Unveiling the role of SPOP in DNA damage response and double strand break repair: From mechanistic insights to clinical application

Abstract

PCa is the most common male cancer in Germany and the second leading cause of male cancer deaths. Genomic instability and DNA repair defects are considered hallmarks of PCa. PCa is mainly characterized by the presence of structural genomic rearrangements with the ERG fusion being the most common, in around 50% of the patients. However, unlike other cancers, point mutations are less common in PCa. Importantly, SPOP point mutations have been revealed in around 10-15 % of PCa, being the most frequent point mutated tumor suppressor gene in PCa. SPOP mutations have been associated with the highest frequency of genomic instabilities and worse prognosis in PCa. However, the mechanism is yet not fully characterized. In this study, we aimed to unveil the mechanism underlying increased genomic instabilities associated with SPOP loss of function. To that end, we established SPOP-KO sublines from LNCaP (AR+) and DU145 (AR-) cells. The results of the current study can be summarized as follows: I. In the AR-negative DU145 cells: 1. SPOP loss results in upregulated global DNA and histone methylation (measured by illumina HumanMethylation array and MS-based proteomics, respectively). 2. This leads to increased chromatin compaction and reduced chromatin accessibility (measured by ATAC-seq). 3. The reduced chromatin accessibility in DU145 SPOP-KO cells results in transcription stress and decreased transcription activity (measured by EU incorporation), due to decreased transcription dynamics, as revealed by profound reduction in RNA pol II phosphorylation at ser2p (a marker of active elongation). 4. Stalled transcription leads to accumulation of the RNA-DNA hybrid transcription intermediates R-loops (measured by IF). 5. The accumulation of unscheduled R-loops and slowed transcription activity result in replication stress (RS) upon colliding with the replication machinery (monitored by DNA fiber assay), leading to DSBs in S-phase (investigated by IF detection of H2AX in EdU+ cells) and chromosomal aberrations. 6. Furthermore, DSBs are generated in G1 and G2 phases upon accumulation of unscheduled R-loops (measured by IF detection of γH2AX in EdU-/CenpF- and EdU-/CenpF+ cells, respectively).

II. In AR-positive (LNCaP) cells: 1. As, a substrate of SPOP, AR transcription signaling is enhanced, as illustrated by increased expression of AR and its downstream target PSA. 2. Given that AR is a transcription factor, its signal stimulation leads to enhanced transcription activity (as measured by increased EU incorporation), which masked the effect of SPOP loss on transcription. 3. Furthermore, no increase in global histone or DNA methylation was observed in these cells. 4. The unscheduled increase in transcription activity leads to accumulation of R-loops, RS and DSBs similar to SPOP-KO DU145 cells.

In conclusion, the current study unveils the role of SPOP in maintaining genomic stability through regulating chromatin accessibility and transcription dynamics to prevent transcription stress, accumulation of R-loops, RS and DSBs.