PRIMA-1 (p53 Reactivation and Induction of Massive Apoptosis) Mediates Anti-Myeloma Effect through the ER Stress Pathway
Authors: Teoh Phaik Ju , Bi Chonglei , Sebastian Sinto , Fonseca Rafael , Chng Wee Joo 
Published: October 2019

Abstract
It is well established that 17p13(del) and p53 mutations represent poor prognosis in MM patients. We have previously shown that p53 is a haploinsufficient tumor suppressor in myeloma and p53 expression level is correlated with functional status of the pathway. In addition, p53 expression levels do not always correlate with p53 genotype. The absence of p53 expression in myeloma cells severely abolishes the integrity of its downstream signaling response, thus, driving disease aggressiveness and drug resistance. There is a pressing need for a more effective treatment strategy for high-risk patients harbouring p53 abnormalities. PRIMA-1 is a first-in-class drug that was identified to specifically kill cancer cells harboring mutant p53. It has shown substantial efficacy in various solid tumors with mutant as well as non-mutant p53. However, the discovery of the anti-myeloma activity of PRIMA-1 is still at its infancy. By utilizing human myeloma cell lines (HMCLs) of different p53 status as our study model, we aim to identify the efficacy of PRIMA-1 in MM and to dissect the mechanisms behind PRIMA-1-induced toxicity. Despite its known ability to reactivate mutant-p53 function, we did not observe any restoration of mutant p53 transcriptional activities. The expression levels of the p53-direct-targets, p21, MDM2 and PUMA, were unchanged upon treatment in the p53-mutant-HMCLs. In fact, PRIMA-1 was found to be able to decrease the cell viability of all the HMCLs, irrespective of their p53 status (Mutant, WT, Null), strongly indicating a p53-independent response. Importantly, PRIMA-1 showed the highest cytotoxicity in a p53-deficient background (IC50 of 16uM in -/-, 55uM in WT/WT and 70uM in mutant HMCLs). This finding was validated when p53 silencing in a WT HMCL significantly enhanced its sensitivity to PRIMA-1. GEP analysis on the two most sensitive cell lines (JJN3 and KMS11) demonstrated significant enrichment of genes related to unfolded (p=1.90E-06 in JJN3 and 6.12E-08 in KMS11) and misfolded protein (p=2.99E-06 in JJN3 and 8.21E-08 in KMS11) responses. Subsequent validation by Western blot and qPCR confirmed the activation of ER stress pathway in response to PRIMA-1, evidenced by the up-regulation of ER stress markers such as GRP78, GADD34 and CHOP. Further investigations then revealed that the PERK/CHOP axis was most important as CHOP knockdown saw an attenuation of MM cells sensitivity to PRIMA-1-induced apoptosis. Recently, it was reported that cells with increased ER stress are more sensitive to bortezomib. Indeed, we showed that PRIMA-1 synergizes with bortezomib, and was able to rescue bortezomib resistance when both compounds were co-incubated in a bortezomib-resistant-HMCL. In conclusion, we identified a novel pathway by which PRIMA-1 exerts its toxicity in MM through induction of ER stress. This may be a novel therapeutic strategy to induce ER stress to sensitize cells to proteasome inhibitor. Owing to the versatility of the drug in targeting myeloma cells with or without p53 abnormalities; PRIMA-1 can potentially be used alone or in combination with other drugs such as bortezomib in a broad range of MM including those with high-risk disease.

Disclosures
No relevant conflicts of interest to declare.