IQGAP1 scaffolding links phosphoinositide kinases to cytoskeletal reorganization.

IQGAP1 is a multidomain scaffold protein that coordinates the direction and impact of multiple signaling pathways by scaffolding its various binding partners. However, the spatial and temporal resolution of IQGAP1 scaffolding remains unclear. Here, we use fluorescence imaging and correlation methods that allow for real-time live-cell changes in IQGAP1 localization and complex formation during signaling. We find that IQGAP1 and PIPKIγ interact on both the plasma membrane and in cytosol. Epidermal growth factor (EGF) stimulation, which can initiate cytoskeletal changes, drives the movement of the cytosolic pool toward the plasma membrane to promote cytoskeletal changes. We also observe that a significant population of cytosolic IQGAP1-PIPKIγ complexes localize to early endosomes, and in some instances form aggregated clusters which become highly mobile upon EGF stimulation. Our imaging studies show that PIPKIγ and PI3K bind simultaneously to IQGAP1, which may accelerate conversion of PI4P to PI(3,4,5)P3 that is required for cytoskeletal changes. Additionally, we find that IQGAP1 is responsible for PIPKIγ association with two proteins associated with cytoskeletal changes, talin and Cdc42, during EGF stimulation. These results directly show that IQGAP1 provides a physical link between phosphoinositides (through PIPKIγ), focal adhesion formation (through talin), and cytoskeletal reorganization (through Cdc42) upon EGF stimulation. Taken together, our results support the importance of IQGAP1 in regulating cell migration by linking phosphoinositide lipid signaling with cytoskeletal reorganization.

Multimodal quantitative imaging of brain cancer in cultured cells.

Fluorescence emission, polarization and subcellular localization of methylene blue (MB) were studied in four cancerous and two normal human brain cell lines. Fluorescence emission and polarization images were acquired and analyzed. The co-localization of MB with mitochondria, lysosomes and nuclei of the cells was evaluated. Glioblastoma cells exhibited significantly higher MB fluorescence polarization compared to normal astrocytes. Preferential accumulation of MB in mitochondria of glioblastoma cells may explain higher fluorescence polarization values in cancer cells as compared to normal. These findings may lead to the development of a quantitative method for the detection of brain cancer in single cells.

ER stress in temozolomide-treated glioblastomas interferes with DNA repair and induces apoptosis.

Glioblastoma multiforme (GBM) is a deadly grade IV brain tumor. Radiation in combination with temozolomide (TMZ), the current chemotherapeutic for GBMs, only provides 12-14 months survival post diagnosis. Because GBMs are dependent on both activation of the DNA damage pathway and the endoplasmic reticulum (ER) stress response, we asked if a novel ER stress inducing agent, JLK1486, increases the efficacy of TMZ.We found that the combination of TMZ+JLK1486 resulted in decreased proliferation in a panel of adherent GBM cells lines and reduced secondary sphere formation in non-adherent and primary lines. Decreased proliferation correlated with increased cell death due to apoptosis. We found prolonged ER stress in TMZ+JLK1486 treated cells that resulted in sustained activation of the unfolded protein response (UPR) through increased levels of BiP, ATF4, and CHOP. In addition, TMZ+JLK1486 treatment caused decreased RAD51 levels, impairing DNA damage repair. Furthermore, we found delayed time to tumor doubling in TMZ+JLK1486 treated mice.Our data shows that the addition of JLK1486 to TMZ increases the efficaciousness of the treatment by decreasing proliferation and inducing cell death. We propose increased cell death is due to two factors. One, prolonged ER stress driving the expression of the pro-apoptotic transcription factor CHOP, and, second, unresolved DNA double strand breaks, due to decreased RAD51 levels. The combination of TMZ+JLK1486 is a potential novel therapeutic combination and suggests an inverse relationship between unresolved ER stress and the DNA damage response pathway.

The PTEN Tumor Suppressor Forms Homodimers in Solution.

