Corrigendum to "Cabozantinib inhibits AXL- and MET-dependent cancer cell migration induced by growth-arrest-specific 6 and hepatocyte growth factor" [Biochem. Biophys. Rep. 21 (2020) 100726].

[This corrects the article DOI: 10.1016/j.bbrep.2020.100726.].

A selective androgen receptor modulator SARM-2f activates androgen receptor, increases lean body mass, and suppresses blood lipid levels in cynomolgus monkeys.

SARM-2f a selective androgen receptor (AR) modulator, increases skeletal muscle mass and locomotor activity in rats. This study aimed to clarify its pharmacological effects in monkeys. In reporter assays, the EC50 values of SARM-2f for rat, monkey, and human AR were 2.5, 3, and 3.6 nmol/L, respectively; those of testosterone were 12, 3.2, and 11 nmol/L, respectively. A single oral administration (10 mg/kg SARM-2f) produced a maximal plasma concentration of 3011 ng/mL, with an area under the 24 hours concentration-time curve of 8152 ng·h/mL in monkeys. Body weight (BW), lean body mass (LBM), and plasma levels of total cholesterol, triglyceride, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, lipoprotein (a), alanine aminotransferase, and asparagine aminotransferase were measured after 4 weeks of treatment with SARM-2f (1, 3, and 10 mg/kg/day, QD, p.o.) or testosterone enanthate (TE; 2 mg/kg/2 weeks, s.c.) in monkeys. BW and LBM were significantly increased by 12% each by SARM-2f at 10 mg/kg, and by 5% and 8%, respectively, by TE, but these effects were not statistically significant. Plasma levels of all lipids were either decreased or showed a tendency to be decreased by SARM-2f. TE decreased the triglyceride level and increased the low-density lipoprotein cholesterol level. Liver marker levels were not changed by either SARM-2f or TE. Our data demonstrated that SARM-2f exerted anabolic effects and produced a lipid profile that differed from that produced by testosterone in monkeys, suggesting that SARM-2f might be useful for diseases such as sarcopenia.

Cabozantinib inhibits AXL- and MET-dependent cancer cell migration induced by growth-arrest-specific 6 and hepatocyte growth factor.

Cabozantinib is known as an inhibitor of receptor tyrosine kinases mainly targeting AXL receptor tyrosine kinase (AXL), MET proto-oncogene-encoded receptor tyrosine kinase (MET), and vascular endothelial growth factor receptor 2. Growth arrest-specific 6 (GAS6) and hepatocyte growth factor (HGF), the natural ligands of AXL and MET, respectively, are associated with the induction of cancer cell proliferation or metastasis. Currently, it is still unclear how cabozantinib regulates cancer cell migration and invasion by inhibiting AXL and MET. This study was conducted to investigate the mechanism underlying the anti-cancer effects of cabozantinib through regulation of AXL and MET signaling. The results of Boyden chamber assays showed that cancer cell migration was induced by GAS6 and HGF in SKOV3 cells in serum-free medium. Combinatorial treatment with GAS6 and HGF exerted an additive effect on cell migration. Furthermore, we examined the role of AXL and MET signaling in cell migration. Short interfering RNA targeting AXL and MET inhibited GAS6- and HGF-induced migration, respectively. Double knockdown of AXL and MET completely suppressed cell migration induced by combination treatment with GAS6 and HGF compared to AXL or MET inhibition alone. Finally, we investigated the effects of cabozantinib on cell migration and invasion. Cabozantinib inhibited AXL and MET phosphorylation and downregulated the downstream mediators, phosphorylated SRC in the presence of both GAS6 and HGF in SKOV3 cells. The cell migration and invasion induced by combined GAS6 and HGF treatment were suppressed by cabozantinib, but not by capmatinib, a selective MET inhibitor. Our data indicate that the GAS6-AXL and HGF-MET signal pathways markedly contribute to cancer cell migration and invasion in an independent manner, suggesting that simultaneous inhibition of these two pathways contributes to the anti-cancer effects of cabozantinib.

Amelioration of sexual behavior and motor activity deficits in a castrated rodent model with a selective androgen receptor modulator SARM-2f.

