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The retinoblastoma tumor suppressor (RB) is a critical regulator of E2F-dependent transcription, controlling a multitude of protumorigenic networks including but not limited to cell-cycle control. Here, genome-wide assessment of E2F1 function after RB loss in isogenic models of prostate cancer revealed unexpected repositioning and cooperation with oncogenic transcription factors, including the major driver of disease progression, the androgen receptor (AR). Further investigation revealed that observed AR/E2F1 cooperation elicited novel transcriptional networks that promote cancer phenotypes, especially as related to evasion of cell death. These observations were reflected in assessment of human disease, indicating the clinical relevance of the AR/E2F1 cooperome in prostate cancer. Together, these studies reveal new mechanisms by which RB loss induces cancer progression and highlight the importance of understanding the targets of E2F1 function.Significance:This study identifies that RB loss in prostate cancer drives cooperation between AR and E2F1 as coregulators of transcription, which is linked to the progression of advanced disease.

Supplementary Figures 1-6

Purpose:DNA-dependent protein kinase catalytic subunit (DNA-PK) is a pleiotropic kinase involved in DNA repair and transcriptional regulation. DNA-PK is deregulated in selected cancer types and is strongly associated with poor outcome. The underlying mechanisms by which DNA-PK promotes aggressive tumor phenotypes are not well understood. Here, unbiased molecular investigation in clinically relevant tumor models reveals novel functions of DNA-PK in cancer.Experimental Design: DNA-PK function was modulated using both genetic and pharmacologic methods in a series of in vitro models, in vivo xenografts, and patient-derived explants (PDE), and the impact on the downstream signaling and cellular cancer phenotypes was discerned. Data obtained were used to develop novel strategies for combinatorial targeting of DNA-PK and hormone signaling pathways.Results:Key findings reveal that (i) DNA-PK regulates tumor cell proliferation; (ii) pharmacologic targeting of DNA-PK suppresses tumor growth both in vitro, in vivo, and ex vivo; (iii) DNA-PK transcriptionally regulates the known DNA-PK–mediated functions as well as novel cancer-related pathways that promote tumor growth; (iv) dual targeting of DNA-PK/TOR kinase (TORK) transcriptionally upregulates androgen signaling, which can be mitigated using the androgen receptor (AR) antagonist enzalutamide; (v) cotargeting AR and DNA-PK/TORK leads to the expansion of antitumor effects, uncovering the modulation of novel, highly relevant protumorigenic cancer pathways; and (viii) cotargeting DNA-PK/TORK and AR has cooperative growth inhibitory effects in vitro and in vivo.Conclusions:These findings uncovered novel DNA-PK transcriptional regulatory functions and led to the development of a combinatorial therapeutic strategy for patients with advanced prostate cancer, currently being tested in the clinical setting.

Table S1, Primer sequences used for gene expression analysis. Table S2, PDE pathological characteristics.

SF1: Analysis of USP22 expression with known drivers of disease progression; SF2: USP22 modulates DNA repair factors expression and survival after DNA damage; SF3: Genetically engineered mouse model of tumor-associated USP22 expression; SF4: The USP22-sensitive ubiquitylome reveals altered modification of DNA repair proteins; SF5: USP22 deubiquitylates the NER protein XPC, modulating foci formation.

Supplementary Figure from A Novel Role for DNA-PK in Metabolism by Regulating Glycolysis in Castration-Resistant Prostate Cancer

Purpose:DNA-dependent protein kinase catalytic subunit (DNA-PKcs, herein referred as DNA-PK) is a multifunctional kinase of high cancer relevance. DNA-PK is deregulated in multiple tumor types, including prostate cancer, and is associated with poor outcomes. DNA-PK was previously nominated as a therapeutic target and DNA-PK inhibitors are currently undergoing clinical investigation. Although DNA-PK is well studied in DNA repair and transcriptional regulation, much remains to be understood about the way by which DNA-PK drives aggressive disease phenotypes.Experimental Design:Here, unbiased proteomic and metabolomic approaches in clinically relevant tumor models uncovered a novel role of DNA-PK in metabolic regulation of cancer progression. DNA-PK regulation of metabolism was interrogated using pharmacologic and genetic perturbation using in vitro cell models, in vivo xenografts, and ex vivo in patient-derived explants (PDE).Results:Key findings reveal: (i) the first-in-field DNA-PK protein interactome; (ii) numerous DNA-PK novel partners involved in glycolysis; (iii) DNA-PK interacts with, phosphorylates (in vitro), and increases the enzymatic activity of glycolytic enzymes ALDOA and PKM2; (iv) DNA-PK drives synthesis of glucose-derived pyruvate and lactate; (v) DNA-PK regulates glycolysis in vitro, in vivo, and ex vivo; and (vi) combination of DNA-PK inhibitor with glycolytic inhibitor 2-deoxyglucose leads to additive anti-proliferative effects in aggressive disease.Conclusions:Findings herein unveil novel DNA-PK partners, substrates, and function in prostate cancer. DNA-PK impacts glycolysis through direct interaction with glycolytic enzymes and modulation of enzymatic activity. These events support energy production that may contribute to generation and/or maintenance of DNA-PK–mediated aggressive disease phenotypes.

