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

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.


INTRODUCTION
B cell lymphoma (BCL) is one of the most frequently diagnosed lymphomas, which has been experiencing a substantial increase in incidence and mortality worldwide [1].Chemotherapy had long been the only systematic treatment of BCL until the first monoclonal antibody (mAb) drug, rituximab, was approved by the US food and drug administration (FDA) to treat BCL in 1997 [2].In 2013, a small molecule targeted drug, Ibrutinib [3], which is a BTK inhibitor, was also approved by US FDA for the therapy of BCL.Nevertheless, the BCL treatment still faces some challenges.For example, 30% -50% of patients succumb to the BCL yet [4].Rituximab has been reported to exert certain acute side effects such as acute infusion reasons, immune reconstitution defects, various infections, reactivation of hepatitis, intestinal perforation, and so on [5,6].Moreover, drug resistant mutations have also been found in BTK, leading that patients originally responding to the Ibrutinib treatment have no response anymore [7,8].Therefore, novel targeted agents for the BCL treatment are urgently demanded at present.BCL has been demonstrated to be a complicated disease with multiple genes dysregulated.Among these genes, of particular importance are Syk, Src, and JAK2, in addition to BTK.Syk is a non-receptor tyrosine kinase, which could be recruited by dually phosphorylated ITAMs on Igα and Igβ after BCR (B cell receptor, BCR) aggregation.Syk phosphorylates several signal intermediates like BTK and BLNK, which then activate downstream signal pathways, including extracellular signal-related kinase (ERK) and NF-κB [9,10].Syk has been demonstrated to be critical for the survival and maintenance of malignant B cells [11,12], and targeted inhibition of Syk has also been shown to be able to abrogate BCR signaling and induce apoptosis of BCL cells [13][14][15].Src is also a nonreceptor tyrosine kinase, and plays important roles in a variety of cellular signal transduction pathways [16].It has been demonstrated to be involved in regulating tumor cell division, motility, adhesion, angiogenesis, and survival [17,18].Recent studies have indicated that activation of Src is highly related to the early stage phenotype of BCL and tumor growth [19].These results highlight Src as a potential target for BCL therapy [18,20].JAK2, which is again a non-receptor tyrosine kinase, plays essential roles in transmitting signals from multiple cytokine receptors, and constitutive activation of JAK2 has been demonstrated to result in myeloproliferative neoplasms [21].Recent studies further showed that mutation of JAK2 was capable of inducing BCL [22,23,24], and inhibition of JAK2 decreased IgM-induced STAT3 phosphorylation and increased apoptosis of tumor cells in a dose-dependent manner, which suggest that JAK2 could be a target for the treatment of BCL [22,25].
Obviously, Syk, Src and JAK2 all are potential targets for BCL treatment.It is reasonable to hypothesize that agents that can simultaneously attack these targets might have enhanced effects on the therapy of BCL.SKLB-850 (Figure 1A) is an orally available multikinase inhibitor potently inhibiting Syk, Src, and JAK2, which was obtained recently by us through a process of computer-aided lead discovery and subsequent structural optimization; the related results will be published elsewhere.The main purpose of this investigation is to perform a comprehensive preclinical evaluation to this compound, including in vitro and in vivo anti-BCL activity, and mechanism of action.

In vitro activities of anti-tumor cell viability of SKLB-850
In vitro anti-viability activities of SKLB-850 against various tumor cell lines were measured using the MTT method.As shown in Table 2, SKLB-850 displayed potent inhibitory activity against human B cell lymphoma cell line Ramos with an IC 50 value of 0.03 μM.It also showed considerable activities against several other human B cell lymphoma or diffuse large B cell lymphoma cell lines including HBL-1 (IC 50 : 0.69μM), SuDHL-6 (IC 50 : 0.90μM), Ly-10 (IC 50 : 0.90μM), LY-1 (IC 50 : 1.60μM), and RAJI (IC 50 : 1.95μM), and relatively weak activities against solid tumor cell lines MDA-MB-231, SW1990, SMMC7721, MCF-7, and CFPAC-1.To human pancreatic cancer cell line HPAC and human non-small cell lung cancer cell lines A549 and H358, SKLB-850 did not exhibit activity, which excluded the possibility that the anti-viability activity of SKLB-850 is due to cytotoxicity.

SKLB-850 induced apoptosis of tumor cells in vitro
The ability of SKLB-850 to induce apoptosis of BCL cells was analyzed by FCM.In this assay, the BCL cell lines Ramos and HBL-1 were selected.As shown in Figure 1B-1E, the percentages of apoptotic cells for both Ramos and HBL-1 cell lines were dosedependently increased after SKLB-850 treatment for 18 hours.R788, which is a Syk inhibitor and is in clinical trials for the treatment of BCL, also induced apoptosis but obviously much weaker compared with SKLB-850 (Supplementary Figure 1).

