The eEF2 kinase coordinates the DNA damage response to cisplatin by supporting p53 activation.

Eukaryotic elongation factor 2 (eEF2) kinase (eEF2K) is a stress-responsive hub that inhibits the translation elongation factor eEF2, and consequently mRNA translation elongation, in response to hypoxia and nutrient deprivation. EEF2K is also involved in the response to DNA damage but its role in response to DNA crosslinks, as induced by cisplatin, is not known. Here we found that eEF2K is critical to mediate the cellular response to cisplatin. We uncovered that eEF2K deficient cells are more resistant to cisplatin treatment. Mechanistically, eEF2K deficiency blunts the activation of the DNA damage response associated ATM and ATR pathways, in turn preventing p53 activation and therefore compromising induction of cisplatin-induced apoptosis. We also report that loss of eEF2K delays the resolution of DNA damage triggered by cisplatin, suggesting that eEF2K contributes to DNA damage repair in response to cisplatin. In support of this, our data shows that eEF2K promotes the expression of the DNA repair protein ERCC1, critical for the repair of cisplatin-caused DNA damage. Finally, using Caenorhabditis elegans as an in vivo model, we find that deletion of efk-1, the worm eEF2K ortholog, mitigates the induction of germ cell death in response to cisplatin. Together, our data highlight that eEF2K represents an evolutionary conserved mediator of the DNA damage response to cisplatin which promotes p53 activation to induce cell death, or alternatively facilitates DNA repair, depending on the extent of DNA damage.

A clinically compatible in vitro drug-screening platform identifies therapeutic vulnerabilities in primary cultures of brain metastases.

PURPOSE: Brain metastases represent the most common intracranial tumors in adults and are associated with a poor prognosis. We used a personalized in vitro drug screening approach to characterize individual therapeutic vulnerabilities in brain metastases. METHODS: Short-term cultures of cancer cells isolated from brain metastasis patients were molecularly characterized using next-generation sequencing and functionally evaluated using high-throughput in vitro drug screening to characterize pharmacological treatment sensitivities. RESULTS: Next-generation sequencing identified matched genetic alterations in brain metastasis tissue samples and corresponding short-term cultures, suggesting that short-term cultures of brain metastases are suitable models for recapitulating the genetic profile of brain metastases that may determine their sensitivity to anti-cancer drugs. Employing a high-throughput in vitro drug screening platform, we successfully screened the cultures of five brain metastases for response to 267 anticancer compounds and related drug response to genetic data. Among others, we found that targeted treatment with JAK3, HER2, or FGFR3 inhibitors showed anti-cancer effects in individual brain metastasis cultures. CONCLUSION: Our preclinical study provides a proof-of-concept for combining molecular profiling with in vitro drug screening for predictive evaluation of therapeutic vulnerabilities in brain metastasis patients. This approach could advance the use of patient-derived cancer cells in clinical practice and might eventually facilitate decision-making for personalized drug treatment.

mTORC1 regulates cell survival under glucose starvation through 4EBP1/2-mediated translational reprogramming of fatty acid metabolism.

Energetic stress compels cells to evolve adaptive mechanisms to adjust their metabolism. Inhibition of mTOR kinase complex 1 (mTORC1) is essential for cell survival during glucose starvation. How mTORC1 controls cell viability during glucose starvation is not well understood. Here we show that the mTORC1 effectors eukaryotic initiation factor 4E binding proteins 1/2 (4EBP1/2) confer protection to mammalian cells and budding yeast under glucose starvation. Mechanistically, 4EBP1/2 promote NADPH homeostasis by preventing NADPH-consuming fatty acid synthesis via translational repression of Acetyl-CoA Carboxylase 1 (ACC1), thereby mitigating oxidative stress. This has important relevance for cancer, as oncogene-transformed cells and glioma cells exploit the 4EBP1/2 regulation of ACC1 expression and redox balance to combat energetic stress, thereby supporting transformation and tumorigenicity in vitro and in vivo. Clinically, high EIF4EBP1 expression is associated with poor outcomes in several cancer types. Our data reveal that the mTORC1-4EBP1/2 axis provokes a metabolic switch essential for survival during glucose starvation which is exploited by transformed and tumor cells.

The long non-coding RNA OTX2-AS1 promotes tumor growth and predicts response to BCL-2 inhibition in medulloblastoma.