Emerging evidence indicates the deubiquitinase USP22 regulates transcriptional activation and modification of target substrates to promote pro-oncogenic phenotypes. Here, in vivo characterization of tumor-associated USP22 upregulation and unbiased interrogation of USP22-regulated functions in vitro demonstrated critical roles for USP22 in prostate cancer. Specifically, clinical datasets validated that USP22 expression is elevated in prostate cancer, and a novel murine model demonstrated a hyperproliferative phenotype with prostate-specific USP22 overexpression. Accordingly, upon overexpression or depletion of USP22, enrichment of cell-cycle and DNA repair pathways was observed in the USP22-sensitive transcriptome and ubiquitylome using prostate cancer models of clinical relevance. Depletion of USP22 sensitized cells to genotoxic insult, and the role of USP22 in response to genotoxic insult was further confirmed using mouse adult fibroblasts from the novel murine model of USP22 expression. As it was hypothesized that USP22 deubiquitylates target substrates to promote protumorigenic phenotypes, analysis of the USP22-sensitive ubiquitylome identified the nucleotide excision repair protein, XPC, as a critical mediator of the USP22-mediated response to genotoxic insult. Thus, XPC undergoes deubiquitylation as a result of USP22 function and promotes USP22-mediated survival to DNA damage. Combined, these findings reveal unexpected functions of USP22 as a driver of protumorigenic phenotypes and have significant implications for the role of USP22 in therapeutic outcomes.Significance:The studies herein present a novel mouse model of tumor-associated USP22 overexpression and implicate USP22 in modulation of cellular survival and DNA repair, in part through regulation of XPC.

Prostate cancer (PCa) is the second leading cause of cancer death for men in the United States. While organ-confined disease has reasonable expectation of cure, metastatic PCa is universally fatal upon recurrence during hormone therapy, a stage termed castration-resistant prostate cancer (CRPC). Until such time as molecularly defined subtypes can be identified and targeted using precision medicine, it is necessary to investigate new therapies that may apply to the CRPC population as a whole.The administration of ascorbate, more commonly known as ascorbic acid or Vitamin C, has proved lethal to and highly selective for a variety of cancer cell types. There are several mechanisms currently under investigation to explain how ascorbate exerts anti-cancer effects. A simplified model depicts ascorbate as a pro-drug for reactive oxygen species (ROS), which accumulate intracellularly and generate DNA damage. It was therefore hypothesized that poly(ADP-ribose) polymerase (PARP) inhibitors, by inhibiting DNA damage repair, would augment the toxicity of ascorbate.ResultsTwo distinct CRPC models were found to be sensitive to physiologically relevant doses of ascorbate. Moreover, additional studies indicate that ascorbate inhibits CRPC growthin vitrovia multiple mechanisms including disruption of cellular energy dynamics and accumulation of DNA damage. Combination studies were performed in CRPC models with ascorbate in conjunction with escalating doses of three different PARP inhibitors (niraparib, olaparib, and talazoparib). The addition of ascorbate augmented the toxicity of all three PARP inhibitors and proved synergistic with olaparib in both CRPC models. Finally, the combination of olaparib and ascorbate was testedin vivoin both castrated and non-castrated models. In both cohorts, the combination treatment significantly delayed tumor growth compared to monotherapy or untreated control.ConclusionsThese data indicate that pharmacological ascorbate is an effective monotherapy at physiological concentrations and kills CRPC cells. Ascorbate-induced tumor cell death was associated with disruption of cellular energy dynamics and accumulation of DNA damage. The addition of PARP inhibition increased the extent of DNA damage and proved effective at slowing CRPC growth bothin vitroandin vivo. These findings nominate ascorbate and PARPi as a novel therapeutic regimen that has the potential to improve CRPC patient outcomes.