SKLB-850 blocked the tumor cell cycle in vitro
FCM was again used to examine the influence of SKLB-850 on the tumor cell cycle.As shown in Figure 2A and 2C, the percentages of Ramos cells in the G2 phase were significantly increased after treatment of SKLB-850 compared with the control.For example, 0.01μg/ mL SKLB-850 treatment led to that the percentage of G2 phase cells increased to 53.90 ± 2.65 % (P<0.01)from 19.64 ± 0.61 % (P<0.01) of the control.Very similarly, the percentages of HBL-1 cells in the G2 phase were also significantly increased after treatment of SKLB-850 compared with the control (see Figure 2B and 2D).In contrast, R788 just had a very weak effect on the percentage of cells in G 2 phase.All the results indicated that SKLB-850 arrested the Ramos and HBL-1 cells in the G2 phase in vitro.

SKLB-850 inhibited the secretion of chemokines CCL3, CCL4, CXCL12 and expression of CD184
We then measured the concentrations of CCL3 and CCL4 in supernatant of Ramos cells cultured in medium, or medium supplemented with anti-IgM and anti-CD40 in the presence or absence of SKLB-850 after 18 hours.CCL3 and CCL4 are chemokines, which appear to correlate with the signaling capacity of the BCR [26].As shown in Figure 3A and 3B, SKLB-850 inhibited the increased secretion of CCL3 and CCL4 stimulated by anti-IgM and anti-CD40.The mean CCL3 and CCL4 concentrations in supernatants of Ramos cells after anti-IgM and CD40 stimulation were 3443.00 ± 134.80 pg/ mL and 134.80 ± 1.90 pg/mL, respectively.Treatment of Ramos cells with 0.1μM SKLB-850 significantly decreased anti-IgM and anti-CD40 induced CCL3 and CCL4 levels to 2303.10 ± 109.40 pg/mL (CCL3, n=3, P<0.01) and 15.20 ± 0.30 pg/mL (CCL4, n=3, P<0.01), respectively.Syk has been demonstrated to be involved in the migration of BCL cells.And BCL cells could migrate toward the chemokine CXCL12 upon activation of its receptor CD184 [27].As displayed in Figure 3C and 3D, SKLB-850 treatment decreased the expression of CD184, and reduced the percentage of Ramos cell chemotaxis toward CXCL12.

In vivo anti-tumor effect of SKLB-850
The in vivo anti-BCL effects of SKLB-850 were investigated in the Ramos and HBL xenograft mouse models with R788 being taken as a positive control.The animals were treated orally once daily for 21 days (Ramos model) or 36 days (HBL-1 model).In the Ramos model, 40 mg/kg or 20 mg/kg SKLB-850 significantly suppressed the tumor growth with tumor inhibition rates of 78.82% ± 9.35% and 73.65% ± 21.22%, respectively (Figure 4A and 4C).Similarly, in the HBL-1 model, SKLB-850 treatment with 40 mg/ kg or 20 mg/kg also substantially inhibited the tumor growth with tumor inhibition rates of 67.96% ± 8.40% and 80.84% ±4.09%, respectively (Figure 4B and 4D).In the two models, R788 also showed some anti-tumor effect, but its potencies are relatively weaker than those of SKLB-850.
A preliminary toxicity evaluation to SKLB-850 was also carried out.As shown in Figure 5A, no significant differences in weights were observed for all the treated groups.Furthermore, there were no obvious changes for the levels of ALT, AST, BUN, and CREA, which are important indexes representing liver injury, in mice treated with SKLB-850 (Figure 5B).Besides, toxic pathologic changes in liver, lung, kidney, spleen, and heart were not detected by microscopic examination (Figure 5C).All the  results indicated that SKLB-850 had very small systemic toxicity.

SKLB-850 induced apoptosis in mouse tumor tissues and B-cell lymphoma samples from BCL patients
The ability of inducing apoptosis of SKLB-850 in tumor tissues was examined by TUNEL.As shown in Figure 6A and 6D, SKLB-850 significantly induced apoptosis in tumor tissues compared with R788 (SKLB-850, 406.00 ± 20.70% of control; R788, 242.20 ± 20.00% of control).We then examined apoptotic tumor cells in B-cell lymphoma samples from BCL patients after SKLB-850 treatment by FCM.As shown in Figure 6C and 6F, SKLB-850 induced cell apoptosis (14.10 ± 4.04%), and R788 had the same effect but relatively weak (7.60 ± 0.52%).To better understand the mechanism of antitumor activities in vivo, Ki67 and CD20 in mouse tumor tissues were detected by immunohischemistry; Ki67 was a marker of proliferation in the diagnosis of tumors and CD20 was highly expressed in B cell lymphomas [28].We observed significant reduction of Ki67 and CD20 expression when treated by SKLB-850, indicating inhibition activities of SKLB-850 in tumor tissues (Figure 6B and 6E).All the data showed that SKLB-850 could induce apoptosis in vivo and B lymphoma cells of patients, decrease expression of Ki67 and CD20, which contribute to the tumor growth suppression.

