Zhi Sheng

A genome-wide RNA interference screen reveals an essential CREB3L2-ATF5-MCL1 survival pathway in malignant glioma with therapeutic implications

Activating transcription factor-5 (ATF5) is highly expressed in malignant glioma and has a key role in promoting cell survival. Here we perform a genome-wide RNAi screen to identify transcriptional regulators of ATF5. Our results reveal an essential survival pathway in malignant glioma, whereby activation of a RAS–mitogen-activated protein kinase or phosphoinositide-3-kinase signaling cascade leads to induction of the transcription factor cAMP response element–binding protein-3–like-2 (CREB3L2), which directly activates ATF5 expression. ATF5, in turn, promotes survival by stimulating transcription of myeloid cell leukemia sequence-1 (MCL1), an antiapoptotic B cell leukemia-2 family member. Analysis of human malignant glioma samples indicates that ATF5 expression inversely correlates with disease prognosis. The RAF kinase inhibitor sorafenib suppresses ATF5 expression in glioma stem cells and inhibits malignant glioma growth in cell culture and mouse models. Our results demonstrate that ATF5 is essential in malignant glioma genesis and reveal that the ATF5-mediated survival pathway described here provides potential therapeutic targets for treatment of malignant glioma.

BCR-ABL suppresses autophagy through ATF5-mediated regulation of mTOR transcription

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-ABL-transformed 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.

A therapeutically targetable mechanism of BCR-ABL–independent imatinib resistance in chronic myeloid leukemia

A large-scale RNA interference screen reveals a new mechanism of imatinib resistance in chronic myeloid leukemia that can be therapeutically targeted. An Irresistible Combination Unlike most cancers, which have variable mutation patterns, chronic myeloid leukemia is normally associated with a specific genetic alteration, which produces a fusion protein called BCR-ABL. Famously, this protein was targeted with the first cancer-specific drug, known as imatinib (Gleevec), which is still the standard therapy for this cancer. Unfortunately, leukemia cells can develop resistance to imatinib, which does not always require mutations in BCR-ABL. Now, Ma et al. have identified a mechanism for imatinib resistance in chronic myeloid leukemia cells that lack mutations in BCR-ABL. The authors also demonstrated that a U.S. Food and Drug Administration–approved drug called trametinib can overcome this resistance and kill leukemia stem cells without harming the nonmalignant precursors that give rise to normal blood cells. 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 screen and identified IM-sensitizing genes (IMSGs) whose knockdown renders BCR-ABL+ cells IM-resistant. In these IMSG knockdown cells, RAF/mitogen-activated protein kinase kinase (MEK)/extracellular signal–regulated kinase (ERK) signaling is sustained after IM treatment because of up-regulation of PRKCH, which encodes the protein kinase C (PKC) family member PKCη, an activator of CRAF. PRKCH is also up-regulated in samples from CML patients with BCR-ABL–independent IM resistance. Combined treatment with IM and trametinib, a U.S. Food and Drug Administration–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.

Connexin 43 Inhibition Sensitizes Chemoresistant Glioblastoma Cells to Temozolomide.

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.

A Phase I Study of the Combination of Sorafenib With Temozolomide and Radiation Therapy for the Treatment of Primary and Recurrent High-Grade Gliomas

PURPOSE Despite recent advances in the management of high-grade and recurrent gliomas, survival remains poor. Antiangiogenic therapy has been shown to be efficacious in the treatment of high-grade gliomas both in preclinical models and in clinical trials. We sought to determine the safety and maximum tolerated dose of sorafenib when combined with both radiation and temozolomide in the primary setting or radiation alone in the recurrent setting. METHODS AND MATERIALS This was a preclinical study and an open-label phase I dose escalation trial. Multiple glioma cell lines were analyzed for viability after treatment with radiation, temozolomide, or sorafenib or combinations of them. For patients with primary disease, sorafenib was given concurrently with temozolomide (75 mg/m(2)) and 60 Gy radiation, for 30 days after completion of radiation. For patients with recurrent disease, sorafenib was combined with a hypofractionated course of radiation (35 Gy in 10 fractions). RESULTS Cell viability was significantly reduced with the combination of radiation, temozolomide, and sorafenib or radiation and sorafenib. Eighteen patients (11 in the primary cohort, 7 in the recurrent cohort) were enrolled onto this trial approved by the institutional review board. All patients completed the planned course of radiation therapy. The most common toxicities were hematologic, fatigue, and rash. There were 18 grade 3 or higher toxicities. The median overall survival was 18 months for the entire population. CONCLUSIONS Sorafenib can be safely combined with radiation and temozolomide in patients with high-grade glioma and with radiation alone in patients with recurrent glioma. The recommended phase II dose of sorafenib is 200 mg twice daily when combined with temozolomide and radiation and 400 mg with radiation alone. To our knowledge, this is the first publication of concurrent sorafenib with radiation monotherapy or combined with radiation and temozolomide.