Sarcopenia and cachexia present characteristic features of a decrease in skeletal muscle mass and strength, anorexia, and lack of motivation. Treatments for these diseases have not yet been established, although selective androgen receptor modulators (SARMs) are considered as therapeutic targets. We previously reported that a novel SARM compound, SARM-2f, exhibits anabolic effect on muscles, with less stimulatory effect on prostate weight compared with testosterone, in rat Hershberger assays and cancer cachexia models. In this study, we studied the mechanism of action for SARM-2f selectivity and also assessed whether the muscle increase by this compound might lead to improvement of muscle function and physical activity. First, we examined the tissue distribution of SARM-2f. Tissue concentration was 1.2-, 1.6-, and 1.9-fold as high as the plasma concentration in the levator ani muscle, brain, and prostate, respectively. This result showed that the tissue-selective pharmacological effect did not depend on SARM-2f concentration in the tissues. The ability of SARM-2f to influence androgen receptor (AR)-mediated transcriptional activation was examined by reporter assays using human normal prostate epithelial cells (PrEC) and skeletal muscle cells (SKMC). SARM-2f exerted higher activity against AR in SKMC than in PrEC. Mammalian two hybrid assays showed different co-factor recruitment patterns between SARM-2f and dihydrotestosterone. Next, we studied the effect of SARM-2f on motivation and physical functions such as sexual behavior and motor activities in castrated rat or mouse models. SARM-2f restored the sexual behavior that was lost by castration in male rats. SARM-2f also increased voluntary running distance and locomotor activities. These results suggest that tissue-specific AR regulation by SARM-2f, but not tissue distribution, might account for its tissue specific androgenic effect, and that the muscle mass increase by SARM-2f leads to improvement of physical function. Together, these findings suggest that SARM-2f might represent an effective treatment for sarcopenia and cachexia.

Intratumor Heterogeneity in Primary Kidney Cancer Revealed by Metabolic Profiling of Multiple Spatially Separated Samples within Tumors.

Metabolic alteration constitutes a hallmark of cancer. Glycolysis and antioxidant pathways in kidney cancer are elevated, with frequent mutation of the VHL gene. Intratumor genetic heterogeneity has been recently demonstrated in kidney cancer. However, intratumor metabolic heterogeneity has not been investigated. Here, we used global metabolomics analysis and tissue slice tracer studies to demonstrate that different portions of a human primary kidney tumor possess different metabolic characteristics and drug sensitivity. Pyruvate levels were elevated and pyruvate metabolism was altered in some tumor sections. These observations indicated that pyruvate metabolism may constitute a possible vulnerability of kidney cancer; indeed, pyruvate stimulated the growth of primary kidney cancer cells and pharmacological inhibition of pyruvate transporters slowed the growth of patient-derived kidney tumors in mice. These findings deepen our understanding of the intratumor metabolic heterogeneity of kidney cancer and may inform novel therapeutic approaches in human kidney cancer.

Synthesis and biological evaluation of novel selective androgen receptor modulators (SARMs). Part II: Optimization of 4-(pyrrolidin-1-yl)benzonitrile derivatives.

We recently reported a class of novel tissue-selective androgen receptor modulators (SARMs), represented by a naphthalene derivative A. However, their pharmacokinetic (PK) profiles were poor due to low metabolic stability. To improve the PK profiles, we modified the hydroxypyrrolidine and benzonitrile substituents of 4-(pyrrolidin-1-yl)benzonitrile derivative B, which had a comparable potency as that of compound A. This optimization led us to further modifications, which improved metabolic stability while maintaining potent androgen agonistic activity. Among the synthesized compounds, (2S,3S)-2,3-dimethyl-3-hydroxylpyrrolidine derivative 1c exhibited a suitable PK profile and improved metabolic stability. Compound 1c demonstrated significant efficacy in levator ani muscle without increasing the weight of the prostate in an in vivo study. In addition, compound 1c showed agonistic activity in the CNS, which was detected using sexual behavior induction assay.

Synthesis and biological evaluation of novel selective androgen receptor modulators (SARMs) Part III: Discovery of 4-(5-oxopyrrolidine-1-yl)benzonitrile derivative 2f as a clinical candidate.

We previously reported that 4-(pyrrolidin-1-yl)benzonitrile derivative 1b was a selective androgen receptor modulator (SARM) that exhibited anabolic effects on organs such as muscles and the central nervous system (CNS), but neutral effects on the prostate. From further modification, we identified that 4-(5-oxopyrrolidine-1-yl)benzonitrile derivative 2a showed strong AR binding affinity with improved metabolic stabilities. Based on these results, we tried to enhance the AR agonistic activities by modifying the substituents of the 5-oxopyrrolidine ring. As a consequence, we found that 4-[(2S,3S)-2-ethyl-3-hydroxy-5-oxopyrrolidin-1-yl]-2-(trifluoromethyl)benzonitrile (2f) had ideal SARM profiles in Hershberger assay and sexual behavior induction assay. Furthermore, 2f showed good pharmacokinetic profiles in rats, dogs, monkeys, excellent nuclear selectivity and acceptable toxicological profiles. We also determined its binding mode by obtaining the co-crystal structures with AR.

Prolyl-tRNA synthetase inhibition promotes cell death in SK-MEL-2 cells through GCN2-ATF4 pathway activation.

