Clinically-observed FOXA1 mutations upregulate SEMA3C through transcriptional derepression in prostate cancer.

FOXA1 is a pioneer transcription factor that is frequently mutated in prostate, breast, bladder, and salivary gland malignancies. Indeed, metastatic castration-resistant prostate cancer (mCRPC) commonly harbour FOXA1 mutations with a prevalence of 35%. However, despite the frequent recurrence of FOXA1 mutations in prostate cancer, the mechanisms by which FOXA1 variants drive its oncogenic effects are still unclear. Semaphorin 3C (SEMA3C) is a secreted autocrine growth factor that drives growth and treatment resistance of prostate and other cancers and is known to be regulated by both AR and FOXA1. In the present study, we characterize FOXA1 alterations with respect to its regulation of SEMA3C. Our findings reveal that FOXA1 alterations lead to elevated levels of SEMA3C both in prostate cancer specimens and in vitro. We further show that FOXA1 negatively regulates SEMA3C via intronic cis elements, and that mutations in FOXA1 forkhead domain attenuate its inhibitory function in reporter assays, presumably by disrupting DNA binding of FOXA1. Our findings underscore the key role of FOXA1 in prostate cancer progression and treatment resistance by regulating SEMA3C expression and suggest that SEMA3C may be a driver of growth and tumor vulnerability of mCRPC harboring FOXA1 alterations.

Dynamic phase separation of the androgen receptor and its coactivators key to regulate gene expression.

Numerous cancers, including prostate cancer (PCa), are addicted to transcription programs driven by specific genomic regions known as super-enhancers (SEs). The robust transcription of genes at such SEs is enabled by the formation of phase-separated condensates by transcription factors and coactivators with intrinsically disordered regions. The androgen receptor (AR), the main oncogenic driver in PCa, contains large disordered regions and is co-recruited with the transcriptional coactivator mediator complex subunit 1 (MED1) to SEs in androgen-dependent PCa cells, thereby promoting oncogenic transcriptional programs. In this work, we reveal that full-length AR forms foci with liquid-like properties in different PCa models. We demonstrate that foci formation correlates with AR transcriptional activity, as this activity can be modulated by changing cellular foci content chemically or by silencing MED1. AR ability to phase separate was also validated in vitro by using recombinant full-length AR protein. We also demonstrate that AR antagonists, which suppress transcriptional activity by targeting key regions for homotypic or heterotypic interactions of this receptor, hinder foci formation in PCa cells and phase separation in vitro. Our results suggest that enhanced compartmentalization of AR and coactivators may play an important role in the activation of oncogenic transcription programs in androgen-dependent PCa.

Structure-Based Study to Overcome Cross-Reactivity of Novel Androgen Receptor Inhibitors.

The mutation-driven transformation of clinical anti-androgen drugs into agonists of the human androgen receptor (AR) represents a major challenge for the treatment of prostate cancer patients. To address this challenge, we have developed a novel class of inhibitors targeting the DNA-binding domain (DBD) of the receptor, which is distanced from the androgen binding site (ABS) targeted by all conventional anti-AR drugs and prone to resistant mutations. While many members of the developed 4-(4-phenylthiazol-2-yl)morpholine series of AR-DBD inhibitors demonstrated the effective suppression of wild-type AR, a few represented by 4-(4-(3-fluoro-2-methoxyphenyl)thiazol-2-yl)morpholine (VPC14368) exhibited a partial agonistic effect toward the mutated T878A form of the receptor, implying their cross-interaction with the AR ABS. To study the molecular basis of the observed cross-reactivity, we co-crystallized the T878A mutated form of the AR ligand binding domain (LBD) with a bound VPC14368 molecule. Computational modelling revealed that helix 12 of AR undergoes a characteristic shift upon VPC14368 binding causing the agonistic behaviour. Based on the obtained structural data we then designed derivatives of VPC14368 to successfully eliminate the cross-reactivity towards the AR ABS, while maintaining significant anti-AR DBD potency.

Development of 2-(5,6,7-Trifluoro-1H-Indol-3-yl)-quinoline-5-carboxamide as a Potent, Selective, and Orally Available Inhibitor of Human Androgen Receptor Targeting Its Binding Function-3 for the Treatment of Castration-Resistant Prostate Cancer.

