Figure 6.
ORP4L inhibition results in T-leukemia cell death. (A) Schematic image depicting the Dox-inducible gRNA lentiviral vectors. Cas9 is constitutively expressed in the cells. Treatment with Dox rapidly induced the sgRNA expression, which activated Cas9 and directed it to the target genomic sequence. (B) The experimental design. T cells were obtained from patients with ATL and maintained in vitro during lentiviral transduction with vectors encoding constitutive Cas9 and the Dox-inducible ORP4L sgRNA. For the in vivo assay, cells were transplanted into B-NDG mice and then monitored for development of T-cell leukemia. To delete the ORP4L gene, Dox was administered via food pellets (625 mg/kg) 10 days after transplantation. (C) Western blot analysis of ORP4L knockout and downstream signaling pathway in the T cells of a patient with ATL (ATL#21) after Dox (1 mg/mL) treatment for 72 hours in vitro. (D) Cell death analysis of T cells in a patient with ATL (ATL#21) transduced with constitutively expressed Cas9 and Dox-inducible ORP4L sgRNA lentiviral vectors with or without treatment with Dox (1 mg/mL) for the indicated times. Mean ± standard deviation (SD; n = 3 experimental repeat; Student t test). (E) Percentage of human CD45+ T cells of patients with ATL in the peripheral blood of B-NDG mouse transplant recipients 10 days after treatment with Dox or no treatment. Mean ± SD (n = 5 mice of each group; Student t test). (F) Kaplan-Meier comparative survival analysis of B-NDG mouse recipients of human T cells from patients with ATL treated with Dox or not treated, as described in panel B (n = 8 mice each group, log-rank test). (G) WT/NF-κB/p53 signaling pathway in T cells of patients with ATL (ATL#21, #26, #27) after LYZ-81 (5 nM) treatment of 18 hours. (H) Cell death analysis of T cells of patients with ATL treated with LYZ-81 (5 nM) for 18 hours. Mean ± SD (n = 3 experimental repeat; Student t test). (I) The experimental design for LYZ-81 therapy after xenotransplantation of human ATL cells in B-NDG mice. Leukemia cells obtained from patients with ATL were transplanted into B-NDG mice. Three weeks later, the mice were randomly assigned to 2 groups and treated with the ORP4L inhibitor LYZ-81 (5.8 mg/kg IV every 2 days ) or vehicle control for 2 weeks. (J) The percentage of transplanted human CD45+ ATL cells in the peripheral blood of B-NDG xenotransplant-recipient mice after a 2-week treatment, with or without LYZ-81. Mean ± SD (n = 5 mice of each group; Student t test). (K) Kaplan-Meier comparative survival analysis of B-NDG mouse recipients of human ATL T-cell xenotransplants and treated with or without LYZ-81 (n = 8 mice each group, log-rank test). *P < .05; ***P < .001.

ORP4L inhibition results in T-leukemia cell death. (A) Schematic image depicting the Dox-inducible gRNA lentiviral vectors. Cas9 is constitutively expressed in the cells. Treatment with Dox rapidly induced the sgRNA expression, which activated Cas9 and directed it to the target genomic sequence. (B) The experimental design. T cells were obtained from patients with ATL and maintained in vitro during lentiviral transduction with vectors encoding constitutive Cas9 and the Dox-inducible ORP4L sgRNA. For the in vivo assay, cells were transplanted into B-NDG mice and then monitored for development of T-cell leukemia. To delete the ORP4L gene, Dox was administered via food pellets (625 mg/kg) 10 days after transplantation. (C) Western blot analysis of ORP4L knockout and downstream signaling pathway in the T cells of a patient with ATL (ATL#21) after Dox (1 mg/mL) treatment for 72 hours in vitro. (D) Cell death analysis of T cells in a patient with ATL (ATL#21) transduced with constitutively expressed Cas9 and Dox-inducible ORP4L sgRNA lentiviral vectors with or without treatment with Dox (1 mg/mL) for the indicated times. Mean ± standard deviation (SD; n = 3 experimental repeat; Student t test). (E) Percentage of human CD45+ T cells of patients with ATL in the peripheral blood of B-NDG mouse transplant recipients 10 days after treatment with Dox or no treatment. Mean ± SD (n = 5 mice of each group; Student t test). (F) Kaplan-Meier comparative survival analysis of B-NDG mouse recipients of human T cells from patients with ATL treated with Dox or not treated, as described in panel B (n = 8 mice each group, log-rank test). (G) WT/NF-κB/p53 signaling pathway in T cells of patients with ATL (ATL#21, #26, #27) after LYZ-81 (5 nM) treatment of 18 hours. (H) Cell death analysis of T cells of patients with ATL treated with LYZ-81 (5 nM) for 18 hours. Mean ± SD (n = 3 experimental repeat; Student t test). (I) The experimental design for LYZ-81 therapy after xenotransplantation of human ATL cells in B-NDG mice. Leukemia cells obtained from patients with ATL were transplanted into B-NDG mice. Three weeks later, the mice were randomly assigned to 2 groups and treated with the ORP4L inhibitor LYZ-81 (5.8 mg/kg IV every 2 days ) or vehicle control for 2 weeks. (J) The percentage of transplanted human CD45+ ATL cells in the peripheral blood of B-NDG xenotransplant-recipient mice after a 2-week treatment, with or without LYZ-81. Mean ± SD (n = 5 mice of each group; Student t test). (K) Kaplan-Meier comparative survival analysis of B-NDG mouse recipients of human ATL T-cell xenotransplants and treated with or without LYZ-81 (n = 8 mice each group, log-rank test). *P < .05; ***P < .001.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal