Anu-Maarit Moilanen

Identification of a Novel RING Finger Protein as a Coregulator in Steroid Receptor-Mediated Gene Transcription

ABSTRACT Using the DNA-binding domain of androgen receptor (AR) as a bait in a yeast two-hybrid screening, we have identified a small nuclear RING finger protein, termed SNURF, that interacts with AR in a hormone-dependent fashion in both yeast and mammalian cells. Physical interaction between AR and SNURF was demonstrated by coimmunoprecipitation from cell extracts and by protein-protein affinity chromatography. Rat SNURF is a highly hydrophilic protein consisting of 194 amino acid residues and comprising a consensus C3HC4 zinc finger (RING) structure in the C-terminal region and a bipartite nuclear localization signal near the N terminus. Immunohistochemical experiments indicated that SNURF is a nuclear protein. SNURF mRNA is expressed in a variety of human and rat tissues. Overexpression of SNURF in cultured mammalian cells enhanced not only androgen, glucocorticoid, and progesterone receptor-dependent transactivation but also basal transcription from steroid-regulated promoters. Mutation of two of the potential Zn2+coordinating cysteines to serines in the RING finger completely abolished the ability of SNURF to enhance basal transcription, whereas its ability to activate steroid receptor-dependent transcription was maintained, suggesting that there are separate domains in SNURF that mediate interactions with different regulatory factors. SNURF is capable of interacting in vitro with the TATA-binding protein, and the RING finger domain is needed for this interaction. Collectively, we have identified and characterized a ubiquitously expressed RING finger protein, SNURF, that may function as a bridging factor and regulate steroid receptor-dependent transcription by a mechanism different from those of previously identified coactivator or integrator proteins.

A TESTIS-SPECIFIC ANDROGEN RECEPTOR COREGULATOR THAT BELONGS TO A NOVEL FAMILY OF NUCLEAR PROTEINS

We have characterized a novel partner for androgen receptor (AR), termed ARIP3, that interacts with the DNA-binding domain/zinc finger region of AR and is predominantly expressed in the testis. Rat ARIP3 is a nuclear protein comprising 572 amino acids. It modulates AR-dependent but not basal transcription, suggesting that ARIP3 acts as an AR transcriptional coregulator. Except for the C-terminal AR-interacting domain, ARIP3 contains distinct regions that are also present in two recently described proteins, a protein inhibitor of activated Stat3 and an RNA helicase II-interacting protein (Gu/RH-II binding protein). Conserved structural features of these proteins indicate the existence of a gene family involved in the regulation of various transcription factors. Collectively, ARIP3 belongs to a novel nuclear protein family and is perhaps the first tissue-specific coregulator of androgen receptor.

Discovery of ODM-201, a new-generation androgen receptor inhibitor targeting resistance mechanisms to androgen signaling-directed prostate cancer therapies.

Activation of androgen receptor (AR) is crucial for prostate cancer growth. Remarkably, also castration-resistant prostate cancer (CRPC) is dependent on functional AR, and several mechanisms have been proposed to explain the addiction. Known causes of CRPC include gene amplification and overexpression as well as point mutations of AR. We report here the pharmacological profile of ODM-201, a novel AR inhibitor that showed significant antitumor activity and a favorable safety profile in phase 1/2 studies in men with CRPC. ODM-201 is a full and high-affinity AR antagonist that, similar to second-generation antiandrogens enzalutamide and ARN-509, inhibits testosterone-induced nuclear translocation of AR. Importantly, ODM-201 also blocks the activity of the tested mutant ARs arising in response to antiandrogen therapies, including the F876L mutation that confers resistance to enzalutamide and ARN-509. In addition, ODM-201 reduces the growth of AR-overexpressing VCaP prostate cancer cells both in vitro and in a castration-resistant VCaP xenograft model. In contrast to other antiandrogens, ODM-201 shows negligible brain penetrance and does not increase serum testosterone levels in mice. In conclusion, ODM-201 is a potent AR inhibitor that overcomes resistance to AR-targeted therapies by antagonizing both overexpressed and mutated ARs. ODM-201 is currently in a phase 3 trial in CRPC.

