222 Background: Androgen indifferent prostate cancer (AIPC) is increasingly common and particularly lethal. Data describing these tumors are sparse and AIPC remains a poorly understood malignancy. This study aims to characterize the clinical and genomic features of AIPC. Our work ultimately seeks to identify biomarkers with diagnostic and therapeutic potential. Methods: Utilizing the Oncology Research Information Exchange Network (ORIEN) database, we queried all prostate cancer (PC) patients, identified metastatic castrate resistant prostate cancer (MCRPC) samples, and aimed to enrich for tumors with features of AIPC using previously described characteristics. Our AIPC cohort included three subgroups: aggressive variant prostate cancer (AVPC) defined as having alterations in at least two of TP53, RB1, PTEN; neuroendocrine PC (NEPC) defined as small cell histology or NEPC signature score ≥ 0.25 (1); and double-negative PC (DNPC), defined as non-NEPC patients with low AR expression/AR signaling score. We compared clinical characteristics and genomic analysis of AIPC vs non-AIPC samples in patients who developed MCRPC. Clinical analysis was done using Wilcoxon rank sum test or Fisher's exact test. Gene expression analysis was performed using DESeq2 and GSEA. Results: Of 1,496 total PC patients available for analysis, we identified 323 (22%) as MCRPC. Of those, 39 (12%) met AIPC criteria (17 AVPC, 13 NEPC, 9 DNPC) and 284 (88%) were non-AIPC. Median age at diagnosis for AIPC was 62 years and 85% were white, compared to 62 years and 87% for non-AIPC. Fifty-seven percent of AIPC patients had ECOG ≥1 at diagnosis vs 16% of non-AIPC. Forty-three percent of AIPC patients had de novo metastatic disease vs 15% for non-AIPC (p=0.003). TMPRSS2-ERG gene fusions were found in a significantly higher proportion of AIPC samples vs non-AIPC (38.5% vs 16%, p=0.014). Homologous recombination deficiency (HRD) and tumor mutational burden (TMB) did not differ between cohorts, but microsatellite instability scores (MSI) were significantly higher in AIPC (p=0.019). Using Gene Set Enrichment Analysis (GSEA), we found that genes defining response to androgens and genes involved in oxidative phosphorylation were the most downregulated, whereas genes involved in epithelial mesenchymal transition (EMT), interferon response, and angiogenesis were significantly upregulated in AIPC vs non-AIPC samples. Conclusions: There was a significantly higher rate of de novo metastasis in the AIPC cohort. The downregulated androgen response and upregulated EMT pathways in AIPC suggest enrichment for androgen indifference with our methodology. Upregulated immune signaling and angiogenesis as well as higher MSI suggest opportunities for therapeutic investigation. Future directions include more focused in vitro and in vivo analysis to identify actionable targets. 1. Beltran H, et al. Nat Med . 2016;22(3):298-305. doi:10.1038/nm.4045.

The androgen receptor (AR) transduces the effects of circulating and tumor-derived androgens to the nucleus through ligand-induced changes in protein conformation, localization, and chromatin engagement. Defining how these events are integrated with signal transduction is critical to understand how AR drives prostate cancer and unveil pathway features that are potentially amenable to therapeutic intervention. We describe a novel post-transcriptional mechanism that controls AR levels on chromatin and gene output based on highly selective, inducible degradation. We find that the mono-ADP-ribosyltransferase PARP7 generates an ADP-ribosyl degron in the DNA-binding domain of AR, which is recognized by the ADP-ribose reader domain in the ubiquitin E3 ligase DTX2 and degraded by the proteasome. Mathematical modeling of the pathway suggested that PARP7 ADP-ribosylates chromatin-bound AR, a prediction that was validated in cells using an AR DNA-binding mutant. Non-conventional ubiquitin conjugation to ADP-ribosyl-cysteine and degradation by the proteasome forms the basis of a negative feedback loop that regulates modules of AR target genes. Our data expand the repertoire of mono-ADP-ribosyltransferases to include gene regulation via highly selective protein degradation.