As the phosphoinositol-3-kinase antagonist in the PI3K pathway, the PTEN tumor suppressor exerts phosphatase activity on diacylphosphatidylinositol triphosphate in the plasma membrane. Even partial loss of this activity enhances tumorigenesis, but a mechanistic basis for this aspect of PTEN physiology has not yet been established. It was recently proposed that PTEN mutations have dominant-negative effects in cancer via PTEN dimers. We show that PTEN forms homodimers in vitro, and determine a structural model of the complex from SAXS and Rosetta docking studies. Our findings shed new light on the cellular control mechanism of PTEN activity. Phosphorylation of the unstructured C-terminal tail of PTEN reduces PTEN activity, and this result was interpreted as a blockage of the PTEN membrane binding interface through this tail. The results presented here instead suggest that the C-terminal tail functions in stabilizing the homodimer, and that tail phosphorylation interferes with this stabilization.

Combined inhibition of Bcl-2/Bcl-xL and Usp9X/Bag3 overcomes apoptotic resistance in glioblastoma in vitro and in vivo.

Despite great efforts taken to advance therapeutic measures for patients with glioblastoma, the clinical prognosis remains grim. The antiapoptotic Bcl-2 family protein Mcl-1 is overexpressed in glioblastoma and represents an important resistance factor to the BH-3 mimetic ABT263. In this study, we show that combined treatment with ABT263 and GX15-070 overcomes apoptotic resistance in established glioblastoma cell lines, glioma stem-like cells and primary cultures. Moreover, this treatment regimen also proves to be advantageous in vivo. On the molecular level, GX15-070 enhanced apoptosis by posttranslational down-regulation of the deubiquitinase, Usp9X, and the chaperone Bag3, leading to a sustained depletion of Mcl-1 protein levels. Moreover, knock-down of Usp9X or Bag3 depleted endogenous Mcl-1 protein levels and in turn enhanced apoptosis induced through Bcl-2/Bcl-xL inhibition. In conclusion, combined treatment with ABT263 and GX15-070 results in a significantly enhanced anti-cancer activity in vitro as well as in vivo in the setting of glioblastoma. Both drugs, ABT263 and GX15-070 have been evaluated in clinical studies which facilitates the translational aspect of taking this combinatorial approach to the clinical setting. Furthermore we present a novel mechanism by which GX15-070 counteracts Mcl-1 expression which may lay a foundation for a novel target in cancer therapy.

Biophysical methods for the characterization of PTEN/lipid bilayer interactions.

PTEN, a tumor suppressor protein that dephosphorylates phosphoinositides at the 3-position of the inositol ring, is a cytosolic protein that needs to associate with the plasma membrane or other subcellular membranes to exert its lipid phosphatase function. Upon membrane association PTEN interacts with at least three different lipid entities: An anionic lipid that is present in sufficiently high concentration to create a negative potential that allows PTEN to interact electrostatically with the membrane, phosphatidylinositol-4,5-bisphosphate, which interacts with PTEN's N-terminal end and the substrate, usually phosphatidylinositol-3,4,5-trisphosphate. Many parameters influence PTEN's interaction with the lipid bilayer, for example, the lateral organization of the lipids or the presence of other chemical species like cholesterol or other lipids. To investigate systematically the different steps of PTEN's complex binding mechanism and to explore its dynamic behavior in the membrane bound state, in vitro methods need to be employed that allow for a systematic variation of the experimental conditions. In this review we survey a variety of methods that can be used to assess PTEN lipid binding affinity, the dynamics of its membrane association as well as its dynamic behavior in the membrane bound state.

Evaluation of novel imidazotetrazine analogues designed to overcome temozolomide resistance and glioblastoma regrowth.

The cellular responses to two new temozolomide (TMZ) analogues, DP68 and DP86, acting against glioblastoma multiforme (GBM) cell lines and primary culture models are reported. Dose-response analysis of cultured GBM cells revealed that DP68 is more potent than DP86 and TMZ and that DP68 was effective even in cell lines resistant to TMZ. On the basis of a serial neurosphere assay, DP68 inhibits repopulation of these cultures at low concentrations. The efficacy of these compounds was independent of MGMT and MMR functions. DP68-induced interstrand DNA cross-links were demonstrated with H2O2-treated cells. Furthermore, DP68 induced a distinct cell-cycle arrest with accumulation of cells in S phase that is not observed for TMZ. Consistent with this biologic response, DP68 induces a strong DNA damage response, including phosphorylation of ATM, Chk1 and Chk2 kinases, KAP1, and histone variant H2AX. Suppression of FANCD2 expression or ATR expression/kinase activity enhanced antiglioblastoma effects of DP68. Initial pharmacokinetic analysis revealed rapid elimination of these drugs from serum. Collectively, these data demonstrate that DP68 is a novel and potent antiglioblastoma compound that circumvents TMZ resistance, likely as a result of its independence from MGMT and mismatch repair and its capacity to cross-link strands of DNA.

