NSD2 is a requisite subunit of the AR/FOXA1 neo-enhanceosome in promoting prostate tumorigenesis.

The androgen receptor (AR) is a ligand-responsive transcription factor that binds at enhancers to drive terminal differentiation of the prostatic luminal epithelia. By contrast, in tumors originating from these cells, AR chromatin occupancy is extensively reprogrammed to drive hyper-proliferative, metastatic, or therapy-resistant phenotypes, the molecular mechanisms of which remain poorly understood. Here, we show that the tumor-specific enhancer circuitry of AR is critically reliant on the activity of Nuclear Receptor Binding SET Domain Protein 2 (NSD2), a histone 3 lysine 36 di-methyltransferase. NSD2 expression is abnormally gained in prostate cancer cells and its functional inhibition impairs AR trans-activation potential through partial off-loading from over 40,000 genomic sites, which is greater than 65% of the AR tumor cistrome. The NSD2-dependent AR sites distinctly harbor a chimeric AR-half motif juxtaposed to a FOXA1 element. Similar chimeric motifs of AR are absent at the NSD2-independent AR enhancers and instead contain the canonical palindromic motifs. Meta-analyses of AR cistromes from patient tumors uncovered chimeric AR motifs to exclusively participate in tumor-specific enhancer circuitries, with a minimal role in the physiological activity of AR. Accordingly, NSD2 inactivation attenuated hallmark cancer phenotypes that were fully reinstated upon exogenous NSD2 re-expression. Inactivation of NSD2 also engendered increased dependency on its paralog NSD1, which independently maintained AR and MYC hyper-transcriptional programs in cancer cells. Concordantly, a dual NSD1/2 PROTAC degrader, called LLC0150, was preferentially cytotoxic in AR-dependent prostate cancer as well as NSD2-altered hematologic malignancies. Altogether, we identify NSD2 as a novel subunit of the AR neo-enhanceosome that wires prostate cancer gene expression programs, positioning NSD1/2 as viable paralog co-targets in advanced prostate cancer.

Cellular reprogramming in vivo initiated by SOX4 pioneer factor activity.

Tissue damage elicits cell fate switching through a process called metaplasia, but how the starting cell fate is silenced and the new cell fate is activated has not been investigated in animals. In cell culture, pioneer transcription factors mediate "reprogramming" by opening new chromatin sites for expression that can attract transcription factors from the starting cell's enhancers. Here we report that SOX4 is sufficient to initiate hepatobiliary metaplasia in the adult mouse liver, closely mimicking metaplasia initiated by toxic damage to the liver. In lineage-traced cells, we assessed the timing of SOX4-mediated opening of enhancer chromatin versus enhancer decommissioning. Initially, SOX4 directly binds to and closes hepatocyte regulatory sequences via an overlapping motif with HNF4A, a hepatocyte master regulatory transcription factor. Subsequently, SOX4 exerts pioneer factor activity to open biliary regulatory sequences. The results delineate a hierarchy by which gene networks become reprogrammed under physiological conditions, providing deeper insight into the basis for cell fate transitions in animals.

Loss of LCMT1 and biased protein phosphatase 2A heterotrimerization drive prostate cancer progression and therapy resistance.

Loss of the tumor suppressive activity of the protein phosphatase 2A (PP2A) is associated with cancer, but the underlying molecular mechanisms are unclear. PP2A holoenzyme comprises a heterodimeric core, a scaffolding A subunit and a catalytic C subunit, and one of over 20 distinct substrate-directing regulatory B subunits. Methylation of the C subunit regulates PP2A heterotrimerization, affecting B subunit binding and substrate specificity. Here, we report that the leucine carboxy methyltransferase (LCMT1), which methylates the L309 residue of the C subunit, acts as a suppressor of androgen receptor (AR) addicted prostate cancer (PCa). Decreased methyl-PP2A-C levels in prostate tumors is associated with biochemical recurrence and metastasis. Silencing LCMT1 increases AR activity and promotes castration-resistant prostate cancer growth. LCMT1-dependent methyl-sensitive AB56αCme heterotrimers target AR and its critical coactivator MED1 for dephosphorylation, resulting in the eviction of the AR-MED1 complex from chromatin and loss of target gene expression. Mechanistically, LCMT1 is regulated by S6K1-mediated phosphorylation-induced degradation requiring the ß-TRCP, leading to acquired resistance to anti-androgens. Finally, feedforward stabilization of LCMT1 by small molecule activator of phosphatase (SMAP) results in attenuation of AR-signaling and tumor growth inhibition in anti-androgen refractory PCa. These findings highlight methyl-PP2A-C as a prognostic marker and that the loss of LCMT1 is a major determinant in AR-addicted PCa, suggesting therapeutic potential for AR degraders or PP2A modulators in prostate cancer treatment.