Androgen-indifferent prostate cancer (AIPC) is increasingly common and particularly lethal. Data describing these tumors are sparse, and AIPC remains a poorly understood malignancy. Utilizing the Oncology Research Information Exchange Network (ORIEN) database, we enriched for tumors with features of AIPC using previously described characteristics. Our AIPC cohort included three subgroups: aggressive variant prostate cancer (AVPC), neuroendocrine PC (NEPC), and double-negative PC (DNPC). Of 1496 total PC patients available for analysis, we identified 323 (22%) as MCRPC. Of those, 39 (12%) met AIPC criteria (17 AVPC, 13 NEPC, 9 DNPC) and 284 (88%) were non-AIPC. Forty-three percent of AIPC patients had de novo metastatic disease vs. 15% for non-AIPC (p = 0.003). Homologous recombination deficiency (HRD) and tumor mutational burden (TMB) did not differ between cohorts, but microsatellite instability scores (MSI) were significantly higher in AIPC (p = 0.019). Using Gene Set Enrichment Analysis (GSEA), we found that genes defining response to androgens and genes involved in oxidative phosphorylation were the most downregulated, whereas genes involved in epithelial–mesenchymal transition (EMT) and immune signaling were significantly upregulated in AIPC vs. non-AIPC. Our study demonstrates the potential for predefined criteria that aim to enrich for AIPC and suggests opportunities for therapeutic investigation.

Deltex proteins are a family of E3 ubiquitin ligases that encode C-terminal RING and DTC domains that mediate interactions with E2 ubiquitin-conjugating enzymes and recognise ubiquitination substrates. DTX3L is unique among the Deltex proteins based on its N-terminal domain architecture. The N-terminal D1 and D2 domains of DTX3L mediate homo-oligomerisation, and the D3 domain interacts with PARP9, a protein that contains tandem macrodomains with ADP-ribose reader function. While DTX3L and PARP9 are known to heterodimerize, they assemble into a high molecular weight oligomeric complex, but the nature of the oligomeric structure, including whether this contributes to the ADP-ribose reader function is unknown. Here, we report a crystal structure of the DTX3L N-terminal D2 domain and show that it forms a tetramer with, conveniently, D2 symmetry. We identified two interfaces in the structure: a major, conserved interface with a surface of 973 Å2 and a smaller one of 415 Å2. Using native mass spectrometry, we observed molecular species that correspond to monomers, dimers and tetramers of the D2 domain. Reconstitution of DTX3L knockout cells with a D1-D2 deletion mutant showed the domain is dispensable for DTX3L-PARP9 heterodimer formation, but necessary to assemble an oligomeric complex with efficient reader function for ADP-ribosylated androgen receptor. Our results suggest that homo-oligomerisation of DTX3L is important for mono-ADP-ribosylation reading by the DTX3L-PARP9 complex and to a ligand-regulated transcription factor.

BBQ and FICZ treatment activates AHR signaling in prostate cancer cells

Androgen signaling in prostate cancer cells involves multisite cysteine ADP-ribosylation of the androgen receptor (AR) by PARP7. The AR modification is read by ADP-ribosyl binding macrodomains in PARP9, but the reason that multiple cysteines are modified is unknown. Here, we use synthetic peptides to show that dual ADP-ribosylation of closely spaced cysteines mediates recognition by the DTX3L/PARP9 complex. Mono and dual ADP-ribosylated cysteine peptides were prepared using a novel solid-phase synthetic strategy utilizing a key, Boc-protected, ribofuranosylcysteine building block. This synthetic strategy allowed us to synthesize fluorescently labeled peptides containing a dual ADP-ribosylation motif. It was found that the DTX3L/PARP9 complex recognizes the dual ADP-ribosylated AR peptide (Kd = 80.5 nM) with significantly higher affinity than peptides with a single ADP-ribose. Moreover, oligomerization of the DTX3L/PARP9 complex proved crucial for ADP-ribosyl-peptide interaction since a deletion mutant of the complex that prevents its oligomer formation dramatically reduced peptide binding. Our data show that features of the substrate modification and the reader contribute to the efficiency of the interaction and imply that multivalent interactions are important for AR-DTX3L/PARP9 assembly.

