Interferon-Inducible Protein 16 (IFI-16) Is Upregulated In Chronic Myeloid Leukemia (CML) and Is Involved In Oncogene-Induced Senescence

IFI-16 is a transcriptional factor induced by IFN-γ and α in human cells and is involved in several cellular processes including cell cycle regulation, telomere length control and cellular senescence. Recently it has been shown that, similar to other oncogenes such as RAS and BRAF in solid tumors, acute expression of BCR-ABL in hematopoietic cells can induce senescence (Wajapayee et al, Blood 2010). This oncogene-induced senescence (OIS) which is a barrier for tumor development, is obviously bypassed by secondary events leading to malignant transformation. The molecular basis of the BCR-ABL-induced senescence in CML cells is not determined. In the UT7 cell line model expressing native (unmutated) BCR-ABL and its T315I-mutated counterpart, we identified by a gene profiling, a major increase of the expression IFI-16 (42-fold and 18-fold increase, respectively) as compared to parental UT7 cells. This major increase of IFI-16 expression in UT7 cells expressing native BCR-ABL was confirmed in western blots, whereas there was no detectable IFI-16 protein in parental UT7 cells. To determine if IFI-16 protein levels were dependent on BCR-ABL expression, we have used doxycycline-inducible BCR-ABL UT7 model. In this TET-Off system, the addition of doxycycline to cultures inhibits the expression of BCR-ABL by day7 and this was accompanied by a concomittant inhibition of IFI-16 expression. As expected, IFN-α (1000 U/ml) induced IFI-16 expression in parental UT7 cells and to a much higher degree in UT-7 BCR-ABL cells. In the UT7-T315I cells, IFN- α showed also a synergistic effect for IFI-16 expression which was less prominent. As IFI-16 expression can be modulated by oxidative stress, we have performed experiments in the presence of N-acetyl-cysteine which did not modify IFI-16 protein levels. To further study the role of IFI-16 in this model, we have transfected UT7 cells and their BCR-ABL expressing counterparts using shRNA-IFI-16 lentiviral vectors allowing silencing of IFI-16 expression. UT7 clones resistant to puromycin were amplified and the clones with reduced or absent expression of IFI-16 were selected for further analyses. In senescence-induction experiments, we have found that native BCR-ABL expressing UT7 silenced for IFI-16 expression, had reduced beta-galactosidase activity suggesting an inhibition of senescence phenotype. We have then checked the localization of IFI-16 in BCR-ABL and BCR-ABL T315I-expressing UT7 cells. In BCR-ABL-UT7 cells. IFI-16 was mainly in the cytoplasm and was relocated to nucleus upon IFN-α treatment. On the other hand, in UT7-T135I cells, IFI-16 was distributed between cytoplasm and nucleus and IFN-α had no effect on IFI-16 localisation. To determine the potential involvement of IFI-16 in human CML, we have analyzed IFI-16 expression by Q-RT-PCR. In a cohort of 40 Chronic Phase (CP) CML patients at diagnosis, the expression of IFI-16 was found to be significantly increased (2-Fold) as compared to controls (n=30). Thus, our results suggest strongly that IFI-16 induction could be one of the mechanisms of OIS in in BCR-ABL expressing cells. We also demonstrate for the first time that this mechanism is operational in primary CML cells. Further exploitation of this senescence pathway could be of therapeutic interest with the goal of enhancing this phenomenon especially at the level of the most primitive, TKI-resistant CML stem cells. Current experiments are underway to study the role of IFI-16 in the most primitive leukemic stem cell compartments.