A unified nomenclature and amino acid numbering for human PTEN.

The tumor suppressor PTEN is a major brake for cell transformation, mainly due to its phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] phosphatase activity that directly counteracts the oncogenicity of phosphoinositide 3-kinase (PI3K). PTEN mutations are frequent in tumors and in the germ line of patients with tumor predisposition or with neurological or cognitive disorders, which makes the PTEN gene and protein a major focus of interest in current biomedical research. After almost two decades of intense investigation on the 403-residue-long PTEN protein, a previously uncharacterized form of PTEN has been discovered that contains 173 amino-terminal extra amino acids, as a result of an alternate translation initiation site. To facilitate research in the field and to avoid ambiguities in the naming and identification of PTEN amino acids from publications and databases, we propose here a unifying nomenclature and amino acid numbering for this longer form of PTEN.

PI3K and Bcl-2 inhibition primes glioblastoma cells to apoptosis through downregulation of Mcl-1 and Phospho-BAD.

UNLABELLED: Glioblastoma multiforme (GBM) is a highly malignant human brain neoplasm with limited therapeutic options. GBMs display a deregulated apoptotic pathway with high levels of the antiapoptotic Bcl-2 family of proteins and overt activity of the phosphatidylinositol 3-kinase (PI3K) signaling pathway. Therefore, combined interference of the PI3K pathway and the Bcl-2 family of proteins is a reasonable therapeutic strategy. ABT-263 (Navitoclax), an orally available small-molecule Bcl-2 inhibitor, and GDC-0941, a PI3K inhibitor, were used to treat established glioblastoma and glioblastoma neurosphere cells, alone or in combination. Although GDC-0941 alone had a modest effect on cell viability, treatment with ABT-263 displayed a marked reduction of cell viability and induction of apoptotic cell death. Moreover, combinatorial therapy using ABT-263 and GDC-0941 showed an enhanced effect, with a further decrease in cellular viability. Furthermore, combination treatment abrogated the ability of stem cell-like glioma cells to form neurospheres. ABT-263 and GDC-0941, in combination, resulted in a consistent and significant increase of Annexin V positive cells and loss of mitochondrial membrane potential compared with either monotherapy. The combination treatment led to enhanced cleavage of both initiator and effector caspases. Mechanistically, GDC-0941 depleted pAKT (Serine 473) levels and suppressed Mcl-1 protein levels, lowering the threshold for the cytotoxic actions of ABT-263. GDC-0941 decreased Mcl-1 in a posttranslational manner and significantly decreased the half-life of Mcl-1 protein. Ectopic expression of human Mcl-1 mitigated apoptotic cell death induced by the drug combination. Furthermore, GDC-0941 modulated the phosphorylation status of BAD, thereby further enhancing ABT-263-mediated cell death. IMPLICATIONS: Combination therapy with ABT-263 and GDC-0941 has novel therapeutic potential by specifically targeting aberrantly active, deregulated pathways in GBM, overcoming endogenous resistance to apoptosis.

Cholesterol stabilizes fluid phosphoinositide domains.

