Jonathan F. Goodwin

Dual roles of PARP-1 promote cancer growth and progression

UNLABELLED PARP-1 is an abundant nuclear enzyme that modifies substrates by poly(ADP-ribose)-ylation. PARP-1 has well-described functions in DNA damage repair and also functions as a context-specific regulator of transcription factors. With multiple models, data show that PARP-1 elicits protumorigenic effects in androgen receptor (AR)-positive prostate cancer cells, in both the presence and absence of genotoxic insult. Mechanistically, PARP-1 is recruited to sites of AR function, therein promoting AR occupancy and AR function. It was further confirmed in genetically defined systems that PARP-1 supports AR transcriptional function, and that in models of advanced prostate cancer, PARP-1 enzymatic activity is enhanced, further linking PARP-1 to AR activity and disease progression. In vivo analyses show that PARP-1 activity is required for AR function in xenograft tumors, as well as tumor cell growth in vivo and generation and maintenance of castration resistance. Finally, in a novel explant system of primary human tumors, targeting PARP-1 potently suppresses tumor cell proliferation. Collectively, these studies identify novel functions of PARP-1 in promoting disease progression, and ultimately suggest that the dual functions of PARP-1 can be targeted in human prostate cancer to suppress tumor growth and progression to castration resistance. SIGNIFICANCE These studies introduce a paradigm shift with regard to PARP-1 function in human malignancy, and suggest that the dual functions of PARP-1 in DNA damage repair and transcription factor regulation can be leveraged to suppress pathways critical for promalignant phenotypes in prostate cancer cells by modulation of the DNA damage response and hormone signaling pathways. The combined studies highlight the importance of dual PARP-1 function in malignancy and provide the basis for therapeutic targeting.

A Hormone–DNA Repair Circuit Governs the Response to Genotoxic Insult

UNLABELLED Alterations in DNA repair promote tumor development, but the impact on tumor progression is poorly understood. Here, discovery of a biochemical circuit linking hormone signaling to DNA repair and therapeutic resistance is reported. Findings show that androgen receptor (AR) activity is induced by DNA damage and promotes expression and activation of a gene expression program governing DNA repair. Subsequent investigation revealed that activated AR promotes resolution of double-strand breaks and resistance to DNA damage both in vitro and in vivo. Mechanistically, DNA-dependent protein kinase catalytic subunit (DNAPKcs) was identified as a key target of AR after damage, controlling AR-mediated DNA repair and cell survival after genotoxic insult. Finally, DNAPKcs was shown to potentiate AR function, consistent with a dual role in both DNA repair and transcriptional regulation. Combined, these studies identify the AR-DNAPKcs circuit as a major effector of DNA repair and therapeutic resistance and establish a new node for therapeutic intervention in advanced disease. SIGNIFICANCE The present study identifies for the fi rst time a positive feedback circuit linking hormone action to the DNA damage response and shows the significant impact of this process on tumor progression and therapeutic response. These provocative findings provide the foundation for development of novel nodes of therapeutic intervention for advanced disease.

Targeting cell cycle and hormone receptor pathways in cancer

The cyclin/cyclin-dependent kinase (CDK)/retinoblastoma (RB)-axis is a critical modulator of cell cycle entry and is aberrant in many human cancers. New nodes of therapeutic intervention are needed that can delay or combat the onset of malignancies. The antitumor properties and mechanistic functions of PD-0332991 (PD; a potent and selective CDK4/6 inhibitor) were investigated using human prostate cancer (PCa) models and primary tumors. PD significantly impaired the capacity of PCa cells to proliferate by promoting a robust G1-arrest. Accordingly, key regulators of the G1-S cell cycle transition were modulated including G1 cyclins D, E and A. Subsequent investigation demonstrated the ability of PD to function in the presence of existing hormone-based regimens and to cooperate with ionizing radiation to further suppress cellular growth. Importantly, it was determined that PD is a critical mediator of PD action. The anti-proliferative impact of CDK4/6 inhibition was revealed through reduced proliferation and delayed growth using PCa cell xenografts. Finally, first-in-field effects of PD on proliferation were observed in primary human prostatectomy tumor tissue explants. This study shows that selective CDK4/6 inhibition, using PD either as a single-agent or in combination, hinders key proliferative pathways necessary for disease progression and that RB status is a critical prognostic determinant for therapeutic efficacy. Combined, these pre-clinical findings identify selective targeting of CDK4/6 as a bona fide therapeutic target in both early stage and advanced PCa and underscore the benefit of personalized medicine to enhance treatment response.

Beyond DNA repair: DNA-PK function in cancer

UNLABELLED The DNA-dependent protein kinase (DNA-PK) is a pivotal component of the DNA repair machinery that governs the response to DNA damage, serving to maintain genome integrity. However, the DNA-PK kinase component was initially isolated with transcriptional complexes, and recent findings have illuminated the impact of DNA-PK-mediated transcriptional regulation on tumor progression and therapeutic response. DNA-PK expression has also been correlated with poor outcome in selected tumor types, further underscoring the importance of understanding its role in disease. Herein, the molecular and cellular consequences of DNA-PK are considered, with an eye toward discerning the rationale for therapeutic targeting of DNA-PK. SIGNIFICANCE Although DNA-PK is classically considered a component of damage response, recent findings illuminate damage-independent functions of DNA-PK that affect multiple tumor-associated pathways and provide a rationale for the development of novel therapeutic strategies.

