Entrectinib, a Pan-TRK, ROS1, and ALK Inhibitor with Activity in Multiple Molecularly Defined Cancer Indications.

Activated ALK and ROS1 tyrosine kinases, resulting from chromosomal rearrangements, occur in a subset of non-small cell lung cancers (NSCLC) as well as other tumor types and their oncogenic relevance as actionable targets has been demonstrated by the efficacy of selective kinase inhibitors such as crizotinib, ceritinib, and alectinib. More recently, low-frequency rearrangements of TRK kinases have been described in NSCLC, colorectal carcinoma, glioblastoma, and Spitzoid melanoma. Entrectinib, whose discovery and preclinical characterization are reported herein, is a novel, potent inhibitor of ALK, ROS1, and, importantly, of TRK family kinases, which shows promise for therapy of tumors bearing oncogenic forms of these proteins. Proliferation profiling against over 200 human tumor cell lines revealed that entrectinib is exquisitely potent in vitro against lines that are dependent on the drug's pharmacologic targets. Oral administration of entrectinib to tumor-bearing mice induced regression in relevant human xenograft tumors, including the TRKA-dependent colorectal carcinoma KM12, ROS1-driven tumors, and several ALK-dependent models of different tissue origins, including a model of brain-localized lung cancer metastasis. Entrectinib is currently showing great promise in phase I/II clinical trials, including the first documented objective responses to a TRK inhibitor in colorectal carcinoma and in NSCLC. The drug is, thus, potentially suited to the therapy of several molecularly defined cancer settings, especially that of TRK-dependent tumors, for which no approved drugs are currently available. Mol Cancer Ther; 15(4); 628-39. ©2016 AACR.

The TPM3-NTRK1 rearrangement is a recurring event in colorectal carcinoma and is associated with tumor sensitivity to TRKA kinase inhibition.

The NTRK1 gene encodes Tropomyosin-related kinase A (TRKA), the high-affinity Nerve Growth Factor Receptor. NTRK1 was originally isolated from a colorectal carcinoma (CRC) sample as component of a somatic rearrangement (TPM3-NTRK1) resulting in expression of the oncogenic chimeric protein TPM3-TRKA, but there has been no subsequent report regarding the relevance of this oncogene in CRC. The KM12 human CRC cell line expresses the chimeric TPM3-TRKA protein and is hypersensitive to TRKA kinase inhibition. We report the detailed characterization of the TPM3-NTRK1 genomic rearrangement in KM12 cells and through a cellular screening approach, the identification of NMS-P626, a novel highly potent and selective TRKA inhibitor. NMS-P626 suppressed TPM3-TRKA phosphorylation and downstream signaling in KM12 cells and showed remarkable antitumor activity in mice bearing KM12 tumors. Finally, using quantitative reverse transcriptase PCR and immunohistochemistry (IHC) we identified the TPM3-NTRK1 rearrangement in a CRC clinical sample, therefore suggesting that this chromosomal translocation is indeed a low frequency recurring event in CRC and that such patients might benefit from therapy with TRKA kinase inhibitors.

Role of HIF-1 and NF-kappaB transcription factors in the modulation of transferrin receptor by inflammatory and anti-inflammatory signals.

Inflammation generates various changes in body iron homeostasis, including iron sequestration in the reticuloendothelial system with ensuing hypoferremia and anemia of chronic disease. Increased iron accumulation is caused by hepcidin-mediated down-regulation of the iron export protein ferroportin and higher iron uptake. However, enhanced iron acquisition by macrophages cannot be accounted for by the previously reported transferrin receptor (TfR1) down-regulation in macrophages exposed to lipopolysaccharide (LPS)/interferon gamma (IFNgamma) because it impairs a major iron uptake mechanism. Because TfR1 is up-regulated by the hypoxia-inducible factor (HIF-1), we investigated the effect of inflammatory and anti-inflammatory signals on HIF-1-mediated TfR1 gene expression. Exposure of mouse macrophages (RAW 264.7 and J774A.1 cells or peritoneal macrophages) to LPS/IFNgamma up-regulated NF-kappaB, which in turn rapidly and transiently activated HIF-1-dependent TfR1 expression and iron uptake. Activation of an anti-inflammatory pathway by pre-exposure to the adenosine A(2A) receptor agonist CGS21680 prevented the inducing effect of LPS/IFNgamma on HIF-1 and TfR1 expression by inhibiting NF-kappaB activity, whereas treatment with CGS21680 alone increased HIF-1-mediated TfR1 expression by means of an NF-kappaB-independent signaling pathway. In conclusion, an interplay of the HIF-1 and NF-kappaB pathways controls TfR1 transcription in inflammation. The consequent changes in TfR1 expression may be involved in modulating iron retention in inflammatory macrophages, thus possibly contributing to the development of hypoferremia in the early phases preceding the down-regulation of macrophage ferroportin by hepcidin.

Adenosine-dependent activation of hypoxia-inducible factor-1 induces late preconditioning in liver cells.