Prostate cancer (PCa) patients undergoing androgen deprivation therapy almost invariably develop castration-resistant prostate cancer (CRPC). Targeting the androgen receptor (AR) Binding Function-3 (BF3) site offers a promising option to treat CRPC. However, BF3 inhibitors have been limited by poor potency or inadequate metabolic stability. Through extensive medicinal chemistry, molecular modeling, and biochemistry, we identified 2-(5,6,7-trifluoro-1H-Indol-3-yl)-quinoline-5-carboxamide (VPC-13789), a potent AR BF3 antagonist with markedly improved pharmacokinetic properties. We demonstrate that VPC-13789 suppresses AR-mediated transcription, chromatin binding, and recruitment of coregulatory proteins. This novel AR antagonist selectively reduces the growth of both androgen-dependent and enzalutamide-resistant PCa cell lines. Having demonstrated in vitro efficacy, we developed an orally bioavailable prodrug that reduced PSA production and tumor volume in animal models of CRPC with no observed toxicity. VPC-13789 is a potent, selective, and orally bioavailable antiandrogen with a distinct mode of action that has a potential as novel CRPC therapeutics.

Development of an Androgen Receptor Inhibitor Targeting the N-Terminal Domain of Androgen Receptor for Treatment of Castration Resistant Prostate Cancer.

Prostate cancer patients undergoing androgen deprivation therapy almost invariably develop castration-resistant prostate cancer. Resistance can occur when mutations in the androgen receptor (AR) render anti-androgen drugs ineffective or through the expression of constitutively active splice variants lacking the androgen binding domain entirely (e.g., ARV7). In this study, we are reporting the discovery of a novel AR-NTD covalent inhibitor 1-chloro-3-[(5-([(2S)-3-chloro-2-hydroxypropyl]amino)naphthalen-1-yl)amino]propan-2-ol (VPC-220010) targeting the AR-N-terminal Domain (AR-NTD). VPC-220010 inhibits AR-mediated transcription of full length and truncated variant ARV7, downregulates AR response genes, and selectively reduces the growth of both full-length AR- and truncated AR-dependent prostate cancer cell lines. We show that VPC-220010 disrupts interactions between AR and known coactivators and coregulatory proteins, such as CHD4, FOXA1, ZMIZ1, and several SWI/SNF complex proteins. Taken together, our data suggest that VPC-220010 is a promising small molecule that can be further optimized into effective AR-NTD inhibitor for the treatment of CRPC.

Stress-Mediated Reprogramming of Prostate Cancer One-Carbon Cycle Drives Disease Progression.

One-carbon (1C) metabolism has a key role in metabolic programming with both mitochondrial (m1C) and cytoplasmic (c1C) components. Here we show that activating transcription factor 4 (ATF4) exclusively activates gene expression involved in m1C, but not the c1C cycle in prostate cancer cells. This includes activation of methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) expression, the central player in the m1C cycle. Consistent with the key role of m1C cycle in prostate cancer, MTHFD2 knockdown inhibited prostate cancer cell growth, prostatosphere formation, and growth of patient-derived xenograft organoids. In addition, therapeutic silencing of MTHFD2 by systemically administered nanoliposomal siRNA profoundly inhibited tumor growth in preclinical prostate cancer mouse models. Consistently, MTHFD2 expression is significantly increased in human prostate cancer, and a gene expression signature based on the m1C cycle has significant prognostic value. Furthermore, MTHFD2 expression is coordinately regulated by ATF4 and the oncoprotein c-MYC, which has been implicated in prostate cancer. These data suggest that the m1C cycle is essential for prostate cancer progression and may serve as a novel biomarker and therapeutic target. SIGNIFICANCE: These findings demonstrate that the mitochondrial, but not cytoplasmic, one-carbon cycle has a key role in prostate cancer cell growth and survival and may serve as a biomarker and/or therapeutic target.

Development of Novel Inhibitors Targeting the D-Box of the DNA Binding Domain of Androgen Receptor.

