Figure 4. The HES4 gene is required to... | American Society of Hematology

Figure 4.

$The HES4 gene is required to maintain growth of established T-cell leukemias. (A) Flow cytometric analysis of GFP+ NGFR+ cell abundance in NOTCH1-ΔE–derived (N-only) established CB leukemias, transduced with lentivectors encoding the wt intracellular Notch1 domain (ICN1), the ICN1-R1984A dimer mutant, or the EV as control, together with a truncated nerve growth factor receptor (tNGFR) as a marker. Two days after the transduction, the NGFR+ cells were sorted using FACS and treated with GSI Compound E (1 μM) or dimethyl sulfoxide (DMSO) as mock control for 4 days. The absolute cell numbers were calculated using an internal beads-based control. The mean values of duplicate experiments are plotted. ∗∗P < .01 (2-way ANOVA). (B) Heat map and hierarchical clustering based on the normalized values of gene expression RNA-seq data derived from human ICN1 (WT), ICN1-R1984A (R1984A), and EV N-only established CB leukemia cells as reported in panel A. Differentially expressed genes, scaled to mean = 0 and SD = 1, are represented (adjusted P value ≤.1). (C) Flow cytometric analysis of GFP+ NGFR+mTagBFP2+ cell abundance of NOTCH1-ΔE-derived (N-only) established CB leukemias, doubly transduced with ICN1-R1984A/tNGFR lentiviruses and HES4/mTagBFP2 or EVs as control. The transduced cells were sorted using FACS and treated with GSI Compound E (1 μM) or DMSO as a mock control for 4 days. The absolute cell numbers were calculated with an internal beads-based control. The mean values of duplicate experiments are plotted. ∗P < .05; ∗∗∗P < .001 (2-way ANOVA). (D) Flow cytometric analysis of the NGFR+mTagBFP2+ cell abundance of PDX transduced with ICN1-R1984A/tNGFR lentiviruses alone or in combination with the HES4/mTagBFP2 vector. Cells transduced with ICN1/tNGFR or EV alone were also included as control. The transduced cells were sorted using FACS and treated with GSI Compound E (1 μM) or DMSO as mock control for 3 days. The absolute cell numbers were calculated using an internal beads-based control. The mean values of duplicate experiments are plotted. ∗∗P < .01; ∗∗∗P < .001 (2-way ANOVA). (E) Abundance of the shRNA-transduced Cherry+ cell fraction, tracked over time in culture by flow cytometry. The CUTLL-1, TALL-1, and RPMI-8402 cell lines were independently transduced with 2 different clones of shRNA/mCherry lentiviral constructs against HES4 gene or scramble control as indicated, sorted using FACS, and cultured in vitro. The graphs report the result of 3 independent experiments performed in triplicate. ∗∗∗P < .001 (2-way ANOVA). (F) Abundance of the HES4-transduced mTagBFP2+ cell fraction, tracked over time in culture by flow cytometry. The DND41 and ALL-SILL cell lines were independently transduced with the HES4/mTagBFP2 lentiviral construct or EVs as control. The graphs report the result of 3 independent experiments performed in triplicate. ∗∗∗P < .001 (2-way ANOVA).$

The HES4 gene is required to maintain growth of established T-cell leukemias. (A) Flow cytometric analysis of GFP+ NGFR+ cell abundance in NOTCH1-ΔE–derived (N-only) established CB leukemias, transduced with lentivectors encoding the wt intracellular Notch1 domain (ICN1), the ICN1-R1984A dimer mutant, or the EV as control, together with a truncated nerve growth factor receptor (tNGFR) as a marker. Two days after the transduction, the NGFR+ cells were sorted using FACS and treated with GSI Compound E (1 μM) or dimethyl sulfoxide (DMSO) as mock control for 4 days. The absolute cell numbers were calculated using an internal beads-based control. The mean values of duplicate experiments are plotted. ∗∗P < .01 (2-way ANOVA). (B) Heat map and hierarchical clustering based on the normalized values of gene expression RNA-seq data derived from human ICN1 (WT), ICN1-R1984A (R1984A), and EV N-only established CB leukemia cells as reported in panel A. Differentially expressed genes, scaled to mean = 0 and SD = 1, are represented (adjusted P value ≤.1). (C) Flow cytometric analysis of GFP+ NGFR+mTagBFP2+ cell abundance of NOTCH1-ΔE-derived (N-only) established CB leukemias, doubly transduced with ICN1-R1984A/tNGFR lentiviruses and HES4/mTagBFP2 or EVs as control. The transduced cells were sorted using FACS and treated with GSI Compound E (1 μM) or DMSO as a mock control for 4 days. The absolute cell numbers were calculated with an internal beads-based control. The mean values of duplicate experiments are plotted. ∗P < .05; ∗∗∗P < .001 (2-way ANOVA). (D) Flow cytometric analysis of the NGFR+mTagBFP2+ cell abundance of PDX transduced with ICN1-R1984A/tNGFR lentiviruses alone or in combination with the HES4/mTagBFP2 vector. Cells transduced with ICN1/tNGFR or EV alone were also included as control. The transduced cells were sorted using FACS and treated with GSI Compound E (1 μM) or DMSO as mock control for 3 days. The absolute cell numbers were calculated using an internal beads-based control. The mean values of duplicate experiments are plotted. ∗∗P < .01; ∗∗∗P < .001 (2-way ANOVA). (E) Abundance of the shRNA-transduced Cherry+ cell fraction, tracked over time in culture by flow cytometry. The CUTLL-1, TALL-1, and RPMI-8402 cell lines were independently transduced with 2 different clones of shRNA/mCherry lentiviral constructs against HES4 gene or scramble control as indicated, sorted using FACS, and cultured in vitro. The graphs report the result of 3 independent experiments performed in triplicate. ∗∗∗P < .001 (2-way ANOVA). (F) Abundance of the HES4-transduced mTagBFP2+ cell fraction, tracked over time in culture by flow cytometry. The DND41 and ALL-SILL cell lines were independently transduced with the HES4/mTagBFP2 lentiviral construct or EVs as control. The graphs report the result of 3 independent experiments performed in triplicate. ∗∗∗P < .001 (2-way ANOVA).

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