Combination of Enzastaurin and Ibrutinib Synergistically Induces Anti-Tumor Effects in Diffuse Large B Cell Lymphoma

Background:Diffuse large B cell lymphoma(DLBCL), the most common form of lymphoma, is characterized by a heterogeneous tumor entity. Although durable remissions can be achieved in more than half of these patients, DLBCL remains a significant clinical challenge, with approximately 30% of patients not being cured. BCR-associated kinases (SYK, BTK, PI3K)inhibitors have exhibited encouraging pre-clinical and clinical effects, as reported by many researchers.Early studies demonstrated that PKCβ inhibitors alter phosphorylation level of BTK, which enhances activation of BTK signaling pathway.Here we aim to investigate whether the combination of PKCβ inhibitor enzastaurin and BTK inhibitor ibrutinib has synergistic anti-tumor effects in DLBCL.

Methods:In vitro cell proliferation was analyzed using Cell Titer-Glo Luminescent Cell Viability Assay. Induction of apoptosis and cell cycle arrest were measured by flow cytometry. Western Blotting analysis was used to detect the essential regulatory enzymes in related signaling pathways. RNA-seq was conducted to evaluate the whole-transcriptome changes brought by co-treatment with low doses of enzastaurin and ibrutinib. The synergistic anti-tumor effect of enzastaurin and ibrutinib was also evaluated in vivo.

Results: Combination of enzastaurin and ibrutinib produced a lasting synergistic effect on the survival and proliferation of ABC (HBL-1,TMD8,SU-DHL-2)and GCB (SU-DHL-6, OCI-LY7)DLBCL cells lines. Exposure of these DLBCL cells to minimally toxic concentration of enzastaurin and ibrutinib resulted in more significant inhibitory activity than each agent alone. In addition, the combination of these two agents led to an enhanced apoptosis and induced G1 phase arrest compared with monotherapy, which were accompanied by marked regulation of apoptosis-related and cell cycle-related proteins. Combination treatment with enzastaurin and ibrutinib, to some extent, also functioned synergistically to inhibit cell migration and invasion compared with single agent. Moreover, combination of enzastaurin and ibrutinib seemed to be more effective in inhibition p-ERK, p-AKT and p-P38 in DLBCL cell lines. In addition, RNA-seq results showed that co-treatment with low doses of enzastaurin and ibrutinib could effectively downregulate BCR, NF-κB, JAK and MAPK related signaling pathways, and the pathway analysis results were consistent with the immunoblotting analysis. Furthermore, the mRNA expression analysis using real-time PCR further indicated that treatment with enzastaurin and ibrutinib significantly decreased the mRNA levels of NOTCH1 which has been recognized as an important oncogenic regulator of hematological malignancy. The combination effect of enzastaurin and ibrutinib in inhibiting proliferation of DLBCL cells probably was realized through suppression of NOTCH1 expression. Finally, the anti-tumor activity of co-treatment with enzastaurin and ibrutinib was also demonstrated using xenograft mice models.

Conclusion:We investigated the combination of enzastaurin and ibrutinib in ABC and GCB DLBCL, demonstrating the co-treatment had synergistic anti-tumor effects in DLBCL. These results provided a sound foundation for an attractive therapeutic treatment, and the simultaneous suppression of BTK and PKCβ might be an new treatment strategy for DLBCL.