PURPOSE: Primary brain tumors are a leading cause of cancer-related death in children, and medulloblastoma is the most common malignant pediatric brain tumor. The current molecular characterization of medulloblastoma is mainly based on protein-coding genes, while little is known about the involvement of long non-coding RNAs (lncRNAs). This study aimed to elucidate the role of the lncRNA OTX2-AS1 in medulloblastoma. METHODS: Analyses of DNA copy number alterations, methylation profiles, and gene expression data were used to characterize molecular alterations of OTX2-AS1 in medulloblastoma tissue samples. In vitro analyses of medulloblastoma cell models and orthotopic in vivo experiments were carried out for functional characterization of OTX2-AS1. High-throughput drug screening was employed to identify pharmacological inhibitors, while proteomics and metabolomics analyses were performed to address potential mechanisms of drug action. RESULTS: We detected amplification and consecutive overexpression of OTX2 and OTX2-AS1 in a subset of medulloblastomas. In addition, OTX2-AS1 promoter methylation was linked to OTX2-AS1 expression. OTX2-AS1 knockout reduced medulloblastoma cell viability and cell migration in vitro and prolonged survival in the D283 orthotopic medulloblastoma mouse xenograft model. Pharmacological inhibition of BCL-2 suppressed the growth of OTX2-AS1 overexpressing medulloblastoma cells in vitro. CONCLUSIONS: Our study revealed a pro-tumorigenic role of OTX2-AS1 in medulloblastoma and identified BCL-2 inhibition as a potential therapeutic approach to target OTX2-AS1 overexpressing medulloblastoma cells.

Integrative multi-omics reveals two biologically distinct groups of pilocytic astrocytoma.

Pilocytic astrocytoma (PA), the most common pediatric brain tumor, is driven by aberrant mitogen-activated protein kinase signaling most commonly caused by BRAF gene fusions or activating mutations. While 5-year overall survival rates exceed 95%, tumor recurrence or progression constitutes a major clinical challenge in incompletely resected tumors. Here, we used similarity network fusion (SNF) analysis in an integrative multi-omics approach employing RNA transcriptomic and mass spectrometry-based proteomic profiling to molecularly characterize PA tissue samples from 62 patients. Thereby, we uncovered that PAs segregated into two molecularly distinct groups, namely, Group 1 and Group 2, which were validated in three non-overlapping cohorts. Patients with Group 1 tumors were significantly younger and showed worse progression-free survival compared to patients with group 2 tumors. Ingenuity pathways analysis (IPA) and gene set enrichment analysis (GSEA) revealed that Group 1 tumors were enriched for immune response pathways, such as interferon signaling, while Group 2 tumors showed enrichment for action potential and neurotransmitter signaling pathways. Analysis of immune cell-related gene signatures showed an enrichment of infiltrating T Cells in Group 1 versus Group 2 tumors. Taken together, integrative multi-omics of PA identified biologically distinct and prognostically relevant tumor groups that may improve risk stratification of this single pathway driven tumor type.

Tacedinaline (CI-994), a class I HDAC inhibitor, targets intrinsic tumor growth and leptomeningeal dissemination in MYC-driven medulloblastoma while making them susceptible to anti-CD47-induced macrophage phagocytosis via NF-kB-TGM2 driven tumor inflammation.