Local accumulation of phosphoinositides (PIPs) is an important factor for a broad range of cellular events including membrane trafficking and cell signaling. The negatively charged phosphoinositide headgroups can interact with cations or cationic proteins and this electrostatic interaction has been identified as the main phosphoinositide clustering mechanism. However, an increasing number of reports show that phosphoinositide-mediated signaling events are at least in some cases cholesterol dependent, suggesting other possible contributors to the segregation of phosphoinositides. Using fluorescence microscopy on giant unilamellar vesicles and monolayers at the air/water interface, we present data showing that cholesterol stabilizes fluid phosphoinositide-enriched phases. The interaction with cholesterol is observed for all investigated phosphoinositides (PI(4)P, PI(3,4)P2, PI(3,5)P2, PI(4,5)P2 and PI(3,4,5)P3) as well as phosphatidylinositol. We find that cholesterol is present in the phosphoinositide-enriched phase and that the resulting phase is fluid. Cholesterol derivatives modified at the hydroxyl group (cholestenone, cholesteryl ethyl ether) do not promote formation of phosphoinositide domains, suggesting an instrumental role of the cholesterol hydroxyl group in the observed cholesterol/phosphoinositide interaction. This leads to the hypothesis that cholesterol participates in an intermolecular hydrogen bond network formed among the phosphoinositide lipids. We had previously reported that the intra- and intermolecular hydrogen bond network between the phosphoinositide lipids leads to a reduction of the charge density at the phosphoinositide phosphomonoester groups (Kooijman et al., 2009). We believe that cholesterol acts as a spacer between the phosphoinositide lipids, thereby reducing the electrostatic repulsion, while participating in the hydrogen bond network, leading to its further stabilization. To illustrate the effect of phosphoinositide segregation on protein binding, we show that binding of the tumor suppressor protein PTEN to PI(5)P and PI(4,5)P2 is enhanced in the presence of cholesterol. These results provide new insights into how phosphoinositides mediate important cellular events.

Glioblastoma multiforme therapy and mechanisms of resistance.

Glioblastoma multiforme (GBM) is a grade IV brain tumor characterized by a heterogeneous population of cells that are highly infiltrative, angiogenic and resistant to chemotherapy. The current standard of care, comprised of surgical resection followed by radiation and the chemotherapeutic agent temozolomide, only provides patients with a 12-14 month survival period post-diagnosis. Long-term survival for GBM patients remains uncommon as cells with intrinsic or acquired resistance to treatment repopulate the tumor. In this review we will describe the mechanisms of resistance, and how they may be overcome to improve the survival of GBM patients by implementing novel chemotherapy drugs, new drug combinations and new approaches relating to DNA damage, angiogenesis and autophagy.

PtdIns(4,5)P2-mediated cell signaling: emerging principles and PTEN as a paradigm for regulatory mechanism.

PtdIns(4,5)P2 (phosphatidylinositol 4,5-bisphosphate) is a relatively common anionic lipid that regulates cellular functions by multiple mechanisms. Hydrolysis of PtdIns(4,5)P2 by phospholipase C yields inositol trisphosphate and diacylglycerol. Phosphorylation by phosphoinositide 3-kinase yields PtdIns(3,4,5)P3, which is a potent signal for survival and proliferation. Also, PtdIns(4,5)P2 can bind directly to integral and peripheral membrane proteins. As an example of regulation by PtdIns(4,5)P2, we discuss phosphatase and tensin homologue deleted on chromosome 10 (PTEN) in detail. PTEN is an important tumor suppressor and hydrolyzes PtdIns(3,4,5)P3. PtdIns(4,5)P2 enhances PTEN association with the plasma membrane and activates its phosphatase activity. This is a critical regulatory mechanism, but a detailed description of this process from a structural point of view is lacking. The disordered lipid bilayer environment hinders structural determinations of membrane-bound PTEN. A new method to analyze membrane-bound protein measures neutron reflectivity for proteins bound to tethered phospholipid membranes. These methods allow determination of the orientation and shape of membrane-bound proteins. In combination with molecular dynamics simulations, these studies will provide crucial structural information that can serve as a foundation for our understanding of PTEN regulation in normal and pathological processes.

Membrane association of the PTEN tumor suppressor: molecular details of the protein-membrane complex from SPR binding studies and neutron reflection.

