Genome-wide CRISPR screening reveals genetic modifiers of mutant EGFR dependence in human NSCLC.

EGFR-mutant NSCLCs frequently respond to EGFR tyrosine kinase inhibitors (TKIs). However, the responses are not durable, and the magnitude of tumor regression is variable, suggesting the existence of genetic modifiers of EGFR dependency. Here, we applied a genome-wide CRISPR-Cas9 screening to identify genetic determinants of EGFR TKI sensitivity and uncovered putative candidates. We show that knockout of RIC8A, essential for G-alpha protein activation, enhanced EGFR TKI-induced cell death. Mechanistically, we demonstrate that RIC8A is a positive regulator of YAP signaling, activation of which rescued the EGFR TKI sensitizing phenotype resulting from RIC8A knockout. We also show that knockout of ARIH2, or other components in the Cullin-5 E3 complex, conferred resistance to EGFR inhibition, in part by promoting nascent protein synthesis through METAP2. Together, these data uncover a spectrum of previously unidentified regulators of EGFR TKI sensitivity in EGFR-mutant human NSCLC, providing insights into the heterogeneity of EGFR TKI treatment responses.

S55746 is a novel orally active BCL-2 selective and potent inhibitor that impairs hematological tumor growth.

Escape from apoptosis is one of the major hallmarks of cancer cells. The B-cell Lymphoma 2 (BCL-2) gene family encodes pro-apoptotic and anti-apoptotic proteins that are key regulators of the apoptotic process. Overexpression of the pro-survival member BCL-2 is a well-established mechanism contributing to oncogenesis and chemoresistance in several cancers, including lymphoma and leukemia. Thus, BCL-2 has become an attractive target for therapeutic strategy in cancer, as demonstrated by the recent approval of ABT-199 (Venclexta™) in relapsed or refractory Chronic Lymphocytic Leukemia with 17p deletion. Here, we describe a novel orally bioavailable BCL-2 selective and potent inhibitor called S55746 (also known as BCL201). S55746 occupies the hydrophobic groove of BCL-2. Its selectivity profile demonstrates no significant binding to MCL-1, BFL-1 (BCL2A1/A1) and poor affinity for BCL-XL. Accordingly, S55746 has no cytotoxic activity on BCL-XL-dependent cells, such as platelets. In a panel of hematological cell lines, S55746 induces hallmarks of apoptosis including externalization of phosphatidylserine, caspase-3 activation and PARP cleavage. Ex vivo, S55746 induces apoptosis in the low nanomolar range in primary Chronic Lymphocytic Leukemia and Mantle Cell Lymphoma patient samples. Finally, S55746 administered by oral route daily in mice demonstrated robust anti-tumor efficacy in two hematological xenograft models with no weight lost and no change in behavior. Taken together, these data demonstrate that S55746 is a novel, well-tolerated BH3-mimetic targeting selectively and potently the BCL-2 protein.

Tessaracoccus rhinocerotis sp. nov., isolated from the faeces of Rhinoceros unicornis.

A novel Gram-stain-positive, non-spore-forming, irregular rod-shaped, non-motile and facultatively anaerobic actinobacterium, designated strain YIM 101269T, was isolated from the faeces of Rhinoceros unicornis living in Yunnan Wild Animal Park, Yunnan province, south-west China. The isolate grew at 10-35 °C, at pH 6-12 and with 0-9 % (w/v) NaCl. The cell-wall peptidoglycan of the organism contained ll-diaminopimelic acid as the diagnostic diamino acid. The polar lipids detected were diphosphatidylglycerol, phosphatidylglycerol, three unidentified polar lipids, one unidentified aminophospholipid and three unknown glycolipids. The major cellar fatty acid was anteiso-C15 : 0.MK-10(H4) was the predominant menaquinone. The DNA G+C content was 69.5 mol%. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain YIM 101269T belonged to the genus Tessaracoccus, closely related to Tessaracoccus flavescens DSM 18582T (97.4 % similarity). Based on the evidence from the present study, strain YIM 101269T is considered to represent a novel species of the genus Tessaracoccus, for which the name Tessaracoccus rhinocerotis sp. nov. is proposed. The type strain is YIM 101269T ( = DSM 27579T = CCTCC AB 2013217T).

Enteractinococcus lamae sp. nov. and Enteractinococcus viverrae sp. nov., isolated from animal faeces.

Two novel actinobacteria, designated strains YIM 101617(T) and YIM 101632(T), were isolated from Lama pacos (alpaca) and Viverra zibetha (civet) faeces in Yunnan Wild Animal Park in Yunnan province, southwestern China. Both strains should be placed in genus Enteractinococcus based on phylogenetic analysis. Based on 16S rRNA gene sequence analysis, strain YIM 101617(T) exhibits high similarity to Enteractinococcus fodinae DSM 22966(T) (97.70 %) and Enteractinococcus coprophilus YIM 100590(T) (97.45 %), whilst YIM 101632(T) exhibits high similarity to Enteractinococcus coprophilus YIM 100590(T) (97.25 %), and the similarity between YIM 101617(T) and YIM 101632(T) is 95.90 %. However, DNA-DNA hybridization values of the two strains with the type strains in the genus Enteractinococcus were low (<70 %). Most morphological and chemotaxonomic characteristics of the two strains were found to be similar to those of species in the genus Enteractinococcus but also some differences were observed. The DNA G+C contents of strains YIM 101617(T) and YIM 101632(T) were determined to be 55.9 and 56.4 mol%, respectively. Based on these data, the two strains are concluded to represent two different novel species in the genus Enteractinococcus. The names Enteractinococcus lamae sp. nov. (type strain YIM 101617(T)=DSM 27612(T)=CCTCC AB 2013230(T)) and Enteractinococcus viverrae sp. nov. (type strain YIM 101632(T)=KCTC 39552(T)=CCTCC AB 2013280(T)) are proposed, respectively.

