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.

DNA-Dependent Protein Kinase Drives Prostate Cancer Progression through Transcriptional Regulation of the Wnt Signaling Pathway.

PURPOSE: Protein kinases are known to play a prominent role in oncogenic progression across multiple cancer subtypes, yet their role in prostate cancer progression remains underexplored. The purpose of this study was to identify kinases that drive prostate cancer progression.Experimental Design: To discover kinases that drive prostate cancer progression, we investigated the association between gene expression of all known kinases and long-term clinical outcomes in tumor samples from 545 patients with high-risk disease. We evaluated the impact of genetic and pharmacologic inhibition of the most significant kinase associated with metastatic progression in vitro and in vivo. RESULTS: DNA-dependent protein kinase (DNAPK) was identified as the most significant kinase associated with metastatic progression in high-risk prostate cancer. Inhibition of DNAPK suppressed the growth of both AR-dependent and AR-independent prostate cancer cells. Gene set enrichment analysis nominated Wnt as the top pathway associated with DNAPK. We found that DNAPK interacts with the Wnt transcription factor LEF1 and is critical for LEF1-mediated transcription. CONCLUSIONS: Our data show that DNAPK drives prostate cancer progression through transcriptional regulation of Wnt signaling and is an attractive therapeutic target in aggressive prostate cancer.

Pleiotropic Impact of DNA-PK in Cancer and Implications for Therapeutic Strategies.

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-PKcs-Mediated Transcriptional Regulation Drives Prostate Cancer Progression and Metastasis.

Emerging evidence demonstrates that the DNA repair kinase DNA-PKcs exerts divergent roles in transcriptional regulation of unsolved consequence. Here, in vitro and in vivo interrogation demonstrate that DNA-PKcs functions as a selective modulator of transcriptional networks that induce cell migration, invasion, and metastasis. Accordingly, suppression of DNA-PKcs inhibits tumor metastases. Clinical assessment revealed that DNA-PKcs is significantly elevated in advanced disease and independently predicts for metastases, recurrence, and reduced overall survival. Further investigation demonstrated that DNA-PKcs in advanced tumors is highly activated, independent of DNA damage indicators. Combined, these findings reveal unexpected DNA-PKcs functions, identify DNA-PKcs as a potent driver of tumor progression and metastases, and nominate DNA-PKcs as a therapeutic target for advanced malignancies.

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.

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.

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.