USP22 regulates oncogenic signaling pathways to drive lethal cancer progression.

Increasing evidence links deregulation of the ubiquitin-specific proteases 22 (USP22) deubitiquitylase to cancer development and progression in a select group of tumor types, but its specificity and underlying mechanisms of action are not well defined. Here we show that USP22 is a critical promoter of lethal tumor phenotypes that acts by modulating nuclear receptor and oncogenic signaling. In multiple xenograft models of human cancer, modeling of tumor-associated USP22 deregulation demonstrated that USP22 controls androgen receptor accumulation and signaling, and that it enhances expression of critical target genes coregulated by androgen receptor and MYC. USP22 not only reprogrammed androgen receptor function, but was sufficient to induce the transition to therapeutic resistance. Notably, in vivo depletion experiments revealed that USP22 is critical to maintain phenotypes associated with end-stage disease. This was a significant finding given clinical evidence that USP22 is highly deregulated in tumors, which have achieved therapeutic resistance. Taken together, our findings define USP22 as a critical effector of tumor progression, which drives lethal phenotypes, rationalizing this enzyme as an appealing therapeutic target to treat advanced disease.

Abstract LB-264: Preclinical evaluation of DNA-PK as a therapeutic target in prostate cancer

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

Abstract 1217: Determining the impact of CRPC-specific p53 mutation on therapeutic response and prostate tumor progression

Prostatic adenocarcinoma (PCa) is the most frequently diagnosed non-cutaneous malignancy and second leading cause of cancer death in men in the US. Although organ-confined disease is manageable, treatment options for disseminated PCa are limited. First-line therapy for metastatic PCa targets the androgen receptor (AR) via androgen deprivation therapy (ADT); however, tumors often recur, displaying reactivation of AR signaling despite continued therapeutic targeting. There is currently no durable treatment for this advanced disease stage, termed castrate-resistant PCa (CRPC). While most localized PCa express wild-type p53, recent high-profile, genome-wide studies identified increased TP53 gene mutation rates in advanced disease (specifically CRPC). Using these data, we noted that the patient-derived, CRPC-specific p53 mutations observed are relatively unique to prostate cancer. These mutations occur in “hotspot” clusters altered in other tumor types, but represent distinct amino acid changes seemingly unique to prostate cancer. While the underlying consequences of these CRPC-specific p53 mutations are unknown, it was hypothesized that their presence in the DNA-binding domain of p53 protein may represent “gain-of-function” (GOF) alterations that direct the transcription factor to alternative, pro-tumorigenic gene targets. Our preliminary findings strongly support this contention and reveal that these p53 mutations are major drivers of CRPC phenotypes. Specifically, our findings demonstrate that mutant p53: 1. Selectively alter p53 activity, based on analyses of PCa-relevant p53 target genes; 2. Compromise DNA damage response; 3. Alter AR signaling and response to AR-directed therapeutics; and 4. Promote transition to castration-resistance. These data are further supported by recent longitudinal studies of patient progression, wherein the R248Q mutation was found to be a key genetic alteration identifying lethal disease. While future investigation will be directed to better understand the specific contribution of individual DDR mutations, it is clear that deregulation of DNA damage repair and response pathways is likely to underlie increased genomic instability that is observed among advanced prostate cancers. Citation Format: Jeffry L. Dean, Jennifer K. Jones, Jonathan F. Goodwin, Karen E. Knudsen. Determining the impact of CRPC-specific p53 mutation on therapeutic response and prostate tumor progression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1217. doi:10.1158/1538-7445.AM2015-1217

Abstract 1859: Discerning the molecular basis of DNA-PK pro-tumorigenic functions and translational capacity as a therapeutic target in prostate cancer

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 and reactivation of AR results in incurable state of disease (castration resistant prostate cancer, CRPC). Thus, there is a significant need to discern the mechanisms by which aberrant AR activity arises, and to develop new means to clinically target advanced disease. Recently, AR activation was found to promote tumor cell survival and proliferation through a feed-forward loop involving the repair factor DNA-dependent protein kinase (DNA-PK). Emerging new data correlates elevated DNA-PK expression with poor prognosis and identifies DNA-PK as a driver of metastatic signaling. Although the role of DNA-PK activity in DNA repair is well characterized, the underpinning mechanisms and consequences of DNA-PK in transcriptional regulation are understudied. Data to be discussed will build on our new preliminary DNA-PK interactome data to identify sequence-specific transcription factors found in complex with DNA-PK. In order to molecularly dissect DNA-PK transcriptional events, the DNA-PK cistrome and transcriptome will be defined using clinically relevant models, resulting in a detailed understanding of DNA-PK mediated transcriptional regulation. The correlation of high expression of DNA-PK with poor prognosis in advanced PCa, as well as the known roles of DNA-PK in cancer-relevant pathways make DNA-PK a viable therapeutic target for PCa. Despite the current clinical assessment of DNA-PK inhibitors in multiple tumor types, there are no existing trials targeting PCa. Additional studies that assess the efficacy of current DNA-PK inhibitors in PCa will be presented and used to determine the best clinical space for their use. Citation Format: Emanuela Dylgjeri, Jonathan Goodwin, Karen Knudsen. Discerning the molecular basis of DNA-PK pro-tumorigenic functions and translational capacity as a therapeutic target in prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1859. doi:10.1158/1538-7445.AM2015-1859

