Combination of Enzastaurin and Ibrutinib synergistically induces anti-tumor effects in diffuse large B cell lymphoma

Abstract

Background Diffuse large B cell lymphoma (DLBCL) is the most common form of lymphoma. 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, and PI3K) inhibitors have exhibited encouraging pre-clinical and clinical effects, as reported by many researchers. Early studies demonstrated that protein kinase C-β (PKCβ) inhibitors alter phosphorylation level the Bruton’s tyrosine kinase (BTK), which leads to enhanced BTK signaling. Here, for the first time, we 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 effects of enzastaurin and ibrutinib were also evaluated in vivo. Results Combination of enzastaurin and ibrutinib produced a lasting synergistic effect on the survival and proliferation of DLBCL cells, including reduction of proliferation, promoting apoptosis, inducting G1 phase arrest, preventing cell invasion and migration, and down-regulating activation of downstream signaling. More importantly, whole-transcriptome changes results showed that combination therapy worked synergistically to regulate whole-transcriptome expression compared with enzastaurin and ibrutinib alone. Co-treatment with low doses of enzastaurin and ibrutinib could effectively downregulate BCR, NF-κB, JAK and MAPK related signaling pathway. Furthermore, the mRNA expression analysis further indicated that co-treatment significantly decreased the mRNA levels of NOTCH1. The combination effect in inhibiting proliferation of DLBCL cells probably was realized through suppression of NOTCH1 expression. Finally, the anti-tumor activity of co-treatment also was demonstrated in vivo. Conclusions Combination of enzastaurin and ibrutinib had synergistic anti-tumor effects in DLBCL, independent of molecular subtype. These results provided a sound foundation for an attractive therapeutic treatment, and the simultaneous suppression of BTK and PKCβ might be a new treatment strategy for DLBCL. Electronic supplementary material The online version of this article (10.1186/s13046-019-1076-4) contains supplementary material, which is available to authorized users.

Oncogenic Mutations of MYD88 and CD79B in Diffuse Large B-Cell Lymphoma and Implications for Clinical Practice

“…mTOR inhibition with everolimus produced an overall response rate of 50% in patients with WM [ 68 ], while PI3K inhibition with parsaclisib produced overall response rates ranging between 20% and 78% in several mutated lymphoma subtypes [ 69 ]. In in vitro assays, enzastaurin, a protein kinase C inhibitor, in combination with BTK inhibition, reduced the proliferation and viability of DLBCL cells both by regulating the PI3K, MAPK, and JAK/STAT pathways and increasing the phosphorylation of the BTK [ 70 ]. Patients with DLBCL are currently being recruited into a randomized, placebo-controlled phase III study in which enzastaurin is combined with R-CHOP (NCT03263026) [ 71 ].…”

LCP1 triggers mTORC2/AKT activity and is pharmacologically targeted by enzastaurin in hypereosinophilia

“…[43][44][45][46][47] Despite limited benefits shown in these tumors in a single treatment, Enza demonstrated manageable side effects and a good hematologic toxicity profile. [43][44][45][46][47][48] Furthermore, more recent studies suggest combining Enza with ibrutinib 49 or all-trans retinoic acid 50 in diffuse large B cell lymphoma and acute promyelocytic leukemia, respectively. In the present study, Enza resulted in a significant reduction of migration, proliferation, viability, and clonogenic growth as well as induction of cell death of aberrant eosinophilic cells (Figures 2 and 3).…”

miR‑144‑3p inhibits the proliferation, migration and angiogenesis of multiple myeloma cells by targeting myocyte enhancer factor 2A

“…A Cell Titer-Glo chemiluminescence cell viability assay (Promega Corp.) was used to detect cell proliferation ( 11 ). The transfected MM cells were seeded into 96-well plates (10,000 cells/well).…”