The p53 Reactivating Drug PRIMA-1Met Induces Apoptosis Of Myeloma Cells Via ROS Production

Patients with multiple myeloma (MM) carrying a del(17)(p13) (del17p) deletion and/or TP53 mutation at diagnosis have a shortened survival whatever the treatment received. By using a large and characterized collection of human myeloma cell lines (HMCLs), we have shown that TP53^{mutated/deleted} cell lines are more resistant to melphalan and nutlin3a (MDM2 inhibitor) than TP53^wild-type HMCLs (Surget S, Cancer Res 2012;72:4562). The absence of a functional p53 pathway prevents the induction of p53 target genes, including pro-apoptotic genes, and therefore impairs apoptosis. In cancer cells, p53 mutations are frequent and highly expressed mutant p53 proteins exert a dominant effect. Pharmacological drugs such as PRIMA-1Met/APR246, which has been recently evaluated in a phase I/II study (Lehmann S J Clin Oncol 2012;30:3633) offers an interesting opportunity to redirect the activity of mutant misfolded p53 proteins. With this aim, the efficiency of PRIMA-1Met was assessed in 22 HMCLs and 16 primary MM samples characterized for del17p. HMCLs were heterogeneously sensitive to PRIMA-1Met, with an LD₅₀ median value of 35 µM (range 4->100 µM). We failed to observe any correlation with HMCL’s TP53 status since the LD₅₀ median values of TP53^wt, TP53^mut and TP53^del HMCLs were not significantly different (p=0.1, Kruskal Wallis test) at respectively 21 µM (range 4-70, n=7), 45 µM (21->100, n=13) and 21 µM (8-33, n=2). Primary MM samples were also heterogeneously sensitive, with a median cell death value induced by 10 µM PRIMA-1Met of 51% (n=16, range 5-100%). The 5 del17p+ samples showed no significant difference in their sensitivity to PRIMA-1Met from the 11 del17p- (p=0.31 Mann Whitney). Moreover, the sensitivity of 2 TP53^wt and 2 TP53^mut HMCLs was unchanged upon p53 silencing, and PRIMA-1Met failed to increase the expression of such p53 target genes as p21, Puma or DR5 (TRAIL-R2) in both TP53^wt and TP53^mut/del HMCLs. By contrast, nutlin3a induced cell death and increased DR5 expression in all TP53^wt but in none of the TP53^mut/del HMCLs (p<0.001, n=22) and both cell death and DR5 expression increase was prevented upon p53 silencing. In primary samples, nutlin3a increased DR5 expression in del17p- (n=9, median fold-increase 1.29) but not in del17p+ samples (n=4, median fold-increase 0.90, p=0.05) while PRIMA-1Met increased DR5 expression in neither del17p- nor del17p+ samples (0.90 versus 0.96, p=0.94). Despite a lack of activation of p53 target genes, PRIMA-1Met induced apoptosis as shown by caspase 3 and PARP cleavages. Interestingly, PRIMA-1Met induced a burst of ROS (shown by DHE staining in flow cytometry), involved in cell death since the ROS scavengers NAC (5 mM), N-acetyl-L-cysteine, as well as 5 mM GSH-MEE (gluthatione reduced ethyl ester, data not shown), strongly inhibited cell death in both HMCLs and primary cells with respective median inhibitions of 98% (n=8, p=0.01) and 93% (n=11, p<0.001 Wilcoxon matched-pairs signed rank test). Reciprocally, 0.5 mM BSO (L-buthionine-sulfoximine), an irreversible inhibitor of gamma-glutamyl cysteine synthetase, which depletes GSH in situ and in turn increases ROS, strongly synergized with 10 µM PRIMA-1Met and overcame resistance to apoptosis in 91% of HMCLs (n=22) and in 100% of primary samples (n=6). The median value of HMCL cell death was respectively 92% for 10µM PRIMA-1Met plus 0.5 mM BSO versus 2% for 10 µM PRIMA-1Met (n=22, p<0.001) and 4% for 0.5 mM BSO (n=22, p<0.001), and that of primary samples was 88% for PRIMA-1Met /BSO versus 22% for PRIMA-1Met (n=6, p=0.03) or 1% for BSO (n=6, p=0.03, Wilcoxon matched-pairs signed rank test). Notably, cell death induced by nutlin3a was not inhibited by NAC nor did BSO synergize with nutlin3a, confirming that nutlin3a and PRIMA-1Met have distinct death inducing pathways. Interestingly, the sensitivity of HMCLs to PRIMA-1Met alone correlated to the expression of an enzyme involved in oxidative stress (p=0.04, Spearman test), the participation of which is currently under investigation. Finally, the 10 µM PRIMA-1Met plus 0.5 mM BSO combination was found to be effective in only 2 of 6 mantle cell lymphoma cell lines, suggesting that it could be cell-type specific. These data show that PRIMA-1Met in combination with BSO has a strong pro-apoptotic activity in MM, independently of the p53 pathway, and thus could be of interest for del17p+ patients refractory to current therapies.