Figure 5.
Impact of targeting EZH2 on APL progression. (A) Experimental scheme of the analysis of RA, GSK, or combination-treated BM. PLZF-RARA TG BM is transplanted into recipient mice. Seven days after, mice are injected for 3 days with GSK or with corn oil (NT) followed by 7 consecutive days of RA (RA). After treatments (NT: corn oil, GSK, RA, GSK and RA) mice are sacrificed, and treated BM are immunophenotyped and reinjected into new recipient mice (tNT, tGSK, tRA, tGSK+RA). At this time treatments are stopped (Ø). Mice are sacrificed 15 to 20 days after the secondary transplantation. (B) (Left) Global levels of PLZF-RARA (black arrow: full length, black star: degraded form), Ezh2, and H3K27me3. Actin is used as loading control. (Right) Bar plots representing the signal intensity of PLZF-RARA (P-RARA), EZH2, and H3K27me3 normalized to the loading control and to the NT condition. ∗P < .05 (3 biological replicates). (C) (Left) Impact of GSK, RA, and combo treatments on PLZF-RARA leukemia at day 17 after the first transplantation (NT, GSK, RA, GSK+RA). (Middle and right) Evaluation of leukemia relapse monitored by FACS analysis (Cd45.2, Cd11b, and Gr1 markers are monitored) at day 13 (d13) and day 18 (d18) after the secondary transplantation (tNT, tGSK, tRA, tGSK+RA). (D) Scheme resuming the protocol to evaluate the effect of MS treatment on PLZF-RARA BM. (E) (Left) Global levels of PLZF-RARA and Ezh2 after RA (0.1 μM) and/or MS (1.25 μM) treatments. Actin is used as loading controls. (Right) Bar plots representing the signal intensity of PLZF-RARA (P-RARA) and EZH2 normalized to the loading control and to the NT condition. ∗P < .05 (2 biological replicates). (F) Cell morphology analyzed by MGG staining (magnification 64×; bar 20 μm). (G) Cell viability of PLZF-RARA TG cells upon GSK or MS treatments monitored by bioluminescence. Results are expressed by percent of living cells and normalized to the untreated condition (NT). Results are expressed as the mean standard deviation of 3 independent experiments (n = 3). ∗P < .05, ∗∗∗P < .001. (H) Survival rate of mice transplanted with untreated (tNT, gray curve, 8 mice) GSK (tGSK, 2.5 μM, blue curve, 5 mice), RA (tRA, 0.1 μM, 5 mice), MS (tMS, 2.5 μM, purple curve, 10 mice), MS and RA (tMS+RA, 1.25 μM MS and 0.1 μM RA, green curve, 5 mice) pretreated PLZF-RARA TG BM. ∗P < .05, ∗∗P < .01. (I) RNA-seq (2 biological replicates) performed on promyelocytes purified from PLZF-RARA TG mice and treated or not (NT) with MS (MS, 2.5 μM), or GSK (2.5 μM) (in vitro treatments). Impact of MS (MS) and GSK on the expression of the most differentially overexpressed genes in the ReP cluster compared with the other clusters. Results are expressed as gene counts z score. (J) Gene set enrichment analysis of t(11;17) APL patients (PLZF-RARA) compared with t(15;17) APL patients (PML-RARA). ReP, NeuRA, the targeting of nonmethyltransferase activities of EZH2 (Non methyl.targeting) and the targeting of methyltransferase activities of EZH2 (Methyl. targeting) enrichment signatures (ESs) are computed. FDR, false discovery rate; NES, normalized enrichment score.

Impact of targeting EZH2 on APL progression. (A) Experimental scheme of the analysis of RA, GSK, or combination-treated BM. PLZF-RARA TG BM is transplanted into recipient mice. Seven days after, mice are injected for 3 days with GSK or with corn oil (NT) followed by 7 consecutive days of RA (RA). After treatments (NT: corn oil, GSK, RA, GSK and RA) mice are sacrificed, and treated BM are immunophenotyped and reinjected into new recipient mice (tNT, tGSK, tRA, tGSK+RA). At this time treatments are stopped (Ø). Mice are sacrificed 15 to 20 days after the secondary transplantation. (B) (Left) Global levels of PLZF-RARA (black arrow: full length, black star: degraded form), Ezh2, and H3K27me3. Actin is used as loading control. (Right) Bar plots representing the signal intensity of PLZF-RARA (P-RARA), EZH2, and H3K27me3 normalized to the loading control and to the NT condition. ∗P < .05 (3 biological replicates). (C) (Left) Impact of GSK, RA, and combo treatments on PLZF-RARA leukemia at day 17 after the first transplantation (NT, GSK, RA, GSK+RA). (Middle and right) Evaluation of leukemia relapse monitored by FACS analysis (Cd45.2, Cd11b, and Gr1 markers are monitored) at day 13 (d13) and day 18 (d18) after the secondary transplantation (tNT, tGSK, tRA, tGSK+RA). (D) Scheme resuming the protocol to evaluate the effect of MS treatment on PLZF-RARA BM. (E) (Left) Global levels of PLZF-RARA and Ezh2 after RA (0.1 μM) and/or MS (1.25 μM) treatments. Actin is used as loading controls. (Right) Bar plots representing the signal intensity of PLZF-RARA (P-RARA) and EZH2 normalized to the loading control and to the NT condition. ∗P < .05 (2 biological replicates). (F) Cell morphology analyzed by MGG staining (magnification 64×; bar 20 μm). (G) Cell viability of PLZF-RARA TG cells upon GSK or MS treatments monitored by bioluminescence. Results are expressed by percent of living cells and normalized to the untreated condition (NT). Results are expressed as the mean standard deviation of 3 independent experiments (n = 3). ∗P < .05, ∗∗∗P < .001. (H) Survival rate of mice transplanted with untreated (tNT, gray curve, 8 mice) GSK (tGSK, 2.5 μM, blue curve, 5 mice), RA (tRA, 0.1 μM, 5 mice), MS (tMS, 2.5 μM, purple curve, 10 mice), MS and RA (tMS+RA, 1.25 μM MS and 0.1 μM RA, green curve, 5 mice) pretreated PLZF-RARA TG BM. ∗P < .05, ∗∗P < .01. (I) RNA-seq (2 biological replicates) performed on promyelocytes purified from PLZF-RARA TG mice and treated or not (NT) with MS (MS, 2.5 μM), or GSK (2.5 μM) (in vitro treatments). Impact of MS (MS) and GSK on the expression of the most differentially overexpressed genes in the ReP cluster compared with the other clusters. Results are expressed as gene counts z score. (J) Gene set enrichment analysis of t(11;17) APL patients (PLZF-RARA) compared with t(15;17) APL patients (PML-RARA). ReP, NeuRA, the targeting of nonmethyltransferase activities of EZH2 (Non methyl.targeting) and the targeting of methyltransferase activities of EZH2 (Methyl. targeting) enrichment signatures (ESs) are computed. FDR, false discovery rate; NES, normalized enrichment score.

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