Mirin, an inhibitor of the Mre11-Rad50-Nbs1 complex required for HR, was used as a control [38]. human stromal cell line hMSC-TERT, and in co-culture with bone marrow mesenchymal stromal cells from a patient with MM (pBMSC). In all cases, the alkylating and the HDACi effect of EDO-S101 were preserved. Figure S5. Different MM cell lines were incubated with 1 and 2.5?M EDO-S101 for 48?h. After propidium iodide staining, the cell cycle profile was analyzed by flow cytometry. Calculation of percentages of cells at each phase did not consider cells at G0. Figure S6. Bcl-2 family proteins studied by Western blot after treatment of MM1S with the indicated doses of EDO-S101 for 48?h. Figure S7. Toxicity profile of mice bearing a subcutaneus plasmacytoma and treated with the indicated drug. The EDO-S101 group showed a reversible 10C20% loss of body weight. Each Fmoc-Lys(Me3)-OH chloride point represents the mean??SD. Figure S8. The combination of EDO-S101 plus bortezomib was also able to improve the effect of single treatments in RPMI-8266, JJN3, and U266 cell lines. Figure S9. Toxicity profile of mice bearing a subcutaneus plasmacytoma and treated with the indicated drugs. The EDO-S101 + Bortezomib group showed a reversible 10C20% loss of body weight. Each point represents the mean??SD. (PPTX 348?kb) 13045_2017_495_MOESM1_ESM.pptx (348K) GUID:?7E0CCC68-2506-4EDB-96BE-C2F4516CB170 Additional file 2: Supplemental material and methods. (DOCX 127?kb) 13045_2017_495_MOESM2_ESM.docx (127K) GUID:?BB77C9F6-0E5D-4ACC-8225-12593B30CBCF KRAS2 Data Availability StatementAll data generated or analyzed during this study are included in this published article [and its Additional files 1 and 2]. Abstract Background Despite recent advances in the treatment of multiple myeloma (MM), the prognosis of most patients Fmoc-Lys(Me3)-OH chloride remains poor, and resistance to traditional and Fmoc-Lys(Me3)-OH chloride new drugs frequently occurs. EDO-S101 is a novel therapeutic agent conceived as the fusion of a histone deacetylase inhibitor radical to bendamustine, with the aim of potentiating its alkylating activity. Methods The efficacy of EDO-S101 was evaluated in vitroex vivo and in vivoalone, and in combination with standard anti-myeloma agents. The underlying mechanisms of action were also evaluated on MM cell lines, patient samples, and different murine models. Results EDO-S101 displayed potent activity in vitro in MM cell lines (IC50 1.6C4.8?M) and ex vivo in cells isolated from MM patients, which was higher than that of bendamustine and independent of the p53 status and previous melphalan resistance. This activity was confirmed in vivo, in a CB17-SCID murine plasmacytoma model and in de novo Vk*MYC mice, leading to a significant survival improvement in both models. In addition, EDO-S101 was the only drug with single-agent activity in the multidrug resistant Vk12653 murine model. Attending to its mechanism of action, the molecule showed both, a HDACi effect (demonstrated by -tubulin and histone hyperacetylation) and a DNA-damaging effect (shown by an increase in H2AX); the latter being again clearly more potent than that of bendamustine. Using Fmoc-Lys(Me3)-OH chloride a reporter plasmid integrated into the genome of some MM cell lines, we demonstrate that, apart from inducing a potent DNA damage, EDO-S101 specifically inhibited the double strand break repair by the homologous recombination pathway. Moreover, EDO-S101 treatment reduced the recruitment of repair proteins such as RAD51 to DNA-damage sites identified as H2AX foci. Finally, EDO-S101 preclinically synergized with bortezomib, both in vitro and in vivo. Conclusion These findings provide rationale for the clinical investigation of EDO-S101 in MM, either as a single agent or in combination with other anti-MM drugs, particularly proteasome inhibitors. Electronic supplementary material The online version of this article (doi:10.1186/s13045-017-0495-y) contains supplementary material, which is available to authorized users. using an automated flow cytometry platform [25]. For the simultaneous evaluation of the efficacy on plasma cells and toxicity in lymphocytes, a different method was employed [18]. The percentage of cells Fmoc-Lys(Me3)-OH chloride at each cycle phase was calculated on the alive cells, not considering sub-G0 (apoptotic) cells in the computation. Microenvironment assays MM1S cells were incubated for 48?h with increasing doses of EDO-S101, together with IL-6 at 1? nM or IGF-1 at 10?nM, and proliferation of MM cells was assessed by.