Purpose: DNA-dependent protein kinase catalytic subunit (DNA-PKcs, herein referred as DNA-PK) is a multifunctional kinase of high cancer relevance. DNA-PK is deregulated in multiple tumor types, including prostate cancer, and is associated with poor outcomes. DNA-PK was previously nominated as a therapeutic target and DNA-PK inhibitors are currently undergoing clinical investigation. Although DNA-PK is well studied in DNA repair and transcriptional regulation, much remains to be understood about the way by which DNA-PK drives aggressive disease phenotypes. Experimental Design: Here, unbiased proteomic and metabolomic approaches in clinically relevant tumor models uncovered a novel role of DNA-PK in metabolic regulation of cancer progression. DNA-PK regulation of metabolism was interrogated using pharmacologic and genetic perturbation using in vitro cell models, in vivo xenografts, and ex vivo in patient-derived explants (PDE). Results: Key findings reveal: (i) the first-in-field DNA-PK protein interactome; (ii) numerous DNA-PK novel partners involved in glycolysis; (iii) DNA-PK interacts with, phosphorylates (in vitro), and increases the enzymatic activity of glycolytic enzymes ALDOA and PKM2; (iv) DNA-PK drives synthesis of glucose-derived pyruvate and lactate; (v) DNA-PK regulates glycolysis in vitro, in vivo, and ex vivo; and (vi) combination of DNA-PK inhibitor with glycolytic inhibitor 2-deoxyglucose leads to additive anti-proliferative effects in aggressive disease. Conclusions: Findings herein unveil novel DNA-PK partners, substrates, and function in prostate cancer. DNA-PK impacts glycolysis through direct interaction with glycolytic enzymes and modulation of enzymatic activity. These events support energy production that may contribute to generation and/or maintenance of DNA-PK–mediated aggressive disease phenotypes.

The tumor suppressor gene TP53 is the most frequently mutated gene in numerous cancer types, including prostate cancer (PCa). Specifically, missense mutations in TP53 are selectively enriched in PCa, and cluster to particular “hot spots” in the p53 DNA binding domain with mutation at the R273 residue occurring most frequently. While this residue is similarly mutated to R273C-p53 or R273H-p53 in all cancer types examined, in PCa selective enrichment of R273C-p53 is observed. Importantly, examination of clinical datasets indicated that TP53 heterozygosity can either be maintained or loss of heterozygosity (LOH) occurs. Thus, to mimic tumor-associated mutant p53, R273C-p53 and R273H-p53 isogenic PCa models were developed in the presence or absence of wild-type p53. In the absence of wild-type p53, both R273C-p53 and R273H-p53 exhibited similar loss of DNA binding, transcriptional profiles, and loss of canonical tumor suppressor functions associated with wild-type p53. In the presence of wild-type p53 expression, both R273C-p53 and R273H-p53 supported canonical p53 target gene expression yet elicited distinct cistromic and transcriptional profiles when compared to each other. Moreover, heterozygous modeling of R273C-p53 or R273H-p53 expression resulted in distinct phenotypic outcomes in vitro and in vivo. Thus, mutant p53 acts in a context-dependent manner to elicit pro-tumorigenic transcriptional profiles, providing critical insight into mutant p53-mediated prostate cancer progression.

The retinoblastoma tumor suppressor (RB) is a critical regulator of E2F-dependent transcription, controlling a multitude of protumorigenic networks including but not limited to cell-cycle control. Here, genome-wide assessment of E2F1 function after RB loss in isogenic models of prostate cancer revealed unexpected repositioning and cooperation with oncogenic transcription factors, including the major driver of disease progression, the androgen receptor (AR). Further investigation revealed that observed AR/E2F1 cooperation elicited novel transcriptional networks that promote cancer phenotypes, especially as related to evasion of cell death. These observations were reflected in assessment of human disease, indicating the clinical relevance of the AR/E2F1 cooperome in prostate cancer. Together, these studies reveal new mechanisms by which RB loss induces cancer progression and highlight the importance of understanding the targets of E2F1 function. Significance: This study identifies that RB loss in prostate cancer drives cooperation between AR and E2F1 as coregulators of transcription, which is linked to the progression of advanced disease.

Loss of the retinoblastoma (RB) tumor suppressor protein is a critical step in reprogramming biological networks that drive cancer progression, although mechanistic insight has been largely limited to the impact of RB loss on cell-cycle regulation. Here, isogenic modeling of RB loss identified disease stage–specific rewiring of E2F1 function, providing the first-in-field mapping of the E2F1 cistrome and transcriptome after RB loss across disease progression. Biochemical and functional assessment using both in vitro and in vivo models identified an unexpected, prominent role for E2F1 in regulation of redox metabolism after RB loss, driving an increase in the synthesis of the antioxidant glutathione, specific to advanced disease. These E2F1-dependent events resulted in protection from reactive oxygen species in response to therapeutic intervention. On balance, these findings reveal novel pathways through which RB loss promotes cancer progression and highlight potentially new nodes of intervention for treating RB-deficient cancers. Significance: This study identifies stage-specific consequences of RB loss across cancer progression that have a direct impact on tumor response to clinically utilized therapeutics. The study herein is the first to investigate the effect of RB loss on global metabolic regulation and link RB/E2F1 to redox control in multiple advanced diseases. This article is highlighted in the In This Issue feature, p. 2113

