The Btk-dependent PIP5K1γ lipid kinase activation by Fas counteracts FasL-induced cell death.

The Fas/FasL system plays a critical role in death by apoptosis and immune escape of cancer cells. The Fas receptor being ubiquitously expressed in tissues, its apoptotic-inducing function, initiated upon FasL binding, is tightly regulated by several negative regulatory mechanisms to prevent inappropriate cell death. One of them, involving the non-receptor tyrosine kinase Btk, was reported mainly in B cells and only poorly described. We report here that Btk negatively regulates, through its tyrosine kinase activity, the FasL-mediated cell death in epithelial cell lines from colon cancer origin. More importantly, we show that Btk interacts not only with Fas but also with the phosphatidylinositol-4-phosphate 5-kinase, PIP5K1γ, which, upon stimulation by Fas ligand, is responsible of a rapid and transient synthesis of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2). This production requires both the presence and the tyrosine kinase activity of Btk, and participates in the negative regulation of FasL-mediated cell death since knocking down PIP5K1γ expression significantly strengthens the apoptotic signal upon FasL engagement. Altogether, our data demonstrate the cooperative role of Btk and PIP5K1γ in a FasL-induced PI(4,5)P2 production, both proteins participating to the threshold setting of FasL-induced apoptotic commitment in colorectal cell lines.

Identification of a lysine-rich region of Fas as a raft nanodomain targeting signal necessary for Fas-mediated cell death.

Fas interaction at the plasma membrane with its lipid and protein environment plays a crucial role in the early steps of Fas signalling induced by Fas ligand binding. Particularly, Fas localisation in the raft nanodomains, ezrin-mediated interaction with the actin cytoskeleton and subsequent internalization are critical steps in Fas-mediated cell death. We identified a lysine-rich region (LRR) in the cytoplasmic, membrane-proximal region of Fas as a key determinant modulating these initial events. Through a genetic approach, we demonstrate that Fas LRR represents another signal additional to palmitoylation targeting Fas to the raft nanodomains, and modulates Fas interaction with the cytoskeleton.

NF-kappaB inhibition triggers death of imatinib-sensitive and imatinib-resistant chronic myeloid leukemia cells including T315I Bcr-Abl mutants.

The Bcr-Abl inhibitor imatinib is the current first-line therapy for all newly diagnosed chronic myeloid leukemia (CML). Nevertheless, resistance to imatinib emerges as CML progresses to an acute deadly phase implying that physiopathologically relevant cellular targets should be validated to develop alternative therapeutic strategies. The NF-kappaB transcription factor that exerts pro-survival actions is found abnormally active in numerous hematologic malignancies. In the present study, using Bcr-Abl-transfected BaF murine cells, LAMA84 human CML cell line and primary CML, we show that NF-kappaB is active downstream of Bcr-Abl. Pharmacological blockade of NF-kappaB by the IKK2 inhibitor AS602868 prevented survival of BaF cells expressing either wild-type, M351T or T315I imatinib-resistant mutant forms of Bcr-Abl both in vitro and in vivo using a mouse xenograft model. AS602868 also affected the survival of LAMA84 cells and of an imatinib-resistant variant. Importantly, the IKK2 inhibitor strongly decreased in vitro survival and ability to form hematopoietic colonies of primary imatinib resistant CML cells including T315I cells. Our data strongly support the targeting of NF-kappaB as a promising new therapeutic opportunity for the treatment of imatinib resistant CML patients in particular in the case of T315I patients. The T315I mutation escapes all currently used Bcr-Abl inhibitors and is likely to become a major clinical problem as it is associated with a poor clinical outcome.

The metabolic perturbators metformin, phenformin and AICAR interfere with the growth and survival of murine PTEN-deficient T cell lymphomas and human T-ALL/T-LL cancer cells.

We show here that the antidiabetic agents metformin and phenformin and the AMPK activator AICAR exert strong anti-tumoural effects on tPTEN-/- lymphoma cells and on human T-ALL cell lines and primary samples. The compounds act by inhibiting tumour metabolism and proliferation and by inducing apoptosis in parallel with an activation of AMPK and an inhibition of constitutive mTOR. In tPTEN-/- cells, the drugs potentiated the anti-leukaemic effects of dexamethasone, and metformin and phenformin synergised with 2-deoxyglucose (2DG) to impair tumour cell survival. In vivo, metformin and AICAR strongly decreased the growth of luciferase-expressing tPTEN-/- cells xenografted in Nude mice, demonstrating that metabolism targeting could be a potent adjuvant strategy for lymphoma/leukaemia treatment.

Pharmacological inhibition of carbonic anhydrase XII interferes with cell proliferation and induces cell apoptosis in T-cell lymphomas.

The membrane-bound carbonic anhydrase isoforms CAIX and CAXII, underpin a pH-regulating system that enables hypoxic tumor cell survival. Here, we observed for the first time an upregulation of CAXII in T-cell acute lymphoblastic leukemia/lymphoma (T-ALL/LL) cells. First we showed that CAXII is overexpressed in thymocytes from tPTEN-/- mice suffering of T lymphoma and that its pharmacological inhibition decreased cell proliferation and induced apoptosis. The same results were observed with the SupT1 human T cell lymphoma line. In addition we observed an upregulation of CAXII in human T-ALL samples supporting the case that CAXII may represent a new therapeutic target for T-ALL/LL.