The Blk pathway functions as a tumor suppressor in chronic myeloid leukemia stem cells

A therapeutic strategy for treating cancer is to target and eradicate cancer stem cells (CSCs) without harming their normal stem cell counterparts. The success of this approach relies on the identification of molecular pathways that selectively regulate CSC function. Using BCR-ABL–induced chronic myeloid leukemia (CML) as a disease model for CSCs, we show that BCR-ABL downregulates the Blk gene (encoding B-lymphoid kinase) through c-Myc in leukemic stem cells (LSCs) in CML mice and that Blk functions as a tumor suppressor in LSCs but does not affect normal hematopoietic stem cells (HSCs) or hematopoiesis. Blk suppresses LSC function through a pathway involving an upstream regulator, Pax5, and a downstream effector, p27. Inhibition of this Blk pathway accelerates CML development, whereas increased activity of the Blk pathway delays CML development. Blk also suppresses the proliferation of human CML stem cells. Our results show the feasibility of selectively targeting LSCs, an approach that should be applicable to other cancers.

Direct Regulation of rRNA Transcription by Fibroblast Growth Factor 2

ABSTRACT Fibroblast growth factor 2 (FGF-2), which is highly expressed in developing tissues and malignant cells, regulates cell growth, differentiation, and migration. Five isoforms (18 to ∼34 kDa) of FGF-2 are derived from alternative initiation codons of a single mRNA. The 18-kDa FGF-2 isoform is released from cells by a nonclassical secretory pathway and regulates gene expression by binding to cell surface receptors. This isoform also localizes to the nucleolus, raising the possibility that it may directly regulate ribosome biogenesis, a rate-limiting process in cell growth. Although several growth factors have been shown to accumulate in the nucleolus, their function and mechanism of action remain unclear. Here we show that 18-kDa FGF-2 interacts with upstream binding factor (UBF), an architectural transcription factor essential for rRNA transcription. The maximal activation of rRNA transcription in vitro by 18-kDa FGF-2 requires UBF. The 18-kDa FGF-2 localizes to rRNA genes and is necessary for the full activation of pre-rRNA synthesis in vivo. Our results demonstrate that 18-kDa FGF-2 directly regulates rRNA transcription.

Transcription and signalling pathways involved in BCR–ABL‐mediated misregulation of 24p3 and 24p3R

Lipocalin 24p3 is a secreted protein that can induce apoptosis in cells containing the 24p3 cell surface receptor, 24p3R. The oncoprotein BCR–ABL activates 24p3 and represses 24p3R expression. Thus, BCR–ABL+ cells synthesise and secrete 24p3, which induces apoptosis in normal 24p3R‐containing cells but not in BCR–ABL+ cells. The cell signalling and transcription factor pathways by which BCR–ABL misregulates expression of 24p3 and 24p3R remain to be elucidated. Here we show that BCR–ABL upregulates 24p3 expression through activation of the JAK/STAT pathway, which culminates in binding of Stat5 to the 24p3 promoter. We find that 24p3R expression is regulated by Runx transcription factors, and that BCR–ABL induces a switch in binding from Runx3, an activator of 24p3R expression, to Runx1, a repressor of 24p3R expression, through a Ras signalling pathway. Finally, we show that repression of 24p3R by BCR–ABL is a critical feature of the mechanism by which imatinib kills BCR–ABL+ cells. Our results reveal diverse signalling/transcription pathways that regulate 24p3 and 24p3R expression in response to BCR–ABL and are directly relevant to the treatment of BCR–ABL+ disease.

Real-Time Visualization of Nanoparticles Interacting with Glioblastoma Stem Cells

Nanoparticle-based therapy represents a novel and promising approach to treat glioblastoma, the most common and lethal malignant brain cancer. Although similar therapies have achieved significant cytotoxicity in cultured glioblastoma or glioblastoma stem cells (GSCs), the lack of an appropriate approach to monitor interactions between cells and nanoparticle-based therapies impedes their further clinical application in human patients. To address this critical issue, we first obtained NOTCH1 positive GSCs from patient-derived primary cultures. We then developed a new imaging approach to directly observe the dynamic nature of nanoparticles at the molecular level using in situ transmission electron microscopy (TEM). Utilizing these tools we were able to visualize real-time movements of nanoparticles interacting with GSCs for the first time. Overall, we show strong proof-of-concept results that real-time visualization of nanoparticles in single cells can be achieved at the nanoscale using TEM, thereby providing a powerful platform for the development of nanotherapeutics.

Expression and purification of a biologically active basic fibroblast growth factor fusion protein.

Basic fibroblast growth factor (bFGF) is a potent mitogen of many cell types and plays an important role in angiogenesis. To help identify proteins that bind to bFGF and mediate its intracellular transport and signaling, we overexpressed and purified a bFGF fusion protein in Escherichia coli. The fusion protein consists of bFGF fused to the C-terminus of glutathione S-transferase (GST). The GST-bFGF fusion protein was purified using SP-Sepharose and glutathione-Sepharose affinity chromatography. The ability of the purified GST-bFGF to stimulate the growth of human umbilical vein endothelial cells (HUVECs) was equivalent to that of purified recombinant 18 kDa bFGF.