Setd2 inactivation sensitizes lung adenocarcinoma to inhibitors of oxidative respiration and mTORC1 signaling.

SETD2 is a tumor suppressor that is frequently inactivated in several cancer types. The mechanisms through which SETD2 inactivation promotes cancer are unclear, and whether targetable vulnerabilities exist in these tumors is unknown. Here we identify heightened mTORC1-associated gene expression programs and functionally higher levels of oxidative metabolism and protein synthesis as prominent consequences of Setd2 inactivation in KRAS-driven mouse models of lung adenocarcinoma. Blocking oxidative respiration and mTORC1 signaling abrogates the high rates of tumor cell proliferation and tumor growth specifically in SETD2-deficient tumors. Our data nominate SETD2 deficiency as a functional marker of sensitivity to clinically actionable therapeutics targeting oxidative respiration and mTORC1 signaling.

Physiological reprogramming in vivo mediated by Sox4 pioneer factor activity.

Tissue damage elicits cell fate switching through a process called metaplasia, but how the starting cell fate is silenced and the new cell fate is activated has not been investigated in animals. In cell culture, pioneer transcription factors mediate "reprogramming" by opening new chromatin sites for expression that can attract transcription factors from the starting cell's enhancers. Here we report that Sox4 is sufficient to initiate hepatobiliary metaplasia in the adult liver. In lineage-traced cells, we assessed the timing of Sox4-mediated opening of enhancer chromatin versus enhancer decommissioning. Initially, Sox4 directly binds to and closes hepatocyte regulatory sequences via a motif it overlaps with Hnf4a, a hepatocyte master regulator. Subsequently, Sox4 exerts pioneer factor activity to open biliary regulatory sequences. The results delineate a hierarchy by which gene networks become reprogrammed under physiological conditions, providing deeper insight into the basis for cell fate transitions in animals.

Oncofusion-driven de novo enhancer assembly promotes malignancy in Ewing sarcoma via aberrant expression of the stereociliary protein LOXHD1.

Ewing sarcoma (EwS) is a highly aggressive tumor of bone and soft tissues that mostly affects children and adolescents. The pathognomonic oncofusion EWSR1::FLI1 transcription factor drives EwS by orchestrating an oncogenic transcription program through de novo enhancers. By integrative analysis of thousands of transcriptomes representing pan-cancer cell lines, primary cancers, metastasis, and normal tissues, we identify a 32-gene signature (ESS32 [Ewing Sarcoma Specific 32]) that stratifies EwS from pan-cancer. Among the ESS32, LOXHD1, encoding a stereociliary protein, is the most highly expressed gene through an alternative transcription start site. Deletion or silencing of EWSR1::FLI1 bound upstream de novo enhancer results in loss of the LOXHD1 short isoform, altering EWSR1::FLI1 and HIF1α pathway genes and resulting in decreased proliferation/invasion of EwS cells. These observations implicate LOXHD1 as a biomarker and a determinant of EwS metastasis and suggest new avenues for developing LOXHD1-targeted drugs or cellular therapies for this deadly disease.

Toppling the HAT to Treat Lethal Prostate Cancer.

In this issue of Cancer Discovery, Welti and colleagues demonstrate a positive correlation between the expression of the histone acetyltransferase paralogs CBP and p300 with increased androgen receptor (AR) signaling and androgen deprivation therapy resistance in advanced prostate cancer. CCS1477, a selective inhibitor of p300/CBP bromodomain, disrupts AR- and MYC-regulated gene expression, suppresses tumor growth in vivo in multiple castration-resistant prostate cancer xenograft models, and modulates biomarker expression in early clinical evaluation, providing a novel therapeutic approach for AR-addicted advanced prostate cancer.See related article by Welti et al., p. 1118.