The constitutively active androgen receptor (AR) splice variant, AR-V7, plays an important role in resistance to androgen deprivation therapy in castration resistant prostate cancer (CRPC). Studies seeking to determine whether AR-V7 is a partial mimic of the AR, or also has unique activities, and whether the AR-V7 cistrome contains unique binding sites have yielded conflicting results. One limitation in many studies has been the low level of AR variant compared to AR. Here, LNCaP and VCaP cell lines in which AR-V7 expression can be induced to match the level of AR, were used to compare the activities of AR and AR-V7. The two AR isoforms shared many targets, but overall had distinct transcriptomes. Optimal induction of novel targets sometimes required more receptor isoform than classical targets such as PSA. The isoforms displayed remarkably different cistromes with numerous differential binding sites. Some of the unique AR-V7 sites were located proximal to the transcription start sites (TSS). A de novo binding motif similar to a half ARE was identified in many AR-V7 preferential sites and, in contrast to conventional half ARE sites that bind AR-V7, FOXA1 was not enriched at these sites. This supports the concept that the AR isoforms have unique actions with the potential to serve as biomarkers or novel therapeutic targets.

Mechanisms regulating DNA repair processes remain incompletely defined. Here, the circadian factor CRY1, an evolutionally conserved transcriptional coregulator, is identified as a tumor specific regulator of DNA repair. Key findings demonstrate that CRY1 expression is androgen-responsive and associates with poor outcome in prostate cancer. Functional studies and first-in-field mapping of the CRY1 cistrome and transcriptome reveal that CRY1 regulates DNA repair and the G2/M transition. DNA damage stabilizes CRY1 in cancer (in vitro, in vivo, and human tumors ex vivo), which proves critical for efficient DNA repair. Further mechanistic investigation shows that stabilized CRY1 temporally regulates expression of genes required for homologous recombination. Collectively, these findings reveal that CRY1 is hormone-induced in tumors, is further stabilized by genomic insult, and promotes DNA repair and cell survival through temporal transcriptional regulation. These studies identify the circadian factor CRY1 as pro-tumorigenic and nominate CRY1 as a new therapeutic target., Cryptochrome 1 (CRY1) is a transcriptional coregulator associated with the circadian clock. Here the authors reveal that CRY1 is hormone-regulated, stabilized by genomic insult, and promotes DNA repair and cell survival through temporal transcriptional regulation.

Emerging evidence indicates the deubiquitinase USP22 regulates transcriptional activation and modification of target substrates to promote pro-oncogenic phenotypes. Here, in vivo characterization of tumor-associated USP22 upregulation and unbiased interrogation of USP22-regulated functions in vitro demonstrated critical roles for USP22 in prostate cancer. Specifically, clinical datasets validated that USP22 expression is elevated in prostate cancer, and a novel murine model demonstrated a hyperproliferative phenotype with prostate-specific USP22 overexpression. Accordingly, upon overexpression or depletion of USP22, enrichment of cell-cycle and DNA repair pathways was observed in the USP22-sensitive transcriptome and ubiquitylome using prostate cancer models of clinical relevance. Depletion of USP22 sensitized cells to genotoxic insult, and the role of USP22 in response to genotoxic insult was further confirmed using mouse adult fibroblasts from the novel murine model of USP22 expression. As it was hypothesized that USP22 deubiquitylates target substrates to promote protumorigenic phenotypes, analysis of the USP22-sensitive ubiquitylome identified the nucleotide excision repair protein, XPC, as a critical mediator of the USP22-mediated response to genotoxic insult. Thus, XPC undergoes deubiquitylation as a result of USP22 function and promotes USP22-mediated survival to DNA damage. Combined, these findings reveal unexpected functions of USP22 as a driver of protumorigenic phenotypes and have significant implications for the role of USP22 in therapeutic outcomes. Significance: The studies herein present a novel mouse model of tumor-associated USP22 overexpression and implicate USP22 in modulation of cellular survival and DNA repair, in part through regulation of XPC.

The circadian clock is a master regulator of mammalian physiology, regulating daily oscillations of crucial biological processes and behaviors. Notably, circadian disruption has recently been identified as an independent risk factor for cancer and classified as a carcinogen. As such, it is imperative to discern the underpinning mechanisms by which circadian disruption alters cancer risk. Emergent data, reviewed herein, demonstrate that circadian regulatory functions play critical roles in several hallmarks of cancer, including control of cell proliferation, cell death, DNA repair, and metabolic alteration. Developing a deeper understanding of circadian-cancer regulation cross-talk holds promise for developing new strategies for cancer interception, prevention, and management.

