Resistance to androgen deprivation therapies and increased androgen receptor (AR) activity are major drivers of castration resistant prostate cancer (CRPC). Although prior work focused on targeting AR directly, co-activators of AR signaling—which may represent new therapeutic targets—are relatively underexplored. Here we demonstrate that the mixed-lineage leukemia (MLL) complex, a well-known driver of MLL-fusion-positive leukemia, acts as a co-activator of AR signaling. AR directly interacts with the MLL complex via the menin MLL subunit. Menin expression is higher in castration resistant prostate cancer compared to hormone naïve prostate cancer and benign prostate and high menin expression correlates with poor overall survival. Treatment with a small molecule inhibitor of the menin-MLL interaction blocks AR signaling and inhibits the growth of castration resistant tumors in vivo in mice. Taken together, this work identifies the MLL complex as a critical co-activator of AR and a potential therapeutic target 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.
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. .
Attempts to target mutant KRAS have been unsuccessful. Here, we report the identification of Smad ubiquitination regulatory factor 2 (SMURF2) and UBCH5 as a critical E3:E2 complex maintaining KRAS protein stability. Loss of SMURF2 either by small interfering RNA/short hairpin RNA (siRNA/shRNA) or by overexpression of a catalytically inactive mutant causes KRAS degradation, whereas overexpression of wild-type SMURF2 enhances KRAS stability. Importantly, mutant KRAS is more susceptible to SMURF2 loss where protein half-life decreases from >12 hours in control siRNA-treated cells to <3 hours on Smurf2 silencing, whereas only marginal differences were noted for wild-type protein. This loss of mutant KRAS could be rescued by overexpressing a siRNA-resistant wild-type SMURF2. Our data further show that SMURF2 monoubiquitinates UBCH5 at lysine 144 to form an active complex required for efficient degradation of a RAS-family E3, β-transducing repeat containing protein 1 (β-TrCP1). Conversely, β-TrCP1 is accumulated on SMURF2 loss, leading to increased KRAS degradation. Therefore, as expected, β-TrCP1 knockdown following Smurf2 siRNA treatment rescues mutant KRAS loss. Further, we identify two conserved proline (P) residues in UBCH5 critical for SMURF2 interaction; mutation of either of these P to alanine also destabilizes KRAS. As a proof of principle, we demonstrate that Smurf2 silencing reduces the clonogenic survival in vitro and prolongs tumor latency in vivo in cancer cells including mutant KRAS-driven tumors. Taken together, we show that SMURF2:UBCH5 complex is critical in maintaining KRAS protein stability and propose that targeting such complex may be a unique strategy to degrade mutant KRAS to kill cancer cells.
We have synthesized and studied the biological and biophysical properties of triazole-linked ribo and xylo locked nucleic acid (LNA). The combination of LNA with the Isobe triazole linkage gave high binding affinity when incorporated at the 3' or 5' termini of oligonucleotides, but low binding affinity at internal positions. Antisense oligonucleotides (ASOs) and siRNAs containing triazole dimers were highly active and nuclease resistant. Surprisingly, the xyloLNA-modified siRNA was the most active of the series.
Two mechanisms dominate the clinical pipeline for oligonucleotide-based gene silencing, namely, the antisense approach that recruits RNase H to cleave target RNA and the RNAi approach that recruits the RISC complex to cleave target RNA. Multiple chemical designs can be used to elicit each pathway. We compare the silencing of the asthma susceptibility gene ADAM33 in MRC-5 lung fibroblasts using four classes of gene silencing agents, two that use each mechanism: traditional duplex small interfering RNAs (siRNAs), single-stranded small interfering RNAs (ss-siRNAs), locked nucleic acid (LNA) gapmer antisense oligonucleotides (ASOs), and novel hexadecyloxypropyl conjugates of the ASOs. Of these designs, the gapmer ASOs emerged as lead compounds for silencing ADAM33 expression: several gapmer ASOs showed subnanomolar potency when transfected with cationic lipid and low micromolar potency with no toxicity when delivered gymnotically. The preferential susceptibility of ADAM33 mRNA to silencing by RNase H may be related to the high degree of nuclear retention observed for this mRNA. Dynamic light scattering data showed that the hexadecyloxypropyl ASO conjugates self-assemble into clusters. These conjugates showed reduced potency relative to unconjugated ASOs unless the lipophilic tail was conjugated to the ASO using a biocleavable linkage. Finally, based on the lead ASOs from (human) MRC-5 cells, we developed a series of homologous ASOs targeting mouse Adam33 with excellent activity. Our work confirms that ASO-based gene silencing of ADAM33 is a useful tool for asthma research and therapy.
The efficacy of radiation therapy for lung cancer is limited by radiation-induced lung toxicity (RILT). Although tumor necrosis factor-alpha (TNF-α) signaling plays a critical role in RILT, the molecular regulators of radiation-induced TNF-α production remain unknown. We investigated the role of a major TNF-α regulator, Tristetraprolin (TTP), in radiation-induced TNF-α production by macrophages. For in vitro studies we irradiated (4 Gy) either a mouse lung macrophage cell line, MH-S or macrophages isolated from TTP knockout mice, and studied the effects of radiation on TTP and TNF-α levels. To study the in vivo relevance, mouse lungs were irradiated with a single dose (15 Gy) and assessed at varying times for TTP alterations. Irradiation of MH-S cells caused TTP to undergo an inhibitory phosphorylation at Ser-178 and proteasome-mediated degradation, which resulted in increased TNF-α mRNA stabilization and secretion. Similarly, MH-S cells treated with TTP siRNA or macrophages isolated from ttp (−/−) mice had higher basal levels of TNF-α, which was increased minimally after irradiation. Conversely, cells overexpressing TTP mutants defective in undergoing phosphorylation released significantly lower levels of TNF-α. Inhibition of p38, a known kinase for TTP, by either siRNA or a small molecule inhibitor abrogated radiation-induced TNF-α release by MH-S cells. Lung irradiation induced TTPSer178 phosphorylation and protein degradation and a simultaneous increase in TNF-α production in C57BL/6 mice starting 24 h post-radiation. In conclusion, irradiation of lung macrophages causes TTP inactivation via p38-mediated phosphorylation and proteasome-mediated degradation, leading to TNF-α production. These findings suggest that agents capable of blocking TTP phosphorylation or stabilizing TTP after irradiation could decrease RILT.
Nuclease-directed genome editing is a powerful tool for investigating physiology and has great promise as a therapeutic approach to correct mutations that cause disease. In its most precise form, genome editing can use cellular homology-directed repair (HDR) pathways to insert information from an exogenously supplied DNA repair template (donor) directly into a targeted genomic location. Unfortunately, particularly for long insertions, toxicity and delivery considerations associated with repair template DNA can limit HDR efficacy. Here, we explore chemical modifications to both double-stranded and single-stranded DNA-repair templates. We describe 5′-terminal modifications, including in its simplest form the incorporation of triethylene glycol (TEG) moieties, that consistently increase the frequency of precision editing in the germlines of three animal models (Caenorhabditis elegans, zebrafish, mice) and in cultured human cells.
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