Oligomerization mediated by the D2 domain of DTX3L is critical for DTX3L-PARP9 reading function of mono-ADP-ribosylated androgen receptor.

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 recognize 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-oligomerization, 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, and assemble into a high molecular weight oligomeric complex, 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-oligomerization of DTX3L is important for the DTX3L-PARP9 complex to read mono-ADP-ribosylation on a ligand-regulated transcription factor.

Oligomerisation mediated by the D2 domain of DTX3L is critical for DTX3L-PARP9 reading function of mono-ADP-ribosylated androgen receptor.

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.

Synthetic Dual Cysteine-ADP Ribosylated Peptides from the Androgen Receptor are Recognized by the DTX3L/PARP9 Complex.

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.

Induction of PARP7 Creates a Vulnerability for Growth Inhibition by RBN2397 in Prostate Cancer Cells.

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.

Reconstitution of the DTX3L-PARP9 complex reveals determinants for high-affinity heterodimerization and multimeric assembly.

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.

Genomic analyses of the metastasis-derived prostate cancer cell lines LNCaP, VCaP, and PC3-AR.

BACKGROUND: The lymph node metastasis-derived LNCaP, the bone metastasis-derived PC3 (skull), and VCaP (vertebral) cell lines are widely used as preclinical models of human prostate cancer (CaP) and have been described in more than 19,000 publications. Here, we report on short-read whole-genome sequencing and genomic analyses of LNCaP, VCaP, and PC3 cells stably transduced with WT AR (PC3-AR). METHODS: LNCaP, VCaP, and PC3-AR cell lines were sequenced to an average depth of more than 30-fold using Illumina short-read sequencing. Using various computational methods, we identified and compared the single-nucleotide variants, copy-number profiles, and the structural variants observed in the three cell lines. RESULTS: LNCaP cells are composed of multiple subpopulations, which results in nonintegral copy number states and a high mutational load when the data is analyzed in bulk. All three cell lines contain pathogenic mutations and homozygous deletions in genes involved in DNA mismatch repair, along with deleterious mutations in cell-cycle, Wnt signaling, and other critical cellular processes. PC3-AR cells have a truncating mutation in TP53 and do not express the p53 protein. The VCaP cells contain a homozygous gain-of-function mutation in TP53 (p.R248W) that promotes cancer invasion, metastasis, and progression and has also been observed in prostate adenocarcinomas. In addition, we detect the signatures of chromothripsis of the q arms of chromosome 5 in both PC3-AR and VCaP cells, strengthening the association of TP53 inactivation with chromothripsis reported in other systems. CONCLUSIONS: Our work provides a resource for genetic, genomic, and biological studies employing these commonly-used prostate cancer cell lines.

ADP-ribosyltransferases, an update on function and nomenclature.

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.

PARP7 mono-ADP-ribosylates the agonist conformation of the androgen receptor in the nucleus.

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.

Androgen signaling uses a writer and a reader of ADP-ribosylation to regulate protein complex assembly.

Androgen signaling through the androgen receptor (AR) directs gene expression in both normal and prostate cancer cells. Androgen regulates multiple aspects of the AR life cycle, including its localization and post-translational modification, but understanding how modifications are read and integrated with AR activity has been difficult. Here, we show that ADP-ribosylation regulates AR through a nuclear pathway mediated by Parp7. We show that Parp7 mono-ADP-ribosylates agonist-bound AR, and that ADP-ribosyl-cysteines within the N-terminal domain mediate recruitment of the E3 ligase Dtx3L/Parp9. Molecular recognition of ADP-ribosyl-cysteine is provided by tandem macrodomains in Parp9, and Dtx3L/Parp9 modulates expression of a subset of AR-regulated genes. Parp7, ADP-ribosylation of AR, and AR-Dtx3L/Parp9 complex assembly are inhibited by Olaparib, a compound used clinically to inhibit poly-ADP-ribosyltransferases Parp1/2. Our study reveals the components of an androgen signaling axis that uses a writer and reader of ADP-ribosylation to regulate protein-protein interactions and AR activity.