The inhibition of the androgen receptor (AR) is an established strategy in prostate cancer (PCa) treatment until drug resistance develops either through mutations in the ligand-binding domain (LBD) portion of the receptor or its deletion. We previously identified a druggable pocket on the DNA binding domain (DBD) dimerization surface of the AR and reported several potent inhibitors that effectively disrupted DBD-DBD interactions and consequently demonstrated certain antineoplastic activity. Here we describe further development of small molecule inhibitors of AR DBD dimerization and provide their broad biological characterization. The developed compounds demonstrate improved activity in the mammalian two-hybrid assay, enhanced inhibition of AR-V7 transcriptional activity, and improved microsomal stability. These findings position us for the development of AR inhibitors with entirely novel mechanisms of action that would bypass most forms of PCa treatment resistance, including the truncation of the LBD of the AR.

Computer-Aided Ligand Discovery for Estrogen Receptor Alpha.

Breast cancer (BCa) is one of the most predominantly diagnosed cancers in women. Notably, 70% of BCa diagnoses are Estrogen Receptor α positive (ERα+) making it a critical therapeutic target. With that, the two subtypes of ER, ERα and ERß, have contrasting effects on BCa cells. While ERα promotes cancerous activities, ERß isoform exhibits inhibitory effects on the same. ER-directed small molecule drug discovery for BCa has provided the FDA approved drugs tamoxifen, toremifene, raloxifene and fulvestrant that all bind to the estrogen binding site of the receptor. These ER-directed inhibitors are non-selective in nature and may eventually induce resistance in BCa cells as well as increase the risk of endometrial cancer development. Thus, there is an urgent need to develop novel drugs with alternative ERα targeting mechanisms that can overcome the limitations of conventional anti-ERα therapies. Several functional sites on ERα, such as Activation Function-2 (AF2), DNA binding domain (DBD), and F-domain, have been recently considered as potential targets in the context of drug research and discovery. In this review, we summarize methods of computer-aided drug design (CADD) that have been employed to analyze and explore potential targetable sites on ERα, discuss recent advancement of ERα inhibitor development, and highlight the potential opportunities and challenges of future ERα-directed drug discovery.

ETS transcription factors as emerging drug targets in cancer.

The ETS family of proteins consists of 28 transcription factors, many of which have been implicated in development and progression of a variety of cancers. While one family member, ERG, has been rigorously studied in the context of prostate cancer where it plays a critical role, other ETS factors keep emerging as potential hallmark oncodrivers. In recent years, numerous studies have reported initial discoveries of small molecule inhibitors of ETS proteins and opened novel avenues for ETS-directed cancer therapies. This review summarizes the state of the art data on therapeutic targeting of ETS family members and highlights the corresponding drug discovery strategies.

Regulation of the unfolded protein response through ATF4 and FAM129A in prostate cancer.

Cancer cells exploit many of the cellular adaptive responses to support their survival needs. One such critical pathway in eukaryotic cells is the unfolded protein response (UPR) that is important in normal physiology as well as disease states, including cancer. Since UPR can serve as a lever between survival and death, regulated control of its activity is critical for tumor formation and growth although the underlying mechanisms are poorly understood. Here we show that one of the main transcriptional effectors of UPR, activating transcription factor 4 (ATF4), is essential for prostate cancer (PCa) growth and survival. Using systemic unbiased gene expression and proteomic analyses, we identified a novel direct ATF4 target gene, family with sequence similarity 129 member A (FAM129A), which is critical in mediating ATF4 effects on prostate tumorigenesis. Interestingly, FAM129A regulated both PERK and eIF2α in a feedback loop that differentially channeled the UPR output. ATF4 and FAM129A protein expression is increased in patient PCa samples compared with benign prostate. Importantly, in vivo therapeutic silencing of ATF4-FAM129A axis profoundly inhibited tumor growth in a preclinical PCa model. These data support that one of the canonical UPR branches, through ATF4 and its target gene FAM129A, is required for PCa growth and thus may serve as a novel therapeutic target.

Computer-Aided Discovery of Small Molecules Targeting the RNA Splicing Activity of hnRNP A1 in Castration-Resistant Prostate Cancer.

The heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is a versatile RNA-binding protein playing a critical role in alternative pre-mRNA splicing regulation in cancer. Emerging data have implicated hnRNP A1 as a central player in a splicing regulatory circuit involving its direct transcriptional control by c-Myc oncoprotein and the production of the constitutively active ligand-independent alternative splice variant of androgen receptor, AR-V7, which promotes castration-resistant prostate cancer (CRPC). As there is an urgent need for effective CRPC drugs, targeting hnRNP A1 could, therefore, serve a dual purpose of preventing AR-V7 generation as well as reducing c-Myc transcriptional output. Herein, we report compound VPC-80051 as the first small molecule inhibitor of hnRNP A1 splicing activity discovered to date by using a computer-aided drug discovery approach. The inhibitor was developed to target the RNA-binding domain (RBD) of hnRNP A1. Further experimental evaluation demonstrated that VPC-80051 interacts directly with hnRNP A1 RBD and reduces AR-V7 messenger levels in 22Rv1 CRPC cell line. This study lays the groundwork for future structure-based development of more potent and selective small molecule inhibitors of hnRNP A1⁻RNA interactions aimed at altering the production of cancer-specific alternative splice isoforms.

IRE1α-XBP1s pathway promotes prostate cancer by activating c-MYC signaling.

Activation of endoplasmic reticulum (ER) stress/the unfolded protein response (UPR) has been linked to cancer, but the molecular mechanisms are poorly understood and there is a paucity of reagents to translate this for cancer therapy. Here, we report that an IRE1α RNase-specific inhibitor, MKC8866, strongly inhibits prostate cancer (PCa) tumor growth as monotherapy in multiple preclinical models in mice and shows synergistic antitumor effects with current PCa drugs. Interestingly, global transcriptomic analysis reveal that IRE1α-XBP1s pathway activity is required for c-MYC signaling, one of the most highly activated oncogenic pathways in PCa. XBP1s is necessary for optimal c-MYC mRNA and protein expression, establishing, for the first time, a direct link between UPR and oncogene activation. In addition, an XBP1-specific gene expression signature is strongly associated with PCa prognosis. Our data establish IRE1α-XBP1s signaling as a central pathway in PCa and indicate that its targeting may offer novel treatment strategies.

Computer-Aided Discovery of Small Molecule Inhibitors of Transcriptional Activity of TLX (NR2E1) Nuclear Receptor.

Orphan nuclear receptor TLX (NR2E1) plays a critical role in the regulation of neural stem cells (NSC) as well as in the development of NSC-derived brain tumors. In the last years, new data have emerged implicating TLX in prostate and breast cancer. Therefore, inhibitors of TLX transcriptional activity may have a significant impact on the treatment of several critical malignancies. However, the TLX protein possesses a non-canonical ligand-binding domain (LBD), which lacks a ligand-binding pocket (conventionally targeted in case of nuclear receptors) that complicates the development of small molecule inhibitors of TLX. Herein, we utilized a rational structure-based design approach to identify small molecules targeting the Atro-box binding site of human TLX LBD. As a result of virtual screening of ~7 million molecular structures, 97 compounds were identified and evaluated in the TLX-responsive luciferase reporter assay. Among those, three chemicals demonstrated 40⁻50% inhibition of luciferase-detected transcriptional activity of the TLX orphan nuclear receptor at a dose of 35 µM. The identified compounds represent the first class of small molecule inhibitors of TLX transcriptional activity identified via methods of computer-aided drug discovery.

Computer-aided drug discovery of Myc-Max inhibitors as potential therapeutics for prostate cancer.