The presence of a transcription activation function in the hormone-binding domain of androgen receptor is revealed by studies in yeast cells

To assess the importance of various regions of the androgen receptor (AR) in transcriptional regulation, we have compared its activation functions (AFs) in yeast and mammalian cells. The receptors amino‐terminal region contains a major transcriptional activator (AF‐1) in both cell types, whereas AF‐2 in the ligand‐binding domain (LBD) is very weak in mammalian cells but clearly functional in the yeast. Hormone‐binding ability of LBD is mandatory for AF‐2 to operate, as illustrated by mutated LBD constructs. The activity of AF‐2 in yeast is severely attenuated when the hinge region is attached to LBD, suggesting that the former region modulates AF‐2 in vivo, probably by presenting an interface for interacting proteins.

Androgen Receptor in Transcriptional Regulation

Male sex steroids (androgens) sculpture a male body in a number of ways, such as development of male sex organs; growth of facial, body, and pubic hair; enlargement of vocal cords (deepening of the voice); loss of hair at temples; production of sperm; development of muscle strength; growth of prostate, and development of masculine behavior. Each of these events is regulated by tissue-specific mechanisms through the androgen receptor that activates and/or represses gene networks to elicit its distinct physiological actions. The principal events leading to hyperplastic and/or hypertrophic responses to androgens are, however, believed to be similar, if not identical, in reproductive and nonreproductive tissues (Berger and Watson 1989). Recent studies in a number of laboratories have delineated the ways by which androgen receptor interacts with specific DNA elements — located within and/or around responsive genes — to increase the rate of gene transcription. Much less information is available, however, about mechanisms by which androgens bring about gene repression. In order to elicit defined biological (androgenic) responses, the receptor protein ought to communicate not only with DNA motifs, but also with other transcription factors or coactivators. Finally, practically nothing is known about the role of androgen receptor protein in the stabilization of specific mRNA species.

Abstract LB-113: Immune-mediated anti-tumor activity with a clinical stage BET bromodomain inhibitor ODM-207 in pre-clinical models

Background: ODM-207 is a potent and selective BET inhibitor that is structurally unrelated to the benzodiazepine-based inhibitors including JQ1, I-BET762, and OTX015. Phase I clinical trials have now been initiated with this agent based on its potent anti-tumor activity in various in vitro and in vivo models of hematologic malignancies and solid tumors. In view of the recent publications implicating a role for BET protein BRD4 in the suppression of PD-L1 expression, an immune checkpoint ligand for PD-1, we sought to evaluate ODM-207 for its effect on immune-mediated anti-tumor efficacy in pre-clinical models. Methods and Results: Mouse splenocytes were stimulated with anti-CD3 and anti-CD28 in the presence or absence of ODM-207 for four days and changes in immune cell population were analyzed by FACS. Results revealed an increase in the level of activated cytotoxic CD8+ T cells as indicated by increased intracellular IFNγ and granzyme B with ODM-207 treatment. After confirming the lack of direct anti-proliferative activity on the mouse colon carcinoma cell line CT26, in vivo evaluation of ODM-207 was carried out in the syngeneic CT26 subcutaneous tumor model established in BALB/c mice. Daily oral administration of ODM-207 at 30 mg/kg was well tolerated in this model and resulted in a statistically significant inhibition of tumor growth. Interestingly, the tumor growth inhibition observed with ODM-207 was comparable to that with a commercially available anti-mouse PD1 antibody. Studies to characterize the immune changes in the tumor and anti-tumor activity of ODM-207 in combination with an anti-mouse PD1 antibody are currently underway and the results will be presented. Conclusions: In summary, these studies demonstrate the anti-tumor activity of BET inhibitor in a syngeneic model of colon carcinoma in the absence of a direct anti-proliferative activity on tumor cells. Observed tumor growth inhibition correlated with the in vitro activation of cytotoxic CD8+ T cells supporting the immune-mediated effect leading to tumor growth inhibition. In view of the remarkable success with the immune-based therapeutic approaches, these findings are relevant in devising appropriate strategies for the continued clinical development of ODM-207. Citation Format: Pratima Deshpande, Ravi Krishna Babu, Prashant Yallappa Vadnal, Mahaboobi Jaleel, Murali Ramachandra, Chandrasekhar Abbineni, Susanta Samajdar, Anu-Maarit Moilanen, Pekka Kallio. Immune-mediated anti-tumor activity with a clinical stage BET bromodomain inhibitor ODM-207 in pre-clinical models [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-113. doi:10.1158/1538-7445.AM2017-LB-113