The structure and function of the PTEN phosphatase is investigated by studying its membrane affinity and localization on in-plane fluid, thermally disordered synthetic membrane models. The membrane association of the protein depends strongly on membrane composition, where phosphatidylserine (PS) and phosphatidylinositol diphosphate (PI(4,5)P(2)) act pronouncedly synergistic in pulling the enzyme to the membrane surface. The equilibrium dissociation constants for the binding of wild type (wt) PTEN to PS and PI(4,5)P(2) were determined to be K(d)∼12 µM and 0.4 µM, respectively, and K(d)∼50 nM if both lipids are present. Membrane affinities depend critically on membrane fluidity, which suggests multiple binding sites on the protein for PI(4,5)P(2). The PTEN mutations C124S and H93R show binding affinities that deviate strongly from those measured for the wt protein. Both mutants bind PS more strongly than wt PTEN. While C124S PTEN has at least the same affinity to PI(4,5)P(2) and an increased apparent affinity to PI(3,4,5)P(3), due to its lack of catalytic activity, H93R PTEN shows a decreased affinity to PI(4,5)P(2) and no synergy in its binding with PS and PI(4,5)P(2). Neutron reflection measurements show that the PTEN phosphatase "scoots" along the membrane surface (penetration <5 Å) but binds the membrane tightly with its two major domains, the C2 and phosphatase domains, as suggested by the crystal structure. The regulatory C-terminal tail is most likely displaced from the membrane and organized on the far side of the protein, ∼60 Å away from the bilayer surface, in a rather compact structure. The combination of binding studies and neutron reflection allows us to distinguish between PTEN mutant proteins and ultimately may identify the structural features required for membrane binding and activation of PTEN.

Resolution of inflammation by N-arachidonoylglycine.

N-arachidonoylglycine (NAgly) is an endogenous signaling lipid that is a member of the eicosanoid super family and is related to anandamide. It shows anti-inflammatory activity in vivo in the mouse peritonitis model where it reduces migration of inflammatory leukocytes following injection of pro-inflammatory agents into the peritoneal cavity. Using cell culture models, including GPR18 transfected HEK-293 cells, evidence is presented that the orphan receptor GPR18 is involved in this action. Increases in free arachidonic acid, and robust stimulation of anti-inflammatory eicosanoids were observed at low micromolar concentrations. These included 15-deoxy-delta-13,14-PGJ(2) and lipoxin A(4) both of which are believed to mediate the resolution stage of inflammation. It was further shown that NAgly might act via GPR18 activation in promoting the number of Trypan Blue stained cells, a possible indicator of programmed cell death. Thus, we hypothesize that NAgly induces the death of inflammatory cells, a process that is considered to be important for the resolution of inflammation.

Exploiting the mitochondrial unfolded protein response for cancer therapy in mice and human cells.

Fine tuning of the protein folding environment in subcellular organelles, such as mitochondria, is important for adaptive homeostasis and may participate in human diseases, but the regulators of this process are still largely elusive. Here, we have shown that selective targeting of heat shock protein-90 (Hsp90) chaperones in mitochondria of human tumor cells triggered compensatory autophagy and an organelle unfolded protein response (UPR) centered on upregulation of CCAAT enhancer binding protein (C/EBP) transcription factors. In turn, this transcriptional UPR repressed NF-κB-dependent gene expression, enhanced tumor cell apoptosis initiated by death receptor ligation, and inhibited intracranial glioblastoma growth in mice without detectable toxicity. These data reveal what we believe to be a novel role of Hsp90 chaperones in the regulation of the protein-folding environment in mitochondria of tumor cells. Disabling this general adaptive pathway could potentially be used in treatment of genetically heterogeneous human tumors.

A mutant form of PTEN linked to autism.

The tumor suppressor, phosphatase, and tensin homologue deleted on chromosome 10 (PTEN), is a phosphoinositide (PI) phosphatase specific for the 3-position of the inositol ring. PTEN has been implicated in autism for a subset of patients with macrocephaly. Various studies identified patients in this subclass with one normal and one mutated PTEN gene. We characterize the binding, structural properties, activity, and subcellular localization of one of these autism-related mutants, H93R PTEN. Even though this mutation is located at the phosphatase active site, we find that it affects the functions of neighboring domains. H93R PTEN binding to phosphatidylserine-bearing model membranes is 5.6-fold enhanced in comparison to wild-type PTEN. In contrast, we find that binding to phosphatidylinositol-4,5-bisphosphate (PI(4,5)P(2)) model membranes is 2.5-fold decreased for the mutant PTEN in comparison to wild-type PTEN. The structural change previously found for wild-type PTEN upon interaction with PI(4,5)P(2), is absent for H93R PTEN. Consistent with the increased binding to phosphatidylserine, we find enhanced plasma membrane association of PTEN-GFP in U87MG cells. However, this enhanced plasma membrane association does not translate into increased PI(3,4,5)P(3) turnover, since in vivo studies show a reduced activity of the H93R PTEN-GFP mutant. Because the interaction of PI(4,5)P(2) with PTEN's N-terminal domain is diminished by this mutation, we hypothesize that the interaction of PTEN's N-terminal domain with the phosphatase domain is impacted by the H93R mutation, preventing PI(4,5)P(2) from inducing the conformational change that activates phosphatase activity.

