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Activating transcription factor 5 (ATF5) is highly expressed in malignant glioma and plays an important role in promoting cell survival. Here we perform a genome-wide RNA interference (RNAi) screen to identify transcriptional regulators of ATF5. Our results reveal an essential survival pathway in malignant glioma, whereby activation of a RAS/MAPK or PI3K signaling cascade leads to induction of the transcription factor CREB3L2, which directly activates ATF5 expression. ATF5, in turn, promotes survival by stimulating transcription of MCL1, an anti-apoptotic BCL2 family member. Analysis of human malignant glioma samples indicates that ATF5 expression inversely correlates with disease prognosis. The RAF inhibitor sorafenib suppresses ATF5 expression in glioma stem cells and inhibits malignant glioma growth in cell culture and mouse xenografts. Our results demonstrate that ATF5 plays an essential role in malignant glioma genesis, and reveal that the ATF5-mediated survival pathway described here provides potential therapeutic targets for treatment of malignant glioma.

Resistance of glioblastoma (GBM) to the front-line chemotherapeutic agent temozolomide (TMZ) continues to challenge GBM treatment efforts. The repair of TMZ-induced DNA damage by O-6-methylguanine-DNA methyltransferase (MGMT) confers one mechanism of TMZ resistance. Paradoxically, MGMT-deficient GBM patients survive longer despite still developing resistance to TMZ. Recent studies indicate that the gap junction protein connexin 43 (Cx43) renders GBM cells resistant to TMZ through its carboxyl terminus (CT). In this study, we report insights into how Cx43 promotes TMZ resistance. Cx43 levels were inversely correlated with TMZ sensitivity of GBM cells, including GBM stem cells. Moreover, Cx43 levels inversely correlated with patient survival, including as observed in MGMT-deficient GBM patients. Addition of the C-terminal peptide mimetic αCT1, a selective inhibitor of Cx43 channels, sensitized human MGMT-deficient and TMZ-resistant GBM cells to TMZ treatment. Moreover, combining αCT1 with TMZ blocked AKT/mTOR signaling, induced autophagy and apoptosis in TMZ-resistant GBM cells. Our findings suggest that Cx43 may offer a biomarker to predict the survival of patients with MGMT-independent TMZ resistance, and that combining a Cx43 inhibitor with TMZ could enhance therapeutic responses in GBM and perhaps other TMZ-resistant cancers.

(2015) A rapid and high content assay that measures cyto-ID-stained autophagic compartments and estimates autophagy flux with potential clinical applications, Autophagy, 11:3, 560-572, DOI: 10.1080/15548627.2015 T he lack of a rapid and quantitative autophagy assay has substantially hindered the development and implementation of autophagy-targeting therapies for a variety of human diseases. To address this critical issue, we developed a novel autophagy assay using the newly developed Cyto-ID fluorescence dye. We first verified that the Cyto-ID dye specifically labels autophagic compartments with minimal staining of lysosomes and endosomes. We then developed a new Cyto-ID fluorescence spectrophotometric assay that makes it possible to estimate autophagy flux based on measurements of the Cyto-ID-stained autophagic compartments. By comparing to traditional autophagy approaches, we found that this assay yielded a more sensitive, yet less variable, quantification of the stained autophagic compartments and the estimate of autophagy flux. Furthermore, we tested the potential application of this autophagy assay in high throughput research by integrating it into an RNA interference (RNAi) screen and a small molecule screen. The RNAi screen revealed WNK2 and MAP3K6 as autophagy-modulating genes, both of which inhibited the MTOR pathway. Similarly, the small molecule screen identified sanguinarine and actinomycin D as potent autophagy inducers in leukemic cells. Moreover, we successfully detected autophagy responses to kinase inhibitors and chloroquine in normal or leukemic mice using this assay. Collectively, this new Cyto-ID fluorescence spectrophotometric assay provides a rapid, reliable quantification of autophagic compartments and estimation of autophagy flux with potential applications in developing autophagy-related therapies and as a test to monitor autophagy responses in patients being treated with autophagymodulating drugs.

PIK3CB/p110β is a biomarker for GBM recurrence and selectively important for GBM cell survival.

