Figure 1 from Disruption of KLHL6 Fuels Oncogenic Antigen Receptor Signaling in B-Cell Lymphoma
Authors: Meriranta Leo , Sorri Selma , Huse Kanutte , Liu Xiaonan , Spasevska Ivana , Zafar Sadia , Chowdhury Iftekhar , Dufva Olli , Sahlberg Eerika , Tandarić Luka , Karjalainen-Lindsberg Marja-Liisa , Hyytiäinen Marko , Varjosalo Markku , Myklebust June H. , Leppä Sirpa 
Published: September 2024

<p>Molecular framework and clinical significance of KLHL6 protein expression in DLBCL. <i>P</i> values for pairwise comparisons indicated as >0.05, ns; <0.05, *; <0.01, **; <0.001, ***; <0.0001, ****. <b>A,</b> Representative microscopy images of IHC staining of the human tonsil for KLHL6. Arrowheads indicate cells with representative GC-like subcellular staining (KLHL6<sup>GC+</sup>). Scale bars, 5,000, 200, and 20 μm. <b>B,</b> Representative IF microscopy image of a GC in a reactive human tonsil (tonsils, <i>n</i> = 3; lymph nodes, <i>n</i> = 4) co-stained for light zone marker CD23, dark zone marker Ki-67, and KLHL6. Scale bar, 200 μm. <b>C</b> and <b>D,</b> IF microscopy images representative of KLHL6 subcellular localization, co-stained with GM130 (Golgi marker) and DAPI (DNA marker, blue) obtained from GC of lymph node and DLBCL tissue at 40× with optical sectioning and Ocily7 cells at 100× with conventional IF microscopy (<b>D</b>). White arrowheads pinpoint examples of colocalization. Scale bar, 10 μm. <b>E,</b> KLHL6 expression in DLBCL cell lines. Top, Western blot lanes with arrowhead showing the size of overexpressed KLHL6 construct (lane not shown). Middle, Heatmap of <i>KLHL6</i> RNA transcript levels by qPCR. Bottom, Representative IF microscopy images of endogenous KLHL6 expression. White arrowheads pinpoint KLHL6<sup>GC+</sup> expression. RQ, relative quantification. Scale bar, 20 μm. <b>F</b> and <b>G,</b> Representative KLHL6 IHC staining patterns in four tumors (<b>F</b>, training set, <i>n</i> = 39) and quantification in the discovery cohort (<b>G</b>, <i>n</i> = 134). Black arrowheads denote KLHL6<sup>GC+</sup>, whereas red arrowheads show additional dispersed puncta. Interpretation of KLHL6 GC-like staining shown below the images. On the right, a scale to quantify KLHL6 immunoreactivity. GC-like pattern with immunoreactivity ≥5% was considered positive. Scale bars, 50 and 10 μm. <b>H,</b> Bar plot of KLHL6<sup>GC+</sup> cases in the discovery cohort (<i>n</i> = 134) according to molecular subtype. Fisher exact test comparing ABC and GCB. <b>I–L,</b> UMAP dimensionality reduction of 624 DLBCLs (<a href="#bib34" target="_blank">34</a>) with RNA sequencing expression data colored according to KLHL6 expression. Next to the plot, inlets of UMAP projections and dot and box plots showing KLHL6 expression within (<b>J</b>) molecular subtypes, (<b>K</b>) B-cell states and (<b>L</b>) BCL6 expression, translocations, and IHC positivity. Mann–Whitney <i>U</i> test comparisons in <b>J</b> and <b>L</b>; Kruskal–Wallis in <b>K</b>. <b>M,</b> Oncoprint of mutations in driver genes most significantly associated with KLHL6<sup>GC</sup> status in the discovery cohort (<i>n</i> = 110). Select MCD drivers and <i>KLHL6</i> shown, genes mutated in ≥5% of the cases considered, multiple testing correction performed with FDR. <b>N–O,</b> Kaplan–Meier survival estimates for overall survival (OS) according to KLHL6<sup>GC+</sup> pattern. Log-rank test. Below the estimates: multivariable Cox regression model’s HRs, 95% confidence intervals, and <i>P</i> values for OS with KLHL6<sup>GC+</sup> and IPI.</p>