Whether TMPRSS2-ERG fusion and TP53 gene alteration coordinately promote prostate cancer (PCa) remains unclear. Here we demonstrate that TMPRSS2-ERG fusion and TP53 mutation / deletion co-occur in PCa patient specimens and this co-occurrence accelerates prostatic oncogenesis. p53 gain-of-function (GOF) mutants are now shown to bind to a unique DNA sequence in the CTNNB1 gene promoter and transactivate its expression. ERG and β-Catenin co-occupy sites at pyrimidine synthesis gene (PSG) loci and promote PSG expression, pyrimidine synthesis and PCa growth. β-Catenin inhibition by small molecule inhibitors or oligonucleotide-based PROTAC suppresses TMPRSS2-ERG- and p53 mutant-positive PCa cell growth in vitro and in mice. Our study identifies a gene transactivation function of GOF mutant p53 and reveals β-Catenin as a transcriptional target gene of p53 GOF mutants and a driver and therapeutic target of TMPRSS2-ERG- and p53 GOF mutant-positive PCa.

Supplementary Data from An Active Learning Framework Improves Tumor Variant Interpretation

Supplementary Figure S1. In panel A, ERG pull-down in HUVEC cells showing no interaction between FOXO1 and ERG. In contrast, AR binding with ERG is detected as expected. In panel B, bioinformatic analysis of 492 prostate cancer cases from TCGA to show the profiles of FOXO1, PTEN and ERG alterations. PTEN and FOXO1 deletions largely do not overlap.

<p>Supplementary Information. File containing Materials and Methods, references for Materials and Methods, a table of PCR primers for genotyping and RT-qPCR studies, and Supplementary Figure legends.</p>

Nucleotide excision repair (NER) neutralizes treatment with platinum (Pt)-based chemotherapy by removing Pt lesions from DNA. Previous study has identified that missense mutation or loss of either of the NER genes Excision Repair Cross Complementation Group 1 and 2 ( ERCC1 and ERCC2 ) leads to improved patient outcomes after treatment with Pt-based chemotherapies. Although most NER gene alterations found in patient tumors are missense mutations, the impact of such mutations in the remaining nearly 20 NER genes is unknown. Towards this goal, we previously developed a machine learning strategy to predict genetic variants in an essential NER scaffold protein, Xeroderma Pigmentosum Complementation Group A (XPA), that disrupt repair activity on a UV-damaged substrate. In this study, we report in-depth analyses of a subset of the predicted NER-deficient XPA variants, including in vitro analyses of purified recombinant protein and cell-based assays to test Pt agent sensitivity in cells and determine mechanisms of NER dysfunction. The most NER deficient variant Y148D had reduced protein stability, weaker DNA binding, disrupted recruitment to damage, and degradation resulting from tumor missense mutation. Our findings demonstrate that tumor mutations in XPA impact cell survival after cisplatin treatment and provide valuable mechanistic insights to further improve variant effect prediction efforts. More broadly, these findings suggest XPA tumor variants should be considered when predicting patient response to Pt-based chemotherapy. Significance A destabilized, readily degraded tumor variant identified in the NER scaffold protein XPA sensitizes cells to cisplatin, suggesting that XPA variants can be used to predict response to chemotherapy.

Supplementary Figures and Tables. /// Supplementary Figure 1. TP53 mutation status in human prostate cancer. /// Supplementary Figure 2. Genotypic and histological characterization of Pten/Trp53/ERG-altered mouse prostates. /// Supplementary Figure 3. Immunohistochemistry and immunofluorescent co-staining of key markers in serially-sectioned mouse prostate tissues. /// Supplementary Figure 4. Transcriptomic characterization of the Pten/Trp53/ERG-mutated mouse prostates. /// Supplementary Figure 5. Western blot analysis of expression of ERG downstream genes and E2F1 targets in human prostate cancer cells. /// Supplementary Figure 6. Analysis of expression of ERG-regulated genes and ERG chromatin binding at these gene loci in human prostate cancer PC-3 cells. /// Supplementary Figure 7. Palbociclib and enzalutamide treatment optimization in human prostate cancer cell lines. /// Supplementary Figure 8. Characterization of LNCaP-RF xenografts and mouse tumor-derived allografts. /// Supplementary Table 1. shRNA sequences. /// Supplementary Table 2. RT-qPCR primer sequences. /// Supplementary Table 3. ChIP-qPCR primer sequences. /// Supplementary Table 4. PCR genotyping primer sequences. /// Supplementary Table 5. Raw staining index scores for AR and ERG IHC of mCRPC human tissue microarray. /// References

Nucleotide excision repair (NER) is a versatile DNA repair pathway essential for the removal of a broad spectrum of structurally diverse DNA lesions arising from a variety of sources, including UV irradiation and environmental toxins. Although the core factors and basic stages involved in NER have been identified, the mechanisms of the NER machinery are not well understood. This review summarizes our current understanding of the mechanisms and order of assembly in the core global genome (GG-NER) pathway.