PARP‐1 holds major functions on chromatin, DNA damage repair and transcriptional regulation, both of which are relevant in the context of cancer. Here, unbiased transcriptional profiling revealed the downstream transcriptional profile of PARP‐1 enzymatic activity. Further investigation of the PARP‐1‐regulated transcriptome and secondary strategies for assessing PARP‐1 activity in patient tissues revealed that PARP‐1 activity was unexpectedly enriched as a function of disease progression and was associated with poor outcome independent of DNA double‐strand breaks, suggesting that enhanced PARP‐1 activity may promote aggressive phenotypes. Mechanistic investigation revealed that active PARP‐1 served to enhance E2F1 transcription factor activity, and specifically promoted E2F1‐mediated induction of DNA repair factors involved in homologous recombination (HR). Conversely, PARP‐1 inhibition reduced HR factor availability and thus acted to induce or enhance “BRCA‐ness”. These observations bring new understanding of PARP‐1 function in cancer and have significant ramifications on predicting PARP‐1 inhibitor function in the clinical setting.

Purpose: DNA-dependent protein kinase catalytic subunit (DNA-PK) is a pleiotropic kinase involved in DNA repair and transcriptional regulation. DNA-PK is deregulated in selected cancer types and is strongly associated with poor outcome. The underlying mechanisms by which DNA-PK promotes aggressive tumor phenotypes are not well understood. Here, unbiased molecular investigation in clinically relevant tumor models reveals novel functions of DNA-PK in cancer. Experimental Design: DNA-PK function was modulated using both genetic and pharmacologic methods in a series of in vitro models, in vivo xenografts, and patient-derived explants (PDE), and the impact on the downstream signaling and cellular cancer phenotypes was discerned. Data obtained were used to develop novel strategies for combinatorial targeting of DNA-PK and hormone signaling pathways. Results: Key findings reveal that (i) DNA-PK regulates tumor cell proliferation; (ii) pharmacologic targeting of DNA-PK suppresses tumor growth both in vitro, in vivo, and ex vivo; (iii) DNA-PK transcriptionally regulates the known DNA-PK–mediated functions as well as novel cancer-related pathways that promote tumor growth; (iv) dual targeting of DNA-PK/TOR kinase (TORK) transcriptionally upregulates androgen signaling, which can be mitigated using the androgen receptor (AR) antagonist enzalutamide; (v) cotargeting AR and DNA-PK/TORK leads to the expansion of antitumor effects, uncovering the modulation of novel, highly relevant protumorigenic cancer pathways; and (viii) cotargeting DNA-PK/TORK and AR has cooperative growth inhibitory effects in vitro and in vivo. Conclusions: These findings uncovered novel DNA-PK transcriptional regulatory functions and led to the development of a combinatorial therapeutic strategy for patients with advanced prostate cancer, currently being tested in the clinical setting.

DNA dependent protein kinase catalytic subunit (DNA-PK) is a multifunctional kinase involved in repairing double-strand DNA breaks through non-homologous end joining (NHEJ), with parallel roles as a master transcriptional regulator of gene networks supporting cell migration and invasion. In prostate cancer (PCa), DNA-PK was found to be highly upregulated and hyperactivated. Strikingly, DNA-PK is the most deregulated kinase in metastatic castration resistant prostate cancer (CRPC), and is independently predictive of metastasis and overall survival in patients with high-risk disease. Combined, these findings highlight the importance of understanding DNA-PK functions beyond DNA repair and transcriptional regulation, which promote the acquisition of aggressive tumor phenotypes. Rapid immunoprecipitation of endogenous proteins (RIME) was performed to identify the DNA-PK interactome, which uncovered novel DNA-PK interactors that play key roles in metabolism. To assess the role of DNA-PK in regulation of metabolism, metabolic profiles of CRPC models were investigated upon DNA-PK modulation. Data presented will describe novel roles of DNA-PK in cancer metabolism and subsequent effects of DNA-PK modulation in key metabolic pathways. Citation Format: Emanuela Dylgjeri, Jonathan Goodwin, Ayesha Shafi, Vishal Kothari, Giorgia Zadra, Erin Seifert, Felix Feng, Karen Knudsen. Novel roles of DNA-PK in metabolic regulation in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1851.

