SR9009

SR9009 induces a REV-ERB dependent anti-small-cell lung cancer effect through inhibition of autophagy

Rationale: The circadian clock coordinates cell proliferation and metabolism and impacts the progression of some diseases, particularly cancer. Pharmacological modulation of the circadian machinery may be an effective therapeutic approach for treating cancer. SR9009 is a specific synthetic agonist of the REV-ERBs, essential circadian clock components. However, the potential efficacy and antitumor mechanism of this drug in small-cell lung cancer (SCLC) remains poorly understood. Methods: Here, we used chemosensitive cells (H69 and H446) and the corresponding chemoresistant cells (H69AR and H446DDP) to assess the efficacy of the REV-ERB agonist SR9009 for the treatment of SCLC in vitro and further validated the antitumor effect in subcutaneous tumor models of SCLC. Then, we determined whether REV-ERBα was correlated with the anti-SCLC effect of SR9009. Chromatin immunoprecipitation (ChIP) sequencing assays were conducted to identify potential DNA sequences directly regulated by REV-ERBα. Autophagy regulation by REV-ERBα and its possible mechanism in SR9009-based SCLC therapy were analyzed. Results: Here, we showed that the REV-ERB agonist SR9009 is specifically lethal to both chemosensitive and chemoresistant SCLC cells. REV-ERBα was involved in the antitumor effect of SR9009 in SCLC. The core autophagy gene Atg5 was identified as a direct downstream target of REV-ERBα and was suppressed by the REV-ERB agonist SR9009 in SCLC. Furthermore, the interaction of REV-ERBα with this autophagy gene impaired autophagy activity, leading to SR9009 cytotoxicity in SCLC cells. Principal conclusions: Our study provided a novel viewpoint indicating that the REV-ERB agonist SR9009 could be a novel and promising therapeutic strategy in first- or second-line SCLC treatment. The anti-SCLC effect of SR9009 is mediated by REV-ERB dependent suppression of autophagy via direct repression of the autophagy gene Atg5.

SR9009 inhibits lethal prostate cancer subtype 1 by regulating the LXRα/FOXM1 pathway independently of REV-ERBs

Perturbations of the circadian clock are linked to multiple diseases, including cancers. Pharmacological activation of REV-ERB nuclear receptors, the core components of the circadian clock, has antitumor effects on various malignancies, while the impact of SR9009 on prostate cancer (PCa) remains unknown. Here, we found that SR9009 was specifically lethal to PCa cell lines but had no cytotoxic effect on prostate cells. SR9009 significantly inhibited colony formation, the cell cycle, and cell migration and promoted apoptosis in PCa cells. SR9009 treatment markedly inhibited prostate cancer subtype 1 (PCS1), the most lethal and aggressive PCa subtype, through FOXM1 pathway blockade, while it had no impacts on PCS2 and PCS3. Seven representative genes, including FOXM1, CENPA, CENPF, CDK1, CCNB1, CCNB2, and BIRC5, were identified as the shared genes involved in the FOXM1 pathway and PCS1. All of these genes were upregulated in PCa tissues, associated with worse clinicopathological outcomes and downregulated after SR9009 treatment. Nevertheless, knockdown or knockout of REV-ERB could not rescue the anticancer effect of SR9009 in PCa. Further analysis confirmed that it was LXRα rather than REV-ERBs which has been activated by SR9009. The expression levels of these seven genes were changed correspondingly after LXRα knockdown and SR9009 treatment. An in vivo study validated that SR9009 restrained tumor growth in 22RV1 xenograft models and inhibited FOXM1 and its targeted gene expression. In summary, SR9009 can serve as an effective treatment option for highly aggressive and lethal PCS1 tumors through mediating the LXRα/FOXM1 pathway independently of REV-ERBs.