Targeting androgen regulation of TMPRSS2 and ACE2 as a therapeutic strategy to combat COVID-19.

Epidemiological data showing increased severity and mortality of COVID-19 in men suggests a potential role for androgen in SARS-CoV-2 infection. Here, we present evidence for the transcriptional regulation of SARS-CoV-2 host cell receptor ACE2 and TMPRSS2 by androgen in mouse and human cells. Additionally, we demonstrate the endogenous interaction between TMPRSS2 and ACE2 in human cells and validate ACE2 as a TMPRSS2 substrate. Furthermore, camostat-a TMPRSS2 inhibitor-blocked the cleavage of pseudotype SARS-CoV-2 surface Spike without disrupting TMPRSS2-ACE2 interaction, thus providing evidence for the first time of a direct role of TMPRSS2 in priming the SARS-CoV-2 Spike, required for viral fusion to the host cell. Importantly, androgen-deprivation, anti-androgens, or camostat attenuated the SARS-CoV-2 S-mediated cellular entry. Together, our data provide a strong rationale for clinical evaluations of TMPRSS2 inhibitors and androgen-deprivation therapy/androgen receptor antagonists alone or in combination with antiviral drugs as early as clinically possible to prevent COVID-19 progression.

Cell Cycle Checkpoints Cooperate to Suppress DNA- and RNA-Associated Molecular Pattern Recognition and Anti-Tumor Immune Responses.

The DNA-dependent pattern recognition receptor, cGAS (cyclic GMP-AMP synthase), mediates communication between the DNA damage and the immune responses. Mitotic chromosome missegregation stimulates cGAS activity; however, it is unclear whether progression through mitosis is required for cancercell-intrinsic activation of anti-tumor immune responses. Moreover, it is unknown whether cell cycle checkpoint disruption can restore responses in cancer cells that are recalcitrant to DNAdamage-induced inflammation. Here, we demonstrate that prolonged cell cycle arrest at the G2-mitosis boundary from either excessive DNA damage or CDK1 inhibition prevents inflammatory-stimulated gene expression and immune-mediated destruction of distal tumors. Remarkably, DNAdamage-induced inflammatory signaling is restored in a RIG-I-dependent manner upon concomitant disruption of p53 and the G2 checkpoint. These findings link aberrant cell progression and p53 loss to an expanded spectrum of damage-associated molecular pattern recognition and have implications for the design of rational approaches to augment anti-tumor immune responses.

The DNA methylation landscape of advanced prostate cancer.

Although DNA methylation is a key regulator of gene expression, the comprehensive methylation landscape of metastatic cancer has never been defined. Through whole-genome bisulfite sequencing paired with deep whole-genome and transcriptome sequencing of 100 castration-resistant prostate metastases, we discovered alterations affecting driver genes that were detectable only with integrated whole-genome approaches. Notably, we observed that 22% of tumors exhibited a novel epigenomic subtype associated with hypermethylation and somatic mutations in TET2, DNMT3B, IDH1 and BRAF. We also identified intergenic regions where methylation is associated with RNA expression of the oncogenic driver genes AR, MYC and ERG. Finally, we showed that differential methylation during progression preferentially occurs at somatic mutational hotspots and putative regulatory regions. This study is a large integrated study of whole-genome, whole-methylome and whole-transcriptome sequencing in metastatic cancer that provides a comprehensive overview of the important regulatory role of methylation in metastatic castration-resistant prostate cancer.

Tumor-Derived Retinoic Acid Regulates Intratumoral Monocyte Differentiation to Promote Immune Suppression.

