Combined CDKN2A, CDKN2B, and TP53 disruption accelerates tumor growth in the Eμ-TCL1 adoptive transfer model. (A) Schematic representation of the CRISPR/Cas9 procedure to target the CDKN2A, CDKN2B, and TP53 genes in primary Eμ-TCL1–derived murine CLL cells. (B) Box plots with data points showing absolute numbers of CD5⁺/CD19⁺ leukemia cells in peritoneal cavity, peripheral blood, and spleen of mice at 21 days after receiving CDKN2A/2B/TP53-targeted or control leukemia cells (n = 6 mice per group). Statistical analysis was performed using the Student t test. (C) Hematoxylin-and-eosin (H&E) staining and immunohistochemistry analysis of proliferating BrdU⁺ and phosphohistone H3⁺ cells in spleens of mice 21 days after receiving CDKN2A/2B/TP53-targeted or control leukemia cells. Images were captured with an Olympus BX53 microscope using an Olympus UC90 camera and CellSens Entry software. Statistical analysis was performed using the Student t test. Top row: scale bars, 500 μm; rows 2-4: scale bars, 20 μm. (D) Indel analysis by amplicon capillary electrophoresis of CDKN2A/2B/TP53-targeted leukemia cells isolated from spleens of mice after the first and second passage. Wild-type alleles are indicated by a red arrow; mutant alleles are indicated by a black arrow or brackets. The mutant/wild-type (M/WT) ratio was calculated by dividing the sum of the peak areas of the mutant alleles with the peak area of the wild-type allele. (E) Amplicon capillary electrophoresis analysis of indels in CDKN2A, CDKN2B, and TP53 in leukemia cells with independent targeting of CDKN2A/2B or TP53. (F) Box plots with data points showing absolute numbers of CD5⁺/CD19⁺ leukemia cells in peritoneal cavity, peripheral blood, and spleen of mice injected with CDKN2A/2B-targeted, TP53-targeted, or control leukemia cells (n = 8 mice per group).