AbstractFor precision medicine to reach its full potential for treatment of cancer and other diseases, protein variant effect prediction tools are needed to characterize variants of unknown significance (VUS) in a patient's genome with respect to their likelihood to influence treatment response and outcomes. However, the performance of most variant prediction tools is limited by the difficulty of acquiring sufficient training and validation data. To overcome these limitations, we applied an iterative active learning approach starting from available biochemical, evolutionary, and functional annotations. With active learning, VUS that are most challenging to classify by an initial machine learning model are functionally evaluated and then reincorporated with the phenotype information in subsequent iterations of algorithm training. The potential of active learning to improve variant interpretation was first demonstrated by applying it to synthetic and deep mutational scanning datasets for four cancer-relevant proteins. The utility of the approach to guide interpretation and functional validation of tumor VUS was then probed on the nucleotide excision repair (NER) protein xeroderma pigmentosum complementation group A (XPA), a potential biomarker for cancer therapy sensitivity. A quantitative high-throughput cell-based NER activity assay was used to validate XPA VUS selected by the active learning strategy. In all cases, active learning yielded a significant improvement in variant effect predictions over traditional learning. These analyses suggest that active learning is well suited to significantly improve interpretation of VUS and cancer patient genomes.Significance:A novel machine learning approach predicts the impact of tumor mutations on cellular phenotypes, overcomes limited training data, minimizes costly functional validation, and advances efforts to implement cancer precision medicine.

DNA synthesis during replication begins with the generation of an ∼10-nucleotide primer by DNA primase. Primase contains a redox-active 4Fe-4S cluster in the C-terminal domain of the p58 subunit (p58C). The redox state of this 4Fe-4S cluster can be modulated via the transport of charge through the protein and the DNA substrate (redox switching); changes in the redox state of the cluster alter the ability of p58C to associate with its substrate. The efficiency of redox switching in p58C can be altered by mutating tyrosine residues that bridge the 4Fe-4S cluster and the nucleic acid binding site. Here, we report the effects of mutating bridging tyrosines to phenylalanines in yeast p58C. High-resolution crystal structures show that these mutations, even with six tyrosines simultaneously mutated, do not perturb the three-dimensional structure of the protein. In contrast, measurements of the electrochemical properties on DNA-modified electrodes of p58C containing multiple tyrosine to phenylalanine mutations reveal deficiencies in their ability to engage in DNA charge transport. Significantly, this loss of electrochemical activity correlates with decreased primase activity. While single-site mutants showed modest decreases in activity compared to that of the wild-type primase, the protein containing six mutations exhibited a 10-fold or greater decrease. Thus, many possible tyrosine-mediated pathways for charge transport in yeast p58C exist, but inhibiting these pathways together diminishes the ability of yeast primase to generate primers. These results support a model in which redox switching is essential for primase activity.

Urinary tract infections are among the most common human bacterial infections and place a significant burden on healthcare systems due to associated morbidity, cost and antibiotic use. Despite being a facultative anaerobe, uropathogenic Escherichia coli , the primary cause of urinary tract infections, requires aerobic respiration to establish infection in the bladder. Here, by combining bacterial genetics with cell culture and murine models of infection, we demonstrate that the widely conserved respiratory quinol oxidase cytochrome bd is required for intracellular infection of urothelial cells. Through a series of genetic, biochemical and functional assays, we show that intracellular oxygen scavenging by cytochrome bd alters mitochondrial physiology by reducing the efficiency of mitochondrial respiration, stabilizing the hypoxia-inducible transcription factor HIF-1 and promoting a shift towards aerobic glycolysis. This bacterially induced rewiring of host metabolism antagonizes apoptosis, thereby protecting intracellular bacteria from urothelial cell exfoliation and preserving their replicative niche. These results reveal the metabolic basis for intracellular bacterial pathogenesis during urinary tract infection and identify subversion of mitochondrial metabolism as a bacterial strategy to facilitate persistence within the urinary tract.

