Polatuzumab vedotin induced activation of B-cell receptor signaling creates a synthetic lethality with BTK and AKT inhibition in Mantle Cell Lymphoma Free

Polatuzumab Vedotin (PV) is an antibody-drug conjugate of anti-CD79B monoclonal antibody linked with monomethyl auristatin E (MMAE), a microtubule inhibitor. PV was FDA approved in the first line treatment of large B-cell lymphomas in 2023. It has shown a great clinical promise also in other non-Hodgkin lymphomas, including within salvage therapy of mantle cell lymphoma (MCL). Binding of PV to CD79B is followed by its internalization and release of MMAE. CD79B is not only a B-cell specific marker, but also a signaling subunit of the B-cell receptor (BCR) complex. CD79B mediates signal transmission from BCR towards proximal mediators (e.g., BTK, Bruton's tyrosine kinase) and second signaling messengers (e.g., Ca2+ and AKT, protein kinase B pathways). Therefore, we hypothesized that PV might affect BCR signaling itself with unknown consequences of BCR signal activation for lymphoma.

We have used 6 MCL cell line models. AKT activity was measured using genetically encoded Förster resonance energy transfer (FRET)-based AKT activity reporter with FRET detection by flow cytometry, including kinetics measurement. Ca2+ response was measured using Indo-1 ratiometric dye. Cell cycle and apoptosis were measured using Click-iT EdU and CellEvent Caspase-3/7 fluorescent dyes, respectively. Cell growth was evaluated using CellTitr-Glo assay or live cell imaging by IncuCyte. Drug synergism in cross-titration experiments was calculated using Synergyfinder.

Treatment of model MCL cell lines with PV induced strong and fast activation of the BCR signaling. PV induced immediate, large and dose dependent increase of AKT activity with maximum reached within two minutes. AKT activation was similar following PV treatment as response to unconjugated anti-CD79B and anti-IgH (immunoglobulin heavy chain) antibodies. Similarly, PV induced strong Ca2+ signaling response comparable to anti-IgH based BCR stimulation. Next, we evaluated whether this anti-CD79B antibody-mediated activation of BCR signaling contributes to PV toxicity by mechanism of activation induced cell death. In contrast to anti-IgH based BCR-stimulation (which was toxic to all tested MCL cell lines), unconjugated anti-CD79B antibody stimulated cell growth and induced corresponding changes in the cell cycle. As expected, PV treatment resulted in accumulation of cells in the G2/M cell cycle phase with apoptosis induction. To verify that BCR signal activation does not contribute to the PV toxicity, we concurrently inhibited BCR signaling. Combined PV treatment accompanied by BTK inhibition, AKT inhibition, or inhibition of Ca2+ response did not reduce PV toxicity. On the contrary, antibody based CD79B stimulation had an opposite effect. Unconjugated anti-CD79B antibody strongly protected MCL cell lines against unconjugated MMAE toxicity. Next, we used live cell imaging to continuously evaluate treatment time dependent changes of half inhibitory concentrations (IC50). As expected, unconjugated MMAE showed a steady and time dependent decrease of IC50 (the same concentration of MMAE was more effective at later timepoints). In contrast, PV IC50 showed an increase within the 24 to 48 hours interval before starting to decrease (the same PV concentration was less effective at later timepoints between 36 to 48 hours in comparison to 24 hours). This observation is consistent with a combined effect of MMAE toxicity and anti-CD79B mediated growth stimulation in PV conjugate. If our conclusion is correct and PV induced BCR signaling stimulate cell growth, it should be highly therapeutically effective to combine PV with BCR signaling inhibitors. Indeed, using a cross-titration-based drug synergy evaluation, we detected a strong and wide synergism between 1) PV and BTK inhibitor ibrutinib, and 2) PV pan AKT inhibitor capivasertib, both pending in vivo mice tumor xenograft model-based confirmation.

We have identified a very important cellular side effect of PV - stimulation of BCR signaling and associated stimulation of cell growth. Importantly, this anti-CD79B mediated cell growth stimulation could be eliminated by combining PV with BCR signaling inhibition. Our data thus provide a strong foundation for future research and preclinical testing of these therapeutic combinations.

Supported by MHCR (DRO - VFN00064165), National Institute for Cancer Research (EXCELES - LX22NPO5102), and MEYSCR (Cooperatio, SVV 260637).