KLHL6 promotes DLBCL sensitivity to CHOP by controlling NOTCH2 levels. (A) 1 × 10⁷ U2932-Cas9 cells with the indicated genotypes were xenografted in the flanks of NSG mice. After the average tumor volumes reached 100 mm³, mice were treated with cyclophosphamide (30 mg/kg), doxorubicin (2.48 mg/kg), and vincristine (0.38 mg/kg) IV on day 1, and prednisone (0.2 mg/kg) was given via oral gavage daily for 5 days. Experimental end point was reached when the tumor volume reached 1000 mm³. Each line represents one tumor in one mouse (n = 5 per group; two-way ANOVA). (B) Kaplan-Meier survival analysis of mice shown in panel A (n = 5 per group; Mantel-Cox test). (C) Principal component analysis for the transcriptomes of tumors with the indicated genotypes. (D) Volcano plot representing the log₂(fold change) over the -log₁₀(P value) of the differentially expressed genes. The red circles represent the significantly upregulated genes, and the blue circles represent the significantly downregulated genes (n = 3; DeSeq2; P ≤ .01). (E) Dot plot showing the GSEA for the transcriptomes in (D). Size of the dots is a function of the P value, and the color represents the normalized enrichment score. (F) Heat map showing the relative expression of MYC-signature genes using the normalized counts from the transcriptome analysis in panel D. (G, left) Immunoblot analysis for the indicated proteins in a panel of primary DLBCL biopsies. (G, right) Quantification of NOTCH2 (TMIC) (x-axis) and KLHL6 (y-axis) protein levels in each DLBCL biopsy specimen. (H) Percentage of KLHL6-mutated vs KLHL6 WT patients with DLBCL grouped based on the clusters, as previously defined.⁶