Nucleotide excision repair (NER) is an essential DNA damage repair pathway that removes bulky DNA lesions formed by exposure to ultraviolet light, environmental toxins, and platinum (Pt)-based chemotherapeutic drugs that are a standard of care for many cancer types. Mutation or decreased NER gene expression in cancer correlates with improved patient survival after Pt-based chemotherapy. However, the impact of most missense mutations in NER genes is unknown, and few approaches exist to reliably identify nonrecurrent passenger mutations with functional consequences. In this study, a multidisciplinary strategy will be developed to predict, validate, and characterize NER-defective mutations in the essential NER scaffold protein Xeroderma Pigmentosum Complementation Group A (XPA). Computational analyses were used to score NER-deficient versus NER-proficient mutations for further study. Predicted NER-deficient XPA mutants are being expressed in human XPA-deficient cells and screened for both NER activity and cisplatin sensitivity. In-depth biophysical and structural studies are being implemented to elucidate mechanisms of dysfunction. Identifying NER-deficient mutations that may sensitize tumors to Pt-based chemotherapies represents a promising strategy to stratify patients for optimal treatment strategies.

Therapy resistance is a significant challenge for prostate cancer treatment in clinic. Although targeted therapies such as androgen deprivation and androgen receptor (AR) inhibition are effective initially, tumor cells eventually evade these strategies through multiple mechanisms. Lineage reprogramming in response to hormone therapy represents a key mechanism that is increasingly observed. The studies in this area have revealed specific combinations of alterations present in adenocarcinomas that provide cells with the ability to transdifferentiate and perpetuate AR-independent tumor growth after androgen-based therapies. Interestingly, several master regulators have been identified that drive plasticity, some of which also play key roles during development and differentiation of the cell lineages in the normal prostate. Thus, further study of each AR-independent tumor type and understanding underlying mechanisms are warranted to develop combinational therapies that combat lineage plasticity in prostate cancer.

Abstract Purpose: Deletions or mutations in PTEN and TP53 tumor suppressor genes have been linked to lineage plasticity in therapy-resistant prostate cancer. Fusion-driven overexpression of the oncogenic transcription factor ERG is observed in approximately 50% of all prostate cancers, many of which also harbor PTEN and TP53 alterations. However, the role of ERG in lineage plasticity of PTEN/TP53–altered tumors is unclear. Understanding the collective effect of multiple mutations within one tumor is essential to combat plasticity-driven therapy resistance. Experimental Design: We generated a Pten-negative/Trp53-mutated/ERG-overexpressing mouse model of prostate cancer and integrated RNA-sequencing with ERG chromatin immunoprecipitation-sequencing (ChIP-seq) to identify pathways regulated by ERG in the context of Pten/Trp53 alteration. We investigated ERG-dependent sensitivity to the antiandrogen enzalutamide and cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitor palbociclib in human prostate cancer cell lines, xenografts, and allografted mouse tumors. Trends were evaluated in TCGA, SU2C, and Beltran 2016 published patient cohorts and a human tissue microarray. Results: Transgenic ERG expression in mice blocked Pten/Trp53 alteration–induced decrease of AR expression and downstream luminal epithelial genes. ERG directly suppressed expression of cell cycle–related genes, which induced RB hypophosphorylation and repressed E2F1-mediated expression of mesenchymal lineage regulators, thereby restricting adenocarcinoma plasticity and maintaining antiandrogen sensitivity. In ERG-negative tumors, CDK4/6 inhibition delayed tumor growth. Conclusions: Our studies identify a previously undefined function of ERG to restrict lineage plasticity and maintain antiandrogen sensitivity in PTEN/TP53–altered prostate cancer. Our findings suggest ERG fusion as a biomarker to guide treatment of PTEN/TP53-altered, RB1-intact prostate cancer. Clin Cancer Res; 24(18); 4551–65. ©2018 AACR.

