Wnt signaling is down-regulated in embryonal rhabdomyosarcoma (ERMS) and contributes to the block of differentiation. Epigenetic mechanisms leading to its suppression are unknown and could pave the way towards novel therapeutic modalities. We demonstrate that EHMT2 suppresses canonical Wnt signaling by activating expression of the Wnt antagonist DKK1. Inhibition of EHMT2 expression or activity in human ERMS cell lines reduced DKK1 expression and elevated canonical Wnt signaling resulting in myogenic differentiation in vitro and in mouse xenograft models in vivo. Mechanistically, EHMT2 impacted Sp1 and p300 enrichment at the DKK1 promoter. The reduced tumor growth upon EHMT2 deficiency was reversed by recombinant DKK1 or LGK974, which also inhibits Wnt signaling. Consistently, among thirteen drugs targeting chromatin modifiers, EHMT2 inhibitors were highly effective in reducing ERMS cell viability. Our study demonstrates that ERMS cells are vulnerable to EHMT2 inhibitors and suggest that targeting the EHMT2-DKK1-b-catenin node holds promise for differentiation therapy.

The transcription factor NF-E2 Related Factor-2 (NRF2) is an important drug target. Activation of NRF2 has chemopreventive effects in cancer and exerts beneficial effects in a number of diseases, including neurodegenerative diseases, inflammatory diseases, hepatosteatosis, obesity and insulin resistance. Hence, there have been great efforts to discover and characterize novel NRF2 activators. One reported NRF2 activator is the labdane diterpenoid andrographolide. In this study, we identified the mechanism through which andrographolide activates NRF2. We showed that andrographolide inhibits the function of KEAP1, a protein that together with CUL3 and RBX1 forms an E3 ubiquitin ligase that polyubiquitinates NRF2. Andrographolide partially inhibits the interaction of KEAP1 with CUL3 in a manner dependent on Cys151 in KEAP1. This suggests that andrographolide forms Michael acceptor dependent adducts with Cys151 in KEAP1 in vivo, leading to inhibition of NRF2 ubiquitination and consequently accumulation of the transcription factor. Interestingly, we also showed that at higher concentrations andrographolide increases NRF2 protein expression in a Cys151 independent, but likely KEAP1 dependent manner, possibly through modification of other Cys residues in KEAP1. In this study we also screened secondary metabolites produced by endophytes isolated from non-flowering plants for NRF2-inducing properties. One of the extracts, ORX 41, increased both NRF2 protein expression and transcriptional activity markedly. These results suggest that endophytes isolated from non-flowering or other plants may be a good source of novel NRF2 inducing compounds.

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The Wnt signaling pathway is down-regulated in embryonal rhabdomyosarcoma (ERMS) and contributes to the block of myogenic differentiation. Epigenetic mechanisms leading to its suppression are unknown and could pave the way towards novel therapeutic modalities. In this study, we demonstrate that the H3K9 lysine methyltransferase G9a suppresses canonical Wnt signaling by activating expression of the Wnt antagonist DKK1. Inhibition of G9a expression or activity reduced DKK1 expression and elevated canonical Wnt signaling resulting in myogenic differentiation in vitro and in vivo. Mechanistically, G9a impacted Sp1 and p300 enrichment at the DKK1 promoter in a methylation-dependent manner. The reduced tumor growth upon G9a deficiency was reversed by recombinant DKK1 or LGK974, which also inhibits Wnt signaling. Consistently, among thirteen drugs targeting chromatin modifiers, G9a inhibitors were highly effective in reducing ERMS cell viability.Together, our study demonstrates that ERMS cells are vulnerable to G9a inhibitors and suggest that targeting the G9a-DKK1-b-catenin node holds promise for differentiation therapy. receptor from the Frizzled (Fzd) family. The co-receptors LRP5/6 facilitate Wnt signaling. Activation of Wnt signaling leads to disruption of the destruction complex [APC, Axin, GSK3b and CK1a] which phosphorylates and degrades b-catenin. Induction of Wnt signaling results in accumulation of nonphosphorylated b-catenin (active b-catenin). b-catenin then translocates to the nucleus where it activates genes in cooperation with TCF/LEF1, but also other transcription factors (15). Neither of the two non-canonical pathways [Planar Cell Polarity pathway (PCP) and the Wnt/calcium signaling pathway] involve b-catenin. DKK1, a secreted protein interacts with LRP5/6 and antagonizes Wnt signaling by preventing LRP5/6 association with Wnt/Fzd complex (16). Despite the relevance of Wnt signaling in ERMS, epigenetic mechanisms leading to its suppression have not been described and could pave the way to development of targeted therapies.G9a, a lysine methyltransferase mediates mono and di-methylation of H3K9 (H3K9me1/2), which is primarily involved in transcriptional repression (17). Recent studies however have shown that G9a can also function as an activator in methylation-independent and dependent ways (18,19). G9a has been proposed to have oncogenic functions and its overexpression in leukaemia, gastric, lung, prostate cancer and alveolar rhabdomyosarcoma causes silencing of tumor suppressor genes through its H3K9me2 activity (18)(19)(20). In this study, we found that canonical Wnt/b-catenin signaling is epigenetically suppressed in ERMS. G9a activates expression of DKK1 in a methylation-dependent manner through an impact on Sp1 and p300 recruitment. Our data indicate the potential of targeting the G9a-DKK1 axis to activate Wnt signaling for the development of novel ERMS therapeutics. Conception and design: A Pal, R Taneja Development of methodology: A Pal, JY Leung, G Ang, VK Rao, HJ Lim, L Pignata Acquisition of...

BRD4, a bromodomain and extraterminal (BET) protein, is deregulated in multiple cancers and has emerged as a promising drug target. However, the function of the two main BRD4 isoforms (BRD4-L and BRD4-S) has not been analyzed in parallel in most cancers. This complicates determining therapeutic efficacy of pan-BET inhibitors. In this study, using functional and transcriptomic analysis, we show that BRD-L and BRD4-S isoforms play distinct roles in embryonal rhabdomyosarcoma. BRD4-L has an oncogenic role and inhibits myogenic differentiation, at least in part, by activating myostatin expression. Depletion of BRD4-L in vivo impairs tumor progression but does not impact metastasis. On the other hand, depletion of BRD4-S has no significant impact on tumor growth, but strikingly promotes metastasis in vivo. Interestingly, BRD4-S loss results in the enrichment of BRD4-L and RNA Polymerase II at integrin gene promoters resulting in their activation. Our work unveils isoform-specific functions of BRD4 and demonstrates that BRD4-S functions as a gatekeeper to constrain the full oncogenic potential of BRD4-L.

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