The immunosuppressive tumor microenvironment (TME) is a major barrier to immunotherapy. Within solid tumors, why monocytes preferentially differentiate into immunosuppressive tumor-associated macrophages (TAMs) rather than immunostimulatory dendritic cells (DCs) remains unclear. Using multiple murine sarcoma models, we find that the TME induces tumor cells to produce retinoic acid (RA), which polarizes intratumoral monocyte differentiation toward TAMs and away from DCs via suppression of DC-promoting transcription factor Irf4. Genetic inhibition of RA production in tumor cells or pharmacologic inhibition of RA signaling within TME increases stimulatory monocyte-derived cells, enhances T cell-dependent anti-tumor immunity, and synergizes with immune checkpoint blockade. Furthermore, an RA-responsive gene signature in human monocytes correlates with an immunosuppressive TME in multiple human tumors. RA has been considered as an anti-cancer agent, whereas our work demonstrates its tumorigenic capability via myeloid-mediated immune suppression and provides proof of concept for targeting this pathway for tumor immunotherapy.

Global Regulation of the Histone Mark H3K36me2 Underlies Epithelial Plasticity and Metastatic Progression.

Epithelial plasticity, reversible modulation of a cell's epithelial and mesenchymal features, is associated with tumor metastasis and chemoresistance, leading causes of cancer mortality. Although different master transcription factors and epigenetic modifiers have been implicated in this process in various contexts, the extent to which a unifying, generalized mechanism of transcriptional regulation underlies epithelial plasticity remains largely unknown. Here, through targeted CRISPR/Cas9 screening, we discovered two histone-modifying enzymes involved in the writing and erasing of H3K36me2 that act reciprocally to regulate epithelial-to-mesenchymal identity, tumor differentiation, and metastasis. Using a lysine-to-methionine histone mutant to directly inhibit H3K36me2, we found that global modulation of the mark is a conserved mechanism underlying the mesenchymal state in various contexts. Mechanistically, regulation of H3K36me2 reprograms enhancers associated with master regulators of epithelial-to-mesenchymal state. Our results thus outline a unifying epigenome-scale mechanism by which a specific histone modification regulates cellular plasticity and metastasis in cancer. SIGNIFICANCE: Although epithelial plasticity contributes to cancer metastasis and chemoresistance, no strategies exist for pharmacologically inhibiting the process. Here, we show that global regulation of a specific histone mark, H3K36me2, is a universal epigenome-wide mechanism that underlies epithelial-to-mesenchymal transition and mesenchymal-to-epithelial transition in carcinoma cells. These results offer a new strategy for targeting epithelial plasticity in cancer.This article is highlighted in the In This Issue feature, p. 747.

Intracrine androgen biosynthesis and drug resistance.

Castration-resistant prostate cancer is the lethal form of prostate cancer and most commonly remains dependent on androgen receptor (AR) signaling. Current therapies use AR signaling inhibitors (ARSI) exemplified by abiraterone acetate, a P450c17 inhibitor, and enzalutamide, a potent AR antagonist. However, drug resistance to these agents occurs within 12-18 months and they only prolong overall survival by 3-4 months. Multiple mechanisms can contribute to ARSI drug resistance. These mechanisms can include but are not limited to germline mutations in the AR, post-transcriptional alterations in AR structure, and adaptive expression of genes involved in the intracrine biosynthesis and metabolism of androgens within the tumor. This review focuses on intracrine androgen biosynthesis, how this can contribute to ARSI drug resistance, and therapeutic strategies that can be used to surmount these resistance mechanisms.

CDK7 Inhibition Suppresses Castration-Resistant Prostate Cancer through MED1 Inactivation.

Metastatic castration-resistant prostate cancer (CRPC) is a fatal disease, primarily resulting from the transcriptional addiction driven by androgen receptor (AR). First-line CRPC treatments typically target AR signaling, but are rapidly bypassed, resulting in only a modest survival benefit with antiandrogens. Therapeutic approaches that more effectively block the AR-transcriptional axis are urgently needed. Here, we investigated the molecular mechanism underlying the association between the transcriptional coactivator MED1 and AR as a vulnerability in AR-driven CRPC. MED1 undergoes CDK7-dependent phosphorylation at T1457 and physically engages AR at superenhancer sites, and is essential for AR-mediated transcription. In addition, a CDK7-specific inhibitor, THZ1, blunts AR-dependent neoplastic growth by blocking AR/MED1 corecruitment genome-wide, as well as reverses the hyperphosphorylated MED1-associated enzalutamide-resistant phenotype. In vivo, THZ1 induces tumor regression of AR-amplified human CRPC in a xenograft mouse model. Together, we demonstrate that CDK7 inhibition selectively targets MED1-mediated, AR-dependent oncogenic transcriptional amplification, thus representing a potential new approach for the treatment of CRPC. SIGNIFICANCE: Potent inhibition of AR signaling is critical to treat CRPC. This study uncovers a driver role for CDK7 in regulating AR-mediated transcription through phosphorylation of MED1, thus revealing a therapeutically targetable potential vulnerability in AR-addicted CRPC.See related commentary by Russo et al., p. 1490.This article is highlighted in the In This Issue feature, p. 1469.