Abstract 1844: Genome wide analysis of AR-cell cycle interplay reveals novel functions in cancer

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA The androgen receptor (AR) plays a vital role in prostate cancer (PCa) due in part to its ability to interact with cell cycle components in order to drive cell cycle transition. Numerous points of cross-talk have been identified, wherein specific components of the cell cycle machinery “feed back” to modulate AR function, and these interactions are thought to be altered in human malignancies. Despite these observations, the majority of genome wide analyses for AR have been performed in cells that have exited cell cycle (G0). Here, the cell cycle dependent AR transcriptome and cistrome was identified, revealing new and unexpected functions for AR in cycling tumor cells. In studies to be discussed, cells were arrested in 5 distinct phases of the cell cycle, stimulated with androgen, and AR activity assessed through gene expression and ChIP-Seq analyses. In AR binding analyses, significant overlap was seen with previously identified sites, but were accompanied by novel binding events that could be segregated into those that are specific to cycling cells and occur in all phases (“cell cycle common”) or show cell cycle stage specific binding (“phase exclusive”). Over 50% of the cell cycle common sites, and up to 95% of the phases exclusive sites were novel AR occupied sites. Additionally, using a “guilty by association” approach to determine potentially AR regulated genes from this novel cistromic data, it was determined that close to 50% of cell cycle common, and 70% of phase exclusive binding uncover novel candidates for AR regulation. Cistrome data was therefore overlaid with microarray data, to prioritize discovery of meaningful, cell cycle specific AR binding events. Analyses to be discussed reveal striking new insight into disease relevant AR function. In sum, these data rigorously demonstrate that AR acts in a cell cycle dependent manner, and that these functions of AR have a major impact on tumor cell phenotypes. Citation Format: Christopher McNair, Jonathan Goodwin, Michael Augello, Alfonso Urbanucci, Matthew Schiewer, Clay Comstock, Adam Ertel, Liguo Wang, Qianben Wang, Ian Mills, Wei Li, Jason Carroll, Karen Knudsen. Genome wide analysis of AR-cell cycle interplay reveals novel functions in cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1844. doi:10.1158/1538-7445.AM2015-1844

Abstract 1071: Impact of AR activation status on the DNA damage response.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Recent evidence implicates the androgen receptor (AR) as a critical modulator of the DNA damage response in prostate cancer. Treatment for locally advanced disease depends on combined radiation therapy and ablation of AR function, and clinical evidence strongly supports the contention that active AR reduces the response to radiotherapy. Moreover, it was reported in vitro that AR activation can induce DNA damage at sites of active transcription, thus providing additional evidence for crosstalk between the DNA damage response and AR pathways. Despite these observations, the underpinning mechanism by which AR alters the response to DNA damage remains unknown. Several lines of evidence will be discussed which illuminate a role for AR in the response to DNA damage. First, investigation in a battery of hormone-therapy sensitive prostate cancer cells (in which AR activity remains dependent on ligand binding) and in castrate resistant tumor cells (reflecting advanced disease wherein AR activity is enhanced) showed that androgen depletion enhances the response to ionizing radiation. Restoration of dihydrotestosterone reduced the response to radiation, thus demonstrating that ligand-induced AR activity promotes a resistance to radiation. Second, the mechanism by which AR promotes radioresistance was addressed. Preliminary findings indicate that in the presence of DNA damage, AR dramatically alters the rate of DNA damage repair independent of significant effects on cell cycle. Moreover, these studies uncovered an unexpected role for androgen-activated AR to alter expression and subsequent activity of genes critical for the DNA damage response, possibly through differential recruitment of AR to loci of key players in the response to damage. Finally, a series radioresistant prostate cancer model systems were developed, wherein the impact of AR on acquired radioresistance is under investigation; early studies in these models indicate that altered AR output is associated with acquired radioresistance. Combined, the studies to be presented reveal novel functions of androgen and AR in controlling the molecular and cellular response to DNA damage, and provide the basis for future studies directed at targeting DNA-damage specific AR activity in the course of human disease. Citation Format: Jonathan F. Goodwin, Matthew J. Schiewer, Felix Y. Feng, Karen E. Knudsen. Impact of AR activation status on the DNA damage response. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1071. doi:10.1158/1538-7445.AM2013-1071