Multitarget kinase inhibitor olverembatinib is active and synergizes with BTK inhibitor acalabrutinib in preclinical models of Mantle Cell Lymphoma

Abstract Background Mantle cell lymphoma (MCL) is a rare subtype of non-Hodgkin lymphoma derived from pre-germinal center B cells. It is an aggressive, largely incurable disease and prone to relapse with poor prognosis. Although the advent of Bruton's tyrosine kinase (BTK) inhibitors has transformed the treatment landscape, the duration of response with monotherapy is limited and drug resistance remains a challenge. Ongoing efforts have been undertaken to develop combination therapies that enhance clinical outcomes. Olverembatinib is a novel multikinase inhibitor that has been approved in China for the treatment of chronic myeloid leukemia. We hypothesized that dual inhibition of Lyn and BTK pathways may enhance antitumor effects in MCL. Aims We sought to investigate the antitumor effect of olverembatinib in combination with approved BTK inhibitor acalabrutinib in MCL preclinical models and to explore potential mechanisms of action. Methods Human MCL cell lines Mino and Rec-1 were used for in vitro studies. The CellTiter-Glo® assay was conducted to evaluate the antiproliferative activities of the agents. Cellular apoptosis and cell cycle arrest were analyzed by flow cytometry. Mechanisms of action were explored by tyrosine phosphoproteomic analysis and confirmed by western blotting. In vivo activity was evaluated in a subcutaneous cell-derived xenograft mouse model. Results Olverembatinib potently inhibited MCL cell proliferation with IC50 values of 15 nM (Mino cell line) and 25 nM (Rec-1 cell line). Olverembatinib in combination with acalabrutinib showed synergistic antiproliferative effects in both cell lines (Loewe score 10.04 in Mino cells and 10.92 in Rec-1 cells); the combination also significantly increased the percentage of apoptotic cells compared with either agent alone (p < 0.001 in Mino and p < 0.01 in Rec-1 cells) and induced G0/G1 cell cycle arrest after 24 hours (p < 0.01 in Mino and p < 0.0001 in Rec-1 cells vs either agent alone). Tyrosine phosphoproteomic analysis showed that olverembatinib inhibited kinase activities of Lyn, Fyn, Lck, Syk, and BTK, leading to the most potent inhibition of the BCR signaling pathway, which is relevant to MCL. Acalabrutinib inhibited only BTK and other Tec-family kinases, such as Tec and Itk. Combination of the two agents produced greater inhibition of BCR and NF-kB pathways. The tyrosine phosphoproteomic results were further confirmed by western blot analysis. Olverembatinib inhibited phosphorylation of Lyn and its downstream BTK, while the combination further downregulated NF-kB activity. Moreover, the combination dramatically suppressed levels of cell cycle-promoting proteins CDK4/6 and increased apoptotic markers, such as cleaved poly(ADP-ribose) polymerase (PARP) and caspase-3. Synergistic antitumor effects are being further evaluated in MCL models in vivo. Conclusions In MCL preclinical models, olverembatinib is effective and synergizes with acalabrutinib to promote antitumor effects. These findings provide a scientific rationale for further clinical evaluation of olverembatinib and its combination with BTK inhibitors for the treatment of patients with MCL.