An Examination of Transcriptional Effects and Stress Responses That Differentiate the Mechanisms of Pt-Based Therapeutics

Abstract

Platinum (Pt) compounds have been a powerful frontline therapy in cancer treatment for nearly 50 years. Despite longstanding interest in determining their precise mechanism of action, there remain a number of key molecular questions relating to the biological activity of these structurally simple molecules. Historically considered to be canonical DNA damaging agents, emerging evidence indicates that Pt complexes can also exert their cytotoxic effects through the inhibition of RNA transcription or other non-DNA cellular targets. The growing mechanistic diversity in this class of chemotherapeutics has important implications for their clinical application, and can help elucidate fundamental biological pathways related to cell survival and proliferation. Here, we describe biochemical approaches to better characterize the mechanism of action for different Pt complexes and characterize a novel oxaliplatin-induced stress response pathway that links DNA damage signaling and ribosome biogenesis. First, we develop a comparative profile of the cellular RNA binding, sequence specificity, and cellular localization of three clinical Pt analogs, functionalized for use with bioorthogonal ‘click’ chemistry. We highlight the utility of this approach for enabling facile enrichment of Pt-bound targets and improving data quality for RNA/DNA sequencing applications. We then illuminate a novel mechanism of action for the widely used Pt drug oxaliplatin, and conclusively establish that it is a potent disruptor of rRNA transcription and nucleolar function at sub-lethal doses, in contrast to its structural relative cisplatin which does neither of these in a therapeutic regime. We further demonstrate that oxaliplatin-induced rRNA silencing and nucleolar stress are mediated through the central DNA damage response kinases ATM/ATR, but surprisingly in the absence of appreciable nucleolar DNA damage. Instead, we find that the DDR signaling to the nucleolus after oxaliplatin treatment resembles a reported in-trans nucleolar DNA damage response pathway. Moreover, we compare oxaliplatin against other small molecule inhibitors of rRNA synthesis and find that oxaliplatin is unique in its reliance upon DNA damage signaling. Taken together, our research presents biochemical tools to investigate the mechanistic diversity of chemotherapeutic agents, and illuminates one such novel mechanism by which small-molecule induced nuclear DNA damage can cause nucleolar stress.