While Myc is an essential regulator of growth in normal cells, it is also frequently associated with cancer progression, therapy-resistance and lethal outcomes in most human cancers. In prostate cancer (PCa), Myc transcription factors are implicated in the pathogenesis and progression of the full spectrum of PCa, from adenocarcinoma to advanced castration-resistant and neuroendocrine phenotypes. Though a high-value therapeutic target, clinically approved anti-Myc drugs have yet to be discovered. To elicit its oncogenic effects, Myc must form a heterodimer with its partner Max, which together bind DNA and activate transcription of a spectrum of target genes that promote cell growth, proliferation, metabolism, and apoptosis while blocking differentiation. In this study, we identified a binding site on the DNA-binding domain of the structurally ordered Myc-Max complex and employed a computer-aided rational drug discovery approach to identify small molecules that effectively inhibit Myc-Max functionality. A large-scale virtual screening protocol implementing structure-based methodologies was utilized to select a set of top-ranked compounds that were subsequently evaluated experimentally and characterized mechanistically for their ability to inhibit Myc-Max transcriptional activity and subsequent downstream functions, to reduce viability in PCa cell lines, disrupt protein-DNA interactions and to induce apoptosis as their mechanism of action. Among compounds identified that effectively inhibit Myc-Max activity with low to mid-micromolar range potency and no or minimal generic cytotoxicity, VPC-70067, a close analog of the previously identified Myc inhibitor 10058-F4, served as proof-of-concept that our in silico drug discovery strategy performed as expected. Compound VPC-70063, of a chemically different scaffold, was the best performer in a panel of in vitro assays, and the forerunner for future hit-to-lead optimization efforts. These findings lay a foundation for developing more potent, specific and clinically optimized Myc-Max inhibitors that may serve as promising therapeutics, alone or in combination with current anti-cancer treatments, for treatment of specific phenotypes or heterogeneous tumors.

Small molecule-induced degradation of the full length and V7 truncated variant forms of human androgen receptor.

The androgen receptor (AR) is a hormone-activated transcription factor that regulates the development and progression of prostate cancer (PCa) and represents one of the most well-established drug targets. Currently clinically approved small molecule inhibitors of AR, such as enzalutamide, are built upon a common chemical scaffold that interacts with the AR by the same mechanism of action. These inhibitors eventually fail due to the emergence of drug-resistance in the form of AR mutations and expression of truncated AR splice variants (e.g. AR-V7) that are constitutively active, signalling the progression of the castration-resistant state of the disease. The urgent need therefore continues for novel classes of AR inhibitors that can overcome drug resistance, especially since AR signalling remains important even in late-stage advanced PCa. Previously, we identified a collection of 10-benzylidene-10H-anthracen-9-ones that effectively inhibit AR transcriptional activity, induce AR degradation and display some ability to block recruitment of hormones to the receptor. In the current work, we extended the analysis of the lead compounds, and used methods of both ligand- and structure-based drug design to develop a panel of novel 10-benzylidene-10H-anthracen-9-one derivatives capable of suppressing transcriptional activity and protein expression levels of both full length- and AR-V7 truncated forms of human androgen receptor. Importantly, the developed compounds efficiently inhibited the growth of AR-V7 dependent prostate cancer cell-lines which are completely resistant to all current anti-androgens.

Selectively targeting the dimerization interface of human androgen receptor with small-molecules to treat castration-resistant prostate cancer.

Prostate cancer (PCa) is a leading cause of death for men in North America. The androgen receptor (AR) - a hormone inducible transcription factor - drives expression of tumor promoting genes and represents an important therapeutic target in PCa. The AR is activated by steroid recruitment to its ligand binding domain (LBD), followed by receptor nuclear translocation and dimerization via the DNA binding domain (DBD). Clinically used small molecules interfere with steroid recruitment and prevent AR-driven tumor growth, but are rendered ineffective by emergence of LBD mutations or expression of constitutively active variants, such as ARV7, that lack the LBD. Both drug-resistance mechanisms confound treatment of this 'castration resistant' stage of PCa (CRPC), characterized by return of AR signalling. Here, we employ computer-aided drug-design to develop small molecules that block the AR-DBD dimerization interface, an attractive target given its role in AR activation and independence from the LBD. Virtual screening on the AR-DBD structure led to development of prototypical compounds that block AR dimerization, inhibiting AR-transcriptional activity through a LBD-independent mechanism. Such inhibitors may potentially circumvent AR-dependent resistance mechanisms and directly target CRPC tumor growth.

Toxic Colors: The Use of Deep Learning for Predicting Toxicity of Compounds Merely from Their Graphic Images.

