Figure 2 from Disruption of KLHL6 Fuels Oncogenic Antigen Receptor Signaling in B-Cell Lymphoma

Recurrent KLHL6 mutations and their impact on subcellular localization in lymphoma cells. A, Lollipop plot of KLHL6 mutations reported in 303 DLBCL tumors. B, Bar plots showing the fraction of different amino acid (AA) changes according to the most recurrently mutated AA residues in DLBCLs. Most common AA replacement per residue highlighted as the left hand side bar. C, Evolutionary conservation of KLHL6 AA sequences in the vicinity of the most frequently mutated AA residues across selected species. D, Predicted three-dimensional (3D) structure of KLHL6 according to in silico modeling using AlphaFold. Protein domains are shown with different color shading, and recurrently mutated AA residues are highlighted. E, 3D model of KLHL6 Kelch domain seen from above with recurrently mutated glutamic acid residues E547 and E568 shown. Inlet highlights colocalization. F and G, Genetic (F) and protein localization (G) features differentially associated with KLHL6 mutations in exon one (encoding the BTB hotspot) and Kelch domain. F, Mutations enriched in other genes co-occurring with different types of KLHL6 mutations in the Lacy and colleagues data set (n = 928; ref. 38). Genes mutated in ≥5% of the patients included in the analysis. Boxed rows indicate enriched genomic drivers (Fisher exact < 0.05), genes with FDR < 0.1 bolded. G, Co-occurrence with KLHL6 staining patterns according to KLHL6GC+ status in the discovery cohort (n = 110). H, Light microscopy images of diagnostic DLBCL tissue sections immunohistochemically stained for KLHL6 from patients with different KLHL6 mutations according to the original variant calls or re-analysis. Annotated AA changes of mutations highlighted per representative figure. Red arrowheads indicate aberrant puncta in tissues with KLHL6L65P mutation. Black arrowheads indicate KLHL6GC+ pattern. Scale bars, 50 μm (10 μm for inlets). I, Representative IF microscopy images of GCB cell lines SuDHL5 and Ocily7 and ABC cell line U2932 with ectopic expression of KLHL6 WT or mutants indicated on the top. Black arrowheads indicate representative KLHL6GC+ patterns and red arrowheads indicate aberrant vesicles of KLHL6L65P. Scale bar, 10 μm. J, Representative IF microscopy images of SuDHL5 cells expressing KLHL6L65P, KLHL6L90F, or KLHL6E547K mutants. Cells co-stained for KLHL6 and Golgi marker GM130. Co-stainings of other mutants shown in Supplementary Fig. S2E. Scale bar, 10 μm.