RBN2397 inhibition of prostate cancer cell growth requires PARP7 over-expression

The ADP-ribosyltransferase PARP7 modulates protein function by conjugating ADP-ribose to the side chains of acceptor amino acids. PARP7 has been shown to affect gene expression in prostate cancer cells and certain other cell types by mechanisms that include transcription factor ADP-ribosylation. Here, we use a recently developed catalytic inhibitor to PARP7, RBN2397, to study the effects of PARP7 inhibition in androgen receptor (AR)-positive and AR-negative prostate cancer cells. We find that RBN2397 has nanomolar potency for inhibiting androgen-induced ADP-ribosylation of the AR. RBN2397 inhibits the growth of prostate cancer cells in culture when cells are treated with ligands that activate the AR, or the aryl hydrocarbon receptor, and induce PARP7 expression. We show that the growth-inhibitory effects of RBN2397 are distinct from its enhancement of IFN signaling recently shown to promote tumor immunogenicity. RBN2397 treatment also induces trapping of PARP7 in a detergent-resistant fraction within the nucleus, which is reminiscent of how inhibitors such as talazoparib affect PARP1 compartmentalization. Because PARP7 is expressed in AR-negative metastatic tumors and RBN2397 can affect cancer cells through multiple mechanisms, PARP7 may be an actionable target in advanced prostate cancer.Significance:RBN2397 is a potent and selective inhibitor of PARP7 that reduces the growth of prostate cancer cells, including a model for treatment-emergent neuroendocrine prostate cancer. RBN2397 induces PARP7 trapping on chromatin, suggesting its mechanism of action might be similar to clinically used PARP1 inhibitors.

FACS analysis of NCI-H660 cells for detection of necrosis and apoptosis

The growth inhibitory effect of RBN2397 in prostate cells is not dependent on TBK1 and JAK1/2 kinase activity

Supplementary Table 1: RBN2397 growth effects in prostate cencer cell lines

The ADP-ribosyltransferase PARP7 modulates protein function by conjugating ADP-ribose to the side chains of acceptor amino acids. PARP7 has been shown to affect gene expression in prostate cancer cells and certain other cell types by mechanisms that include transcription factor ADP-ribosylation. Here, we use a recently developed catalytic inhibitor to PARP7, RBN2397 (NCT04053673), to study the effects of PARP7 inhibition in androgen receptor-positive (AR+) and androgen receptor-negative (AR-) prostate cancer cells. Ribon Therapeutics developed RBN2397 as a first-in-class mono-ADP-ribosyltransferase inhibitor, and showed that it blocks PARP7 negative regulation of TBK1 [1]. We find that RBN2397 has nanomolar potency for inhibiting androgen-induced ADP-ribosylation of the androgen receptor. RBN2397 inhibits the growth of prostate cancer cells in culture when cells are treated with ligands that activate the androgen receptor (PC3-AR, VCaP, CWR22Rv1), or the aryl hydrocarbon receptor (PC3, DU145, NCI-H660), and induce PARP7 expression. We show that the growth inhibitory effects of RBN2397 are distinct from its enhancement of interferon signaling recently shown to promote tumor immunogenicity in lung cancer models [1]. Chemical inhibitors to PARP1 exert effects on cells by blocking enzyme function, but also via cytotoxic effects attributed to stabilizing PARP1-chromatin interactions in a process termed trapping [2]. Drug-induced trapping of PARP1 can be detected biochemically by immunoblotting the detergent-resistant chromatin fraction. We found that RBN2397 treatment of AR+ and AR- prostate cancer cells induces biochemical trapping of PARP7 within the nucleus, which was also detected by confocal microscopy. Potential therapeutic benefits of RBN2397 are likely to depend on the level of PARP7 expression, given its induction is necessary for growth inhibitory effects of RBN2397 in cell culture. As a first step towards evaluating whether PARP7 levels in human prostate cancer may be actionable with RBN2397, we used computational methods to analyze PARP7 gene expression data from primary prostate tumors and metastatic AR+ and AR- prostate tumors. To assess PARP7 mRNA levels, we used data from the online resource recount3, which uniformly reprocesses publicly available RNA-seq datasets using a Monorail analysis pipeline. Using the level of PARP7 expression in VCaP cells that confers sensitivity to RBN2397 as a threshold, 50% of primary tumors, 41% of metastatic AR- and 11% of AR+ tumors are predicted to have PARP7 expression levels that are sufficient for a response to RBN2397. Because RBN2397 can inhibit the growth of castration-resistant and neuroendocrine prostate cancer cells, PARP7 may be an actionable target in advanced prostate cancer. 1. Gozgit, J.M., et al., PARP7 negatively regulates the Type I interferon response in cancer cells and its inhibition triggers antitumor immunity. Cancer Cell, 2021. 39(9): p. 1214-1226 e10 2. Murai, J., et al., Trapping of PARP1 and PARP2 by Clinical PARP Inhibitors. Cancer Res, 2012. 72(21): p. 5588-99. Citation Format: Chunsong Yang, Krzysztof Wierbilowicz, Natalia M. Dworak, Song Yi Bae, Sachi B. Tengse, Nicki Abianeh, Justin M. Drake, Tarek Abbas, Aakrosh Ratan, David Wotton, Bryce M. Paschal. Induction of PARP7 creates a vulnerability for growth inhibition by RBN2397 in prostate cancer cells [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B072.