The majority of computational methods for predicting toxicity of chemicals are typically based on "nonmechanistic" cheminformatics solutions, relying on an arsenal of QSAR descriptors, often vaguely associated with chemical structures, and typically employing "black-box" mathematical algorithms. Nonetheless, such machine learning models, while having lower generalization capacity and interpretability, typically achieve a very high accuracy in predicting various toxicity endpoints, as unambiguously reflected by the results of the recent Tox21 competition. In the current study, we capitalize on the power of modern AI to predict Tox21 benchmark data using merely simple 2D drawings of chemicals, without employing any chemical descriptors. In particular, we have processed rather trivial 2D sketches of molecules with a supervised 2D convolutional neural network (2DConvNet) and demonstrated that the modern image recognition technology results in prediction accuracies comparable to the state-of-the-art cheminformatics tools. Furthermore, the performance of the image-based 2DConvNet model was comparatively evaluated on an external set of compounds from the Prestwick chemical library and resulted in experimental identification of significant and previously unreported antiandrogen potentials for several well-established generic drugs.

Cheminformatics Driven Development of Novel Therapies for Drug Resistant Prostate Cancer.

Androgen receptor (AR) is a master regulator of prostate cancer (PCa), and therefore is a pivotal drug target for the treatment of PCa including its castration-resistance form (CRPC). The development of acquired resistance is a major challenge in the use of the current antiandrogens. The recent advancements in inhibiting AR activity with small molecules specifically designed to target areas distinct from the receptor's androgen binding site are carefully discussed. Our new classes of AR inhibitors of AF2 and BF3 functional sites and DBD domains designed using cheminformatics techniques are promising to circumvent various AR-dependent resistance mechanisms.

20(S)-protopanaxadiol regio-selectively targets androgen receptor: anticancer effects in castration-resistant prostate tumors.

We have explored the effects of 20(S)-protopanaxadiol (aPPD), a naturally derived ginsenoside, against androgen receptor (AR) positive castration resistant prostate cancer (CRPC) xenograft tumors and have examined its interactions with AR. In silico docking studies for aPPD binding to AR, alongside transactivation bioassays and in vivo efficacy studies were carried out in the castration-resistant C4-2 xenograft model. Immunohistochemical (IHC) and Western blot analyses followed by evaluation of AR, apoptotic, cell cycle and proliferative markers in excised tumors was performed. The growth of established CRPC tumors was inhibited by 53% with aPPD and a corresponding decrease in serum PSA was seen compared to controls. The IHC data revealed that Ki-67 was significantly lower for aPPD treated tumors and was associated with elevated p21 and cleaved caspase-3 expression, compared to vehicle treatment. Furthermore, aPPD decreased AR protein expression in xenograft tumors, while significantly upregulating p27 and Bax protein levels. In vitro data supporting this suggests that aPPD binds to and significantly inhibits the N-terminal or the DNA binding domains of AR. The AR androgen binding site docking score for androgen (dihydrotestosterone) was -11.1, while that of aPPD was -7.1. The novel findings described herein indicate aPPD potently inhibits PCa in vivo partly via inhibition of a site on the AR N-terminal domain. This manifested as cell cycle arrest and concurrent induction of apoptosis via an increase in Bax, cleaved-caspase-3, p27 and p21 expression.

Benzothiophenone Derivatives Targeting Mutant Forms of Estrogen Receptor-α in Hormone-Resistant Breast Cancers.

Estrogen receptor-α positive (ERα⁺) breast cancers represent 75% of all invasive breast cancer cases, while de novo or acquired resistance to ER-directed therapy is also on the rise. Numerous factors contribute to this phenomenon including the recently-reported ESR1 gene mutations such as Y537S, which amplifies co-activator interactions with ERα and promotes constitutive activation of ERα function. Herein, we propose that direct targeting of the activation function-2 (AF2) site on ERα represents a promising alternative therapeutic strategy to overcome mutation-driven resistance in breast cancer. A systematic computer-guided drug discovery approach was employed to develop a potent ERα inhibitor that was extensively evaluated by a series of experiments to confirm its AF2-specific activity. We demonstrate that the developed small-molecule inhibitor effectively prevents ERα-coactivator interactions and exhibits a strong anti-proliferative effect against tamoxifen-resistant cells, as well as downregulates ERα-dependent genes and effectively diminishes the receptor binding to chromatin. Notably, the identified lead compound successfully inhibits known constitutively-active, resistance-associated mutant forms of ERα observed in clinical settings. Overall, this study reports the development of a novel class of ERα AF2 inhibitors, which have the potential to effectively inhibit ERα activity by a unique mechanism and to circumvent the issue of mutation-driven resistance in breast cancer.