mTOR is a serine-threonine protein kinase that plays a central role in regulating critical cellular processes. Consistent with its primary target being the translation machinery, mTOR is predominantly localized in the cytoplasm. However, a nuclear localization of mTOR has been found in rhabdomyosarcomas and HCT8 colon carcinoma cells. Furthermore, mTOR becomes nuclear in HEK293 cells treated with leptomycin B, a specific inhibitor of nuclear export receptor Crm1, suggesting that mTOR may be a cytoplasmic-nuclear shuttling protein. Aims of this study is to evaluate cellular localization of mTOR in multiple myeloma (MM) cell lines and primary MM cells and to evaluate the role of pomalidomide (CC-4047) in regulating the mTOR pathway.
We used OPM-2 and RPMI-8226 MM cell lines and primary MM cells. For primary myeloma specimens, plasmacell population was positively selected by the immunomagnetic method using CD138+ microbeads. Proliferation was evaluated by MTT assay on OPM-2 and RPMI-8226 cells following incubation with pomalidomide at concentrations ranging from 0.01 to 10 μM. Apoptosis was assessed by flow cytometry for the detection of annexin V-positive cells in both MM cell lines and in plasmacells from 3 MM patients. Cellular localization of mTOR protein was also evaluated using a confocal scanning microscopy in RPMI-8226 and OPM-2 cells and in plasmacells from 4 MM patients in basal conditions and after pomalidomide treatment. Immunohistochemistry with antibody against phospho-mTOR was performed on bone marrow sections of 92 MM patients. Furthermore, RPMI-8226 and OPM-2 cells untreated or treated with the drug, were fractionated and both cytoplasmic and nuclear fractions were analysed by Western blotting with specific antibodies for mTOR and pospho-mTOR.
MTT assay performed on MM cell lines demonstrated that pomalidomide has a dose-dependent activity. Pomalidomide 1 μM at 48 hours inhibited proliferation of OPM-2 and RPMI-8226 cells with 50% and 40% decrease in cell numbers, respectively. Only minor increase of apoptosis could be detected in RPMI-8226 and OPM-2 cells incubated with pomalidomide at varying concentrations for 24, 48 and 72 hours. Pomalidomide 1 μM was effective in plasmacells from 3 MM patients at 24 hours with 23%, 33% and 26% annexin-V positive cells (versus 11%,18% and 3% of annexin-V positive cells cultured with media alone, respectively). Immunofluorescence assays with mTOR antibody, demonstrated that mTOR protein is distributed throughout the cytoplasm and the nucleus at baseline in both MM cell lines and in plasmacells of 3 out 4 MM patients. A clearly increase of the nuclear mTOR protein was detected after pomalidomide treatment in RPMI-8226 and OPM-2 cells (10 μM at 48 hours) and in plasmacells from 3 MM patients (1 μM at 24 hours) (2 with nuclear mTOR localization at baseline and 1 without it). Immunohistochemistry performed on bone marrow sections evidenced that 41 out 92 MM samples (44.4%) stained positive for cytoplasmic phospho-mTOR. A nuclear phospho-mTOR staining was also demonstrated in 11 cases (12%). All patients but one showed both nuclear and cytoplasmic phospho-mTOR staining. Cytoplasmic and nuclear distribution of mTOR and pospho-mTOR was also evidenced by Western blotting in RPMI-8226 and OPM-2 cells. As expected, the mTOR and phospho-mTOR protein levels were significantly higher in the cytoplasm when compared to the nucleus. Treatment with pomalidomide 10 μM at 48 hours increased the nuclear mTOR and phospho-mTOR expression levels in the nucleus with a concomitant decrease of the cytoplasmic phospho-mTOR protein amount.
In RPMI-8226 and OPM-2 cell lines and in a fraction of primary MM cells, mTOR is distributed throughout the cell cytoplasm and in some nucleus. The anti-myeloma activity of pomalidomide may be mediated by the downregulation of the mTOR pathway with a nuclear shuttling of mTOR protein and a reduction of the cytoplasmic phospho-mTOR. Further studies are needed to establish the mechanism of mTOR shuttling, to evaluate the role of pomalidomide in regulating the mTOR pathway and to assess the potential synergism between pomalidomide and mTOR inhibitors.
Guglielmelli:Celgene: This study was sponsored by Celgene Other, Honoraria, Research Funding.
Asterisk with author names denotes non-ASH members.