Protein translation is highly activated in cancer tissues through oncogenic mutations and amplifications, and this can support survival and aberrant proliferation. Therefore, blocking translation could be a promising way to block cancer progression. The process of charging a cognate amino acid to tRNA, a crucial step in protein synthesis, is mediated by tRNA synthetases such as prolyl tRNA synthetase (PRS). Interestingly, unlike pan-translation inhibitors, we demonstrated that a novel small molecule PRS inhibitor (T-3861174) induced cell death in several tumor cell lines including SK-MEL-2 without complete suppression of translation. Additionally, our findings indicated that T-3861174-induced cell death was caused by activation of the GCN2-ATF4 pathway. Furthermore, the PRS inhibitor exhibited significant anti-tumor activity in several xenograft models without severe body weight losses. These results indicate that PRS is a druggable target, and suggest that T-3861174 is a potential therapeutic agent for cancer therapy.

Usefulness of Endobronchial Ultrasonography With a Guide Sheath and Virtual Bronchoscopic Navigation for Ground-Glass Opacity Lesions.

BACKGROUND: Endobronchial ultrasonography with guide sheath (EBUS-GS) could be useful for diagnosing ground-glass opacity (GGO) predominant-type lesions in the peripheral lung. Furthermore, several studies have reported that transbronchial biopsy using EBUS-GS and virtual bronchoscopic navigation (VBN) was safe and effective for diagnosing small peripheral lung lesions. Our objectives were to diagnose solitary peripheral GGO predominant-type lesions by transbronchial biopsy using EBUS-GS and VBN under radiographic fluoroscopic guidance, and to evaluate the clinical factors associated with diagnostic yield. METHODS: The medical records of 169 patients with GGO predominant-type lesions who underwent transbronchial biopsy using EBUS-GS and VBN under radiographic fluoroscopic guidance were retrospectively reviewed. RESULTS: Endobronchial ultrasonography images could be obtained for 156 (92%) of 169 GGO predominant-type lesions, and 116 (69%) were successfully diagnosed by this method (20 of 31 pure GGO lesions [65%]; 96 of 138 mixed GGO predominant-type lesions [70%]). The mean size of diagnosed lesions was significantly larger than that of nondiagnosed lesions (22 mm versus 18 mm, p < 0.01). Regarding diagnostic yield based on computed tomography sign, cases with presence of a bronchus leading directly to a lesion had significantly higher diagnostic yield than the other lesions (p < 0.01). CONCLUSIONS: The addition of VBN to EBUS-GS could be useful in clinical practice for diagnosing GGO predominant-type lesions in the peripheral lung.

A Novel LSD1 Inhibitor T-3775440 Disrupts GFI1B-Containing Complex Leading to Transdifferentiation and Impaired Growth of AML Cells.

Dysregulation of lysine (K)-specific demethylase 1A (LSD1), also known as KDM1A, has been implicated in the development of various cancers, including leukemia. Here, we describe the antileukemic activity and mechanism of action of T-3775440, a novel irreversible LSD1 inhibitor. Cell growth analysis of leukemia cell lines revealed that acute erythroid leukemia (AEL) and acute megakaryoblastic leukemia cells (AMKL) were highly sensitive to this compound. T-3775440 treatment enforced transdifferentiation of erythroid/megakaryocytic lineages into granulomonocytic-like lineage cells. Mechanistically, T-3775440 disrupted the interaction between LSD1 and growth factor-independent 1B (GFI1B), a transcription factor critical for the differentiation processes of erythroid and megakaryocytic lineage cells. Knockdown of LSD1 and GFI1B recapitulated T-3775440-induced transdifferentiation and cell growth suppression, highlighting the significance of LSD1-GFI1B axis inhibition with regard to the anti-AML effects of T-3775440. Moreover, T-3775440 exhibited significant antitumor efficacy in AEL and AMKL xenograft models. Our findings provide a rationale for evaluating LSD1 inhibitors as potential treatments and indicate a novel mechanism of action against AML, particularly AEL and AMKL. Mol Cancer Ther; 16(2); 273-84. ©2016 AACR.

Prevention of body weight loss and sarcopenia by a novel selective androgen receptor modulator in cancer cachexia models.