Functional and Mechanistic Interrogation of BET Bromodomain Degraders for the Treatment of Metastatic Castration-resistant Prostate Cancer.

PURPOSE: The bromodomain and extraterminal (BET)-containing proteins (BRD2/3/4) are essential epigenetic coregulators for prostate cancer growth. BRD inhibitors have shown promise for treatment of metastatic castration-resistant prostate cancer (mCRPC), and have been shown to function even in the context of resistance to next-generation AR-targeted therapies such as enzalutamide and abiraterone. Their clinical translation, however, has been limited by off-target effects, toxicity, and rapid resistance. EXPERIMENTAL DESIGN: We have developed a series of molecules that target BET bromodomain proteins through their proteasomal degradation, improving efficacy and specificity of standard inhibitors. We tested their efficacy by utilizing prostate cancer cell lines and patient-derived xenografts, as well as several techniques including RNA-sequencing, mass spectroscopic proteomics, and lipidomics. RESULTS: BET degraders function in vitro and in vivo to suppress prostate cancer growth. These drugs preferentially affect AR-positive prostate cancer cells (22Rv1, LNCaP, VCaP) over AR-negative cells (PC3 and DU145), and proteomic and genomic mechanistic studies confirm disruption of oncogenic AR and MYC signaling at lower concentrations than BET inhibitors. We also identified increases in polyunsaturated fatty acids (PUFA) and thioredoxin-interacting protein (TXNIP) as potential pharmacodynamics biomarkers for targeting BET proteins. CONCLUSIONS: Compounds inducing the pharmacologic degradation of BET proteins effectively target the major oncogenic drivers of prostate cancer, and ultimately present a potential advance in the treatment of mCRPC. In particular, our compound dBET-3, is most suited for further clinical development.

Epigenetic Regulation of Chromatin in Prostate Cancer.

Epigenetics refers to mitotically/meiotically heritable mechanisms that regulate gene transcription without a need for changes in the DNA code. Covalent modifications of DNA, in the form of methylation, and histone post-translational modifications, in the form of acetylation and methylation, constitute the epigenetic code of a cell. Both DNA and histone modifications are highly dynamic and often work in unison to define the epigenetic state of a cell. Most epigenetic mechanisms regulate gene transcription by affecting localized/genome-wide transitions between heterochromatin and euchromatin states, thereby altering the accessibility of the transcriptional machinery and in turn, reduce/increase transcriptional output. Altered chromatin structure is associated with cancer progression, and epigenetic plasticity primarily governs the resistance of cancer cells to therapeutic agents. In this chapter, we specifically focus on regulators of histone methylation and acetylation, the two well-studied chromatin post-translational modifications, in the context of prostate cancer.

EWS/ETS-Driven Ewing Sarcoma Requires BET Bromodomain Proteins.

