Men who develop metastatic castration-resistant prostate cancer (CRPC) invariably succumb to the disease. The development and progression to CRPC following androgen ablation therapy is predominantly driven by unregulated androgen receptor (AR) signaling1-3. Despite the success of recently approved therapies targeting AR signaling such as abiraterone4-6 and second generation anti-androgens MDV3100 (enzalutamide)7,8, durable responses are limited, presumably due to acquired resistance. Recently JQ1 and I-BET, two selective small molecule inhibitors that target the amino-terminal bromodomains of BRD4, have been shown to exhibit anti-proliferative effects in a range of malignancies9-12. Here we show that AR signaling-competent CRPC cell lines are preferentially sensitive to BET bromodomain inhibition. BRD4 physically interacts with the N-terminal domain of AR and can be disrupted by JQ111,13. Like the direct AR antagonist, MDV3100, JQ1 disrupted AR recruitment to target gene loci. In contrast to MDV3100, JQ1 functions downstream of AR, and more potently abrogated BRD4 localization to AR target loci and AR-mediated gene transcription including induction of TMPRSS2-ERG and its oncogenic activity. In vivo, BET bromodomain inhibition was more efficacious than direct AR antagonism in CRPC xenograft models. Taken together, these studies provide a novel epigenetic approach for the concerted blockade of oncogenic drivers in advanced prostate cancer.

Next generation anti-androgen therapies such as enzalutamide and abiraterone have had a profound impact on the management of metastatic castration-resistant prostate cancer (mCRPC). However, mCRPC patients invariably develop resistance to these agents. Here, a series of clonal cell lines were developed from enzalutamide-resistant prostate tumor xenografts to study the molecular mechanism of resistance and test their oncogenic potential under various treatment conditions. Androgen receptor (AR) signaling was maintained in these cell lines which acquired potential resistance mechanisms including expression of AR-variant 7 (AR-v7) and glucocorticoid receptor (GR). BET bromodomain inhibitors were shown previously to attenuate AR signaling in mCRPC; here, we demonstrate the efficacy of BET inhibitors in enzalutamide-resistant prostate cancer models. AR antagonists, enzalutamide and ARN509 exhibit enhanced prostate tumor growth inhibition when combined with BET inhibitors, JQ1 and OTX015, respectively. Taken together, these data provide a compelling pre-clinical rationale to combine BET inhibitors with AR antagonists to subvert resistance mechanisms. Implications Therapeutic combinations of BET inhibitors and AR antagonists may enhance the clinical efficacy in the treatment of mCRPC.

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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. , 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. 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. .

Both KRAS and EGFR are essential mediators of pancreatic cancer development and interact with Argonaute 2 (AGO2) to perturb its function. Here, in a mouse model of mutant KRAS-driven pancreatic cancer, loss of AGO2 allows precursor lesion (PanIN) formation yet prevents progression to pancreatic ductal adenocarcinoma (PDAC). Precursor lesions with AGO2 ablation undergo oncogene-induced senescence with altered microRNA expression and EGFR/RAS signaling, bypassed by loss of p53. In mouse and human pancreatic tissues, PDAC progression is associated with increased plasma membrane localization of RAS/AGO2. Furthermore, phosphorylation of AGO2Y393 disrupts both the wild-type and oncogenic KRAS-AGO2 interaction, albeit under different conditions. ARS-1620 (G12C-specific inhibitor) disrupts the KRASG12C-AGO2 interaction, suggesting that the interaction is targetable. Altogether, our study supports a biphasic model of pancreatic cancer development: an AGO2-independent early phase of PanIN formation reliant on EGFR-RAS signaling, and an AGO2-dependent phase wherein the mutant KRAS-AGO2 interaction is critical for PDAC progression.

Lung cancer is the deadliest malignancy in the United States. Non–small cell lung cancer (NSCLC) accounts for 85% of cases and is frequently driven by activating mutations in the gene encoding the KRAS GTPase (e.g., KRASG12D). Our previous work demonstrated that Argonaute 2 (AGO2)—a component of the RNA-induced silencing complex (RISC)—physically interacts with RAS and promotes its downstream signaling. We therefore hypothesized that AGO2 could promote KRASG12D-dependent NSCLC in vivo. To test the hypothesis, we evaluated the impact of Ago2 knockout in the KPC (LSL-KrasG12D/+;p53f/f;Cre) mouse model of NSCLC. In KPC mice, intratracheal delivery of adenoviral Cre drives lung-specific expression of a stop-floxed KRASG12D allele and biallelic ablation of p53. Simultaneous biallelic ablation of floxed Ago2 inhibited KPC lung nodule growth while reducing proliferative index and improving pathological grade. We next applied the KPHetC model, in which the Clara cell–specific CCSP-driven Cre activates KRASG12D and ablates a single p53 allele. In these mice, Ago2 ablation also reduced tumor size and grade. In both models, Ago2 knockout inhibited ERK phosphorylation (pERK) in tumor cells, indicating impaired KRAS signaling. RNA sequencing (RNA-seq) of KPC nodules and nodule-derived organoids demonstrated impaired canonical KRAS signaling with Ago2 ablation. Strikingly, accumulation of pERK in KPC organoids depended on physical interaction of AGO2 and KRAS. Taken together, our data demonstrate a pathogenic role for AGO2 in KRAS-dependent NSCLC. Given the prevalence of this malignancy and current difficulties in therapeutically targeting KRAS signaling, our work may have future translational relevance.

