MYD88 ^L265P Augments Proximal B-Cell Receptor Signaling in Large B-Cell Lymphomas Via an Interaction with DOCK8

Diffuse large B-cell lymphoma (DLBCL) is a genetically heterogeneous disease comprised of five subtypes including a subset of poor-prognosis activated B cell (ABC)-enriched tumors with frequent MYD88^L265P mutations, often in association with CD79B alterations (Cluster 5 DLBCLs) (Nat. Med. 2018; 24:679-690). Primary central nervous system lymphomas (PCNSLs) and primary testicular lymphomas (PTLs) have similar genetic signatures including recurrent MYD88^L265P mutations and concurrent CD79B alterations (Blood 2016; 127: 869-81). These findings prompted us to evaluate a potential role for MYD88^L265P in proximal B-cell receptor (BCR) signaling, in addition to its defined function as an intermediary in the Toll-Like Receptor (TLR) pathway and downstream NF-kB activation.

In previous studies by Jabara et al., wild-type (WT) MYD88 was found to be constitutively associated with the DOCK8 adapter and the PYK2 tyrosine kinase in normal B-cells (Nat. Immunol. 2012; 13:612-20). In this setting, physiologic ligation of TLR9 with CpG oligodeoxynucleotides (CpG) induced PYK2-mediated phosphorylation of DOCK8, recruitment of Src kinases, including LYN, and downstream activation of the proximal BCR pathway member, spleen tyrosine kinase (SYK) (Nat. Immunol. 2012; 13:612-20). We postulated that mutated MYD88^L265P might similarly augment proximal BCR signaling in DLBCLs in the absence of physiologic (CpG-induced) TLR9 signaling. Using three DLBCL cell lines (OCI-Ly1, SU-DHL4 and OCI-Ly7) with intact BCR signaling and WT endogenous MYD88 and CD79B, we first established that physiologic CpG activation of TLR signaling induced the phosphorylation of PYK2 and the proximal BCR signaling components, SYK and Bruton's tyrosine kinase (BTK). Thereafter, we genetically engineered these three DLBCL cell lines to express MYD88 ^L265P or MYD88 ^WT, alone or in association with CD79B ^Y196F. In all three cell lines, the co-expression of MYD88 ^L265P and CD79B ^Y196F significantly increased magnitude and duration of SYK and BTK phosphorylation following BCR crosslinking. These findings highlight the likely role of MYD88^L265P in CD79B^Y196F-associated proximal BCR signaling in DLBCL.

To elucidate the potential role of the DOCK8 adapter in MYD88 ^L265P-augmented BCR signaling, we first assessed the colocalization of MYD88 ^WT or MYD88 ^L265P with DOCK8 in the same three genetically engineered DLBCL cell lines using proximity ligation assays (PLA), which detect protein-protein interactions at less than 40 nm in situ. In each of these cell lines, we detected significantly increased co-localized MYD88 ^L265P/DOCK8 signals in comparison to MYD88 ^WT/DOCK8 signals (p<.0001, all). Additionally, there were significantly increased co-localized DOCK8/LYN signals in DLBCL cell lines that expressed MYD88 ^L265P rather than MYD88 ^WT (p<.0001, all). These data provide the first direct evidence of an enhanced association between MYD88 ^L265P, DOCK8 and LYN in BCR-dependent DLBCLs and a basis for enhanced BCR signaling in primary tumors with concurrent MYD88^L265P and CD79B genetic alterations.

We next analyzed the consequences of MYD88 ^L265P-associated, DOCK8-dependent increased proximal BCR signaling by depleting DOCK8 in BCR-dependent DLBCL cells with endogenous MYD88^L265P/CD79B^Y196F alterations (HBL1 and TMD8) or endogenous unmutated MYD88 ^WT/CD79B ^WT (OCI-Ly1 and SU-DHL4). ShRNA-mediated DOCK8 knockdown (KD) significantly decreased BCR-mediated phosphorylation of SYK and BTK in MYD88^L265P/CD79B^Y196F DLBCL cell lines but not in lines with MYD88 ^WT/CD79B ^WT, highlighting the specific role of DOCK8 in MYD88 ^L265P-associated proximal BCR signaling. Of great interest, DOCK8 KD selectively decreased the proliferation of MYD88^L265P/CD79B^Y196F, but not MYD88^WT/CD79B^WT, DLBCLs (p<.004, HBL1 and p<.009, TMD8; p = non sig., OCI-Ly1 and SU-DHL4). Additionally, DOCK8 KD significantly increased the efficacy of chemical PI3Kα/δ (copanlisib) and BTK (ibrutinib) inhibition in MYD88^L265P/CD79B^Y196F DLBCLs (HBL1 and TMD8). Taken together, these data identify DOCK8 as an intermediary in MYD88^L265P-driven proximal BCR signaling and a possible treatment target in LBCLs with co-occurring MYD88^L265P/CD79B^Y196F mutations.