM LSK cells; Mann-Whitney U test. (H) Kaplan-Meier survival curves of AE/K secondary recipient mice. Irradiated (13 Gy, single dose) 6- to 8-week-old recipients of 2 × 10⁶ bone marrow cells from AE/K CsA (n = 7) or NaCl 0.9% (n = 8) treated primary recipients; Mantel-Cox test. (I) Spleen weight of MA9 primary recipient mice treated with CsA or diluent control (NaCl 0.9%), n.s., not significant. (J) Histologic analysis of liver, lung, and spleen morphology after onset of AML in MA9 primary recipient mice after treatment with CsA or diluent control. Scale bars, 100 μm. (K) Immunophenotypic analysis of GFP⁺LSK cells in the bone marrow of MA9 primary recipient mice. (L) Survival of MA9-transformed secondary recipient mice. Irradiated (13 Gy, single dose) 6- to 8-week-old recipients of 2 × 10⁶ bone marrow cells from MA9 CsA or NaCl 0.9% treated primary recipient mice (n = 8 CsA; n = 8 NaCl 0.9%), Mantel-Cox test. (M-N) Number of engrafted mice per dilution in the NaCl- vs CsA-treated cohort. LSC frequency was 1/5801 for NaCl-treated recipients (95% confidence interval, 1/2182-15,427) and 1/12,1901 for CsA-treated recipients (95% confidence interval, 1/39,911-372,329), P = .000026 using Poisson analysis; n = 5 mice per dilution and treatment, analysis was performed using ELDA (Extreme Limiting Dilution Assay) software.⁴⁴ (O) Colony formation of primary human AE/t(8;21) AML cells (n = 6 individual patients). Colony number per sample following pharmacologic inhibition with CsA (5, 10 μM) compared with diluent control (NaCl 0.9%). (P) Representative pictures of colonies from t(8;21) AML bone marrow cells after pharmacological inhibition with cyclosporin A compared with diluent control (NaCl). Scale bars, 200 μm. (Q) Colony count of BM cells derived from 3 independent healthy donors. Colony number per sample following pharmacologic inhibition with CsA (5 μM) compared with diluent control (NaCl 0.9%). (R) Number of hCD4⁺ hCD13⁺ cells per 1 × 10⁶ bone marrow (BM) cells after treatment with CsA (n = 4 mice) compared with diluent control (NaCl 0.9%; n = 4 mice). (S) Pie charts depicting engraftment of t(8;21) AML cells (%) after treatment with CsA or diluent control.