Post-Transcriptional Regulation of PARP7 Protein Stability Is Controlled by Androgen Signaling.

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.

Role of SPTSSB-Regulated de Novo Sphingolipid Synthesis in Prostate Cancer Depends on Androgen Receptor Signaling.

Anti-androgens are a common therapy in prostate cancer (PCa) targeting androgen receptor (AR) signaling. However, these therapies fail due to selection of highly aggressive AR-negative cancer cells that have no therapeutic options available. We demonstrate that elevating endogenous ceramide levels with administration of exogenous ceramide nanoliposomes (CNLs) was efficacious in AR-negative cell lines with limited efficacy in AR-positive cells. This effect is mediated through reduced de novo sphingolipid synthesis in AR-positive cells. We show that anti-androgens elevate de novo generation of sphingolipids via SPTSSB, a rate-limiting mediator of sphingolipid generation. Moreover, pharmacological inhibition of AR increases the efficacy of CNL in AR-positive cells through de novo synthesis, while SPTSSB knockdown limited CNL's efficacy in AR-negative cells. Alluding to clinical relevance, SPTSSB is upregulated in patients with advanced PCa after anti-androgens treatment. These findings emphasize the relevance of AR regulation upon sphingolipid metabolism and the potential of CNL as a PCa therapeutic.

An epilepsy-associated mutation in the nuclear import receptor KPNA7 reduces nuclear localization signal binding.

KPNA7 is a member of the Importin-α family of nuclear import receptors. KPNA7 forms a complex with Importin-ß and facilitates the translocation of signal-containing proteins from the cytoplasm to the nucleus. Exome sequencing of siblings with severe neurodevelopmental defects and clinical features of epilepsy identified two amino acid-altering mutations in KPNA7. Here, we show that the E344Q substitution reduces KPNA7 binding to nuclear localization signals, and that this limits KPNA7 nuclear import activity. The P339A substitution, by contrast, has little effect on KPNA7 binding to nuclear localization signals. Given the neuronal phenotype described in the two patients, we used SILAC labeling, affinity enrichment, and mass spectrometry to identify KPNA7-interacting proteins in human induced pluripotent stem cell-derived neurons. We identified heterogeneous nuclear ribonucleoproteins hnRNP R and hnRNP U as KPNA7-interacting proteins. The E344Q substitution reduced binding and KPNA7-mediated import of these cargoes. The c.1030G > C allele which generates E344Q is within a predicted CTCF binding site, and we found that it reduces CTCF binding by approximately 40-fold. Our data support a role for altered neuronal expression and activity of KPNA7 in a rare type of pediatric epilepsy.

Characterization of the Importin-ß binding domain in nuclear import receptor KPNA7.

The KPNA family of mammalian nuclear import receptors are encoded by seven genes that generate isoforms with 42-86% identity. KPNA isoforms have the same protein architecture and share the functional property of nuclear localization signal (NLS) recognition, however, the tissue and developmental expression patterns of these receptors raise the question of whether subtle differences in KPNA isoforms might be important in specific biological contexts. Here, we show that KPNA7, an isoform with expression mostly limited to early development, can bind Importin-ß (Imp-ß) in the absence of NLS cargo. This result contrasts with Imp-ß interactions with other KPNA family members, where affinity is regulated by NLS cargo as part of a cooperative binding mechanism. The Imp-ß binding (IBB) domain, which is highly conserved in all KPNA family members, generally serves to occlude the NLS binding groove and maintain the receptor in an auto-inhibited 'closed' state prior to NLS contact. Cooperative binding of NLS cargo and Imp-ß to KPNA results in an 'open'state. Characterization of KPNA2-KPNA7 chimeric proteins suggests that features of both the IBB domain and the core structure of the receptor contribute to the extent of IBB domain accessibility for Imp-ß binding, which likely reflects an 'open' state. We also provide evidence that KPNA7 maintains an open-state in the nucleus. We speculate that KPNA7 could function within the nucleus by interacting with NLS-containing proteins.

