Celecoxib inhibits proliferation and survival of chronic myelogeous leukemia (CML) cells via AMPK-dependent regulation of ß-catenin and mTORC1/2.

CML is effectively treated with tyrosine kinase inhibitors (TKIs). However, the efficacy of these drugs is confined to the chronic phase of the disease and development of resistance to TKIs remains a pressing issue. The anti-inflammatory COX2 inhibitor celecoxib has been utilized as anti-tumour drug due to its anti-proliferative activity. However, its effects in hematological malignancies, in particular CML, have not been investigated yet. Thus, we tested biological effects and mechanisms of action of celecoxib in Philadelphia-positive (Ph+) CML and ALL cells.We show here that celecoxib suppresses the growth of Ph+ cell lines by increasing G1-phase and apoptotic cells and reducing S- and G2-phase cells. These effects were independent of COX2 inhibition but required the rapid activation of AMP-activated protein kinase (AMPK) and the consequent inhibition mTORC1 and 2. Treatment with celecoxib also restored GSK3ß function and led to down-regulation of ß-catenin activity through transcriptional and post-translational mechanisms, two effects likely to contribute to Ph+ cell growth suppression by celecoxib.Celecoxib inhibited colony formation of TKI-resistant Ph+ cell lines including those with the T315I BCR-ABL mutation and acted synergistically with imatinib in suppressing colony formation of TKI-sensitive Ph+ cell lines. Finally, it suppressed colony formation of CD34+ cells from CML patients, while sparing most CD34+ progenitors from healthy donors, and induced apoptosis of primary Ph+ ALL cells.Together, these findings indicate that celecoxib may serve as a COX2-independent lead compound to simultaneously target the mTOR and ß-catenin pathways, key players in the resistance of CML stem cells to TKIs.

Nicotinamide phosphoribosyltransferase (NAMPT/PBEF/visfatin) is a tumoural cytokine released from melanoma.

High plasma levels of nicotinamide phosphoribosyltransferase (NAMPT), traditionally considered an intracellular enzyme with a key role in NAD synthesis, have been reported in several oncological, inflammatory and metabolic diseases. We now show that eNAMPT can be actively released by melanoma cells in vitro. We analysed the mechanisms of its release, and we found both classical and non-classical pathway involvement. eNAMPT released by melanoma cells, in our hands, has paracrine and autocrine effects: it activates MAPK, AKT and NF-κB pathways and increases colony formation in anchorage-independent conditions. eNAMPT also induces M1 polarization in human monocytes. Last, we demonstrate, for the first time in any cancer type, that eNAMPT levels in plasma of tumour-bearing mice increase and that this increase can be reconducted to the tumour itself. This provides an important cue on previous observations that eNAMPT is increased in patients with cancer. Moreover, silencing NAMPT in melanoma cells leads to a reduction in the tumour growth rate. Our findings extend the basis to consider eNAMPT as a cytokine involved in tumour progression.

A chemical screen identifies the chemotherapeutic drug topotecan as a specific inhibitor of the B-MYB/MYCN axis in neuroblastoma.

The transcription factor MycN is the prototypical neuroblastoma oncogene and a potential therapeutic target. However, its strong expression caused by gene amplification in about 30% of neuroblastoma patients is a considerable obstacle to the development of therapeutic approaches aiming at eliminating its tumourigenic activity. We have previously reported that B-Myb is essentially required for transcription of the MYCN amplicon and have also shown that B-MYB and MYCN are engaged in a feed forward loop promoting the survival/proliferation of neuroblastoma cells. We postulated that pharmacological strategies breaking the B-MYB/MYCN axis should result in clinically desirable effects. Thus, we implemented a high throughput chemical screen, using a curated library of ~1500 compounds from the National Cancer Institute, whose endpoint was the identification of small molecules that inhibited B-Myb. At the end of the screening, we found that the compounds pinafide, ellipticine and camptothecin inhibited B-Myb transcriptional activity in luciferase assays. One of the compounds, the topoisomerase-1 inhibitor camptothecin, is of considerable clinical interest since its derivatives topotecan and irinotecan are currently used as first and second line treatment agents for various types of cancer, including neuroblastoma. We found that neuroblastoma cells with amplification of MYCN are more sensitive than MYCN negative cells to camptothecin and topotecan killing. Campothecin and topotecan caused selective down-regulation of B-Myb and MycN expression in neuroblastoma cells. Notably, forced overexpression of B-Myb could antagonize the killing effect of topotecan and camptothecin, demonstrating that the transcription factor is a key target of the drugs. These results suggest that camptothecin and its analogues should be more effective in patients whose tumours feature amplification of MYCN and/or overexpression of B-MYB.

Expression, purification and preliminary crystallographic studies on the catalytic region of the nonreceptor tyrosine kinase Fes.

The proto-oncogene tyrosine protein kinase c-fps/fes encodes a structurally unique protein (Fes) of the nonreceptor protein-tyrosine kinase (PTK) family. Its expression has been demonstrated in myeloid haematopoietic cells, vascular endothelial cells and in neurons. In human-derived and murine-derived cell lines, the activated form of this kinase can induce cellular transformation; moreover, it has been shown that Fes is involved in the regulation of cell-cell and cell-matrix interactions mediated by adherens junctions and focal adhesions. The N-terminus of Fes contains the FCH (Fps/Fes/Fer/CIP4 homology) domain, which is unique to the Fes/Fer kinase family. It is followed by three coiled-coil domains and an SH2 (Src-homology 2) domain. The catalytic region (Fes-CR) is located at the C-terminus of the protein. The successful expression, purification and crystallization of the catalytic part of Fes (Fes-CR) are described.