Figure 5. ERCC3 is a therapeutic target in MM.
A/NER proficiency in primary multiple myeloma samples. CD138 positive cells from MM patients' bone marrow were purified with anti CD138 microbeads, cultured overnight and exposed to UV-C. Each bar represents the normalized remaining DNA-damage percentage 2 hours after UV-C exposure for each sample. A control MMCL was processed as a positive control for each experiment with primary samples. A minimum of 107 nuclei was analyzed in each experiment (range from 107 to 7390 nuclei). Similar to MM cell lines, primary samples are featured by a heterogeneous NER efficiency. 8 samples from newly diagnosed (white bars)/ and 4 samples from relapse/refractory myeloma patients (grey bars) were evaluated. Cytogenetic information was available for all except one sample. Sample MM6 is featured by the t(4;14 translocation whereas other samples have a standard cytogenetic risk. 8 samples were evaluated with the DDB2 proteo-probe and 4 samples with the 6-4 pthoproducts antibody.
B, C/spironolactone inhibits NER in primary myeloma cells. Primary myeloma cells were incubated with DMSO, or spironolactone (10 μM) overnight before NER evaluation. The figure represents the persistence of DNA damage signal 120 minutes after exposure to UV (A.F.U: arbitrary fluorescent unit). The pictures show the persistence of high DNA-damage signal in presence of spironolactone confirming NER inhibition.
D,E/Spironolactone and triptolide increase sensitivity to alkylating agents in primary MM cells but not in healthy peripheral blood mononuclear circulating cells (PBMC). The figure represents the impact of spironolactone (10 μM) or triptolide (10 nanoM) on melphalan in respectively 7 (3 newly diagnosed and 4 relapse/refractory) and 4 primary MM samples (3 newly diagnosed and 1 relapse/refractory) and in 5 and 4 healthy PBMC. Each dot represents a distinct MM or PBMC sample. Viability was evaluated by Celltiterglo. The figure shows the significant increased sensitivity to melphalan (25 μM) in primary MM cells (D) whereas no significant increase was observed in PBMC. Spironolactone itself impacts the viability of both MM cells and PBMC but does not significantly increase melphalan sensitivity in healthy PBMC. (Sp.=spironolactone, mel= melphalan).
F/ triptolide has significant anti-myeloma activity as a single agent. Triptolide anti-myeloma activity was evaluated in 3 PBMC (blue line), 4 primary myeloma samples (red line) and 3 MM cell lines (green line). Cells were cultured with different doses of triptolide (0 to 250 nanoM) for 24 hours and cell viability was measured with Celltiterglo. The figure shows that myeloma cells are more sensitive than PBMC to triptolide.