TP53 Is Not Involved in the Apoptosis Induced by RITA

Abstract 1848

Patients with a p53 deletion or mutation are resistant to the current therapeutic arsenal. There is an obvious need to find drugs able to kill p53 abnormal myeloma cells. RITA (reactivating tumor apoptosis) is a good candidate because it kills tumor cells expressing a wild type or a mutated p53. RITA binds to p53 and allows mutated p53 to recover a functional transcriptional conformation, activating thus target genes of the p53 pathway, including pro-apoptotic molecules such as Noxa and Puma. We have assessed RITA efficiency on 31 myeloma cell lines (HMCLs), 9 expressing a wt p53 and 22 expressing no p53 or an abnormal p53 (punctual mutation, exon deletion, intron insertion). The efficiency of RITA was compared with that of Nutlin3a, a MDM2 inhibitor activating the p53 pathway through a non-genotoxic stress. Nutlin3a kills all p53^Wt HMCLs with a lethal dose 50 between 3,000 and 10,000nM (median value 8,000 nM) but none p53^Abn HMCLs (p<0.001). Cell cycle analysis showed a G1 arrest and a subG1 peak. In good agreement, Nutlin3a induced p21 and Noxa accumulation. To confirm a p53 involvement, silenced p53 HMCLs were obtained by lentivirus strategy. As expected, (incomplete) silencing of p53 in two p53^Wt HMCLs, NCI-H929 and XG6, prevented Nutlin3a killing (p<0.01). In contrast to Nutlin3a, RITA killed both p53^Wt (median value 3,000; range 40->20,000) and p53^Abn (median value 5500; range 10->20,000) HMCLs without significant difference (p=0.24). CCND1+ cell lines, t(11;14)+, were more sensitive to RITA (median value 600; range 10->20,000, n=10) than t(11;14)- cell lines, (median value 6,500; range 40->20,000, n=21), (p=0.0235). Among the 9 p53^Wt HMCLs, only 2 (22%) had a LD₅₀ inferior to 1,000 nM (40nM and 90nM), all the others having a LD₅₀ value superior to 2,000 nM (median value 3,000 nM; range 2,000->20,000 nM). Among the 22 p53^Abn HMCLs, 5 (23%) had a LD₅₀ inferior to 1,000 nM (median value 100nM; range 10–400nM), all the others having a LD₅₀ value superior to 1,000 nM (median value 7,000 nM; range 1,000->20,000 nM). These results showed that the killing induced by RITA was not significantly related to p53 status. Indeed, silencing of p53 in two RITA sensitive p53^Abn HMCLs did not prevent RITA killing, confirming the lack of p53 involvement. Moreover, in contrast to Nutlin3a, RITA did not commonly induce an increase of expression of the p53 targets (p21, Noxa and Puma) in the sensitive HMCLs. Killing by RITA was associated to the activation of caspases, the appearance of a subG1 peak and a cell cycle arrest in G2M phase, suggesting a mitotic catastrophe. Ongoing works aim at demonstrating the involvement of mitotic catastrophe in both p53^Wt and p53^Abn HMCLs and at identifying the molecular target(s) of RITA, especially in t(11;14)+ cell lines.

While Nutlin3a appears efficient only for cells expressing a normal p53, RITA is efficient for 23% of myeloma cell lines irrespectively of p53 status. Because patients with del17p or expressing an abnormal p53 are resistant to current therapies, RITA could be of interest for them.