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.

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 (ROS) 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.Research.

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.

Oligomerization of G protein-coupled receptors is a recognized mode of regulation of receptor activities, with alternate oligomeric states resulting in different signaling functions. The CXCR4 chemokine receptor is a G protein-coupled receptor that is post-translationally modified by tyrosine sulfation at three sites on its N-terminus (Y7, Y12, Y21), leading to enhanced affinity for its ligand, stromal cell derived factor (SDF-1, also called CXCL12). The complex has been implicated in cancer metastasis and is a therapeutic target in cancer treatment. Using molecular dynamics simulation of NMR-derived structures of the CXCR4 N-terminus in complex with SDF-1, and calculations of electrostatic binding energies for these complexes, we address the role of tyrosine sulfation in this complex. Our results show that sulfation stabilizes the dimeric state of the CXCR4:SDF-1 complex through hydrogen bonding across the dimer interface, conformational changes in residues at the dimer interface, and an enhancement in electrostatic binding energies associated with dimerization. These findings suggest a mechanism through which post-translational modifications such as tyrosine sulfation might regulate downstream function through modulation of the oligomeric state of the modified system.