326 Background: The administration of ascorbate has proved lethal to and highly selective for a variety of cancer cell types; however, despite an increasingly impressive body of evidence, there has not been a robust effort to translate the observed in vitro and in vivo outcomes to the clinic. This is partially due to the fact that the mechanism by which ascorbate exerts its anti-cancer effect is still under investigation. A simplified model depicts ascorbate as a pro-drug for reactive oxygen species (ROS), which accumulate intracellularly and generate DNA damage. It was therefore hypothesized that poly(ADP-ribose) polymerase (PARP) inhibitors (PARPi), by inhibiting DNA damage repair, would augment the toxicity of ascorbate. Methods: In vitro and in vivo models systems queried for anti-tumor effects of PARP inhibitors and ascorbate. Results: Two distinct castration-resistant prostate cancer (CRPC) models were sensitive to ascorbate at physiologically attainable concentrations. These in vitro models were then subjected to treatment with three different PARP inhibitors (olaparib, niraparib, and talazoparib) alone and in combination with ascorbate. The addition of a sub-lethal dose of ascorbate significantly increased cell death across a range of doses for all three PARP inhibitors. A combination index was generated for olaparib and ascorbate in both CRPC models; the results suggest a strongly synergistic relationship between olaparib and ascorbate. Use of a CRPC in vivo model demonstrated that the combination of olaparib and ascorbate significantly increased tumor doubling time compared to vehicle controls and monotherapy. This in vivo efficacy was even more profound in an additional model using castrated mice to mimic the effect of hormone therapy. Additional mechanistic studies are in progress to further investigate the potential for ascorbate and olaparib combination therapy. Conclusions: Ultimately, these data suggest the combination of ascorbate and PARP inhibitors could be an effective treatment for CRPC.

Androgen ablation therapy represents the first line of therapeutic intervention in men with advanced or recurrent prostate tumors. However, the incomplete efficacy and lack of durable response to this clinical strategy highlights an urgent need for alternative treatment options to improve patient outcomes. Targeting the molecular chaperone heat shock protein 90 (Hsp90) represents a potential avenue for therapeutic intervention as its inhibition results in the coordinate blockade of multiple oncogenic signaling pathways in cancer cells. Moreover, Hsp90 is essential for the stability and function of numerous client proteins, a number of which have been causally implicated in the pathogenesis of prostate cancer, including the androgen receptor (AR). Here, we examined the preclinical activity of ganetespib, a small molecule inhibitor of Hsp90, in a panel of prostate cancer cell lines. Ganetespib potently decreased viability in all lines, irrespective of their androgen sensitivity or receptor status, and more effectively than the ansamycin inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG). Interestingly, while ganetespib exposure decreased AR expression and activation, the constitutively active V7 truncated isoform of the receptor was unaffected by Hsp90 inhibition. Mechanistically, ganetespib exerted concomitant effects on mitogenic and survival pathways, as well as direct modulation of cell cycle regulators, to induce growth arrest and apoptosis. Further, ganetespib displayed robust antitumor efficacy in both AR-negative and positive xenografts, including those derived from the 22Rv1 prostate cancer cell line that co-expresses full-length and variant receptors. Together these data suggest that further investigation of ganetespib as a new therapeutic treatment for prostate cancer patients is warranted.

Metastatic prostate cancer (PCa) is primarily an androgen-dependent disease, which is treated with androgen deprivation therapy (ADT). Tumors usually develop resistance (castration-resistant PCa [CRPC]), but remain androgen receptor (AR) dependent. Numerous mechanisms for AR-dependent resistance have been identified including expression of constitutively active AR splice variants lacking the hormone-binding domain. Recent clinical studies show that expression of the best-characterized AR variant, AR-V7, correlates with resistance to ADT and poor outcome. Whether AR-V7 is simply a constitutively active substitute for AR or has novel gene targets that cause unique downstream changes is unresolved. Several studies have shown that AR activation alters cell metabolism. Using LNCaP cells with inducible expression of AR-V7 as a model system, we found that AR-V7 stimulated growth, migration, and glycolysis measured by ECAR (extracellular acidification rate) similar to AR. However, further analyses using metabolomics and metabolic flux assays revealed several differences. Whereas AR increased citrate levels, AR-V7 reduced citrate mirroring metabolic shifts observed in CRPC patients. Flux analyses indicate that the low citrate is a result of enhanced utilization rather than a failure to synthesize citrate. Moreover, flux assays suggested that compared to AR, AR-V7 exhibits increased dependence on glutaminolysis and reductive carboxylation to produce some of the TCA (tricarboxylic acid cycle) metabolites. These findings suggest that these unique actions represent potential therapeutic targets.

