S140: ibrutinib sensitizes cll cells to venetoclax by interrupting tlr9-induced cd40 upregulation and protein translation

Topic: 5. Chronic lymphocytic leukemia and related disorders - Biology & Translational Research Background: Chronic lymphocytic leukemia (CLL) cells upregulate Bcl-2 proteins within the lymph node (LN) microenvironment. Signaling via B-cell receptor, Toll-like receptors and CD40 can separately and collectively reduce sensitivity to the Bcl-2 inhibitor venetoclax. Time-limited treatment with venetoclax plus the BTK-inhibitor ibrutinib results in deep remissions. Resistance eventually develops against both drugs when applied continuously as single agents. In combination, these drugs can theoretically work synergistically to prevent or delay resistance, but this has not yet been studied mechanistically. Secondly, details of how this successful drug combination affects LN-related signaling are not yet completely clear. Aims: To analyze the relation between LN-mediated signaling, and the potential to induce venetoclax resistance. Methods: Samples obtained from the HOVON141/VISION phase 2 clinical trial were studied at baseline, after two cycles of lead-in ibrutinib monotherapy, and at timepoints of minimal residual disease analyses under combination therapy (month 9, 12, 15). Flow cytometry was used to study changes in CD40, CD95, phospho-S6, and Bcl-2 family members including Bcl-2, Bcl-XL, Mcl-1 and Bfl-1. Venetoclax sensitivity was examined after stimulating CLL cells in vitro with stimuli that mimic the LN microenvironement (Haselager Blood 2020). NF-κB activity was determined by ELISA kit (TransAm, Active Motive) and gene expression by qPCR. Global protein synthesis was measured using the Click-iT Plus OPP Protein Synthesis Assay Kit (ThermoFisher). Results: Two cycles of ibrutinib monotherapy resulted in decreased protein expression of Bcl-2, Bcl-XL, Mcl-1 and also Bfl-1 in circulating CLL cells. Strikingly, at this timepoint CD40-induced venetoclax resistance was strongly attenuated, as was the expression of CD40 itself (p≤0.0001). The changes in Bcl-2 proteins, or CD40-induced venetoclax resistance could not be related to MRD status at later timepoints. In addition, the dampening effect on CD40-induced venetoclax resistance could not be reproduced by in vitro ibrutinib treatment, demonstrating it occurs in vivo, as an indirect consequence of ibrutinib treatment and egress of CLL cells from the LN niche to the peripheral blood (PB). Secondly, NF-κB signaling downstream of CD40, determined by DNA binding activity of p65 and p52, and target gene induction (Bcl-XL, Bfl-1) remained intact in PB CLL cells after two months of ibrutinib treatment. The same was true for pS6, a marker of Akt-mTOR activity that mediates Mcl-1 expression. In search of signals that could affect CD40 expression and signaling in vivo, various LN-related signals were then tested. Surprisingly, BCR stimulation had only a minor effect (p≤0.05), various cytokines/chemokines had no effect, but TLR ligands and especially TLR9 stimulation via CpG led to clearly increased CD40 expression (p≤0.0001). Importantly, TLR9 triggering reverted the effects of ibrutinib treatment on CD40-mediated venetoclax resistance. Moreover, this occurred by restoring/ inducing overall protein translation. Thus providing the explanation that after ibrutinib treatment CD40/NF-kB signaling itself was intact, while Bcl-2 family proteins that confer venetoclax resistance were lacking. Conclusion:Together, these findings identify a novel effect of ibrutinib: interruption of TLR9-induced CD40 upregulation and translation of pro-survival proteins. This mechanism further inhibits priming of CLL cells in the LN microenvironment for venetoclax resistance.Keywords: Chronic lymphocytic leukemia, CD40, ibrutinib, Venetoclax