Overcoming p53 Loss with a Multi-Targeted Small Molecule Inhibitor in Multiple Myeloma.
Authors: Cirstea Diana , Hideshima Teru , Santo Loredana , Vallet Sonia , Pozzi Samantha , Patel Kishan , Ikeda Hiroshi , Loferer Hannes , Perrone Giulia , Gorgun Gullu , Hu Yiguo , Munshi Nikhil C. , Anderson Kenneth C. , Raje Noopur 
Published: October 2019

Abstract
Abstract 4915
Multiple studies have highlighted the critical role of mutation and loss of p53 function in multiple myeloma (MM) when acquiring a more aggressive phenotype and refractoriness to treatment. Therefore, agents capable of overcoming p53 mutational status are important in the context of MM therapeutics. We have previously reported the in vitro and in vivo anti-MM activity of the multi-targeted small molecule inhibitor RGB-286638. Using a human MM cell xenograft model in SCID mice we demonstrated that RGB-286638 inhibited tumor growth and prolonged survival. Our data confirmed suppression of CDK1/cyclin B, CDK4, 6/Cyclin D1, D3, and CDK2/Cyclin E complexes in MM.1S MM cells containing wt-p53, which was correlated with rapid downregulation of Rb phosphorylation, resulting in effective G2/M cell cycle blockage and increased sub-G1phase. RGB-286638 induced dose and time-dependent inhibition of RNA pol II phosphorylation as an early event promptly followed by p53 induction. Moreover, RGB-286638 treatment was associated with p53 phosphorylation at ser 15, indicative of DNA damage followed by apoptosis, evidenced by caspases 8, 9 and 3 cleavage and confirmed by Annexin V/PI staining. All together these data suggested that RGB-286638-induced RNA pol II inhibition triggers cytotoxicity in MM cells via p53-dependent apoptosis. Interestingly, RGB-286638 demonstrated cytotoxic activity even in p53-deficient conventional drug-resistant RPMI 8226/Dox 40 MM cells. RGB-286638 treatment of RPMI 8226/Dox40 MM cells showed increased PARP response associated with enhanced NAD depletion followed by increased ATP consumption. Furthermore, concomitant assessment of RGB-286638-induced ATP depletion versus cytotoxicity demonstrated more than 60% ATP loss preceded cell death in RPMI 8226/Dox40 but not in MM.1S. This data suggests the role of either p53-mediated apoptosis (when active) or PARP-induced NAD/ATP depletion and bioenergetic crisis (when absent). Interestingly, the knockdown of p53 did not rescue MM.1S cells from RGB 286638-induced death, suggesting the existence of alternative p53-independent pathways through which RGB-286638 exerts its cytotoxic activity. Ongoing studies are addressing the molecular effects of p53 silencing in MM cells. In addition, dissecting the mechanism of RGB-286638 p53-independent cytotoxicity in MM cells will provide insights for future therapeutic strategies in patients with aggressive MM and associated mutated/deleted-p53.

Disclosures
Loferer: GPC Biotech AG: Employment. Munshi:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Millennium: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis : Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Anderson:Millenium: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding. Raje:Celgene: Research Funding; Novartis: Research Funding; AstraZeneca: Research Funding.