Although rituximab in the combination of CHOP chemotherapy has been widely used as the standard treatment for several kinds of B-cell non-Hodgkin lymphoma (B-NHL), a great number of B-NHL patients treated with this immunotherapy still develop primary and secondary resistance. Recently Bruton’s tyrosine kinase (Btk) inhibitor ibrutinib showed promising therapeutic effect in relapsed/refractory CLL and B-cell NHL, which provided essential alternatives for these patients.

Angioimmunoblastic T cell lymphoma (AITL) is a distinct subtype of peripheral T cell lymphoma and associated with poor outcomes. The activation status of T cell receptor (TCR) signaling has recently become a focus of attention in terms of the therapeutic targets. However, the molecular pathogenesis mechanisms and novel therapeutic targets are largely unknown. Antibodies specific to phosphorylated ZAP70, ITK and PLCγ1 were used to identify the activation status of intracellular proteins involved in TCR signaling in AITL patients. Malignant T cell lymphoma cells were transduced with a lentiviral construct containing ITK shRNA for cellular and functional assays. The antitumor effects of the selective ITK inhibitor BMS-509744 were determined in vitro and in vivo. Immunohistochemistry staining showed that more than half of the AITL patients (n = 38) exhibited continuously activated intracellular TCR signaling pathway. Patients positive for phosphorylated ITK showed a lower rate of complete response (20% vs. 75%, P = 0.004) and a shorter progression-free survival (5.17 months vs. 25.1 months, P = 0.022) than patients negative for phosphorylated ITK. Genetic and pharmacological cellular ITK inhibition significantly compromised the proliferation, invasion and migration of malignant T cells. The selective ITK inhibitor BMS-509744 also induced the pro-apoptotic effects and G2/M phase cell cycle arrest in vitro and in vivo. Finally, inhibition of ITK synergistically enhanced the antitumor effect of vincristine and doxorubicin on malignant T cell lymphoma cell lines. Our findings suggest that ITK may be a novel candidate therapeutic target for the treatment of patients with ITK-expressing malignant T-cell lymphomas.

Background: Upregulation of H3K27me3 induced by EZH2 overexpression or somatic heterozygous mutations were implicated in lymphomagenesis. It has been demonstrated that several EZH2-target agents have notable therapeutic effects in EZH2-mutant B-cell lymphoma patients. Here we present a novel highly selective EZH2 inhibitor SHR2554 and possible combination strategy in diffuse large B-cell lymphoma (DLBCL). Methods: Cell proliferation, cell cycle and apoptosis were analyzed by CellTiter-Glo Luminescent Cell Viability Assay and flow cytometry. Western Blot was used to detect the expression of related proteins. The gene expression profiling post combination treatment was analyzed by RNA-Seq. Finally, CDX and PDX models were used to evaluate the synergistic anti-tumor effects of the combination treatment in vivo. Results: The novel EZH2 inhibitor SHR2554 inhibited proliferation and induced G1 phase arrest in EZH2-mutant DLBCL cell lines. The combination of EZH2 inhibitor SHR2554 with histone deacetylase (HDAC) inhibitor chidamide (hereafter referred to as HBI8000) exerted synergistic anti-proliferative activity in vitro and in vivo. Gene expression profile analysis revealed dramatic inhibition of the DNA replication process in combined treatment. Conclusions: SHR2554, a potent, highly selective small molecule inhibitor of EZH2, inhibited EZH2-mutant DLBCL more significantly in vitro and in vivo. The combination of HDAC inhibitor HBI8000 with EZH2 inhibitor SHR2554 exhibited dramatic anti-tumor activity in both mutant and wild-type DLBCL, which may become a potential therapeutic modality for the treatment of DLBCL patients.

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. 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. 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. 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.

Mantle cell lymphoma (MCL) is an incurable B cell-derived malignant tumor with a median overall survival of 4–5 years. Mer tyrosine kinase (MerTK) has been reported to be aberrantly expressed in leukemia, melanoma, and gastric cancer, and plays a pivotal role in the process of oncogenesis. This study assessed the role of MerTK in MCL for the first time. Immunohistochemistry and western blot were performed to figure out expression of MerTK in MCL. MerTK inhibition by either shRNA or treatment with UNC2250 (a MerTK-selective small molecular inhibitor) was conducted in MCL cell lines. MCL-cell-derived xenograft models were established to evaluate the anti-tumor effects of UNC2250 in vivo. MerTK was ectopically expressed in four of six MCL cell lines. Sixty-five of 132 (48.9%) MCL patients showed positive expression of MerTK. MerTK inhibition by either shRNA or treatment with UNC2250 decreased activation of downstream AKT and p38, inhibited proliferation and invasion in MCL cells, and sensitized MCL cells to treatment with vincristine in vitro and doxorubicin in vitro and in vivo. UNC2250 induced G2/M phase arrest and prompted apoptosis in MCL cells, accompanied by increased expression of Bax, cleaved caspase 3 and poly (ADP-ribose) polymerase, and decreased expression of Bcl-2, Mcl-1 and Bcl-xL. Moreover, UNC2250 delayed disease progression in MCL-cell-derived xenograft models. Our data prove that ectopic MerTK may be a novel therapeutic target in MCL, and further pre-clinical or even clinical studies of UNC2250 or new MerTK inhibitors are essential and of great significance.