Detection of ADP-Ribosylation of the Androgen Receptor Using the Recombinant Macrodomain AF1521 from Archaeoglobus fulgidus.

ADP-ribosylation is a posttranslational modification generated by members of the superfamily of ADP-ribosyltransferases, known as the Parp enzymes. Depending on the superfamily member, Parp enzymes can mono- or poly-ADP-ribosylate a protein substrate. Parp superfamily members confer regulation to a variety of biological processes that include cell signaling, DNA repair, transcription, and stress responses. Here, we describe biochemical methods for detection of ADP-ribose conjugated to the androgen receptor (AR) using the archaeal macrodomain, AF1521, from Archaeoglobus fulgidus. The utility of AF1521 is based on its highly selective recognition of ADP-ribose conjugated to protein. AF1521 immobilized on beads can be used to enrich for ADP-ribosylated proteins, which in our application results in recovery of ADP-ribosylated AR from prostate cancer cell extracts. We engineered tandem AF1521 macrodomains and found this improves the recovery of ADP-ribosylated AR under native conditions, and it enabled development of an assay for detection of ADP-ribosylation on blots. Thus, AF1521 can be used to query ADP-ribosylation of protein under both native and denaturing conditions. Our assays should prove useful for understanding how ADP-ribosylation regulates AR function.

Towards improving proximity labeling by the biotin ligase BirA.

The discovery and validation of protein-protein interactions provides a knowledge base that is critical for defining protein networks and how they underpin the biology of the cell. Identification of protein interactions that are highly transient, or sensitive to biochemical disruption, can be very difficult. This challenge has been met by proximity labeling methods which generate reactive species that chemically modify neighboring proteins. The most widely used proximity labeling method is BioID, which features a mutant biotin ligase BirA(Arg118Gly), termed BirA*, fused to a protein of interest. Here, we explore how amino acid substitutions at Arg118 affect the biochemical properties of BirA. We found that relative to wild-type BirA, the Arg118Lys substitution both slightly reduced biotin affinity and increased the release of reactive biotinyl-5'-AMP. BioID using a BirA(Arg118Lys)-Lamin A fusion enabled identification of PCNA as a lamina-proximal protein in HEK293T cells, a finding that was validated by immunofluorescence microscopy. Our data expand on the concept that proximity labeling by BirA fused to proteins of interest can be modulated by amino acid substitutions that affect biotin affinity and the release of biotinyl-5'-AMP.

Fluorescence-based quantification of nucleocytoplasmic transport.

The sequestration of DNA within the membrane-bound nucleus is a defining characteristic of eukaryotic cells. Replication and transcription are therefore restricted to the nucleus, however, the regulation of these events relies on cytoplasmic processes including protein synthesis and signal transduction pathways. Because a variety of cellular activities depend on nuclear transport, researchers from diverse fields have found it useful to examine the nuclear localization of proteins of interest. Here we present some important technical considerations for studying nuclear and cytoplasmic localization, and provide guidance for quantifying protein levels using fluorescence microscopy and ImageJ software. We include discussion of the use of regions of interest and image segmentation for quantification of protein localization. Nucleocytoplasmic transport is fundamentally important for controlling protein levels and activity in the nucleus or cytoplasm, and quantitative analysis can provide insight into how biological output is achieved.

A nuclear lamina-chromatin-Ran GTPase axis modulates nuclear import and DNA damage signaling.

