Figure 3.
Figure 3. EZH2 depletion induces chemotherapy resistance in T-ALL. (A) CCRF-CEM cells were transduced with Cas9 and EZH2 targeting gRNA, single-cell cloned, and next-generation sequencing was used to identify a clone with EZH2 haploinsufficiency (EZH2 mutant clone E). Western blot analysis was performed to assess expression of the indicated proteins in basal conditions or following transduction with doxycycline-induced constructs encoding WT EZH2 or a catalytically defective triple mutant. One representative experiment is shown, which was repeated independently. (B) BH3 profiling was performed on the cells shown in A. Results shown are the mean ± SEM of n = 3 biological replicates. Significance was assessed by one-way ANOVA with Tukey adjustment for multiple comparisons. P = 0.017 for parental Cas9 versus EZH2 mutant clone; P = 0.004 for clone E transduced with EZH2 WT versus EZH2 catalytic mutant. Bar charts represent the mean of three biological replicates, and the experiment was repeated independently. (C) EZH2 mutant clone E cells or their parental Cas9 controls were treated with the indicated doses of vincristine for 48 h and then released from chemotherapy. Viable cell counts were obtained by trypan blue exclusion at the indicated time points. Significance assessed by Welch t test (P = 0.07 for untreated EZH2 mutant versus untreated parental Cas9; P = 0.006 for vincristine-treated EZH2 mutant versus vincristine-treated parental Cas9). Results shown are the mean ± SEM of n = 3 biological replicates. Representative data of at least two independent experiments shown. (D) EGFP-transduced EZH2 mutant clone E cells were mixed at 1:1 ratio with tdTomato-transduced parental Cas9 controls. The resultant pool of cells was treated with vehicle control or the indicated chemotherapeutics for 48 h and subsequently released from chemotherapy. Relative abundance of each clone was assessed by flow cytometry analysis 4 d after chemotherapy release. Results are normalized to the abundance of each clone in nonchemotherapy-treated controls. Significance assessed by Welch t test, with P < 0.0001 for vincristine, P = 0.0281 for etoposide, P = 0.0003 for cytarabine, P = 0.0008 for doxorubicin, and P = 0.0004 for methotrexate. Results shown are the mean ± SEM of n = 3 biological replicates, and the experiment was repeated independently. (E) Experimental design to assess relative fitness of CCRF-CEM cells transduced with gRNAs targeting the catalytic domain of EZH2, or the AAVS1 safe-harbor genomic locus, in control or chemotherapy-treated conditions. (F) CCRF-CEM cells manipulated as shown in E were treated with 30 nM vincristine or vehicle for 48 h, released from vincristine for 12 d, and gRNA representation was assessed by next-generation sequencing. Relative abundance of each gRNA was normalized to its abundance in vehicle-treated controls. Significance assessed by Welch t test; P = 0.0001. (G) Experimental design to assess in vivo chemosensitivity of EZH2 mutant clone E cells or their parental Cas9 controls in NRG-immunodeficient mice. (H) FACS analysis of splenic cells harvested from mice after treatment as indicated in G. Each bar is the mean of five independent mice. Significance was assessed by Welch t test; P = 0.0026. (I) Representative FACS plots from mice analyzed in H. Control mice without fluorescent leukemia are shown as the negative control for setting FACS gates. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001; n.s., P > 0.05.

EZH2 depletion induces chemotherapy resistance in T-ALL. (A) CCRF-CEM cells were transduced with Cas9 and EZH2 targeting gRNA, single-cell cloned, and next-generation sequencing was used to identify a clone with EZH2 haploinsufficiency (EZH2 mutant clone E). Western blot analysis was performed to assess expression of the indicated proteins in basal conditions or following transduction with doxycycline-induced constructs encoding WT EZH2 or a catalytically defective triple mutant. One representative experiment is shown, which was repeated independently. (B) BH3 profiling was performed on the cells shown in A. Results shown are the mean ± SEM of n = 3 biological replicates. Significance was assessed by one-way ANOVA with Tukey adjustment for multiple comparisons. P = 0.017 for parental Cas9 versus EZH2 mutant clone; P = 0.004 for clone E transduced with EZH2 WT versus EZH2 catalytic mutant. Bar charts represent the mean of three biological replicates, and the experiment was repeated independently. (C) EZH2 mutant clone E cells or their parental Cas9 controls were treated with the indicated doses of vincristine for 48 h and then released from chemotherapy. Viable cell counts were obtained by trypan blue exclusion at the indicated time points. Significance assessed by Welch t test (P = 0.07 for untreated EZH2 mutant versus untreated parental Cas9; P = 0.006 for vincristine-treated EZH2 mutant versus vincristine-treated parental Cas9). Results shown are the mean ± SEM of n = 3 biological replicates. Representative data of at least two independent experiments shown. (D) EGFP-transduced EZH2 mutant clone E cells were mixed at 1:1 ratio with tdTomato-transduced parental Cas9 controls. The resultant pool of cells was treated with vehicle control or the indicated chemotherapeutics for 48 h and subsequently released from chemotherapy. Relative abundance of each clone was assessed by flow cytometry analysis 4 d after chemotherapy release. Results are normalized to the abundance of each clone in nonchemotherapy-treated controls. Significance assessed by Welch t test, with P < 0.0001 for vincristine, P = 0.0281 for etoposide, P = 0.0003 for cytarabine, P = 0.0008 for doxorubicin, and P = 0.0004 for methotrexate. Results shown are the mean ± SEM of n = 3 biological replicates, and the experiment was repeated independently. (E) Experimental design to assess relative fitness of CCRF-CEM cells transduced with gRNAs targeting the catalytic domain of EZH2, or the AAVS1 safe-harbor genomic locus, in control or chemotherapy-treated conditions. (F) CCRF-CEM cells manipulated as shown in E were treated with 30 nM vincristine or vehicle for 48 h, released from vincristine for 12 d, and gRNA representation was assessed by next-generation sequencing. Relative abundance of each gRNA was normalized to its abundance in vehicle-treated controls. Significance assessed by Welch t test; P = 0.0001. (G) Experimental design to assess in vivo chemosensitivity of EZH2 mutant clone E cells or their parental Cas9 controls in NRG-immunodeficient mice. (H) FACS analysis of splenic cells harvested from mice after treatment as indicated in G. Each bar is the mean of five independent mice. Significance was assessed by Welch t test; P = 0.0026. (I) Representative FACS plots from mice analyzed in H. Control mice without fluorescent leukemia are shown as the negative control for setting FACS gates. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001; n.s., P > 0.05.

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