Signal Transducer and Activator of Transcription (STAT)-3 Activates Nuclear Factor (NF)-κB in Primary Myelofibrosis (PMF) Bone Marrow Cells

Abstract 1742

Primary myelofibrosis (PMF) is a stem cell–derived hematologic malignancy, characterized by an expansion of one or more myeloid lineage resulting in bone marrow (BM) hypercellularity, magakaryocyte proliferation with atypia, granulocytic proliferation, and reticulin and/or collagen fibrosis. An acquired activating mutation in Janus kinase 2 at codon V617F (JAK2^V617F) is detected in BM cells of the majority of patients with PMF. Constitutively activated JAK2 induces phosphorylation and activation of STAT3. Phosphorylated STAT3 forms heterodimers, translocates to the nucleus, binds to DNA, activates STAT3-target genes, and induces production of cytokines that interact with the BM microenvironment. Hematopoietic stroma derived soluble factors provide PMF cells with survival advantage (Manshouri et al. Cancer Res 71: 3831, 2011) and, as reported previously, most of these factors activate NF-κB in a variety of cell types. NF-κB plays an important role in the survival and proliferation of normal and neoplastic cells. In several hematologic malignancies, the NF-κB p65/p50 dimers were found to be activated to variable degrees. The activation of NF-κB is mediated by either the canonical pathway or the alternative pathway. The canonical pathway is typically activated by extracellular signals that activate the β subunit of the IκB kinase (IKK) complex (IKKβ) that induces the phosphorylation and degradation of the NF-κB inhibitor IκBα. Following IκBα degradation, NF-κB heterodimers translocate to the nucleus and bind to DNA. We have recently found that in chronic lymphocytic leukemia (CLL) constitutively activated STAT3 induces the production of unphsophorylated (U) STAT3. U-STAT3 binds to the NF-κB dimers p65/p50 in competition with IκB and the U-STAT3/NF-κB complex shuttles to the nucleus where NF-κB binds to DNA and activates NF-κB-regulated genes (Liu et al. Mol Cancer Res 9: 507, 2011). Because in PMF constitutively activated JAK2 induces phosphorylation of STAT3 and this activated form of STAT3 induces the production of U-STAT3, we wondered whether, like in CLL, U-STAT3 activates NF-κB in PMF. To determine whether NF-κB is constitutively activated in PMF we obtained BM low density cells from untreated patients with PMF. First we studied low-density BM cells of 11 patients with PMF using the electrophoretic mobility shift assay (EMSA). Cells of all samples bound to a p65/NF-κB DNA-labeled probe and the addition of an unlabelled (cold) p65/NF-κB probe attenuated or completely eliminated the binding. Typically, NF-κB-DNA binding appears and disappears due to repeated degradation and re-synthesis of IκB and the consequent activation and inactivation of NF-κB, respectively. Because we found that NF-κB is constitutively activated in all PMF BM samples we hypothesized that, like in CLL cells, activation of NF-κB in PMF cells is induced by an IκB-unrelated mechanism as reported by Yang J et al. (Cancer Res 65:939, 2005). By using immunoprecipitation of two different PMF BM samples we determined that STAT3 binds to the RelA/p65 NF-κB protein, and by using EMSA we found that anti-STAT3, similar to anti- NF-κB p65 antibodies, attenuated the binding of PMF BM cell extract to the NF-κB DNA probe. Taken together, our data suggest that U-STAT3 binds the NF-κB dimers p65/p50 and constitutively activates NF-κB in PMF.