Figure 7.
The NMRE is essential for the population growth of BMP-like cells. (A–C) qRT-PCR for MYCN expression in LOUCY cells (A), CUTLL3 cells (B), and THP-6 cells (C) that were transduced with DOX-inducible dCas9-KRAB, Tet3G, and sgRNA directed against the NMRE and treated with DOX for 3 days; n = 3/group. (D–F) Growth assay of LOUCY cells (D), CUTLL3 cells (E), and THP-6 cells (F) that were transduced in A–C. Fold expansion = trypan blue-negative cell counts normalized to day 0 trypan blue-negative cell counts (day 0 = 3 days after DOX addition). Negative sgRNA control (−) = non-targeting. Positive essential sgRNA control (+) = RPL34; n = 3/group. (G) CUTLL3 expressing the dCas9-KRAB protein was infected with the LRCherry2.1 vector expressing mCherry and the indicated sgRNAs. The percentage of mCherry+ cells was analyzed over time by flow cytometry and normalized to day 4 after infection; n = 1/group. (H) RNA was extracted from sorted CUTLL3 cells described in G at day 7 after infection and analyzed for MYCN by qRT-PCR; n = 3/group. (I and J) Jurkat (I) and CUTLL1 (J) mature T-ALL cells expressing dCas9-KRAB protein were transduced with the indicated sgRNAs. The percentage of mCherry+ cells was assessed over time by flow cytometry and normalized to day 4 after infection; n = 1/group. (K and L) CUTLL3 cells were co-transduced with Mycn and either shControl or shZMIZ1 (15) and then measured for population growth (K) or Mycn qRT-PCR analysis (L). Fold expansion = trypan blue-negative cell counts normalized to day 0 trypan blue-negative cell counts; n = 3/group. (M–O) LOUCY cells transduced with indicated sgRNAs were injected into NSG mice and 2 wk later treated with DOX in drinking water to activate dCas9-KRAB-GFP. Pan-essential = RPL8 promoter. Representative GFP/hCD45.2 flow cytometry plots at 11–12 wk after injection (M), GFP+/hCD45+ blast counts (N) and survival (log rank test P values, O) were measured; n = 16 (control), 11 (BCL2e), 8 NMRE), 10 (MYBe), and 16 (pan-essential)/group. Unless noted otherwise, P values were based on two-sided t tests for two samples and one-way ANOVA for more than two samples. *P < 0.05; **P < 0.01; ***P < 0.001. Refer to the image caption for details.

The NMRE is essential for the population growth of BMP-like cells. (A–C) qRT-PCR for MYCN expression in LOUCY cells (A), CUTLL3 cells (B), and THP-6 cells (C) that were transduced with DOX-inducible dCas9-KRAB, Tet3G, and sgRNA directed against the NMRE and treated with DOX for 3 days; n = 3/group. (D–F) Growth assay of LOUCY cells (D), CUTLL3 cells (E), and THP-6 cells (F) that were transduced in A–C. Fold expansion = trypan blue-negative cell counts normalized to day 0 trypan blue-negative cell counts (day 0 = 3 days after DOX addition). Negative sgRNA control (−) = non-targeting. Positive essential sgRNA control (+) = RPL34; n = 3/group. (G) CUTLL3 expressing the dCas9-KRAB protein was infected with the LRCherry2.1 vector expressing mCherry and the indicated sgRNAs. The percentage of mCherry+ cells was analyzed over time by flow cytometry and normalized to day 4 after infection; n = 1/group. (H) RNA was extracted from sorted CUTLL3 cells described in G at day 7 after infection and analyzed for MYCN by qRT-PCR; n = 3/group. (I and J) Jurkat (I) and CUTLL1 (J) mature T-ALL cells expressing dCas9-KRAB protein were transduced with the indicated sgRNAs. The percentage of mCherry+ cells was assessed over time by flow cytometry and normalized to day 4 after infection; n = 1/group. (K and L) CUTLL3 cells were co-transduced with Mycn and either shControl or shZMIZ1 (15) and then measured for population growth (K) or Mycn qRT-PCR analysis (L). Fold expansion = trypan blue-negative cell counts normalized to day 0 trypan blue-negative cell counts; n = 3/group. (M–O) LOUCY cells transduced with indicated sgRNAs were injected into NSG mice and 2 wk later treated with DOX in drinking water to activate dCas9-KRAB-GFP. Pan-essential = RPL8 promoter. Representative GFP/hCD45.2 flow cytometry plots at 11–12 wk after injection (M), GFP+/hCD45+ blast counts (N) and survival (log rank test P values, O) were measured; n = 16 (control), 11 (BCL2e), 8 NMRE), 10 (MYBe), and 16 (pan-essential)/group. Unless noted otherwise, P values were based on two-sided t tests for two samples and one-way ANOVA for more than two samples. *P < 0.05; **P < 0.01; ***P < 0.001.

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