The oncoprotein BCR-ABL transforms myeloid progenitor cells and is responsible for the development of chronic myeloid leukemia (CML). In transformed cells, BCR-ABL suppresses apoptosis as well as autophagy, a catabolic process in which cellular components are degraded by the lysosomal machinery. The mechanism by which BCR-ABL suppresses autophagy is not known. Here we report that in both mouse and human BCR-ABLtransformed cells, activating transcription factor 5 (ATF5), a prosurvival factor, suppresses autophagy but does not affect apoptosis. We find that BCR-ABL, through PI3K/AKT/FOXO4 signaling, transcriptionally up-regulates ATF5 expression and that ATF5, in turn, stimulates transcription of mammalian target of rapamycin (mTOR; also called mechanistic target of rapamycin), a well-established master negative-regulator of autophagy.Previous studies have shown that the BCR-ABL inhibitor imatinib mesylate induces both apoptosis and autophagy, and that the resultant autophagy modulates the efficiency by which imatinib kills BCR-ABL-transformed cells. We demonstrate that imatinib-induced autophagy is because of inhibition of the BCR-ABL/PI3K/ AKT/FOXO4/ATF5/mTOR pathway that we have identified in this study. (Blood. 2011; 118(10):2840-2848) Introduction BCR-ABL is an oncogene derived from the translocation between chromosomes 9 and 22 that can transform myeloid progenitor cells and which drives the development of chronic myeloid leukemia (CML; reviewed in Melo and Barnes 1 ). BCR-ABL encodes a constitutively active protein tyrosine kinase that exerts its oncogenic function by activating a cascade of intracellular signaling pathways, which leads to increased survival and proliferation and limited dependence on growth factors. For example, BCR-ABL stimulates PI3K/AKT signaling, which in turn suppresses forkhead O (FOXO) transcription factors, resulting in increased proliferation and survival. 2,3 FOXO transcription factors have been shown to play an important role in CML. 2 Imatinib mesylate, also called Gleevec or STI571, has revolutionized the treatment of CML and is now the standard first-line therapy provided to CML patients (reviewed in Druker 4 ). Imatinib is a relatively specific tyrosine kinase inhibitor that targets the ATP-binding domain of BCR-ABL and suppresses its enzymatic activity. Inhibition of BCR-ABL tyrosine kinase activity by imatinib results in induction of cell death, because of both a decrease of intracellular survival signals and a relative increase in proapoptotic signals.Recently, it has been found that treatment of BCR-ABLtransformed cells with imatinib also induces autophagy. 5 Autophagy is a degradative process that results in the breakdown of intracellular organelles and proteins within lysosomes. Intriguingly, autophagy can contribute to either survival or death dependent on cellular context. [6][7][8] The finding that imatinib induces autophagy in BCR-ABL-transformed cells implies that BCR-ABL suppresses autophagy through a pathway (or pathways) that remains to be identified.Activating...

Resistance to the BCR-ABL inhibitor imatinib mesylate (IM) poses a major problem for the treatment of chronic myeloid leukemia (CML). IM resistance often results from a secondary mutation in BCR-ABL that interferes with drug binding. However, in many instances there is no mutation in BCR-ABL, and the basis of such BCR-ABL-independent IM resistance remains to be elucidated. To gain insight into BCR-ABL-independent IM resistance mechanisms, we performed a large-scale RNA interference (RNAi) screen and identified IM-sensitizing genes (IMSGs) whose knockdown renders BCR-ABL+ cells IM-resistant. In these IMSG knockdown cells, RAF/MEK/ERK signaling is sustained after IM treatment due to upregulation of PRKCH, which encodes the protein kinase C (PKC) family member PKCη, an activator of CRAF. PRKCH is also upregulated in samples from CML patients with BCR-ABL-independent IM resistance. Combined treatment with IM and trametinib, an FDA-approved MEK inhibitor, synergistically kills BCR-ABL+ IMSG knockdown cells and prolongs survival in mouse models of BCR-ABL-independent IM-resistant CML. Finally, we showed that CML stem cells contain high levels of PRKCH and this contributes to their intrinsic IM resistance. Combined treatment with IM and trametinib synergistically kills CML stem cells with negligible effect on normal hematopoietic stem cells. Collectively, our results identify a therapeutically targetable mechanism of BCR-ABL-independent IM resistance in CML and CML stem cells.

The development of new nanoparticles as next-generation diagnostic and therapeutic ("theranostic") drug platforms is an active area of both chemistry and cancer research. Although numerous gadolinium (Gd) containing metallofullerenes as diagnostic magnetic resonance imaging (MRI) contrast agents have been reported, the metallofullerene cage surface, in most cases, consists of negatively charged carboxyl or hydroxyl groups that limit attractive forces with the cellular surface. It has been reported that nanoparticles with a positive charge will bind more efficiently to negatively charged phospholipid bilayer cellular surfaces, and will more readily undergo endocytosis. In this paper, we report the preparation of a new functionalized trimetallic nitride template endohedral metallofullerene (TNT EMF), Gd3N@C80(OH)x(NH2)y, with a cage surface bearing positively charged amino groups (-NH3(+)) and directly compare it with a similar carboxyl and hydroxyl functionalized derivative. This new nanoparticle was characterized by X-ray photoelectron spectroscopy (XPS), dynamic light scattering (DLS), and infrared spectroscopy. It exhibits excellent (1)H MR relaxivity. Previous studies have clearly demonstrated that the cytokine interleukin-13 (IL-13) effectively targets glioblastoma multiforme (GBM) cells, which are known to overexpress IL-13Rα2. We also report that this amino-coated Gd-nanoplatform, when subsequently conjugated with interleukin-13 peptide IL-13-Gd3N@C80(OH)x(NH2)y, exhibits enhanced targeting of U-251 GBM cell lines and can be effectively delivered intravenously in an orthotopic GBM mouse model.