Cell cycle distributions of cell lines treated with RBN2397 plus androgen or plus AHR agonist

Extendend analysis of PARP7 expression in prostate cancer

Ubiquitination and ADP-ribosylation are post-translational modifications that play major roles in pathways including the DNA damage response and viral infection. The enzymes responsible for these modifications are therefore potential targets for therapeutic intervention. DTX3L is an E3 Ubiquitin ligase that forms a heterodimer with PARP9. In addition to its ubiquitin ligase activity, DTX3L-PARP9 also acts as an ADP-ribosyl transferase for Gly76 on the C-terminus of ubiquitin. NAD+-dependent ADP-ribosylation of ubiquitin by DTX3L-PARP9 prevents ubiquitin from conjugating to protein substrates. To gain insight into how DTX3L-PARP9 generates these post-translational modifications, we produced recombinant forms of DTX3L and PARP9 and studied their physical interactions. We show the DTX3L D3 domain (230-510) mediates the interaction with PARP9 with nanomolar affinity and an apparent 1 : 1 stoichiometry. We also show that DTX3L and PARP9 assemble into a higher molecular weight oligomer, and that this is mediated by the DTX3L N-terminal region (1-200). Lastly, we show that ADP-ribosylation of ubiquitin at Gly76 is reversible in vitro by several Macrodomain-type hydrolases. Our study provides a framework to understand how DTX3L-PARP9 mediates ADP-ribosylation and ubiquitination through both intra- and inter-subunit interactions.

The nuclear lamina is a network of proteins within the nucleus that provides structural support to the nuclear membrane, and helps organize the arrangement of interphase chromatin. There is a wealth of information on the proteins that comprise the nuclear lamina, but our understanding of how these proteins physically contact and regulate the membrane and interphase chromatin is incomplete. What is clear is that there are subtle differences in the nuclear lamina in different cell types and tissues, and that mutations in genes that encode nuclear lamina proteins can lead to human disease.

Ubiquitination and ADP-ribosylation are post-translational modifications that play major roles in pathways like DNA damage response and infection, making them attractive targets for therapeutic intervention. DTX3L, an E3 ubiquitin ligase, forms a heterodimer with PARP9. The complex has ubiquitin ligase activity and also ADP-ribosylates the C-terminus of ubiquitin on Gly 76 . NAD + -dependent ADP-ribosylation of ubiquitin by DTX3L-PARP9 prevents ubiquitin from conjugating to protein substrates. By using individually produced proteins, we have studied the interaction between DTX3L and PARP9. We identify that the D3 domain (230 – 510) of DTX3L mediates interaction with PARP9 with nanomolar affinity and an apparent 1:1 stoichiometry. Our results also suggest the formation of a higher molecular weight oligomer mediated by the N-terminus of DTX3L (1-200). Furthermore, we show that ADP-ribosylation of ubiquitin at Gly 76 is a reversible modification that can be removed by several macrodomain-type hydrolases. Our study provides a framework to understand how DTX3L-PARP9 mediates ADP-ribosylation and ubiquitination in an inter-regulatory manner.

