BackgroundInterferon Regulatory Factor 5 is a transcription factor that regulates the expression of genes involved in the response to viral infection and in the stimulation of the immune system. Moreover, multiple studies have demonstrated that it negatively regulates cell growth and oncogenesis, favoring cell differentiation and apoptosis.Thyroid carcinoma represents 98% of all thyroid malignancies and has shown a steady increase in incidence in both the USA and western European countries.FindingsWe investigated the expression, localization and function of IRF5 in thyroid cancer cells and found that it is highly expressed in both primary and immortalized thyroid carcinomas but not in normal thyrocytes. IRF5 levels were variably modulated by Interferon alpha but IRF5 only localized in the cytoplasmic compartment, thus failing to induce p21 expression as previously reported in different cell models. Furthermore, ectopic IRF5 increased both the proliferation rate and the clonogenic potential of malignant thyroid cells, protecting them from the cytotoxic effects of DNA-damaging agents. These results were directly attributable to IRF5, as demonstrated by the reduction in colony-forming ability of thyroid cancer cells after IRF5 silencing. An IRF5-dependent induction of endogenous B-Raf observed in all thyroid cancer cells might contribute to these unexpected effects.ConclusionsThese findings suggest that, in thyroid malignancies, IRF5 displays tumor-promoting rather than tumor-suppressor activities.

2165 Poster Board II-142 Introduction: Interferon Regulatory Factors (IRFs) comprise ten helix-turn-helix transcription factors that are pivotal modulators of the immune response against viral infections and cancer. IRF-5, a direct target of the p53 tumor suppressor, is known to bind an extensive number of gene promoters regulating cell growth, differentiation and apoptosis. Increased or reduced levels of IRF-5 have been reported in several solid and hematologic malignancies but there is currently no evidence concerning IRF-5 and Chronic Myeloid Leukemia (CML). We therefore wanted to investigate if alterations in IRF-5 expression and function were involved in the pathogenesis of CML. Patients and Methods: Expression studies and colony-forming assays after lentiviral transduction were carried out on primary cells isolated from 10 CML patients (pts) at diagnosis and 6 healthy donors (controls). Experiments on immortalized cell lines were performed using K562, KYO-1 (CML lines) and HL60 (negative control). Results: RT-PCR and immunoblot assays showed that IRF-5 is expressed in primary hematopoietic cells derived from both CML pts and healthy donors as well as in immortalized CML cell lines. We also found that IRF-5 immunoprecipitates with the BCR-ABL oncoprotein of CML and that BCR-ABL induces its tyrosine phosphorylation. Exposure to the semi-specific BCR-ABL inhibitor Imatinib mesylate (IM) reduced the interaction between IRF-5 and BCR-ABL and decreased IRF-5 tyrosine phosphorylation. Cell fractionation experiments employing the nuclear export inhibitor Leptomycin B showed that, in CML cells, IRF-5 is almost exclusively localized in the cell cytoplasm because of excessive nuclear export. Interestingly, both IM and Interferon (IFN) treatment radically modified IRF-5 intracellular distribution restoring its nuclear localization. A computational sequence analysis identified tyrosine 104 as part of a consensus for the BCR-ABL kinase. Hence, we generated an IRF-5 Y104F mutant that would no longer be a substrate for BCR-ABL catalytic activity. Indeed, compared to native IRF-5, the Y104F mutant displayed low levels of tyrosine phosphorylation that remained unchanged after IM treatment. Furthermore, only a fraction of BCR-ABL immunoprecipitated with the Y104F construct that, in cell fractionation experiments, localized predominantly in the nuclear compartment. Finally, both CML cell lines and primary CD34-positive cells lentivirally transduced with IRF-5 Y104F showed a remarkable reduction in cell proliferation in methylcellulose colony-forming assays. This result was not observed after transduction with the gfp control. Conclusions: In CML cells, BCR-ABL associates with IRF-5 and causes its phosphorylation on tyrosine 104. This event favors IRF-5 nuclear export thereby abrogating its function as a transcriptional regulator. Inactivation of BCR-ABL catalytic activity by IM treatment or exposure to IFN restores IRF-5 nuclear localization. Furthermore, expression of an IRF-5 Y104F mutant that can't be phosphorylated by BCR-ABL reduces the proliferative activity of both primary and immortalized CML cells. Taken together these data suggest that IRF-5 is involved in the pathogenesis of CML and that IM or IFN-based treatments can restore IRF-5 localization and function antagonizing the proliferation of the leukemic clones. Disclosures: No relevant conflicts of interest to declare.