Figure 5 from Disruption of KLHL6 Fuels Oncogenic Antigen Receptor Signaling in B-Cell Lymphoma

Recurrent KLHL6 mutations alter levels of BCR components and impact responses to BCR stimulation. A, 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′)2 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, Differences in KLHL6 interactome between the conditions. Y axis represents the LFC of KLHL6 interacting proteins against EmGFP control purifications in the DESeq2 model from Fig. 3B. C and D, Box and dot plots of non-normalized spectral counts of (C) KLHL7 and (D) UBR4 with induced interaction with KLHL6 bait upon stimulation. P values calculated with the Mann–Whitney U test. E and H, Western blot analyses of CD79A, CD79B, and IgH (IgM or IgG) in (E) SuDHL5 cells transduced with EmGFP control and nontagged KLHL6 WT or V5-tagged KLHL7 and KLHL26 overexpression constructs (F) 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; G) SuDHL5 cells transduced with KLHL6 WT and mutant constructs and (H) SuDHL4, U2932, and HBL1 cell lines transduced with WT and mutant KLHL6 constructs. I and J, Impacts of different KLHL6 constructs on the surface levels of all BCR components in SuDHL5 (n = 4), Ocily7, SuDHL4, and U2932 (n = 5) cell lines measured with flow cytometry. Values on y-axis (I) and the color fill (J) represent arcsinh ratio of MFI normalized to expression in parental cells. P values calculated with two-sided one-sample t test and (J) corrected for multiple testing with the Bonferroni method. K, Induction of SYK kinase phosphorylation measured by phospho-flow in various lymphoma cells expressing indicated KLHL6 mutants with different anti–IgH F(ab′)2 concentrations. Data pooled from 5 to 6 independent experiments. P values were calculated with one-way ANOVA followed by the Dunnett multiple comparisons test against the unstimulated control. L, 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. M, 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′)2 anti-IgM or F(ab′)2 anti-IgG. Viability normalized to control treatment with unspecific goat IgG antibody. P values are calculated using the two-sided t test against EmGFP-expressing cells.