Gamma-secretase inhibitors enhance temozolomide treatment of human gliomas by inhibiting neurosphere repopulation and xenograft recurrence.

Malignant gliomas are treated with a combination of surgery, radiation, and temozolomide (TMZ), but these therapies ultimately fail due to tumor recurrence. In glioma cultures, TMZ treatment significantly decreases neurosphere formation; however, a small percentage of cells survive and repopulate the culture. A promising target for glioma therapy is the Notch signaling pathway. Notch activity is upregulated in many gliomas and can be suppressed using gamma-secretase inhibitors (GSI). Using a neurosphere recovery assay and xenograft experiments, we analyzed if the addition of GSIs with TMZ treatment could inhibit repopulation and tumor recurrence. We show that TMZ + GSI treatment decreased neurosphere formation and inhibited neurosphere recovery. This enhancement of TMZ treatment occurred through inhibition of the Notch pathway and depended on the sequence of drug administration. In addition, ex vivo TMZ + GSI treatment of glioma xenografts in immunocompromised mice extended tumor latency and survival, and in vivo TMZ + GSI treatment blocked tumor progression in 50% of mice with preexisting tumors. These data show the importance of the Notch pathway in chemoprotection and repopulation of TMZ-treated gliomas. The addition of GSIs to current treatments is a promising approach to decrease brain tumor recurrence.

Global targeting of subcellular heat shock protein-90 networks for therapy of glioblastoma.

Drug discovery for complex and heterogeneous tumors now aims at dismantling global networks of disease maintenance, but the subcellular requirements of this approach are not understood. Here, we simultaneously targeted the multiple subcellular compartments of the molecular chaperone heat shock protein-90 (Hsp90) in a model of glioblastoma, a highly lethal human malignancy in urgent need of fresh therapeutic strategies. Treatment of cultured or patient-derived glioblastoma cells with Shepherdin, a dual peptidomimetic inhibitor of mitochondrial and cytosolic Hsp90, caused irreversible collapse of mitochondria, degradation of Hsp90 client proteins in the cytosol, and tumor cell killing by apoptosis and autophagy. Stereotactic or systemic delivery of Shepherdin was well tolerated and suppressed intracranial glioma growth via inhibition of cell proliferation, induction of apoptosis, and reduction of angiogenesis in vivo. These data show that disabling Hsp90 cancer networks in their multiple subcellular compartments improves strategies for drug discovery and may provide novel molecular therapy for highly recalcitrant human tumors.

Sorafenib exerts anti-glioma activity in vitro and in vivo.

Despite conventional treatment strategies glioblastoma, the most common malignant primary brain tumor, has a bad prognosis with median survival times of 12-15 months. In this study, the efficacy of sorafenib (Nexavar, BAY43-9006), a multikinase inhibitor, on glioblastoma cells was evaluated both in vitro and in vivo. Treatment of established or patient-derived glioblastoma cells with low concentrations of sorafenib caused a dramatic dose dependent inhibition of proliferation (IC(50), 1.5 microM) and induction of apoptosis and autophagy. Sorafenib inhibited phosphorylation of signal transducer and activator of transcription 3 (Stat3) and expression of cyclins, D and E. In contrast, AKT was not modulated by sorafenib. Most important, systemic delivery of sorafenib was well tolerated, and significantly suppressed intracranial glioma growth via inhibition of cell proliferation, induction of apoptosis and autophagy, and reduction of angiogenesis. Furthermore, intracranial growth inhibition by sorafenib was accompanied by a significant reduction in ph-Stat3 (Tyr 705) levels. In summary, sorafenib has potent anti-glioma activity in vitro and in vivo.

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.