A novel orally available Syk/Src/Jak2 inhibitor, SKLB-850, showed potent anti-tumor activities in B cell lymphoma (BCL) models

Abstract

B cell lymphoma (BCL) is the most frequently diagnosed type of non-Hodgkin lymphoma (NHL), and accounts for about 4% of all cancers in the USA. Kinases spleen tyrosine kinase (Syk), Src, and Janus kinase 2 (JAK2) have been thought as potential targets for the treatment of BCL. We have recently developed a multikinase inhibitor, SKLB-850, which potently inhibits Syk, Src, and JAK2. The aim of this study is to investigate the anti-BCL activities and mechanisms of action of SKLB-850 both in vitro and in vivo. Our results showed that SKLB-850 significantly inhibited BCL cell proliferation, and induced apoptosis of BCL cells. It could considerably decrease the secretion of chemokines CCL3, CCL4, and CXCL12. Oral administration of SKLB-850 considerably suppressed the tumor growth in BCL xenograft models (Ramos and HBL-1) in a dose-dependent manner. Immunohistochemistry of tumor tissues showed that SKLB-850 efficiently inhibited the activation of Syk/ERK, Src/FAK and JAK2/Stat3 pathways. Collectively, SKLB-850 could be a promising agent for the treatment of BCL, hence deserving further study.

Keywords: B cell lymphoma; SKLB-850; multikinase inhibitor.

Figure 1. The chemical structure of SKLB-850 and its anti-tumor effects in vitro

(A) The chemical structure of SKLB-850. (B and C) Apoptosis of Ramos cells induced by SKLB-850. Cells were treated by vehicle, R788 or indicated concentrations of compounds. After 18 hours, Cell were harvested and stained with AnnexinV-FITC and propidium iodide, then the apoptosis cells were analyzed by FCM. (D and E) Apoptosis of HBL-1 cells induced by SKLB-850. Cells were treated by vehicle, R788 or indicated concentrations of compounds. After 24 hours, Cell were harvested and stained with AnnexinV-FITC and propidium iodide, then the apoptosis cells were analyzed by FCM. The apoptotic cells in SKLB-850 group were significantly more than that in R788 and vehicle groups. Columns, mean; bars, SD. ^##P<0.01, or ^#P<0.05, SKLB-850 group versus vehicle group.

Figure 2. Cell cycle analysis of Ramos and HBL-1 cells induced by SKLB-850, R788 DMSO or control

Tumor cells were treated by vehicle, R788 or indicated concentrations of compounds. After 18 hours, cell were harvested and stained with AnnexinV-FITC and propidium iodide, and then analyzed by FCM. The G2 cells in SKLB-850 groups were significantly more than that in R788 and vehicle groups. (A and C) Cell cycle analysis of Ramos cells. (B and D) Cell cycle analysis of HBL-1 cells. The percent of G2 cells in SKLB-850 groups (P < 0.01) was significantly more than that in R788, DMSO and NS groups. Columns, mean; bars, SD.^##P < 0.01, 850 group versus DMSO or blank control group.

Figure 3. SKLB-850 inhibited anti-IgM and anti-CD40-induced secretion of the chemokines CCL3, CCL4, chemotaxis, and CD184 expression

(A and B) SKLB-850 inhibited anti-IgM and anti-CD40-induced secretion of the chemokines CCL3, CCL4. (C) SKLB-850 inhibited anti-IgM and anti-CD40-induced BCL cell chemotaxis toward the chemokines CXCL12. (D) SKLB-850 inhibited anti-IgM and anti-CD40-induced the CD184 expression of Ramos cells. Data represent the mean±SEM. ^**P<0.01, or ^*P<0.05, SKLB-850 group versus control group.

Figure 4. Anti-tumor activities of SKLB-850 in vivo

(A and B) RAMOS or HBL-1 tumor-bearing NOD/SCID mice were treated as described with SKLB-850 at 40 mg/kg or 20 mg/kg, R788 or with vehicle. The treatment with SKLB-850 significantly inhibited tumor growth versus vehicle control (n =6; ANOVA; ^*P < 0.05, ^**P < 0.01, R788 or SKLB-850 vs. vehicle control). (C and D) The tumor weight of Ramos and HBL-1 tumors with treatment of SKLB-850 or R788, respectively. E: Representative photographs of subcutaneous tumors in Ramos model and HBL-1 model.

Figure 5. Cytotoxicity evaluation of SKLB-850

(A) Mouse body weight with treatment of vehicle, R788, or 850 was monitored once every three days. (B) Level of ALT, AST, BUN, and CREA of serum in vehicle, R788 or SKLB-850 group. (C) H&E staining of heart, liver, spleen, lung, and kidney tissues in blank, vehicle, R788, or SKLB-850 group.

Figure 6. SKLB-850 inhibited tumor growth by inducing apoptosis of tumor tissues from the Ramos model and BCL samples from patients

(A and D) TUNEL immunofluorescent staining of tumor tissues from the Ramos model (n-6 mice per group). (B and E) Immunohistochemical staining analyses of Ki67 and CD20 in tumor tissues from the Ramos model. (C and F) Apoptosis of tumor cells from the BCL patients induced by SKLB-850. Tumor tissues from BCL patients were treated with a series of concentrations of SKLB-850 or R788, and stained with AnnexinV-FITC and propidium iodide, then analyzed with a flow cytometer. N=3, Columns, mean; bars, SD. ^##P<0.01, or ^#P<0.05, SKLB-850 group versus vehicle group.

Figure 7. Western blot and histochemical analyses of tumor tissues from the Ramos model

(A) SKLB-850 inhibited Syk/ERK, Src/FAK and JAK2/STAT3 signaling pathways. The Ramos cells were incubated for 24 hours in medium containing vehicle, SKLB-850, or R788, and then lysed for western blot assay. (B) H&E staining and immunohistochemical staining of NF-κB, p-Src, p-STAT3 and p-Syk in tumor tissues isolated from vehicle, R788 or SKLB-850 treated groups. SKBL-850 inhibited p-JAK2, p-FAK, p-Src, p-ERK and p-Syk expression in RAMOS cells.