AbstractE26 transformation-specific transcription factor ERG is aberrantly overexpressed in approximately 50% of all human prostate cancer due to TMPRSS2-ERG gene rearrangements. However, mice with prostate-specific transgenic expression of prostate cancer–associated ERG alone fail to develop prostate cancer, highlighting that ERG requires other lesions to drive prostate tumorigenesis. Forkhead box (FOXO) transcription factor FOXO1 is a tumor suppressor that is frequently inactivated in human prostate cancer. Here, we demonstrate that FOXO1, but not other FOXO proteins (FOXO3 and FOXO4), binds and inhibits the transcriptional activity of prostate cancer–associated ERG independently of FOXO1 transcriptional activity. Knockdown of endogenous FOXO1 increased invasion of TMPRSS2–ERG fusion–positive VCaP cells, an effect completely abolished by ERG knockdown. Patient specimen analysis demonstrated that FOXO1 and ERG protein expression inversely correlated in a subset of human prostate cancer. Although human ERG transgene expression or homozygous deletion of Foxo1 alone in the mouse prostate failed to promote tumorigenesis, concomitant ERG transgene expression and Foxo1 deletion resulted in upregulation of ERG target genes, increased cell proliferation, and formation of high-grade prostatic intraepithelial neoplasia. Overall, we provide biochemical and genetic evidence that aberrantly activated ERG cooperates with FOXO1 deficiency to promote prostate tumorigenesis and cell invasion. Our findings enhance understanding of prostate cancer etiology and suggest that the FOXO1–ERG signaling axis can be a potential target for treatment of prostate cancer. Cancer Res; 77(23); 6524–37. ©2017 AACR.

Prostate cancer (PCa) that becomes resistant to hormone castration and next-generation androgen receptor (AR)-targeted therapies, called castration-resistant prostate cancer (CRPC), poses a significant clinical challenge. A better understanding of PCa progression and key molecular mechanisms could bring novel therapies to light. One potential therapeutic target is ERG, a transcription factor aberrantly up-regulated in PCa due to chromosomal rearrangements between androgen-regulated gene TMPRSS2 and ERG. Here we show that the most common PCa-associated truncated ERG T1-E4 (ERG.39), encoded by fusion between TMPRSS2 exon 1 and ERG exon 4, binds to bromodomain-1 (BD1) of bromodomain containing protein 4 (BRD4), a member of the bromodomain and extraterminal domain (BET) family. This interaction is partially abrogated by BET inhibitors JQ1 and iBET762. Meta-analysis of published ERG (T1-E4) and BRD4 chromatin immunoprecipitation-sequencing (ChIP-seq) data demonstrates overlap in a substantial portion of their binding sites. Gene expression profile analysis shows some ERG-BRD4 co-target genes are upregulated in CRPC compared to hormone-naive counterparts. We provide further evidence that ERG-mediated invasion of PCa cells was significantly enhanced by an acetylation-mimicking mutation in ERG that augments the ERG-BRD4 interaction. Our findings reveal that PCa-associated ERG can interact and co-occupy with BRD4 in the genome, and suggest this druggable interaction is critical for ERG-mediated cell invasion and PCa progression.

Although the majority of prostate cancer (PCa) cases are now detected and treated at early stages, PCa remains the second leading cause of cancer death in American men (1). In the case of more advanced tumors, androgen deprivation can prevent cancer progression temporarily, but this inevitably leads to castration-resistant prostate cancer (CRPC).

The transcription factor ERG is important during development for vasculogenesis and angiogenesis, blood vessel integrity, and maintenance of hematopoietic stem cells. In human adults, ERG is only expressed in endothelial cells and performs similar roles as in development to mediate blood vessel formation. However, aberrant overexpression of ERG in the adult contributes to diseases like prostate cancer, and the molecular and cellular mechanisms of ERG in tumorigenesis remain largely unclear. Studies of ERG-positive prostate cancers have shown that ERG promotes cell invasion and metastasis and contributes to poor patient prognosis. Together, these studies reveal ERG-mediated cell invasion as a potential link between normal ERG function in development and oncogenic ERG function in prostate cancer.

Tissues and organs undergo constant physical perturbations and individual cells must respond to mechanical forces to maintain tissue integrity. However, molecular interactions underlying mechano-transduction are not fully defined at cell-cell junctions. This is in part due to weak and transient interactions that are likely prevalent in force-induced protein complexes. Using in situ proximal biotinylation by the promiscuous biotin ligase BirA tagged to α-catenin and a substrate stretch cell chamber, we sought to identify force-dependent molecular interactions surrounding α-catenin, an actin regulator at the sites of cadherin mediated cell-cell adhesion. While E-cadherin, β-catenin, vinculin and actin localize with α-catenin at cell-cell contacts in immuno-fluorescent staining, only β-catenin and plakoglobin were biotinylated, suggesting that this proximal biotinylation is limited to the molecules that are in the immediate vicinity of α-catenin. In mechanically stretched samples, increased biotinylation of non-muscle myosin IIA, but not myosin IIB, suggests close spatial proximity between α-catenin and myosin IIA during substrate stretching. This force-induced biotinylation diminished as myosin II activity was inhibited by blebbistatin. Taken together, this promising technique enables us to identify force sensitive complexes that may be essential for mechano-responses in force bearing cell adhesion.