Figure 5 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>Recurrent KLHL6 mutations alter levels of BCR components and impact responses to BCR stimulation. <b>A,</b> Strategy to investigate KLHL6 interactome upon BCR engagement. SuDHL5 cells with ectopic expression of Strep-tagged KLHL6 bait were treated at RT for 60 seconds with anti–human IgM F(ab′)<sub>2</sub> fragment or nonspecific goat IgG antibody. Snap-frozen lysates were affinity-purified, and the interacting proteins were identified with mass spectrometry (AP-MS). SuDHL5 cells expressing Strep-tagged EmGFP were stimulated and analyzed as a control by AP-MS. <b>B,</b> Differences in KLHL6 interactome between the conditions. <i>Y</i> axis represents the LFC of KLHL6 interacting proteins against EmGFP control purifications in the DESeq2 model from <a href="#fig3" target="_blank">Fig. 3B</a>. <b>C</b> and <b>D,</b> Box and dot plots of non-normalized spectral counts of (<b>C</b>) KLHL7 and (<b>D</b>) UBR4 with induced interaction with KLHL6 bait upon stimulation. <i>P</i> values calculated with the Mann–Whitney <i>U</i> test. <b>E</b> and <b>H,</b> Western blot analyses of CD79A, CD79B, and IgH (IgM or IgG) in (<b>E</b>) SuDHL5 cells transduced with EmGFP control and nontagged KLHL6 WT or V5-tagged KLHL7 and KLHL26 overexpression constructs (<b>F</b>) Cas9-expressing SuDHL4 cells transduced with KLHL6 (KLHL6.1 and KLHL6.4) or KLHL7 (KLHL7.3 and KLHL7.4) targeting sgRNAs or control sgRNAs (MYC and Chr2; <b>G</b>) SuDHL5 cells transduced with KLHL6 WT and mutant constructs and (<b>H</b>) SuDHL4, U2932, and HBL1 cell lines transduced with WT and mutant KLHL6 constructs. <b>I</b> and <b>J,</b> Impacts of different KLHL6 constructs on the surface levels of all BCR components in SuDHL5 (<i>n</i> = 4), Ocily7, SuDHL4, and U2932 (<i>n</i> = 5) cell lines measured with flow cytometry. Values on <i>y</i>-axis (<b>I</b>) and the color fill (<b>J</b>) represent arcsinh ratio of MFI normalized to expression in parental cells. <i>P</i> values calculated with two-sided one-sample <i>t</i> test and (<b>J</b>) corrected for multiple testing with the Bonferroni method. <b>K,</b> Induction of SYK kinase phosphorylation measured by phospho-flow in various lymphoma cells expressing indicated KLHL6 mutants with different anti–IgH F(ab′)<sub>2</sub> concentrations. Data pooled from 5 to 6 independent experiments. <i>P</i> values were calculated with one-way ANOVA followed by the Dunnett multiple comparisons test against the unstimulated control. <b>L,</b> Balloon plot of anti–IgM-induced or anti–IgG-induced (10 µg/mL) signaling in parental and genetically modified SUDHL5, Ocily7, SuDHL4, and U2932 cells overexpressing KLHL6 WT and mutant constructs measured by phospho-flow. Color fill shows the mean phosphorylated levels of SYK, BTK, and PLCγ as arcsinh ratio of MFI normalized to expression unstimulated cells. Statistical significance according to the one-way ANOVA test followed by the Dunnett multiple comparisons test. <b>M,</b> Cell viability (Cell titer Glo) in various cell lines with different KLHL6 genetic manipulations after 48 hours of treatment in indicated concentrations of F(ab′)<sub>2</sub> anti-IgM or F(ab′)<sub>2</sub> anti-IgG. Viability normalized to control treatment with unspecific goat IgG antibody. <i>P</i> values are calculated using the two-sided <i>t</i> test against EmGFP-expressing cells.</p>