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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

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.

Prostatic adenocarcinoma (PCa) is the most frequently diagnosed non-cutaneous malignancy, and the third leading cause of cancer death in males in the United States. A crucial component to the development and progression of PCa is the activity of the androgen receptor (AR). As such, targeting the AR-signaling axis through androgen deprivation therapy (ADT) is the first line of therapy for PCa. However, cells invariably become resistant to this therapy and men relapse with the incurable form of the disease termed, castration-resistant prostate cancer (CRPC). In addition to AR, another principal component aiding in the progression of disease is the retinoblastoma tumor suppressor protein (RB). RB functions to repress tumor development by negatively regulating the activity of the E2F family of transcription factors, preventing cell cycle progression. RB is lost in roughly 30% of CRPC tumors and is sufficient to induce a CRPC phenotype in hormone-sensitive cells under ADT conditions. Analyses into the molecular significance of RB loss on disease progression identified a potential cooperation between AR and the RB-E2F1 signaling axis. Biological assessment performed in isogenic RB knockdown (i.e. hormone-sensitive and CRPC models) identified distinct functional consequences of RB loss depending on AR status and disease state. Transcriptome analysis identified divergently regulated gene signatures between disease stages in the presence of AR activation, which were not seen under ADT conditions, implicating a unique role of AR in transcriptional regulation with the loss of RB. Data to be discussed will also include further comparison of the E2F1 and AR cistromes in the absence of RB to identify the mechanism by which the AR-RB-E2F1 signaling axis function in promoting the progression of disease. Citation Format: Amy C. Mandigo, Chris McNair, Matthew J. Schiewer, Karen E. Knudsen. RB loss reprograms AR and E2F1 signaling in models of prostate cancer progression [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 5217.

269 Background: PARP-1 holds major functions on chromatin, DNA damage repair and transcriptional regulation, both of which are relevant in the context of cancer. Previously, it was determined that PARP-1 ins involved in regulation of androgen receptor activity. Methods: Here, unbiased transcriptional profiling revealed the downstream transcriptional profile of PARP-1 enzymatic activity.Results: 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”. Conclusions: 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.

The retinoblastoma tumor suppressor (RB) is mechanistically linked to suppression of E2F1-mediated cell cycle regulation. Abrogation of RB function is associated with poor clinical outcome across various tumor types, which frequently elicit a preference for either RB depletion or functional inactivation, yet the basis for selectivity is unknown. Here, examination of RB pathway alterations in advanced prostate cancer revealed that cyclin dependent kinase (CDK)/cyclin/CDKi alterations are infrequent, and identify RB loss as the major mechanism of pathway disruption in human disease. Furthermore, RB status was readily traced through cell-free DNA analyses in human specimens, thus identifying new ways to assign RB status in the clinical setting. Strikingly, RB depletion in human disease was not associated with a higher Ki67 index, indicating a role for the RB/E2F1 pathway in regulating processes distinct from cell cycle control and associated with lethal-stage disease. Subsequent mechanistic investigation utilized isogenic prostate cancer models, wherein RB could be differentially inactivated through depletion or through hormone-induced, CDK-mediated phosphorylation. Unbiased molecular interrogation uncovered a novel E2F1 cistrome and downstream engagement of transcriptional networks exclusively observed after RB loss, with binding specificity divergent from canonically described E2F1 binding patterns. Additionally, E2F1 cistrome alterations elicited by RB depletion were seen to be distinct from those after phosphorylation-induced RB functional inactivation, providing needed insight into the basis of selectivity for RB loss versus CDK-mediated inactivation observed in human disease. Analyses of human CRPC tumor samples further underscored the clinical relevance of RB loss-induced gene expression programs, which were significantly correlated with reprogrammed E2F1 binding identified herein. Taken together, the studies presented are the first to identify the consequences of RB loss, demonstrating molecular distinction from RB inactivation and illustrating the clinical relevance of RB loss-induced E2F rewiring. Citation Format: Christopher McNair, Kexin Xu, Amy C. Mandigo, Matteo Benelli, Benjamin Leiby, Daniel Rodrigues, Johan Lindberg, Henrik Gronberg, Bram De Laere, Luc Dirix, Tapio Visakorpi, Fugen Li, Felix Y. Feng, Johann de Bono, Francesca Demichelis, Mark A Rubin, Myles Brown, Karen E. Knudsen. Differential impact of RB pathway status on E2F1 reprogramming and disease progression in human prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr IA03.

Prostate adenocarcinoma (PCa) is the most highly diagnosed non-cutaneous malignancy in the United States and the third most lethal among men in this category. First line therapies targeting disruption of the androgen receptor (AR) signaling axis are initially effective, however within 2-3 years patients commonly relapse, due in part to reactivation of the receptor, and progress to the ultimately fatal form of the disease (castrate resistant prostate cancer, CRPC). Interestingly, recent examination of CRPC identified a major mechanism of progression as loss of the retinoblastoma (RB) protein. Along with its critical role in controlling cell cycle progression, RB is known to have important tumor suppressor functions, and has been shown in PCa to be lost exclusively in late stage disease. Additionally, loss of RB has been shown to correlate with increases in both AR and E2F1 expression, via E2F dependent mechanisms. Despite the vital role RB loss has been shown to play in this fatal stage of disease, the molecular underpinnings remain undefined. Thus, in order to elucidate these CRPC specific alterations, the current study utilizes isogenic models of RB loss in combination with genome wide binding and transcriptional studies. Data presented herein demonstrate that loss of RB is frequent in CRPC, however this phenomenon is not correlated with changes in proliferative indices, suggested a role for RB loss outside of cell cycle control. Further, RB loss induces genome wide transcriptional changes in PCa cells in castrate conditions, including up-regulation in Myc, E2F, and DNA-repair related pathways. Additionally, RB loss significantly expands E2F1 binding capacity, while maintaining the majority of the RB-intact E2F1 cistrome. Strikingly, while the current RB/E2F1 paradigm suggests that E2F1 exclusively occupies promoter regions of DNA in order to regulate transcriptional changes, RB loss induces sweeping reprogramming of E2F1 occupied regions, with a marked increase in enhancer-bound E2F1. Further, motif analyses suggest divergence away from canonical E2F1 binding motifs after RB loss specifically in regions of expanded E2F1 binding, and additionally suggest likely interaction of novel E2F1 co-factors under RB loss conditions. Finally, interrogation of a CRPC patient tumor cohort showed predictive capacity for RB-loss induced E2F1 binding and differentially expressed gene signature to select for RB loss in patient samples, indicating a novel E2F1 driven set of targets vital for CRPC transition in human disease. Together, these data present the first insight into E2F1 activity resulting from RB loss, and the role these changes play in progression to CRPC. Citation Format: Christopher McNair, Kexin Xu, Amy Mandigo, Matteo Benelli, Benjamin Leiby, Daniel Rodrigues, Johan Lindberg, Henrik Gronberg, Tapio Visakorpi, Fugen Li, Johann De Bono, Francesca Demichelis, Mark Rubin, Myles Brown, Karen E. Knudsen. RB loss-induced genome wide E2F1 reprogramming drive advanced 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-085. doi:10.1158/1538-7445.AM2017-LB-085