Crispr‐cas9 Genetic Screens Uncover a B Cell Receptor‐myd88 Superpathway in Diffuse Large B Cell Lymphoma
Year: 2017
Type: conference-abstract
Abstract: Introduction: The activated B cell like (ABC) subtype of diffuse large B cell lymphoma (DLBCL) frequently relies on signaling from the B cell receptor (BCR) and mutant isoforms of MYD88 to maintain oncogenic levels of NF-κB activity essential to its survival. These pathways are commonly mutated in ABC DLBCL. A recent clinical trial of ibrutinib, an inhibitor of the BCR proximal kinase BTK, in relapsed/refractory DLBCL patients reported exceptional responses in ABC DLBCL patients co-expressing mutations in both MYD88 and CD79B. These clinical data suggested the BCR and MYD88 signaling pathways may be functional coupled. Methods: We performed genome-wide CRISPR-Cas9-based lethality (dropout) screens in 8 DLBCL lines to discover modulators of BCR signaling that are required for cell viability or proliferation. Essential genes were subjected to further genetic, biochemical and image analysis, including the proximity ligation assay (PLA) to visualize protein-protein interactions in situ. Results: Our CRISPR screens identified TLR9 as the only MYD88 binding receptor required in MYD88-mutant ABC cell lines. Surprisingly, this requirement was limited to ABC models that relied on both the MYD88 L265P mutant isoform and chronic active BCR signaling, leading us to explore a link between chronic active BCR signaling and TLR9. We found that TLR9 and IgM co-immunoprecipitate in ABC lines. Moreover, the association between TLR9 and MYD88 L265P is dependent upon active BCR signaling. Using the PLA, we visualized BCR and TLR9 association with each other in Lamp1+ endolysosomes. To advance these findings, we fused the MYD88 L265P isoform to BioID2, a promiscuous biotin ligase that will attach biotin on neighboring proteins. When this construct was expressed in ABC DLBCLs, we unexpectedly observed that MYD88 was in close association with CARD11 and MALT1, components of the CBM adapter complex that is assembled downstream of the BCR. Employing the PLA, we show that endogenous MYD88 interacts with all members of the CBM complex in ABC lines, and that these interactions were dependent on the BCR and TLR9. Finally, we visualized MYD88 signaling in cells expressing the MYD88-L265P-BioID2 construct. We observed MYD88 signaling in large-scale complexes juxtaposed to Lamp1+ vesicles. These complexes contained active NF-κB signaling and were disrupted by BTK inhibition with ibrutinib. Conclusions: We demonstrate that TLR9 and IgM facilitate the assembly of a superpathway of signaling adapters including MYD88 and the CBM complex on endolysosomal membranes to cooperatively drive oncogenic activation of NF-κB in ABC DLBCL. These supercomplexes apparently create a hyperaddiction phenotype given the frequent responses to ibrutinib in ABC tumors that harbor CD79B and MYD88 L265P mutations. Keywords: B-cell receptor (BCR); diffuse large B-cell lymphoma (DLBCL); MYD88.
Source: Hematological Oncology
DOI: 10.1002/hon.2437_4
Institutions: National Institutes of Health, National Cancer Institute
Language: English
Subfield: Pathology and Forensic Medicine
Field: Medicine
Domain: Health Sciences
Open Access status: bronze
OpenAlex ID: W2621557729