Identification and Characterization of TPRKB Dependency in TP53 Deficient Cancers

Abstract

Tumor protein 53 (TP53) is a transcription factor involved in regulating various facets of cellular functionality from its canonical functions in DNA damage response, cell cycle arrest, and apoptosis, to newer roles in metabolism, protein translation, and more. TP53 is also the most frequently altered gene in human cancer, and identification of vulnerabilities imposed by TP53 alterations may enable development of effective therapeutic approaches. Through analyzing shRNA-screening data, we identified TP53RK binding protein (TPRKB) as the most significant vulnerability in TP53-mutated cancer cell lines. To date, TPRKB’s only known role is as a poorly characterized member of the transfer RNA (tRNA)-modifying Endopeptidase-like and Kinase associated to transcribed Chromatin/Kinase, Endopeptidase and Other Proteins of small Size (EKC/KEOPS) complex, responsible for depositing the t6A37 modification on all ANN decoding tRNAs. In vitro and in vivo, across multiple benign-immortalized and cancer cell lines, we confirmed that TPRKB knockdown in TP53-null, TP53-mutated, and Mouse double minute 2 homolog (MDM2; an E3-ubiquitin ligase for TP53)-amplified cells significantly inhibited proliferation, with minimal effect in TP53 wild-type cells. Furthermore, we used isogenic cell lines to demonstrate TP53 reintroduction into TP53-null cells resulted in loss of TPRKB sensitivity, while deletion of TP53 in wild-type cells enhanced sensitivity, confirming specificity for TP53 status. Sensitivity was accompanied by cell cycle arrest and reduced expression of anti-apoptotic proteins – B-cell lymphoma 2 (BCL2) and BCL2 like 1 (BCL2L1). Depletion of other EKC/KEOPS complex members exhibited TP53-independent effects, supporting novel, complex-independent functions of TPRKB. To explore mechanisms surrounding this sensitivity, we characterized several protein-protein interactions in this context. We demonstrate dynamic regulation of TPRKB, whereby TP53 indirectly mediates TPRKB degradation through the proteasome, while TP53 Regulating Kinase (TP53RK or PRPK), an interacting member of the EKC/KEOPS complex, directly stabilizes TPRKB. Furthermore, through co-immunoprecipitation followed by mass spectrometry (IP:MS) analysis we identify and validate that TPRKB interacts with another tRNA-modifying complex, tRNA methyltransferase 6/tRNA methyltransferase 61A (TRMT6/TRMT61A), responsible for the m1A58 tRNA modification. However, knockdown of TRMT6/TRMT61A in our characterized cell lines showed no proliferative differences and m1A levels in TPRKB depleted cells were unaltered, leaving the functional consequence of the interaction unknown. Nonetheless, TPRKB depletion was accompanied by a TP53-dependent reduction in protein translation and general reductions in other tRNA modifications (t6A, ms2t6A, m3C, and m3U), Polymerase RNA III DNA directed polypeptide G 32kD-like (POLR3GL; involved in transcription of tRNA) expression, and altered sensitivity to tRNA and ribosomal RNA (rRNA) polymerase inhibitors. Together, these results identify a unique and specific requirement of TPRKB in a variety of TP53-deficient cancers, and implicate TPRKB in several aspects of protein translation. Future studies aimed at elucidating the mechanism for TPRKB sensitivity are critical to explore the potential for therapeutic targeting in TP53-deficient cancer.