Notch1 hyperactivation induces cell cycle–related gene expression in CLL cells. (A) Sample clustering based on correlation of gene expression (Pearson correlation of 1000 most variable genes) in sorted splenic CLL cells (CD19⁺B220⁻) from IgH.TEμ;RNIC ^lox/+ controls (in blue) or from IgH.TEμ;RNIC^{Δ/+ CD19-Cre} and IgH.TEμ;RNIC^{Δ/+ mb1-Cre} mice (in red) is illustrated. (B) Pathways from Hallmark collection (MSigDB) significantly induced or repressed (false discovery rate < 0.05) as result of GSEA are shown. Genes are ranked according to differential expression in sorted CLL cells comparing IgH.TEμ;RNIC^{Δ/+ CD19-Cre} and IgH.TEμ;RNIC^{Δ/+ mb1-Cre} mice to IgH.TEμ;RNIC^lox/+ mice. P values were obtained by permutation test (n = 100 000) and FDR adjusted.⁵⁴  (C) Detailed GSEA plots for E2F (left) and MYC target genes (right) from the Hallmark collection are depicted. (D) RNA expression of E2F target genes that are significantly induced (log fold-change > 1 and adjusted P value < .05) in IgH.TEμ;RNIC^{Δ/+ CD19-Cre} and IgH.TEμ;RNIC^{Δ/+ mb1-Cre} mice compared with IgH.TEμ;RNIC^lox/+ controls are shown. (E) RNA expression of Notch target genes Hes1, Hey1, Hey2, HeyL, and c-Myc assessed by qRT-PCR and normalized to the expression of Hprt is shown from sorted splenic CLL cells (CD19⁺B220⁻) of IgH.TEμ;control (n = 7 composed of n = 3 IgH.TEμ^mb1-Cre and n = 4 IgH.TEμ;RNIC^lox/+ mice, blue dots) and of IgH.TEμ;RNIC^lox/+mb1-Cre mice (n = 6, red squares). P values were calculated using unpaired Student t test: Hes1 P < .0001, Hey1 P = .0126, Hey2 P = .0077, HeyL P = .0075, c-Myc P = .0117. (F) RNA expression of Ccna2, Ccnb2, Ccne2, Cdc6, MCM5, MCM6, MCM7, and Dbf4 assessed by qRT-PCR and normalized to the expression of Hprt is depicted for the same samples used in panel A. P values were calculated using unpaired Student t test: Ccna2 P < .0001, Ccnb2 P = .0002, Ccne2 P = .0042, Cdc6 P = .0066, MCM5 P = .0025, MCM6 P = .0147, MCM7 P = .0093, Dbf4 P = .0003. (G) Protein expression of Cyclin A2, Cyclin E2, and c-Myc assessed by immunoblotting from sorted splenic CLL (CD19⁺B220⁻) cells of IgH.TEμ;control (n = 2 IgH.TEμ;RNIC^lox/+ and n = 2 IgH.TEμ^mb1-Cre) and IgH.TEμ;RNIC^{lox/+ mb1-Cre} mice (n = 4) is shown. (H) Representative immunohistochemical staining for c-Myc of spleen (top) and LN (bottom) sections from 7-month-old WT control, IgH.TEμ, and IgH.TEμ;RNIC^{Δ/+ mb1-Cre} mice is shown. Quantification of c-Myc⁺ stained cells in WT (n = 4), IgH.TEμ (n = 4) and IgH.TEμ;RNIC^{Δ/+ mb1-Cre} (n = 3) is depicted on the right.