The catalytic subunit of DNA-PK regulates transcription and splicing of AR in advanced prostate cancer
Beth Adamson, … , Johann S. de Bono, Luke Gaughan
Beth Adamson, … , Johann S. de Bono, Luke Gaughan
Published September 26, 2023
Citation Information: J Clin Invest. 2023;133(22):e169200. https://doi.org/10.1172/JCI169200.
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Research Article Endocrinology Oncology

The catalytic subunit of DNA-PK regulates transcription and splicing of AR in advanced prostate cancer

Abstract

Aberrant androgen receptor (AR) signaling drives prostate cancer (PC), and it is a key therapeutic target. Although initially effective, the generation of alternatively spliced AR variants (AR-Vs) compromises efficacy of treatments. In contrast to full-length AR (AR-FL), AR-Vs constitutively activate androgenic signaling and are refractory to the current repertoire of AR-targeting therapies, which together drive disease progression. There is an unmet clinical need, therefore, to develop more durable PC therapies that can attenuate AR-V function. Exploiting the requirement of coregulatory proteins for AR-V function has the capacity to furnish tractable routes for attenuating persistent oncogenic AR signaling in advanced PC. DNA-PKcs regulates AR-FL transcriptional activity and is upregulated in both early and advanced PC. We hypothesized that DNA-PKcs is critical for AR-V function. Using a proximity biotinylation approach, we demonstrated that the DNA-PK holoenzyme is part of the AR-V7 interactome and is a key regulator of AR-V–mediated transcription and cell growth in models of advanced PC. Crucially, we provide evidence that DNA-PKcs controls global splicing and, via RBMX, regulates the maturation of AR-V and AR-FL transcripts. Ultimately, our data indicate that targeting DNA-PKcs attenuates AR-V signaling and provide evidence that DNA-PKcs blockade is an effective therapeutic option in advanced AR-V–positive patients with PC.

Authors

Beth Adamson, Nicholas Brittain, Laura Walker, Ruaridh Duncan, Sara Luzzi, Pasquale Rescigno, Graham Smith, Suzanne McGill, Richard J.S. Burchmore, Elaine Willmore, Ian Hickson, Craig N. Robson, Denisa Bogdan, Juan M. Jimenez-Vacas, Alec Paschalis, Jonathan Welti, Wei Yuan, Stuart R. McCracken, Rakesh Heer, Adam Sharp, Johann S. de Bono, Luke Gaughan

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Figure 2

DNA-PKcs inhibition represses growth of AR-V–expressing PC cell lines.

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DNA-PKcs inhibition represses growth of AR-V–expressing PC cell lines. (...
(A) The TCGA data set was analyzed to compare PRKDC expression in matched normal and tumor samples (n = 51) and in localized (n = 49) and metastatic (n = 27) PC from a publicly available microarray data set (Grasso et al., ref. 10). **P < 0.01. (B) CWR22Rv1-AR-EK cells grown in serum-containing media and CWR22Rv1 and VCaP cells grown in steroid-depleted media supplemented with 10 M enzalutamide (Enz) were treated with increasing concentrations of NU7441 for 96 hours prior to cell count. Data were normalized to the untreated (NT) control arm (–Enz/–DHT group) and are representative of 3 independent repeats ± SEM. One-way ANOVA using Bonferroni’s post hoc analysis was used to determine the statistical significance for CWR22Rv1-AR-EK and 2-way ANOVA was used for CWR22Rv1, LNCaP, and VCaP cells. *P < 0.05, **P < 0.01, ***P < 0.001. (C) Representative structures of DNA-PKcs inhibitors NU5455 and AZD7648 are shown adjacent to cell count data from CWR22Rv1-AR-EK cells treated with 1 mM NU7441, NU5455, and AZD7648 for 24 hours. Data represent an average of 3 repeats ± SEM (*P < 0.05, **P < 0.01). (D) CWR22Rv1-AR-EK cells were treated with increasing concentrations of AZD7648, NU7441, and NU5455 for 120 hours before harvesting for an SRB proliferation assay. Data are shown as the mean ± SEM across 3 independent repeats that included 3 technical replicates for each experimental arm.