P-073: PDZ proteins, SCRIB and DLG1, regulate CD86 surface expression, myeloma growth, and survival

Abstract

<h3>Background</h3> We have previously demonstrated that the cytoplasmic region of CD86 confers a survival phenotype in myeloma cells and regulates IRF4 and Integrin β7 expression. Here, we show that the cytoplasmic tail is important for surface expression of CD86. <h3>Methods</h3> We transfected HEK293T with either full length CD86 (CD86FL) or a mutant lacking the cytoplasmic tail (CD86TL). CD86FL can properly traffic to the cell surface while CD86TL cannot effectively export from the Golgi apparatus. We developed additional truncation mutants of the CD86 cytoplasmic tail and identified that several regions of the tail are required for proper trafficking out of the Golgi. While the truncation mutants traffic CD86 more effectively than CD86TL, they never fully phenocopy CD86FL surface expression. One specific region for proper transport is a three amino acid-long PDZ binding motif at the C-terminus of the tail. Using BioID analysis, we identified two PDZ-domain containing proteins, SCRIB and DLG1 as proximal to the CD86 cytoplasmic tail. <h3>Results</h3> Using the CoMMpass dataset, we identified that high SCRIB expression is a poor prognostic indicator for progression free (p=0.00022) and overall (p=0.000132) survival. We observed co-localization of SCRIB and DLG1 with CD86 in myeloma using confocal microscopy. Using a doxycycline-inducible CRISPR-Cas9 system, we deleted SCRIB and DLG1 in two myeloma cell lines, KMS18 and RPMI8226. Ablation of SCRIB or DLG1 decreased CD86 surface expression 3 days following activation of the Cas9 enzyme via doxycycline addition. Additionally, we found that knockout of SCRIB or DLG1 results in significantly decreased cell proliferation and viability in these cell lines. SCRIB and DLG1 have been classically studied in the context of apical-basal polarity indicating a possible role for regulating CD86 expression in time and space. We found decreased localization of SCRIB/DLG1 with CD86 at areas of cell-cell contact, presumably where CD86 binds to CD28 to signal for myeloma cell survival. These regions of contact have increased surface expression of CD86 relative to areas where there is no cell contact. This is only prevalent in myeloma cells, and we observed uniform distribution of expression of CD86 throughout the membrane of CD86FL-transfected HEK293T. This suggests that SCRIB and DLG1 transport CD86 to the cell surface, and binding with CD28 may help to stabilize CD86 surface expression. Ablation of SCRIB and DLG1 also decreases IRF4 and Integrin β7 expression, suggesting that SCRIB and DLG1 may contribute to CD86-mediated growth and survival signaling. <h3>Conclusion</h3> Our data supports a role for PDZ proteins as regulators of myeloma cell signaling and viability. Study of PDZ-proteins and their binding motifs may be warranted as numerous surface proteins in myeloma cells such as ICAM-1 and CD138 contain PDZ-binding motifs. The PDZ-binding motif may represent a specific region that can effectively be targeted and may be an attractive strategy for novel therapeutics.