Hodgkin Lymphoma (HL) and Primary Mediastinal B-cell Lymphoma (PMBL) exhibit similar molecular features and pathogenesis. Both lymphoid malignancies shared similar cytogenetic abnormalities, namely 9p and 2p gains (Bentz et al., Genes Chromosomes Cancer, 2001; Joos et al., Int J Cancer 2003), and exhibit higher Janus Kinase 2 (JAK2) transcript levels with increased JAK2 activity (Green et al., Blood, 2010), suggesting aberrant activity of JAK2 and Signal Transducer and Activator of Transcription (STAT) pathways, which may 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 exhibit dysregulation of the JAK1/JAK2 pathway, for reasons such as the presence of the JAK2 V617F mutation; this drug also inhibits JAK2/STAT5 signaling in vitro and in murine model of MPNs (Quintas-Cardama et al., Blood, 2010). Ruxolitinib is associated with marked and durable clinical benefits in patients with myelofibrosis (Verstovsek et al., NEJM, 2010).
Given the rationale for JAK2 inhibition in lymphoma, we designed the studies described here to evaluate the effects of Ruxolitinib on JAK2/STAT signaling pathways, cell proliferation and apoptosis in HDLM-2 (HL) and Karpas-1106P (PMBL) lymphomas.
Both HDLM-2 and Karpas-1106P cells were obtained from the DSMZ, Germany and maintained in RPMI with 20% FBS. Ruxolitinib was generously provided by Incyte Corporation, and for cytokine stimulation, Interlukin-4 (IL-4) was purchased from Invitrogen. Mono- or poly-clonal antibodies for western blotting were from Cell signaling Technology and Santa Cruz Biotech, respectively. For the effects on proliferative and apoptosis, Cell titer 96 Aqueous One solution cell proliferation assay (MTS) (Promega) and Caspase-Glo 3/7 assay (Promega) were used according to the manufacturer's instruction. Briefly, HDLM-2 and Karpas-1106P cells (0.5×106/ml) were seeded into 24-well plated and treated with vehicle (DMSO) alone or Ruxolitinib at various concentrations for 48 and 72 hours and measured by Clarity Luminescence microplate reader (BioTek) and statistical significance on this study was determined by one-tailed Student t-test.
Ruxolitinib treatment resulted in the dose dependent inhibition of JAK2-dependent pSTAT3 (IC50: 35nM for HDLM-2; IC50: 90nM for Karpas-1106P) and pSTAT5 (IC50: 28nM for HDLM-2; IC50: 98nM for Karpas-1106P) activation. In addition, the same inhibitor potency for pSTAT6 in both HDLM-2 and Karpas-1106P cells (IC50:20nM) was observed with Ruxolitinib. The level of STAT6 phosphorylation in Karpas-1106P cells was further enhanced significantly by 10ng/ml IL-4 treatment for 10 minutes and also increased by a dose-dependent reduction of Ruxolitinib (25–400nM).
The effects of Ruxolitinib on cell proliferation by MTS assay demonstrated antiproliferative effects in a dose-dependent (1–100uM) manner (p<0.05) for up to 72 hours. Consistent with the anti-proliferative effect of Ruxolitinib, Ruxolitinib induced cell death was observed with increasing doses (1–100uM) (p<0.05) in both HL and PMBL cells. The cleavage of poly adenosine diphosphate ribose polymerase (PARP), another hallmark of apoptosis, was substantially increased by Ruxolitinib in the same dose-dependent manner. We examined the effects of Ruxolitinib on the expression of anti-apoptotic genes to enhance our understanding of the effect on apoptosis, expression of two anti-apoptotic genes, Bcl-xL and Mcl-1were inhibited in a dose-dependent manner 72hours after Ruxolitinib treatment of HDLM-2 cells. These results suggested that Ruxolitinib decreases cancer cell survival by inducing programmed cell death via down-regulating the expression of anti-apoptotic genes.
Taken altogether, Ruxolitinib demonstrated efficay against HDLM-2 and Karpas-1106P cells with constitutively active JAK2 signaling and effectively blocked STAT signaling in both HL and PMBL. Ruxolitinib significantly induced antiproliferative effects as well as apoptosis in HL and PMBL.
Ruxolitinib may be a future potential targeted agent for the treatment of HL and PMBL lymphomas, and in vivo efficacy of Ruxolitinib will be evaluated in NOD/SCID mouse models of HL and PMBL lymphomas.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.