Abstract
BACKGROUND: Primary Mediastinal large B-cell lymphoma (PMBL) and Hodgkin lymphoma (HL) are two of the most common malignancies among adolescents and young adults (AYA). PMBL and HL share similar molecular features by gene expression profiling and pathogenesis (Rosenwald et al., J Exp Med., 2003). HL represents only approximately 4-5% of all cancers in children younger than 15 years of age, but is the most common cancer in the 15-35 yrs AYA group. While the prognosis is excellent with AYA HL, there are significant late effects secondary to chemoradiotherapy and therefore new targeted agents are needed to avoid these morbid late effects in HL (Hochberg/Cairo et al., Br. J. Haem., 2009). Frequent gains of chromosome 9p exhibit higher Janus Kinase 2 (JAK2) transcript levels with increased JAK2 activity (Bentz et al., Genes Chromosomes Cancer, 2001), suggesting aberrant activity of JAK2 and Signal Transducer and Activator of Transcription (STAT) pathways, which may in part play an important role in the pathogenesis of HL and PMBL. Ruxolitinib is a potent and selective JAK1/JAK2 inhibitor against myeloproliferative neoplasms (MPNs) that consistently exhibits dysregulation of the JAK1/JAK2 pathway, including those MPNs with a JAK2V617F mutation. Ruxolitinb also inhibits JAK2/STAT5 signaling in vitro and in a murine model of MPN (Quintas-Cardama et al., Blood, 2010).
OBJECTIVES: We hypothesize that ruxolitinib may potentially function as targeted therapeutic agent for both PMBL and HL and therefore, investigated the efficacy of ruxolitinib in PMBL and HL cells xenografted into NSG mice.
METHODS: Cell proliferation and apoptosis analysis were assessed using MTS and Caspase-3/7 assay (Promega), respectively. The expression of protein was examined by immunoblotting and statistical significance was determined by Student t-test. Karpas-1106P PMBL cell line and L-428 HL cell line were stably transfected with a firefly luciferase expression plasmid (ffluc-zeo), kindly provided by Laurence Cooper MD, PhD. The six to eight weeks old Female NSG (NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ) mice were from Jackson laboratory. The ffluc-zeo NSG mice were irradiated (2.5 Gy) and then were subcutaneously injected with 1x106 ffluc-zeo Karpas-1106P or L-428 tumor cells. Tumor burden and tumor progression were monitored by bioluminescence imaging (BLI) using the Xenogen IVIS-200 (Caliper Life Sciences) for up to 60 days. Mice were orally gavaged with either vehicle or ruxolitinib (45.0mg/kg or 90.0mg/kg) (generously provided by Incyte Corporation, Wilmington, DE, USA) for 21 days. Survival rates were analyzed by the Kaplan-Meier method and differences evaluated by log-rank test using the Prism Version 6.0 software.
RESULTS: We observed that ruxolitinib significantly decreased the phosphorylation of STAT3, -5 and -6 in Karpas-1106P PMBL and L-428 HL cells. The reduction in cell proliferation by 10uM ruxolitinib are 41% in Karpas-1106P (p<0.04) and 19% in L-428 (p<0.03), respectively. In addition, ruxolitinib significantly induced Caspase-3/7 activity (p<0.05) and increased cleavage of PARP and caspase-3 fragments (p<0.001). In PMBL and HL xenografted mice models, we observed a significant decrease in tumor luminescence intensity following ruxolitinib treated PMBL (45.0mg/kg and 90.0mg/kg, p<0.05) and HL xenografted NSG mice at day 21 and 28 (45.0mg/kg, p<0.05) compared to control mice (Figure 1A). Ruxolitinib (45.0mg/kg) treated xenograft NSG mice had a significantly prolonged survival in PMBL (p<0.0001) and HL (p=0.0001) compared to control mice (Figure 1B).
CONCLUSIONS: Ruxolitinib significantly inhibited JAK2 enzymatic activity by the abrogation of the phosphorylation of STAT3, STAT5 and STAT6, and ruxolitinib showed significant anti-proliferative effects. Ruxolitinib (45mg/kg) significantly inhibited tumor progression and significantly prolonged survival in PMBL and HL xenografted NSG mice compared to control mice. Ruxolitinib may be a potential adjuvant agent in the treatment of PMBL and HL. Future studies will focus on whether the addition of ruxolitinib to chemoimmunotherapy improves the survival in a NSG PMBL and HL xenograft mouse models.
No relevant conflicts of interest to declare.
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