SKLB-850 inhibited tumor growth via blocking Syk/ERK, Src/FAK and JAK2/STAT3 signaling pathways
To understand the mechanism of antitumor activities in vivo, pivotal protein activities of Syk/ERK, Src/FAK and JAK2/STAT3 signaling pathways were detected by Western blot analysis.In Figure 7A and 7B, SKLB-850 effectively inhibited Syk, Src, JAK2 phosphorylation, and down-regulated the phosphorylation levels of ERK, FAK and Stat3 at concentrations between 0.01μMl and 1μM/ ml in Ramos cell.These results imply that mechanisms of anti-tumor action were related with inhibition of Syk/ ERK, Src/FAK and JAK2/STAT3 signaling pathways.Immunohistochemistry assays were performed using tumor tissues isolated from the Ramos tumor model at the end of treatment.As shown in Figure 7B, phosphorylations of Syk, Src/FAK and JAK2/STAT3 were inhibited in SKLB-850 treatment group.In addition, phosphorylation of NF-κB p65, one of the proteins in Syk-regulated signaling pathways, was also inhibited by SKLB-850 in tumor tissues.

Kinase inhibition assay
The IC 50 values of SKLB-850 for kinase inhibition in vitro were measured by radiometric assays conducted by Kinase Profiler service provided by Millipore as described in Pan's study [29].

Cell proliferation assay
Cell proliferation was measured using the MTT assay as previously described [30].Various cells including were treated with indicated concentrations of SKLB-850 for 72 hours.R788 (Selleck.cn)was served as a positive control.Each assay was replicated 3 times.

Apoptosis assay
Flow cytometry (FCM) assays were performed to investigate the apoptosis of Ramos and HBL-1 cells induced by SKLB-850.Ramos and HBL-1 cell cultures in plates were treated with indicated concentrations of SKLB-850 or R788 for 24 hours.Medium without treatment reagents were added as control.Cells were harvested and washed with PBS, stained with 2.5μL AnnexinV-FITC and 2.5μL propidium iodide (Beyotime Institute of Biotechnology).Cell apoptosis was analyzed with a flow cytometer (BD FACSCalibur, BD, USA).

Cell cycle analysis
Ramos and HBL-1 cells cultured in 6-well plates were treated with DMSO, R788, SKLB-850 or medium.After 18 hours, cells were harvested and washed with PBS, fixed with 70% ethanol for 30 min, stained with 200 μL PI (50 μg/mL PI with 1 mg/mL RNase I, and 0.1% Triton X-100) for 30 min.Cell cycle was analyzed with a flow cytometer (BD FACSCalibur, BD, USA).

Enzyme-linked immunosorbent assays
To evaluate the effect of SKLB-850 on CCL3 and CCL4 secretion after stimulation of Ramos cells with anti-IgM (30 μg/ml) and anti-CD40 (10 μg/ml), chemokine levels were measured in supernatants of activated BCL cells, as previously described [31].After 18 hours of anti-IgM and anti-CD40 co-culture, supernatants were harvested and assayed for CCL3 and CCL4 by quantitative enzyme-linked immunosorbent assays according to the manufacturer's instructions (Quantikine, R&D Systems, Minneapolis, MN, USA).
Chemotaxis assay was performed as previously described [31].Briefly, 1×10 6 Ramos cells were cultured in RPMI 1640 containing 0.5% bovine serum albumin and were pre-incubated for 30 minutes with SKLB-850, R788 or DMSO, and then they were added to the inserts of transwell chambers (5μm poresize; Corning).Inserts were then transferred into wells containing CXCL12 (200ng/ mL).After 12 hours, cell count was determined for the upper and lower well in duplicates using a flow cytometer (Novocyte Technologies, China).Chemotaxis indices were calculated as: [number of cells in the lower chamber×100] / [number of cells in the lower and upper chambers].The chemotaxis index with SKLB-850 was calculated as percentage of the DMSO index.

Flow cytometry
The expression of surface molecules was analyzed by flow cytometry, using the following monoclonal antibody CD184 (CXCR4), and the relevant isotype controls mAbs (BD biosciences).A total of 10 6 mononuclear cells were incubated for 30 minutes at 4°C with saturating concentrations of mAbs, and the cells were washed twice and analyzed with a flow cytometer (BD FACSCalibur, BD, USA).

Western blot analysis
For Ramos cell immunoplot studies, cancer cells were incubated with vehicle, R788, or SKLB-850 for 24 hours, and then lysed in RIPA buffer (Beyotime, China) containing Roche protease inhibitor cocktail, and the protein concentrations were determined by the Bradford method.Proteins were separated by gel electrophoresis on 8-12% SDS-PAGE gels and probed with specific antibodies

Figure 1 :
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 :
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 6 :
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.