BCR-ABL Tyrosine Kinase Activity Modulates the Phosphorylation, Localization and Function of Interferon Regulatory Factor 5 (irf-5) in Chronic Myeloid Leukemia (CML) Cells.

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.

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).

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.

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.

No relevant conflicts of interest to declare.