Poly-ADP-ribose polymerases (PARPs) are enzymes that catalyze ADP-ribosylation and play critical roles in normal and disease settings. The PARP family member, PARP7, is a mono-ADP-ribosyltransferase that has been suggested to play a tumor suppressive role in breast, ovarian, and colorectal cancer. Here, we have investigated how androgen signaling regulates PARP7 homeostasis in prostate cancer cells, where PARP7 is a direct target gene of AR. We found that the PARP7 protein is extremely short-lived, with a half-life of 4.5 min. We show that in addition to its transcriptional regulation by AR, PARP7 is subject to androgen-dependent post-transcriptional regulation that increases its half-life to 25.6 min. This contrasts with PARP1, PARP2, PARP9, and PARP14, which do not display rapid turnover and are not regulated by androgen signaling. Androgen- and AR-dependent stabilization of PARP7 leads to accumulation in the nucleus, which we suggest is a major site of action. Mutations in the catalytic domain, the Cys3His1 zinc finger, and WWE (tryptophan–tryptophan–glutamate) domains in PARP7 each reduce the degradation rate of PARP7, suggesting the overall structure of the protein is tuned for its rapid turnover. Our finding that PARP7 is regulated by AR signaling both transcriptionally and post-transcriptionally in prostate cancer cells suggests the dosage of PARP7 protein is subject to tight regulation.

ADP‐ribosylation, a modification of proteins, nucleic acids, and metabolites, confers broad functions, including roles in stress responses elicited, for example, by DNA damage and viral infection and is involved in intra‐ and extracellular signaling, chromatin and transcriptional regulation, protein biosynthesis, and cell death. ADP‐ribosylation is catalyzed by ADP‐ribosyltransferases (ARTs), which transfer ADP‐ribose from NAD+ onto substrates. The modification, which occurs as mono‐ or poly‐ADP‐ribosylation, is reversible due to the action of different ADP‐ribosylhydrolases. Importantly, inhibitors of ARTs are approved or are being developed for clinical use. Moreover, ADP‐ribosylhydrolases are being assessed as therapeutic targets, foremost as antiviral drugs and for oncological indications. Due to the development of novel reagents and major technological advances that allow the study of ADP‐ribosylation in unprecedented detail, an increasing number of cellular processes and pathways are being identified that are regulated by ADP‐ribosylation. In addition, characterization of biochemical and structural aspects of the ARTs and their catalytic activities have expanded our understanding of this protein family. This increased knowledge requires that a common nomenclature be used to describe the relevant enzymes. Therefore, in this viewpoint, we propose an updated and broadly supported nomenclature for mammalian ARTs that will facilitate future discussions when addressing the biochemistry and biology of ADP‐ribosylation. This is combined with a brief description of the main functions of mammalian ARTs to illustrate the increasing diversity of mono‐ and poly‐ADP‐ribose mediated cellular processes.

We recently described a signal transduction pathway that contributes to androgen receptor (AR) regulation based on site-specific ADP-ribosylation by PARP7, a mono-ADP-ribosyltransferase implicated in several human cancers. ADP-ribosylated AR is recognized by PARP9/DTX3L, a heterodimeric complex that contains an ADP-ribose reader (PARP9) and a ubiquitin E3 ligase (DTX3L). Here, we have characterized the cellular and biochemical requirements for AR ADP-ribosylation by PARP7. We found that the reaction requires nuclear localization of PARP7 and an agonist-induced conformation of AR. PARP7 contains a Cys3His1-type zinc finger (ZF), which also is critical for AR ADP-ribosylation. The Parp7 ZF is required for efficient nuclear import by a nuclear localization signal encoded in PARP7, but rescue experiments indicate the ZF makes a contribution to AR ADP-ribosylation that is separable from the effect on nuclear transport. ZF mutations do not detectably reduce PARP7 catalytic activity and binding to AR, but they do result in the loss of PARP7 enhancement of AR-dependent transcription of the MYBPC1 gene. Our data reveals critical roles for AR conformation and the PARP7 ZF in AR ADP-ribosylation and AR-dependent transcription.