SR9009 improves heart function after pressure overload independent of cardiac REV-ERB

The core clock component REV-ERB is essential for heart function. Previous studies show that REV-ERB agonist SR9009 ameliorates heart remodeling in the pressure overload model with transverse aortic constriction (TAC). However, it is unknown whether SR9009 indeed works through cardiac REV-ERB, given that SR9009 might target other proteins and that REV-ERB in non-cardiac tissues might regulate cardiac functions indirectly. To address this question, we generated the REV-ERBα/β cardiac-specific double knockout mice (cDKO). We found that REV-ERB cardiac deficiency leads to profound dilated cardiac myopathy after TAC compared to wild-type (WT) control mice, confirming the critical role of REV-ERB in protecting against pressure overload. Interestingly, the cardioprotective effect of SR9009 against TAC retains in cDKO mice. In addition, SR9009 administered at the time points corresponding to the peak or trough of REV-ERB expression showed similar cardioprotective effects, suggesting the REV-ERB-independent mechanisms in SR9009-mediated post-TAC cardioprotection. These findings highlight that genetic deletion of REV-ERB in cardiomyocytes accelerates adverse cardiac remodeling in response to pressure overload and demonstrated the REV-ERB-independent cardioprotective effect of SR9009 upon pressure overload.

SR9009 Regulates Acute Lung Injury in Mice Induced by Sepsis

Rev-Erbα is a nuclear heme receptor, transcriptional repressor, and critical component of the molecular clock that drives daily rhythms of metabolism. However, the roles of Rev-Erbα in acute lung injury (ALI) remain unclarified. Hence, the effect of Rev-Erbα on lung injury of sepsis mice is investigated here. The mice sepsis model is established using lipopolysaccharide (LPS) injection, and the expression levels of proinflammatory cytokines, such as tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and interleukin-10 (IL-10) in both RAW246.7 cells and lung tissues, are tested. The inflammatory response is obviously enhanced in LPS-constructed sepsis mice and alleviated by SR9009 agonist treatment. Cell-based experiments reveal that pharmacological activation of Rev-Erbα via SR9009 attenuates the LPS-induced inflammatory response by suppressing TLR4-regulated NF-κB activation. Sepsis induces the increase in W/D ratio; promotes the levels of malondialdehyde (MDA), lactic acid (LA), and superoxide dismutase (SOD); and inhibits the levels of glutathione (GSH), whereas SR9009 treatment could effectively yield beneficial effects on metabolism. In addition, SR9009 treatment ameliorates acidosis and hypoxemia by efficiently decreasing arterial PaCO₂ and increasing arterial PaO₂, SO₂, HCO₃ ^-, lactic acid concentration, and blood PH. These findings confirm that SR9009 treatment can alleviate the sepsis-induced lung injury and targeting Rev-Erbα may represent a promising approach for the prevention and management of ALI.

REV-ERBα Agonist SR9009 Promotes a Negative Energy Balance in Goldfish

REV-ERBα (nr1d1, nuclear receptor subfamily 1 group D member 1) is a transcriptional repressor that in mammals regulates nutrient metabolism, and has effects on energy homeostasis, although its role in teleosts is poorly understood. To determine REV-ERBα's involvement in fish energy balance and metabolism, we studied the effects of acute and 7-day administration of its agonist SR9009 on food intake, weight and length gain, locomotor activity, feeding regulators, plasma and hepatic metabolites, and liver enzymatic activity. SR9009 inhibited feeding, lowering body weight and length gain. In addition, the abundance of ghrelin mRNA decreased in the intestine, and abundance of leptin-aI mRNA increased in the liver. Hypocretin, neuropeptide y (npy), and proopiomelanocortin (pomc) mRNA abundance was not modified after acute or subchronic SR9009 administration, while hypothalamic cocaine- and amphetamine-regulated transcript (cartpt-I) was induced in the subchronic treatment, being a possible mediator of the anorectic effects. Moreover, SR9009 decreased plasma glucose, coinciding with increased glycolysis and a decreased gluconeogenesis in the liver. Decreased triglyceride levels and activity of lipogenic enzymes suggest a lipogenesis reduction by SR9009. Energy expenditure by locomotor activity was not significantly affected by SR9009. Overall, this study shows for the first time in fish the effects of REV-ERBα activation via SR9009, promoting a negative energy balance by reducing energetic inputs and regulating lipid and glucose metabolism.