Activating mutations in RAS GTPases drive nearly 30% of all human cancers. Our prior work described an essential role for Argonaute 2 (AGO2), of the RNA-induced silencing complex, in mutant KRAS-driven cancers. Here, we identified a novel endogenous interaction between AGO2 and RAS in both wild-type (WT) and mutant HRAS/NRAS cells. This interaction was regulated through EGFR-mediated phosphorylation of Y393-AGO2, and utilizing molecular dynamic simulation, we identified a conformational change in pY393-AGO2 protein structure leading to disruption of the RAS binding site. Knockdown of AGO2 led to a profound decrease in proliferation of mutant HRAS/NRAS-driven cell lines but not WT RAS cells. These cells demonstrated oncogene-induced senescence (OIS) as evidenced by beta-galactosidase staining and induction of multiple downstream senescence effectors. Mechanistically, we discovered that the senescent phenotype was mediated via induction of reactive oxygen species. Intriguingly, we further identified that loss of AGO2 promoted a novel feed forward pathway leading to inhibition of the PTP1B phosphatase and activation of EGFR-MAPK signaling, consequently resulting in OIS. Taken together, our study demonstrates that the EGFR-AGO2-RAS signaling axis is essential for maintaining mutant HRAS and NRAS-driven malignancies.

. CC-BY 4.0 International license peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/227264 doi: bioRxiv preprint first posted online Nov. 30, 2017; Mutations in RAS account for over 30% of all cancers and over 90% of pancreatic cancer harbor KRAS mutations, a disease with a dismal overall 5-year survival rate of only 7% 9 .The KRAS GTPase transduces extracellular mitogenic signals by cycling between an active GTP-bound and an inactive GDP-bound state. Recurrent driver mutations in KRAS decrease intrinsic GTPase activity thereby accumulating in its active GTP-bound form. Constitutively active KRAS leads to aberrant signaling activities through interactions with multiple effector proteins [10][11][12] ;p48Cre alleles (Extended Data Fig. 1a).Further, qRT-PCR analysis showed significant reduction in AGO2 expression in . CC-BY 4.0 International license peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/227264 doi: bioRxiv preprint first posted online Nov. 30, 2017; AGO2fl/fl ;KRAS G12D;p48Cre mice (Extended Data Fig. 1b), confirming Cre-mediated mutant KRAS activation with concomitant loss of AGO2 expression in the pancreas.Histological analysis of the pancreas from mice with Cre-mediated AGO2 ablation (AGO2 fl/fl ; p48Cre), showed normal morphology (Fig. 1b, left panels) with no differences in pancreatic weight compared to pancreata from AGO2 +/+ ; p48Cre mice (Extended Data Fig. 2).This suggests that loss of AGO2 in the acinar cells of the exocrine compartment, does not interfere with gross pancreas development. Immunohistochemical (IHC) staining with antibodies specific to AGO2 (Extended Data Fig. 3 Data Fig. 4). ;p48Cre) had survived at the cut-off time point of 500 days (Fig. 1f). PDAC was observed in pancreata of all mice that express AGO2 (wild type or heterozygous expression) but mice deficient for AGO2 developed only early PanIN precursor lesions without progression to PDAC (Fig. 1g) ;p48Cre rarely showed abnormal pathologies, and never frank adenocarcinoma or metastases (Fig. 1g). While all metastatic lesions from mice expressing AGO2 showed PDAC, analysis of lungs with abnormal pathologies in two of the ;p48Cre mice, IHC analysis showed increased levels of AGO2 in PDAC and metastatic tissues as compared to early PanIN lesions (Fig. 2a). This suggests that AGO2 Surprisinglyprotein levels are elevated with disease progression. To extend these analyses to human pancreatic cancer, we performed a systematic IHC analysis of a human pancreatic tissue microarray (TMA), containing 44 duplicated pancreatic tissue cores, including PanIN, PDAC and metastatic PDAC samples. We observed a remarkable increase in AGO2 expression in PDAC and metastatic PDAC cells compared to PanINs (Fig. 2b), scored as statistically significant (Fig. 2c). Furthermore, AGO2 staining appeared to be particularly intense along the membranes o...