UNLABELLED: The cellular mechanisms by which ischemic preconditioning increases liver tolerance to ischemia/reperfusion injury are still poorly understood. This study investigated the role of the hypoxia-inducible factor-1 (HIF-1) in the protection associated with the late phase of liver preconditioning. Late preconditioning was induced in primary cultured rat hepatocytes by a transient (10 minute) hypoxic stress or by 15 minutes incubation with the adenosine A(2A) receptors agonist CGS21680 24 hours before exposure to 90 minutes of hypoxia in a serum-free medium. Late preconditioning induced the nuclear translocation of HIF-1 and the expression of carbonic anhydrase IX (CAIX), a HIF-1-regulated transmembrane enzyme that catalyzes bicarbonate production. Such effects were associated with prevention of hepatocyte killing by hypoxia and the amelioration of intracellular acidosis and Na+ accumulation. The inhibition of PKC-mediated and PI3-kinase-mediated signals with, respectively, chelerythrine and wortmannin abolished HIF-1 activation and blocked both CAIX expression and the protective action of late preconditioning. CAIX expression was also prevented by interfering with the transcriptional activity of HIF-1 using a dominant negative HIF-1beta subunit. The inhibition of CAIX with acetazolamide or the block of bicarbonate influx with disodium-4-acetamido-4'-isothiocyanato-stilben-2,2'-disulfonate also reverted the protective effects of late preconditioning on intracellular acidosis and Na+ accumulation. CONCLUSION: The stimulation of adenosine A(2A) receptors induced late preconditioning in liver cells through the activation of HIF-1. HIF-1-induced expression of CAIX increases hepatocyte tolerance to ischemia by maintaining intracellular Na+ homeostasis. These observations along with the importance of HIF-1 in regulating cell survival indicates HIF-1 activation as a possible key event in liver protection by late preconditioning.

Adenosine A2a receptor-mediated, normoxic induction of HIF-1 through PKC and PI-3K-dependent pathways in macrophages.

Adenosine released by cells in injurious or hypoxic environments has tissue-protecting and anti-inflammatory effects, which are also a result of modulation of macrophage functions, such as vascular endothelial growth factor (VEGF) production. As VEGF is a well-known target of hypoxia-inducible factor 1 (HIF-1), we hypothesized that adenosine may activate HIF-1 directly. Our studies using subtype-specific adenosine receptor agonists and antagonists showed that by activating the A(2A) receptor, adenosine treatment induced HIF-1 DNA-binding activity, nuclear accumulation, and transactivation capacity in J774A.1 mouse macrophages. Increased HIF-1 levels were also found in adenosine-treated mouse peritoneal macrophages. The HIF-1 activation induced by the A(2A) receptor-specific agonist CGS21680 required the PI-3K and protein kinase C pathways but was not mediated by changes in iron levels. Investigation of the molecular basis of HIF-1 activation revealed the involvement of transcriptional and to a larger extent, translational mechanisms. HIF-1 induction triggered the expression of HIF-1 target genes involved in cell survival (aldolase, phosphoglycerate kinase) and VEGF but did not induce inflammation-related genes regulated by HIF-1, such as TNF-alpha or CXCR4. Our results show that the formation of adenosine and induction of HIF-1, two events which occur in response to hypoxia, are linked directly and suggest that HIF-1 activation through A(2A) receptors may contribute to the anti-inflammatory and tissue-protecting activity of adenosine.

Up regulation of IL-6 by ischemic preconditioning in normal and fatty rat livers: association with reduction of oxidative stress.

We analyzed the role of IL-6 in the protection that ischemic preconditioning (IP) exerts against hepatic ischemia reperfusion-mediated (I/R) oxidative damage, particularly in fatty livers. IP-related IL-6 up-regulation during reperfusion in steatotic and non-steatotic livers was correlated with reduced indices of liver damage, as also demonstrated by pharmacological modulation of IL-6. IP activated NF-kB and HSF during ischemia (Isc), whereas AP-1 activity was unaffected. IP blunted the activation of STAT3 and stress-responsive genes, such as NF-kB, AP-1 and heme oxygenase (HO-1) during reperfusion. The role of reduced oxidative stress in hepatoprotection of fatty livers was further demonstrated by the fact that: (i) IP prevented the decrease of glutathione levels and the increase of lipid peroxidation; (ii) the anti-oxidant GSH-ester prevented lipid peroxidation and necrosis. In conclusion, IP modulates the activity of transcription factors and triggers IL-6 production; this may prevent hepatic I/R damage in a oxidative stress-dependent way, particularly in fatty livers.

Hepatocyte growth factor signaling regulates transactivation of genes belonging to the plasminogen activation system via hypoxia inducible factor-1.

Hepatocyte growth factor (HGF) plays an important role in tumor growth and progression also by regulating invasive/metastatic phenotype and angiogenesis. Here we report that a molecular mechanism possibly contributing to these functions of HGF may be hypoxia inducible factor-1 (HIF-1)-dependent expression of genes of the plasminogen activation system. The following findings support this conclusion: (1) HGF enhanced the activity of a luciferase reporter construct under the control of multiple HIF-1 responsive elements (HRE) in HepG2 cells, and the cotransfection of the dominant negative for the beta-subunit (ARNT) prevented this increase; (2) HGF activated uPA and PAI-1 promoters through HIF-1 activity regulated by PI3K/JNK1 transducers, as demonstrated by cotransfection with the reporter gene promoters and the dominant negative for ARNT, p85 subunit of PI3K or JNK1; (3) hypoxia was additive to HGF in increasing reporter vector activities, but probably through different transduction pathways; (4) JNK1 wild-type expression vector increased HIF-1alpha protein expression probably in a phosphorylated state and, thus, functional for transactivating activity; and (5) c-Jun did not seem to be involved in the activation of the luciferase construct containing multiple HREs because it was not prevented by expression of TAM-67, which is the dominant negative mutant form for c-Jun.