Ex vivo drug sensitivity screening predicts response to temozolomide in glioblastoma patients and identifies candidate biomarkers.

BACKGROUND: Patient-derived glioma stem-like cells (GSCs) have become the gold-standard in neuro-oncological research; however, it remains to be established whether loss of in situ microenvironment affects the clinically-predictive value of this model. We implemented a GSC monolayer system to investigate in situ-in vitro molecular correspondence and the relationship between in vitro and patient response to temozolomide (TMZ). METHODS: DNA/RNA-sequencing was performed on 56 glioblastoma tissues and 19 derived GSC cultures. Sensitivity to TMZ was screened across 66 GSC cultures. Viability readouts were related to clinical parameters of corresponding patients and whole-transcriptome data. RESULTS: Tumour DNA and RNA sequences revealed strong similarity to corresponding GSCs despite loss of neuronal and immune interactions. In vitro TMZ screening yielded three response categories which significantly correlated with patient survival, therewith providing more specific prediction than the binary MGMT marker. Transcriptome analysis identified 121 genes related to TMZ sensitivity of which 21were validated in external datasets. CONCLUSION: GSCs retain patient-unique hallmark gene expressions despite loss of their natural environment. Drug screening using GSCs predicted patient response to TMZ more specifically than MGMT status, while transcriptome analysis identified potential biomarkers for this response. GSC drug screening therefore provides a tool to improve drug development and precision medicine for glioblastoma.

CIC reduces xCT/SLC7A11 expression and glutamate release in glioma.

Capicua (CIC) is an important downstream molecule of RTK/RAS/MAPK pathway. The regulatory mechanism of CIC underlying tumorigenesis in oligodendroglioma, where CIC is frequently mutated, has yet to be fully elucidated. Using patient-derived glioma lines, RNA-sequencing and bioinformatic analysis of publicly available databases, we investigated how CIC loss- or gain-of-function regulates its downstream targets, cell proliferation and glutamate release. Our results indicate an increased frequency of CIC truncating mutations in oligodendroglioma during progression. In vitro, CIC modulation had a modest effect on cell proliferation in glioma lines, and no significant changes in the expression of ETV1, ETV4 and ETV5. Transcriptional repression of known CIC targets was observed in gliomas expressing non-phosphorylatable CIC variant on Ser173 which was unable to interact with 14-3-3. These data outline a mechanism by which the repressor function of CIC is inhibited by 14-3-3 in gliomas. Using transcriptional profiling, we found that genes related to glutamate release were upregulated because of CIC depletion. In addition, loss of CIC leads to increased extracellular glutamate. Consistent with this, CIC restoration in an oligodendroglioma line reduced the levels of extracellular glutamate, neuronal toxicity and xCT/SLC7A11 expression. Our findings may provide a molecular basis for the prevention of glioma-associated seizures.

Stimulation of the hepatoportal nerve plexus with focused ultrasound restores glucose homoeostasis in diabetic mice, rats and swine.

Peripheral neurons that sense glucose relay signals of glucose availability to integrative clusters of neurons in the brain. However, the roles of such signalling pathways in the maintenance of glucose homoeostasis and their contribution to disease are unknown. Here we show that the selective activation of the nerve plexus of the hepatic portal system via peripheral focused ultrasound stimulation (pFUS) improves glucose homoeostasis in mice and rats with insulin-resistant diabetes and in swine subject to hyperinsulinemic-euglycaemic clamps. pFUS modulated the activity of sensory projections to the hypothalamus, altered the concentrations of metabolism-regulating neurotransmitters, and enhanced glucose tolerance and utilization in the three species, whereas physical transection or chemical blocking of the liver-brain nerve pathway abolished the effect of pFUS on glucose tolerance. Longitudinal multi-omic profiling of metabolic tissues from the treated animals confirmed pFUS-induced modifications of key metabolic functions in liver, pancreas, muscle, adipose, kidney and intestinal tissues. Non-invasive ultrasound activation of afferent autonomic nerves may represent a non-pharmacologic therapy for the restoration of glucose homoeostasis in type-2 diabetes and other metabolic diseases.

Identification of the GTPase-activating protein DEP domain containing 1B (DEPDC1B) as a transcriptional target of Pitx2.

Pitx2 is a bicoid-related homeobox transcription factor implicated in regulating left-right patterning and organogenesis. However, only a limited number of Pitx2 downstream target genes have been identified and characterized. Here we demonstrate that Pitx2 is a transcriptional repressor of DEP domain containing 1B (DEPDC1B). The first intron of the human and mouse DEP domain containing 1B genes contains multiple consensus DNA-binding sites for Pitx2. Chromatin immunoprecipitation assays revealed that Pitx2, along with histone deacetylase 1, was recruited to the first intron of Depdc1b. In contrast, RNAi-mediated depletion of Pitx2 not only enhanced the acetylation of histone H4 in the first intron of Depdc1b, but also increased the protein level of Depdc1b. Luciferase reporter assays also showed that Pitx2 could repress the transcriptional activity mediated by the first intron of human DEPDC1B. The GAP domain of DEPDC1B interacted with nucleotide-bound forms of RAC1 in vitro. In addition, exogenous expression of DEPDC1B suppressed RAC1 activation and interfered with actin polymerization induced by the guanine nucleotide exchange factor TRIO. Moreover, DEPDC1B interacted with various signaling molecules such as U2af2, Erh, and Salm. We propose that Pitx2-mediated repression of Depdc1b expression contributes to the regulation of multiple molecular pathways, such as Rho GTPase signaling.

Intramolecular interactions between the Dbl homology (DH) domain and the carboxyl-terminal region of myosin II-interacting guanine nucleotide exchange factor (MyoGEF) act as an autoinhibitory mechanism for the regulation of MyoGEF functions.

We have reported previously that nonmuscle myosin II-interacting guanine nucleotide exchange factor (MyoGEF) plays an important role in the regulation of cell migration and cytokinesis. Like many other guanine nucleotide exchange factors (GEFs), MyoGEF contains a Dbl homology (DH) domain and a pleckstrin homology domain. In this study, we provide evidence demonstrating that intramolecular interactions between the DH domain (residues 162-351) and the carboxyl-terminal region (501-790) of MyoGEF can inhibit MyoGEF functions. In vitro and in vivo pulldown assays showed that the carboxyl-terminal region (residues 501-790) of MyoGEF could interact with the DH domain but not with the pleckstrin homology domain. Expression of a MyoGEF carboxyl-terminal fragment (residues 501-790) decreased RhoA activation and suppressed actin filament formation in MDA-MB-231 breast cancer cells. Additionally, Matrigel invasion assays showed that exogenous expression of the MyoGEF carboxyl-terminal region decreased the invasion activity of MDA-MB-231 cells. Moreover, coimmunoprecipitation assays showed that phosphorylation of the MyoGEF carboxyl-terminal region by aurora B kinase interfered with the intramolecular interactions of MyoGEF. Furthermore, expression of the MyoGEF carboxyl-terminal region interfered with RhoA localization during cytokinesis and led to an increase in multinucleation. Together, our findings suggest that binding of the carboxyl-terminal region of MyoGEF to its DH domain acts as an autoinhibitory mechanism for the regulation of MyoGEF activation.