Prostatic adenocarcinoma (PCa) is dependent on androgen receptor (AR) signaling at all stages of disease, as AR activation induces both cell proliferation and survival. Though organ–confined disease can be treated, the response is transient. Reactivation of AR leads to castration resistant prostate cancer (CRPC), which remains fatal. Previously published studies demonstrate that AR activation promotes tumor cell survival and proliferation through a feed-forward loop involving the DNA repair factor DNA-dependent protein kinase (DNA-PK). Recent data from our lab has highlighted the role of DNA-PK in DNA damage repair as well as transcriptional regulation of pro-metastatic signaling. Strikingly, DNA-PK is the most deregulated kinase in metastatic CRPC. DNAPK is highly elevated and hyperactivated, and is an independent predictor of metastasis and overall survival in patients with high-risk disease. Concordantly, DNAPK suppression attenuated DNA-PK functions in NHEJ and transcriptional regulation. DNA-PK inhibition is sufficient to prevent proliferation in vitro and ex vivo, as well as tumor metastasis in vivo. Combined these findings highlight importance of DNA-PK functions as a transcriptional regulator and a mediator of NHEJ DNA repair and nominate it as a therapeutic target in PCa. Data presented here will interrogate the translational capacity of a specific DNA-PK inhibitor, a dual DNA-PK/TOR kinase inhibitor and a specific TOR kinase inhibitor in CRPC models. Currently, CC-115, a dual kinase inhibitor, is the only DNA-PK inhibitor in Phase1/2 trial in combination with Enzalutamide in PCa. Unbiased transcriptomic analyses were performed and will be used to identify mechanisms of action that lead to suppression of PCa growth in vitro and ex vivo. These findings will provide insight in the effectiveness of DNA-PK inhibitors as a single agent or combinatorial treatments and provide rational for its placement in the correct clinical space. Citation Format: Emanuela Dylgjeri, Jonathan F. Goodwin, Christopher M. McNair, Ayesha A. Shafi, Vishal Kothari, Felix Feng, Dana Rathkop, Karen Knudsen. Preclinical evaluation of DNA-PK as a therapeutic target in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-264. doi:10.1158/1538-7445.AM2017-LB-264

Prostate cancer (PCa) is the most common non-cutanenous cancer and the third leading cause of cancer-related death in American men. Androgen receptor (AR) is a hormone-activated transcription factor that plays an important role in both the development and progression of PCa. Androgen deprivation therapy is a common first-line therapy for disseminated disease. However, virtually all tumors become resistant to such therapy and the tumor recurs. Thus, there is a vital need for the development of novel, more effective drugs. One major hurdle in this aspect is the lack of adequate preclinical models. Current models do not effectively recapitulate the heterogeneity and the microenvironment of human PCa tumors, significantly hindering the ability to accurately predict therapeutic response. Our collaborative group has utilized and characterized a method to culture patient tumors ex vivo, termed Patient Derived eXplant (PDeX). This approach maintains the integrity of the native tumor microenvironment, tumor tissue morphology, and molecular signaling. Importantly, our PDeX model can be manipulated both chemically (drugs/compounds) and genetically (shRNA) in order to determine specific reactions and mechanisms of response on individual tumor growth. Furthermore, with this model we can quantitatively assess drug efficacy on numerous parameters (i.e. AR levels, Ki67 staining, apoptosis screening, and desmoplasmic indices). Data to be discussed will assess the variances in response to AR-directed therapeutics and underlying mechanisms of action, while also utilizing TME characteristics as a means to predict response to therapy. In addition, we can potentially identify clinically relevant subpopulations of patients and molecularly profile their cultured tissue to uncover new pathways for therapeutic intervention. Thus, the PDeX model allows for a comprehensive evaluation of individual tumors in their native microenvironment to ultimately develop more effective therapies. This study will have transformative clinical impact discerning novel metrics for the inclusion of precision medicine for advanced PCa. Citation Format: Ayesha A. Shafi, Matthew J. Schiewer, Renee de Leeuw, Peter A. McCue, Leonard G. Gomella, Costas D. Lallas, Edouard J. Trabulsi, Theresa Hickey, Neelima Shah, Edna Cukierman, Lisa M. Butler, Wayne Tilley, Ganesh Raj, Karen E. Knudsen. PDeX (Patient Derived eXplant) models to determine the basis for response to targeted agents in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-109. doi:10.1158/1538-7445.AM2017-LB-109

Prostate cancer (PCa) is an androgen-dependent disease, and tumors that are resistant to androgen ablation therapy often remain androgen receptor (AR) dependent. Among the contributors to castration-resistant PCa are AR splice variants that lack the ligand-binding domain (LBD). Instead, they have small amounts of unique sequence derived from cryptic exons or from out of frame translation. The AR-V7 (or AR3) variant is constitutively active and is expressed under conditions consistent with CRPC. AR-V7 is reported to regulate a transcriptional program that is similar but not identical to that of AR. However, it is unknown whether these differences are due to the unique sequence in AR-V7, or simply to loss of the LBD. To examine transcriptional regulation by AR-V7, we have used lentiviruses encoding AR-V7 (amino acids 1-627 of AR with the 16 amino acids unique to the variant) to prepare a derivative of the androgen-dependent LNCaP cells with inducible expression of AR-V7. An additional cell line was generated with regulated expression of AR-NTD (amino acids 1-660 of AR); this mutant lacks the LBD but does not have the AR-V7 specific sequence. We find that AR and AR-V7 have distinct activities on target genes that are co-regulated by FOXA1. Transcripts regulated by AR-V7 were similarly regulated by AR-NTD, indicating that loss of the LBD is sufficient for the observed differences. Differential regulation of target genes correlates with preferential recruitment of AR or AR-V7 to specific cis-regulatory DNA sequences providing an explanation for some of the observed differences in target gene regulation.