The EWS/ETS fusion transcription factors drive Ewing sarcoma (EWS) by orchestrating an oncogenic transcription program. Therapeutic targeting of EWS/ETS has been unsuccessful; however, identifying mediators of the EWS/ETS function could offer new therapeutic options. Here, we describe the dependency of EWS/ETS-driven transcription upon chromatin reader BET bromdomain proteins and investigate the potential of BET inhibitors in treating EWS. EWS/FLI1 and EWS/ERG were found in a transcriptional complex with BRD4, and knockdown of BRD2/3/4 significantly impaired the oncogenic phenotype of EWS cells. RNA-seq analysis following BRD4 knockdown or inhibition with JQ1 revealed an attenuated EWS/ETS transcriptional signature. In contrast to previous reports, JQ1 reduced proliferation and induced apoptosis through MYC-independent mechanisms without affecting EWS/ETS protein levels; this was confirmed by depleting BET proteins using PROTAC-BET degrader (BETd). Polycomb repressive complex 2 (PRC2)-associated factor PHF19 was downregulated by JQ1/BETd or BRD4 knockdown in multiple EWS lines. EWS/FLI1 bound a distal regulatory element of PHF19, and EWS/FLI1 knockdown resulted in downregulation of PHF19 expression. Deletion of PHF19 via CRISPR-Cas9 resulted in a decreased tumorigenic phenotype, a transcriptional signature that overlapped with JQ1 treatment, and increased sensitivity to JQ1. PHF19 expression was also associated with worse prognosis in patients with EWS. In vivo, JQ1 demonstrated antitumor efficacy in multiple mouse xenograft models of EWS. Together these results indicate that EWS/ETS requires BET epigenetic reader proteins for its transcriptional program and can be mitigated by BET inhibitors. This study provides a clear rationale for the clinical utility of BET inhibitors in treating EWS.Significance: These findings reveal the dependency of EWS/ETS transcription factors on BET epigenetic reader proteins and demonstrate the potential of BET inhibitors for the treatment of EWS. Cancer Res; 78(16); 4760-73. ©2018 AACR.

Resistance to BET Inhibitor Leads to Alternative Therapeutic Vulnerabilities in Castration-Resistant Prostate Cancer.

BRD4 plays a major role in the transcription networks orchestrated by androgen receptor (AR) in castration-resistant prostate cancer (CRPC). Several BET inhibitors (BETi) that displace BRD4 from chromatin are being evaluated in clinical trials for CRPC. Here, we describe mechanisms of acquired resistance to BETi that are amenable to targeted therapies in CRPC. BETi-resistant CRPC cells displayed cross-resistance to a variety of BETi in the absence of gatekeeper mutations, exhibited reduced chromatin-bound BRD4, and were less sensitive to BRD4 degraders/knockdown, suggesting a BRD4-independent transcription program. Transcriptomic analysis revealed reactivation of AR signaling due to CDK9-mediated phosphorylation of AR, resulting in sensitivity to CDK9 inhibitors and enzalutamide. Additionally, increased DNA damage associated with PRC2-mediated transcriptional silencing of DDR genes was observed, leading to PARP inhibitor sensitivity. Collectively, our results identify the therapeutic limitation of BETi as a monotherapy; however, our BETi resistance data suggest unique opportunities for combination therapies in treating CRPC.

The depalmitoylase APT1 directs the asymmetric partitioning of Notch and Wnt signaling during cell division.

Asymmetric cell division results in two distinctly fated daughter cells. A molecular hallmark of asymmetric division is the unequal partitioning of cell fate determinants. We have previously established that growth factor signaling promotes protein depalmitoylation to foster polarized protein localization, which, in turn, drives migration and metastasis. We report protein palmitoylation as a key mechanism for the asymmetric partitioning of the cell fate determinants Numb and ß-catenin through the activity of the depalmitoylating enzyme APT1. Using point mutations, we showed that specific palmitoylated residues on Numb were required for its asymmetric localization. By live-cell imaging, we showed that reciprocal interactions between APT1 and the Rho family GTPase CDC42 promoted the asymmetric localization of Numb and ß-catenin to the plasma membrane. This, in turn, restricted Notch- or Wnt-responsive transcriptional activity to one daughter cell. Moreover, we showed that altering APT1 abundance changed the transcriptional signatures of MDA-MB-231 triple receptor-negative breast cancer cells, similar to changes in Notch and ß-catenin-mediated Wnt signaling. We also showed that loss of APT1 depleted a specific subpopulation of tumorigenic cells in colony formation assays. Together, our findings suggest that APT1-mediated depalmitoylation is a major mechanism of asymmetric cell division that maintains Notch- and Wnt-associated protein dynamics, gene expression, and cellular functions.