The Ran GTPase regulates nuclear import and export by controlling the assembly state of transport complexes. This involves the direct action of RanGTP, which is generated in the nucleus by the chromatin-associated nucleotide exchange factor, RCC1. Ran interactions with RCC1 contribute to formation of a nuclear:cytoplasmic (N:C) Ran protein gradient in interphase cells. In previous work, we showed that the Ran protein gradient is disrupted in fibroblasts from Hutchinson-Gilford progeria syndrome (HGPS) patients. The Ran gradient disruption in these cells is caused by nuclear membrane association of a mutant form of Lamin A, which induces a global reduction in heterochromatin marked with Histone H3K9me3 and Histone H3K27me3. Here, we have tested the hypothesis that heterochromatin controls the Ran gradient. Chemical inhibition and depletion of the histone methyltransferases (HMTs) G9a and GLP in normal human fibroblasts reduced heterochromatin levels and caused disruption of the Ran gradient, comparable to that observed previously in HGPS fibroblasts. HMT inhibition caused a defect in nuclear localization of TPR, a high molecular weight protein that, owing to its large size, displays a Ran-dependent import defect in HGPS. We reasoned that pathways dependent on nuclear import of large proteins might be compromised in HGPS. We found that nuclear import of ATM requires the Ran gradient, and disruption of the Ran gradient in HGPS causes a defect in generating nuclear γ-H2AX in response to ionizing radiation. Our data suggest a lamina-chromatin-Ran axis is important for nuclear transport regulation and contributes to the DNA damage response.

Recurrent mutations at estrogen receptor binding sites alter chromatin topology and distal gene expression in breast cancer.

BACKGROUND: The mutational processes underlying non-coding cancer mutations and their biological significance in tumor evolution are poorly understood. To get better insights into the biological mechanisms of mutational processes in breast cancer, we integrate whole-genome level somatic mutations from breast cancer patients with chromatin states and transcription factor binding events. RESULTS: We discover that a large fraction of non-coding somatic mutations in estrogen receptor (ER)-positive breast cancers are confined to ER binding sites. Notably, the highly mutated estrogen receptor binding sites are associated with more frequent chromatin loop contacts and the associated distal genes are expressed at higher level. To elucidate the functional significance of these non-coding mutations, we focus on two of the recurrently mutated estrogen receptor binding sites. Our bioinformatics and biochemical analysis suggest loss of DNA-protein interactions due to the recurrent mutations. Through CRISPR interference, we find that the recurrently mutated regulatory element at the LRRC3C-GSDMA locus impacts the expression of multiple distal genes. Using a CRISPR base editor, we show that the recurrent C→T conversion at the ZNF143 locus results in decreased TF binding, increased chromatin loop formation, and increased expression of multiple distal genes. This single point mutation mediates reduced response to estradiol-induced cell proliferation but increased resistance to tamoxifen-induced growth inhibition. CONCLUSIONS: Our data suggest that ER binding is associated with localized accumulation of somatic mutations, some of which affect chromatin architecture, distal gene expression, and cellular phenotypes in ER-positive breast cancer.

Genomic analysis of DNA repair genes and androgen signaling in prostate cancer.

BACKGROUND: The cellular effects of androgen are transduced through the androgen receptor, which controls the expression of genes that regulate biosynthetic processes, cell growth, and metabolism. Androgen signaling also impacts DNA damage signaling through mechanisms involving gene expression and transcription-associated DNA damaging events. Defining the contributions of androgen signaling to DNA repair is important for understanding androgen receptor function, and it also has translational implications. METHODS: We generated RNA-seq data from multiple prostate cancer lines and used bioinformatic analyses to characterize androgen-regulated gene expression. We compared the results from cell lines with gene expression data from prostate cancer xenografts, and patient samples, to query how androgen signaling and prostate cancer progression influences the expression of DNA repair genes. We performed whole genome sequencing to help characterize the status of the DNA repair machinery in widely used prostate cancer lines. Finally, we tested a DNA repair enzyme inhibitor for effects on androgen-dependent transcription. RESULTS: Our data indicates that androgen signaling regulates a subset of DNA repair genes that are largely specific to the respective model system and disease state. We identified deleterious mutations in the DNA repair genes RAD50 and CHEK2. We found that inhibition of the DNA repair enzyme MRE11 with the small molecule mirin inhibits androgen-dependent transcription and growth of prostate cancer cells. CONCLUSIONS: Our data supports the view that crosstalk between androgen signaling and DNA repair occurs at multiple levels, and that DNA repair enzymes in addition to PARPs, could be actionable targets in prostate cancer.