Abstract
Introduction: Diffuse large B-cell lymphoma (DLBCL) is driven by B-cell receptor (BCR) signaling. Activated B-cell (ABC)-derived cases require NF-kB activation by the BCR-activated CARD11-BCL10-MALT1 (CBM) complex. BCL10 mutations promoting CBM activation are found in the LymphGen BN2/Chapuy C1 molecular subgroup characterized by BCL6 translocations and NOTCH2 mutations. These cases achieve no benefit from addition of the BTK inhibitor (BTKi) ibrutinib to frontline therapy. Correspondingly, BCL10 mutations, functioning downstream from BTK in the BCR signaling cascade, mediate BTKi resistance. Here, we decode key new details of mutant BCL10-driven lymphomagenesis.
Methods: We developed tet-on expression systems of BCL10 gain-of-function mutants (R58Q and S136X). We examined gene-expression profiling (GEP), cytokine arrays, and transcription factor enrichment by ENCODE ChIP-X Enrichment Analysis Version 3 (ChEA3). We employed drug screening to interrogate impacts on a variety of therapeutic classes using epigenetic Library (TargetMol). We examined activities of a drugs alone and in combination in vitro by measuring cell viability and in xenograft models (NOD scid gamma mice) by measuring tumor volume post treatments. We engineered the activating BCL10-S136X truncation as a cre-inducible conditional allele at the murine Rosa26 locus (called RSB mice) and crossed it later with the IµBCL6 transgenic background to generate an accurate immunocompetent BN2/C1 mouse model.
Results: GEP revealed reprogramming converging on NF-kB activation reinforced by cytokines (CCL17, CCL4, IL32, CXCL10, TNFa, TNFb). Cytokines upregulated by the mutants were also significantly upregulated in BCL10-mutant patient tumors including CCL22 (p=0.0011) and IL7 (p=0.0013). Further, ELISA confirmed the activation of TNFb (p<0.01) and TNFa( p<0.01). Drug screening revealed numerous additional classes besides BTKis whose activities are thwarted by BCL10-mutant activities. These include the BCL2 inhibitor venetoclax, an FDA-approved drug with remarkable activities in other B-cell non-Hodgkin lymphomas but largely inactive in DLBCL. We also found in BCL10-mutant samples significantly higher expression of BCL2L1 (S136X p=0.0001, R58Q P=0.0002), BCL2A1(S136X p=0.0011, R58Q p=0.0005), and BCL2 itself (S136X p=0.0001, R58Q p=0.0074). We find the BCL10 mutant-driven cytokine-reinforced positive feedback loop of lymphomagenesis activates multiple additional pathways converging on diffuse activation of oncogenic transcription factors. BCL10 truncation mediated up-regulation of anti-apoptotic genes promotes increased mitochondrial membrane potential, a key factor underlying multidrug, including venetoclax, resistance, but further investigation revealed this is overcome by the addition of the potent non-covalent BTK inhibitor pirtobrutinib. 24 hours of pirtobrutinib treatment drastically reduced the transcript levels of BCL2 family genes in BCL10 mutants comparing to vector; BCL2 (vector: p=0.0325, R58Q: p=0.005, S136X: p=0.0006), BCL2L1 (vector: p=ns, R58Q: p=0.0484, S136X: p=0.0007),BCL2A1 (vector: p=0.0087, R58Q: p=0.0121, S136X: p=0.0005). Venetoclax+pirtobrutinib synergized and potently killed BCL10-mutant tumors in vitro (Bliss synergy score_ RIVA; vector =7.96, R58Q=9.03, S136X=13.82) and in vivo. RSB x MB-1/cre crosses did not impact survival of double-het offspring but promoted an activation block thwarting germinal center responses to antigen. Crossing these mice into the IµBCL6 transgenic background that replicates a defining chromosomal translocation of BN2/C1 cases resulted in tumors resembling DLBCL or marginal zone lymphoma with 100% penetrance and compromised overall survival (p<0.0001, Mantel-Cox test).
Conclusion:The combination of venetoclax and pirtobrutinib synergistically overcomes BCL10-driven resistance and effectively kills BCL10-mutant DLBCL tumors in vitro and in vivo, suggesting BTK retains key roles protecting DLBCL tumors from apoptosis even when dispensable for CBM activation. Genetically accurate mutant-BCL10 mice revealed a novel germinal-center activation deficit and facilitated generation of valuable preclinical models to further interrogate biology of the BN2/C1 molecular subgroup through activation of the BCL6 oncogene.
Landgren:Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Membership on independent data monitoring committees; Merck: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Membership on independent data monitoring committees; Theradex: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Membership on independent data monitoring committees.; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Membership on independent data monitoring committees; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Membership on independent data monitoring committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Membership on independent data monitoring committees; Adaptive: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Membership on independent data monitoring committees. Maura:Sanofi: Consultancy, Honoraria; Medidata: Consultancy, Honoraria.