In the United States, prostate cancer (PCa) is the most commonly diagnosed non-cutaneous cancer in males and the second leading cause of cancer-related death for men. The prostate is an androgen-dependent organ and PCa is an androgen-dependent disease. Androgen action is mediated by the androgen receptor (AR), a hormone activated transcription factor. The primary treatment for metastatic PCa is androgen deprivation therapy (ADT). For the most part, tumors respond to ADT, but most become resistant to therapy within two years. There is persuasive evidence that castration resistant (also termed castration recurrent) PCa (CRPC) remains AR dependent. Recent studies have shown that there are numerous factors that contribute to AR reactivation despite castrate serum levels of androgens. These include changes in AR expression and structure through gene amplification, mutation, and alternative splicing. Changes in steroid metabolism, cell signaling, and coregulator proteins are also important contributors to AR reactivation in CRPC. Most AR targeted therapies have been directed at the hormone binding domain. The finding that constitutively active AR splice variants that lack the hormone binding domain are frequently expressed in CRPC highlights the need to develop therapies that target other portions of AR. In this review, the role of AR in normal prostate, in PCa, and particularly the mechanisms for its reactivation subsequent to ADT are summarized. In addition, recent clinical trials and novel approaches to target AR are discussed.

Prostate cancer (PCa) is the most common non-cutanenous cancer and the second leading cause of cancer-related death in American men. Androgen receptor (AR) is a hormone-activated transcription factor that plays an important role in both the development and progression of PCa. Androgen deprivation therapy is a common first-line therapy. However, many patients become resistant to such therapy and the tumor recurs. Thus, there is a vital need for the development of novel, more effective drugs. One major hurdle in this aspect is the lack of adequate preclinical models. Current models do not effectively recapitulate the heterogeneity and the microenvironment of human PCa tumors hindering the ability to accurately predict therapeutic response. Our lab has generated a method to culture patient tumors ex vivo termed patient derived explant (PDeX). This approach maintains the integrity of the native tumor microenvironment, tumor tissue morphology, and molecular signaling. Intriguingly, our PDEX model can be targeted with various therapeutic agents or lentiviral-based shRNAs to determine specific responses on individual tumor growth. Furthermore, with this model we can quantitatively assess drug efficacy on numerous parameters (i.e. AR levels, Ki67 staining, apoptosis screening). Data to be discussed will assess the variances in response to AR-directed therapeutics and underlying mechanisms of action. In addition, we can potentially identify clinically relevant subpopulations of patients and molecularly profile their cultured tissue to uncover new pathways for therapeutic intervention. Thus, the PDeX model allows for a comprehensive evaluation of individual tumors in their native microenvironment to ultimately develop more effective therapies. Citation Format: Ayesha Shafi, Matthew Schiewer, Renee de Leeuw, Peter McCue, Ruth Birbe, Leonard Gomella, Costas Lallas, Edouard Trabulsi, Ganesh Raj, Karen Knudsen. Developing PDeX (Patient Derived Explant) to determine the basis for response to AR-directed therapeutics. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr A12.

Androgen ablation therapy is the most common treatment for advanced prostate cancer (PCa), but most patients will develop castration-resistant prostate cancer (CRPC), which has no cure. CRPC is androgen-depletion resistant but androgen receptor (AR) dependent. AR is a nuclear receptor whose transcriptional activity is regulated by hormone binding to the ligand-binding domain (LBD). Constitutively active AR splice variants that lack LBDs often are expressed in CRPC. The expression of these variants indicates that methods to inhibit AR activity that do not rely on inactivating the LBD are needed. Heat shock protein 90 (Hsp90), a potential therapeutic target in PCa, is an AR chaperone crucial for proper folding, hormone binding and transcriptional activity of AR. We generated LNCaP cell lines with regulated expression of the AR-V7 variant as well as a cell line expressing artificially truncated AR (termed AR-NTD) to characterize splice variant function. Using an Hsp90 inhibitor, Geldanamycin (GA), and an AR-Hsp90-FKBP52 specific inhibitor, MJC13, we sought to determine if the AR variants also require Hsp90 and associated co-chaperone, FKBP52, for their activity. GA inhibits AR transcriptional activity but has little effect on AR-V7 activity. Moreover, GA decreases the stability of AR protein, with no effect on AR-V7 levels. Full-length AR activity is strongly inhibited by MJC13 while AR-V7 is unaffected. Thus, the variants are resistant to inhibitors of the Hsp90-AR heterocomplex. Although Hsp90 inhibitors will continue to inhibit growth promoting kinases and signaling through activated full-length AR in CRPC, AR signaling through variants will be retained.