Cancer cachexia is a syndrome that impairs the quality of life and overall survival of patients, and thus the effectiveness of anticancer agents. There are no effective therapies for cancer cachexia due to the complexity of the syndrome, and insufficient knowledge of its pathogenesis results in difficulty establishing appropriate animal models. Previously, promising results have been obtained in clinical trials using novel agents including the ghrelin receptor agonist anamorelin, and the selective androgen receptor modulator (SARM) enobosarm to treat cachexia in patients with cancer. The present study examined the pharmacological effects of SARM-2f, a novel non-steroidal small molecule SARM, in animal models. SARM-2f increased body and skeletal muscle weight without significantly increasing the weight of the seminal vesicles or prostates of the castrated male rats. In the mice with tumor necrosis factor α-induced cachexia, SARM-2f and TP restored body weight, carcass weight, and food consumption rate. In the C26 and G361 cancer cachexia animal models, body and carcass weight, lean body mass, and the weight of the levator ani muscle were increased by SARM-2f and TP treatments. Tissue selectivity of SARM-2f was also observed in these animal models. The results demonstrate the anabolic effects of SARM-2f in a cytokine-induced cachexia model and other cancer cachexia models, and suggest that SARM-2f may be a novel therapeutic option for cachexia in patients with cancer.

An inhibitor of apoptosis protein antagonist T-3256336 potentiates the antitumor efficacy of the Nedd8-activating enzyme inhibitor pevonedistat (TAK-924/MLN4924).

Inhibitors of apoptosis proteins (IAPs) are antiapoptotic regulators that block cell death, and are frequently overexpressed in several human cancers, where they facilitate evasion of apoptosis and promote cell survival. IAP antagonists are also known as second mitochondria-derived activator of caspase (SMAC)-mimetics, and have recently been considered as novel therapeutic agents for inducing apoptosis, alone and in combination with other anticancer drugs. In this study, we showed that T-3256336, the orally available IAP antagonist has synergistically enhances the antiproliferative effects of the NEDD8-activating enzyme (NAE) inhibitor pevonedistat (TAK-924/MLN4924), and these effects were attenuated by a TNFα-neutralizing antibody. In the present mechanistic analyses, pevonedistat induced TNFα mRNA and triggered IAP antagonist-dependent extrinsic apoptotic cell death in cancer cell lines. Furthermore, synergistic effects of the combination of T-3256336 and pevonedistat were demonstrated in a HL-60 mouse xenograft model. Our findings provide mechanistic evidence of the effects of IAP antagonists in combination with NAE inhibitors, and demonstrate the potential of a new combination therapy for cancer.

Brain testosterone deficiency leads to down-regulation of mitochondrial gene expression in rat hippocampus accompanied by a decline in peroxisome proliferator-activated receptor-γ coactivator 1α expression.

Age-related decrease of testosterone levels in blood and brain is believed to be associated with neurodegenerative diseases such as Alzheimer's disease. However, the effect of testosterone on brain function is not well understood. Therefore, we investigated the impact of testosterone deprivation on mitochondrial gene expression in the brain of male gonadectomized (GDX) rats. We found that peripheral castration led to testosterone deficiency in the brain and caused a significant reduction in protein and mRNA expression of genes encoded by mitochondrial DNA, namely NADPH dehydrogenase subunit 1, subunit 4, cytochrome b, and cytochrome c oxidase subunit 1 and subunit 3 in the hippocampus. In addition, gene expression of peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α), which is a master regulator of mitochondrial biogenesis, and its downstream transcriptional factors, nuclear respiratory factors 1 and 2 and mitochondrial transcription factors A and B2, were also decreased in the hippocampus of GDX rats. These reductions in the expression of mitochondrial gene and transcriptional coactivators and factors were recovered by androgen replacement. These findings indicate that androgen plays an important role in mitochondrial gene expression in the hippocampus.

Antitumor activity and pharmacodynamic biomarkers of a novel and orally available small-molecule antagonist of inhibitor of apoptosis proteins.

Inhibitor of apoptosis proteins (IAP), which are key regulators of apoptosis, are inhibited by second mitochondria-derived activator of caspase (SMAC). Small-molecule IAP antagonists have recently been reported as novel therapeutic treatments for cancer. In this study, we showed that the octahydro-pyrrolo[1,2-a]pyrazine derivative, T-3256336, is a novel and orally available small-molecule IAP antagonist. T-3256336 selectively binds to and antagonizes protein interactions involving cellular IAP-1 (cIAP-1), cIAP-2, and X-linked IAP (XIAP). T-3256336 induced the rapid proteasomal degradation of cIAP-1 and activated TNF-α-dependent extrinsic apoptosis signaling in cultured cells. In a MDA-MB-231-Luc breast cancer xenograft model, T-3256336 induced cIAP-1 degradation, TNF-α production, and caspase activation in tumors, which resulted in strong antitumor activities. T-3256336 induced increases in the plasma levels of TNF-α and fragmented cytokeratin-18, which correlated with the antitumor potency in MDA-MB-231-Luc xenograft models. This study provided further insights into biomarkers of IAP antagonists. Furthermore, our data provided evidence that T-3256336 is a promising new anticancer drug worthy of further evaluation and development.