ABC-DLBCLs are among the most aggressive DLBCLs. Genome sequencing studies identified BCL10 gain-of-function mutations in DLBCL mostly within the ABC-DLBCLs. ABC-DLBCLs were recently split into further subtypes according to their genomic characteristics, among which BCL10 somatic mutations were most common in BN2/Cluster 1 cases. These are likely transformed marginal zone lymphomas and were recently suggested to be sensitive to ibrutinib treatment.
Pooling publicly available data we noted that most BCL10 mutations are missense or nonsense SNVs affecting its CARD domain, MALT1 binding domain, and the C-terminal S/T rich domain. We generated a large panel of mutant BCL10 constructs including the top hotspot missense mutation R58Q (CARD domain) and nonsense truncation mutations such as E140X. We found that almost all mutants induced aberrantly strong induction of NF-κB activity in lymphoma cells as compared to WT BCL10, indicating that they induce gain of function.
BCL10 forms a high order complex with CARD11 and MALT1 (CBM signalosome) downstream of BCR signaling. Normally, CARD11 activation induces polymerization of BCL10 which induces MALT1 activity and downstream NF-κB signaling. To investigate the impact of BCL10 mutants on CBM complex formation we performed fluorescence polarization and filamentation formation assays with purified WT and mutant BCL10 species. Both BCL10R58Q and BCL10E140X manifested faster and even spontaneous polarization compared to BCL10WT. BCL10R58Q formed thicker and more heavily bundled filaments (~20 nm) that provide greater surface area to dock signaling proteins, whereas filaments formed by BCL10E140X had the normal ~10 nm structure. Even though the BCL10E140X deletes the canonical MALT1 binding site, the mutant filament still featured robust MALT1 recruitment. Cryo-EM studies revealed that BCL10R58Q mutant gains new interactions within the filament structure that could explain the observed stabilization and bundling effects. Notably, cryo-EM structure of the BCL10E140X mutant in complex with MALT1 showed that it retains its interaction with MALT1 in the filament form despite its predicted lack of interaction with MALT1 in the monomeric form due to the C-terminal deletion.
To gain further functional insight we performed mass spectrometry to identify proteins interacting with WT, BCL10R58Q and BCL10E140X. BCL10R58Q featured gain of many novel protein interactors including NF-κB2 and TAB1 etc. consistent with bundled filament formation enabling more signaling protein recruitment. However, the BCL10E140X interactome was quite different and most notably featured loss of binding to negative regulators of non-canonical NF-κB. NF-κB2 (p100/p52) level was indeed elevated in the presence of this mutant. In addition, both BCL10 mutant classes showed aberrant activation of canonical and non-canonical NF-κB activation (IkBa/p65 and p52) through distinct mechanisms.
As a functional readout of BCL10 function, we generated a MALT1 GloSensor reporter DLBCL lines to detect MALT1 protease activity. Indeed, both classes of mutations showed potent induction of MALT1 protease activity (GloSensor), enhanced cleavage of canonical MALT1 target proteins (Western Blot), expression of canonical NF-κB target genes (QPCR) such as IL6 and IL10. In striking contrast to BCL10WT, and consistent with our structural data showing spontaneous polymerization of BCL10 mutants, we found that CARD11 knockdown did not impair MALT1 activation, NF-κB signaling, or cell growth in ABC-DLBCL lines expressing both BCL10 mutants.
This CARD11 independence was concerning, since it suggests that BCL10 mutant lymphomas might be resistant to drugs targeting upstream components of the BCR signaling pathway such as ibrutinib. Indeed, expression of BCL10R58Q and BCL10E140X (but not BCL10WT) in various ABC-DLBCL cell lines abrogated the ability of ibrutinib to inhibit MALT1 (GloSensor), NF-κB activity (Reporter), cell growth (Growth inhibition) as well as proliferation. Collectively, we find that BCL10 mutations induce aberrant canonical and non-canonical NF-κB activity through novel and structurally distinct biochemical mechanisms that are at least partially dependent on MALT1. BCL10 mutation should be considered as a biomarker for ibrutinib resistance in ABC-DLBCL, so that alternative targeted therapies can be prioritized for these patients.
Disclosures
Fontan: Johnson & Johnson: Current Employment. Melnick:Epizyme: Consultancy; Jubilant: Consultancy; Constellation: Consultancy; Janssen: Research Funding; Daiichi Sankyo: Research Funding.
Evaluation of NLR Family CARD Domain Containing 3 and NLR Family CARD Domain Containing 5 Gene Expression in Interferon Gamma–Treated Mesenchymal Stem Cells from Wharton’s Jelly of Human Umbilical Cord
