Orelabrutinib and Venetoclax Show Synergistic Lethality in Double-Hit Lymphoma By Interfering with the Crosstalk between the PI3K/AKT and p38/MAPK Signaling

Double-hit lymphoma (DHL) is considered a distinct entity in the heterogeneous group of high-grade B-cell lymphoma with translocations of MYC and Bcl-2 and/or Bcl-6. Currently, R-CHOP remains the mainstay of treatment. Still, patients with rearrangements of MYC and Bcl-2 and/or Bcl-6 usually have a poor prognosis when receiving standard regimens, and accumulating studies have demonstrated an increased risk of the central nervous system (CNS) implication and progression. Considering that there is no proper care to manage this condition, is an urgent need for more effective strategies to improve dismal outcomes.

Orelabrutinib is an oral, potent, irreversible, and highly selective BTK inhibitors (Dhillon, 2021). Clinical studies have demonstrated that the daily dosing regimen of orelabrutinib can achieve sustained BTK inhibition within 24 hours. Recent research found that adding orelabrutinib to rituximab improved the antitumor effect of B-cell lymphoma (Hui Yu, 2021). However, data on the efficacy and safety of orelabrutinib in DHL treatment is still lacking. Tumor response to single-agent therapy is often limited by the ability of cells to bypass the target through alternative pathways or by acquired mutations in the mark or its pathway. To overcome these limitations, we have identified and reported the synergistic lethality by a novel regimen targeting DHL containing orelabrutinib with venetoclax (ABT-199), which is a Bcl-2 inhibitor. Thus, this combination could be an opportunity for DHL patients with elusive therapies and dismal outcomes.

To investigate whether combined regimen could potentiate the effect of venetoclax against DHL in preclinical models, we tested the anti-lymphoma effect in four DHL cell lines (TMD8, LR, MCA, and WILL-2). It observed that the combination of orelabrutinib with venetoclax significantly enhanced the anti-DHL effect, manifesting in a dose-dependent manner in significant loss of cell viability. Cell apoptosis of DHL cells and primary B-lymphoma cells was also significantly increased surprisingly. To gain more clues about how the combined regimen affects DHL cell proliferation, we determined G0/G1 phase increased in a time- and dose-dependent manner in the relatively resistant cell lines MCA and WILL-2, the somewhat sensitive cell line TMD8 and LR accumulation in the G2/M phase is more prominent. consistently, two agents inhibited the expression of essential proteins for cell cycle G2/M DNA damage signaling Cyclin B1 and CDC2. Further, we analyzed mitochondrial membrane potential and determined that co-administration of orelabrutinib and venetoclax strongly induced more mitochondrial membrane depolarization.

Notably, orelabrutinib treatment alters genome-wide gene expression in DHL cells. Combined Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed that orelabrutinib affected several important biological processes, including B cell receptor signaling, MAPK signaling, PI3K/AKT signaling, and apoptosis regulation. we next performed western blotting to characterize the protein level changes in different DHL cell lines after the combined regimen. The results were observed that the protein levels of BTK, Mcl-1, PI3K p110, PI3K p85, AKT, and phosphorylated p85, AKT were significantly decreased in the two-drug combination group, accompanied by the central molecules of the downstream NF-kappa B pathway, p105, and p65, and their phosphorylated proteins were also significantly decreased. Similar to pathway enrichment analyses, p38 MAPK and its well-known downstream targets c-Myc and TP53 were also significantly decreased.

Taken together, our study demonstrates that orelabrutinib and venetoclax can synergistically inhibit DHL cell proliferation and promote apoptosis through caspase-mediated mitochondrial apoptosis. Furthermore, this synergistic lethal effect is closely related to the interfering crosstalk of PI3K/AKT and p38/MAPK signaling pathways. For patients with aggressive, refractory DHL with MYC translocations and simultaneous Bcl-2 and/or Bcl-6 translocations, a chemotherapy-free regimen of orelabrutinib and venetoclax may provide another option for DHL.

No relevant conflicts of interest to declare.