Nuclear Receptor NR2E3 Activates Both Wild-Type and Mutated p53 in Multiple Myeloma Cells

Background: Tumor cell plasticity and genome instability contribute to tumor heterogeneity, leading to drug resistance and relapse in cancer patients. Despite great progress in improving 5-year survival rate in multiple myeloma (MM), relapsed and refractory MM (RRMM) remains a major challenge. Given that p53 mutations account for 8% of MM cases, we constructed 50 nonsynonymous mutations of p53 detected in MM patients which include 37 mutations in MMRF database and 13 from published studies. We aim to investigate their functions and interactions with a new tumor suppressor NR2E3, nuclear receptor subfamily 2 group E member 3. We previously demonstrated that NR2E3 stimulates p53 acetylation and its small-molecule agonist 11a represses cancer cells bearing p53 signaling in NCI-60 cancer cell panel.

Results: Our study of TCGA and “All of US” databases uncovered the association of NR2E3 nonsynonymous mutations with 4 cancers including MM. We constructed all NR2E3 mutations detected in MM patients, and NR2E3^R334W lost the capability of activating p53 transactivity. In contrast, high expression of NR2E3 correlated to superior disease-free and overall survivals of 33 cancers. Co-expressing NR2E3 with p53 mutation partially rescued a subgroup, including 4 hotspot mutations, of 50 p53 mutations in p53-responsive reporter assay. For example, p53^R175H was activated by NR2E3 through stimulating the acetylation of p53^R175H at K317/K386 and/or K373/K382. Activated p53^R175H increased the expressions of its downstream tumor-suppressing genes, such as p21, Puma, DDIT3 and ATF3.

NR2E3's agonist 11a enhanced p53 acetylation and transactivation of downstream tumor-suppressing genes. Disrupting NR2E3 by RNAi or overexpression modulated 11a-stimulated p53 acetylation and transactivities. Interestingly, 11a synergized with FDA-approved HDAC inhibitor Romidepsin in killing MM1S myeloma cells that express p53^WT and with a new small-molecule FOXM1 inhibitor NB73 in killing OPM2 myeloma cells that carry homozygous alleles of p53^R175H.

FOXM1 is a master transcription factor of metabolism and cell cycle progression in Forkhead box family, and a critical molecular vulnerability in RRMM. Our recent study demonstrated that FOXM1 inhibitor NB73 synergizes with BCL2 inhibitor Venetoclax in killing non-t(11;14) myeloma cell lines via transcriptional inhibition of MYC pathway. The ChIP-seq study also resolved the DNA binding sites of FOXM1 on the promoters of both p53 and NR2E3 in myeloma cell lines. Degrading FOXM1 protein by NB73 decreased the binding of FOXM1 to the promoters of NR2E3 and p53, enhanced their transcriptions and protein levels in myeloma cell lines. For instance, these NB73-treated OPM2 cells expressed more NR2E3^WT and p53^R175H. The addition of 11a stimulated NR2E3^WT which further rescued p53^R175H, leading to intensive cell apoptosis.

Ongoing experiments: Rescuing p53 mutations by NR2E3 and its agonist 11a is a very interesting observation. U266 cells bearing t(11;14) translocation are resistant to Venetoclax. We found that U266 cells express NR2E3^WT, p53^A161T and substantial amount of FOXM1. NR2E3 activated p53^A161T, and 11a synergized with NB73 but not Venetoclax in killing U266 cells, which may provide a new therapy to t(11;14)-positive myeloma. Moreover, we are conducting the pharmacokinetics study of 11a in 3 mice using oral gavage route, with which we will be able to perform the pharmacodynamics studies of 11a alone and in combination with NB73, Romidepsin or other FDA-approved anti-cancer drugs in NSG mice engrafted with myeloma cell lines bearing various molecular signatures, including p53 mutations. Detailed mechanisms are studied with multifaceted techniques such as ChIP-Seq and molecular/cellular tools.

Significance & conclusion: Nuclear receptors are the main targets of drug development. NR2E3 used to be a retinal photoreceptor-specific transcription factor, but the expression of NR2E3 has been detected in various tissues in human and mouse with new antibodies, which has led to interesting findings in multiple cancers over the past few years. We showed for the first time that NR2E3 is transcriptionally repressed by FOXM1 and pharmaceutically stimulated to activate p53 by its agonist in myeloma cells. Our studies will provide a new drug candidate for MM that is notoriously difficult to cure and burdened with large societal health care expenditures and high mortality rates.

Katzenellenbogen:Celcuity: Membership on an entity's Board of Directors or advisory committees. Katzenellenbogen:Celcuity: Membership on an entity's Board of Directors or advisory committees.