Sphingobacterium rhinocerotis sp. nov., isolated from the faeces of Rhinoceros unicornis.

A novel Gram-negative, strictly aerobic, short rod-shaped, non-motile bacterium, designated YIM 101302(T), was isolated from the faeces of Rhinoceros unicornis dwelling in the Yunnan Wild Animal Park, Yunnan province, South-West China. The 16S rRNA gene sequence analysis revealed a clear affiliation of strain YIM 101302(T) to the genus Sphingobacterium. The newly isolated bacterium was found to be closely related to Sphingobacterium composti T5-12(T) (97.1% 16S rRNA sequence identity) and Sphingobacterium alimentarium WCC 4521(T) (95.6% 16S rRNA sequence identity) forming a distinct clade with these two species. Polar lipids of strain YIM 101302(T) were identified as phosphatidylethanolamine, phosphatidylmethylethanolamine, phosphatidylglycerol, phosphatidylinositol, an unidentified aminophospholipid, and three unidentified polar lipids; the predominant menaquinone as MK-7 and the major fatty as iso-C15:0. The genomic DNA G+C content was determined to be 38.9 mol%. The DNA-DNA hybridization values between strain YIM 101302(T) and S. composti T5-12(T), was 53.6 ± 5.8%. These results indicates that strain YIM 101302(T) represents a novel species of the genus Sphingobacterium, for which the name Sphingobacterium rhinocerotis sp. nov. is proposed. The type strain is YIM 101302(T) (=CCTCC AB 2013218(T) = KCTC 42533(T)).

FGFR-Mediated Reactivation of MAPK Signaling Attenuates Antitumor Effects of Imatinib in Gastrointestinal Stromal Tumors.

UNLABELLED: Activating mutations in either KIT or PDGFRA are present in approximately 90% of gastrointestinal stromal tumors (GIST). Although treatment with the KIT and PDGFR inhibitor imatinib can control advanced disease in about 80% of GIST patients, the beneficial effect is not durable. Here, we report that ligands from the FGF family reduced the effectiveness of imatinib in GIST cells, and FGF2 and FGFR1 are highly expressed in all primary GIST samples examined. The combination of KIT and FGFR inhibition showed increased growth inhibition in imatinib-sensitive GIST cell lines and improved efficacy in patient-derived GIST xenografts. In addition, inhibition of MAPK signaling by imatinib was not sustained in GIST cells. An ERK rebound occurred through activation of FGF signaling, and was repressed by FGFR1 inhibition. Downregulation of Sprouty proteins played a role in the imatinib-induced feedback activation of FGF signaling in GIST cells. SIGNIFICANCE: We here show that FGFR-mediated reactivation of the MAPK pathway attenuates the antiproliferation effects of imatinib in GISTs. The imatinib-induced ERK rebound can be repressed by the FGFR inhibitor BGJ398, and combined KIT and FGFR inhibition leads to increased efficacy in vitro and in patient-derived xenografts.

Tracking the inflammatory response in stroke in vivo by sensing the enzyme myeloperoxidase.

Inflammation can extend ischemic brain injury and adversely affect outcome in experimental animal models. A key difficulty in translating animal studies to humans is the lack of a definitive method to confirm and track inflammation in the brain in vivo. Myeloperoxidase (MPO), a key inflammatory enzyme secreted by activated neutrophils and macrophages/microglia, can generate highly reactive oxygen species to cause additional damage in cerebral ischemia. We report here that a functional, enzyme-activatable MRI agent can accurately track the oxidative activity of MPO noninvasively in stroke in living animals. We found that MPO is widely distributed in ischemic tissues, correlates positively with infarct size, and is detected even 3 weeks postinfarction. The peak level of MPO activity, determined by activation of the MPO-sensing agent in vivo and confirmed by MPO activity and quantitative RT-PCR assays, occurred on day 3 after ischemia. Both neutrophils and macrophages/microglia contribute to secrete MPO in the ischemic brain, although neutrophils peak earlier (days 1-3) whereas macrophages/microglia are most abundant later (days 3-7). In contrast to the conventional MRI agent diethylenetriamine-pentatacetate gadolinium, which reports blood-brain barrier disruption, MPO imaging is able to additionally track MPO activity and confirm inflammation on the molecular level in vivo, information that was previously only possible to obtain on ex vivo brain sections and impossible to assess in living human patients. Our findings could allow efficient noninvasive serial screening of therapies targeting inflammation and the use of MPO imaging as an imaging biomarker to risk-stratify patients.

Cerebrolysin enhances functional recovery following focal cerebral infarction in rats.

BACKGROUND: Cerebrolysin, a preparation derived from porcine brain, contains a mixture of neurotrophic peptides. We tested the effects of Cerebrolysin in a model of stroke recovery in rats. METHODS: Cerebrolysin (1.0, 2.5, or 5.0 ml/kg) was administered once daily intraperitoneally for 21 days, starting 24 hours after focal cerebral infarction (stroke) due to middle cerebral artery occlusion in mature rats. RESULTS: Enhancement of sensorimotor recovery, as assessed by forelimb and hindlimb placing and body swing tests, was seen with Cerebrolysin treatment, especially at the 2.5 ml/kg dose. At this dose, enhanced recovery was found when Cerebrolysin treatment was begun at 24 or 48 (but not 72 hours) after stroke onset. There were no effects on body weight or infarct volume when Cerebrolysin was administered in this manner. CONCLUSIONS: These results suggest that Cerebrolysin may be a useful treatment for enhancing neurological recovery after stroke.