BACKGROUND: While major advances have been made in improving the quality of life and survival of children with most forms of medulloblastoma (MB), those with MYC-driven tumors (Grp3-MB) still suffer significant morbidity and mortality. There is an urgent need to explore multimodal therapeutic regimens which are effective and safe for children. Large-scale studies have revealed abnormal cancer epigenomes caused by mutations and structural alterations of chromatin modifiers, aberrant DNA methylation, and histone modification signatures. Therefore, targeting epigenetic modifiers for cancer treatment has gained increasing interest, and inhibitors for various epigenetic modulators have been intensively studied in clinical trials. Here, we report a cross-entity, epigenetic drug screen to evaluate therapeutic vulnerabilities in MYC amplified MB, which sensitizes them to macrophage-mediated phagocytosis by targeting the CD47-signal regulatory protein α (SIRPα) innate checkpoint pathway. METHODS: We performed a primary screen including 78 epigenetic inhibitors and a secondary screen including 20 histone deacetylase inhibitors (HDACi) to compare response profiles in atypical teratoid/rhabdoid tumor (AT/RT, n=11), MB (n=14), and glioblastoma (n=14). This unbiased approach revealed the preferential activity of HDACi in MYC-driven MB. Importantly, the class I selective HDACi, CI-994, showed significant cell viability reduction mediated by induction of apoptosis in MYC-driven MB, with little-to-no activity in non-MYC-driven MB, AT/RT, and glioblastoma in vitro. We tested the combinatorial effect of targeting class I HDACs and the CD47-SIRPa phagocytosis checkpoint pathway using in vitro phagocytosis assays and in vivo orthotopic xenograft models. RESULTS: CI-994 displayed antitumoral effects at the primary site and the metastatic compartment in two orthotopic mouse models of MYC-driven MB. Furthermore, RNA sequencing revealed nuclear factor-kB (NF-κB) pathway induction as a response to CI-994 treatment, followed by transglutaminase 2 (TGM2) expression, which enhanced inflammatory cytokine secretion. We further show interferon-γ release and cell surface expression of engulfment ('eat-me') signals (such as calreticulin). Finally, combining CI-994 treatment with an anti-CD47 mAb targeting the CD47-SIRPα phagocytosis checkpoint enhanced in vitro phagocytosis and survival in tumor-bearing mice. CONCLUSION: Together, these findings suggest a dynamic relationship between MYC amplification and innate immune suppression in MYC amplified MB and support further investigation of phagocytosis modulation as a strategy to enhance cancer immunotherapy responses.

Elevated FSP1 protects KRAS-mutated cells from ferroptosis during tumor initiation.

Oncogenic KRAS is the key driver oncogene for several of the most aggressive human cancers. One key feature of oncogenic KRAS expression is an early increase in cellular reactive oxygen species (ROS) which promotes cellular transformation if cells manage to escape cell death, mechanisms of which remain incompletely understood. Here, we identify that expression of oncogenic as compared to WT KRAS in isogenic cellular systems renders cells more resistant to ferroptosis, a recently described type of regulated necrosis. Mechanistically, we find that cells with mutant KRAS show a specific lack of ferroptosis-induced lipid peroxidation. Interestingly, KRAS-mutant cells upregulate expression of ferroptosis suppressor protein 1 (FSP1). Indeed, elevated levels of FSP1 in KRAS-mutant cells are responsible for mediating ferroptosis resistance and FSP1 is upregulated as a consequence of MAPK and NRF2 pathway activation downstream of KRAS. Strikingly, FSP1 activity promotes cellular transformation in soft agar and its overexpression is sufficient to promote spheroid growth in 3D in KRAS WT cells. Moreover, FSP1 expression and its activity in ferroptosis inhibition accelerates tumor onset of KRAS WT cells in the absence of oncogenic KRAS in vivo. Consequently, we find that pharmacological induction of ferroptosis in pancreatic organoids derived from the LsL-KRASG12D expressing mouse model is only effective in combination with FSP1 inhibition. Lastly, FSP1 is upregulated in non-small cell lung cancer (NSCLC), colorectal cancer (CRC) and pancreatic ductal adenocarcinoma (PDAC) as compared to the respective normal tissue of origin and correlates with NRF2 expression in PDAC patient datasets. Based on these data, we propose that KRAS-mutant cells must navigate a ferroptosis checkpoint by upregulating FSP1 during tumor establishment. Consequently, ferroptosis-inducing therapy should be combined with FSP1 inhibitors for efficient therapy of KRAS-mutant cancers.

The HHIP-AS1 lncRNA promotes tumorigenicity through stabilization of dynein complex 1 in human SHH-driven tumors.

Most lncRNAs display species-specific expression patterns suggesting that animal models of cancer may only incompletely recapitulate the regulatory crosstalk between lncRNAs and oncogenic pathways in humans. Among these pathways, Sonic Hedgehog (SHH) signaling is aberrantly activated in several human cancer entities. We unravel that aberrant expression of the primate-specific lncRNA HedgeHog Interacting Protein-AntiSense 1 (HHIP-AS1) is a hallmark of SHH-driven tumors including medulloblastoma and atypical teratoid/rhabdoid tumors. HHIP-AS1 is actively transcribed from a bidirectional promoter shared with SHH regulator HHIP. Knockdown of HHIP-AS1 induces mitotic spindle deregulation impairing tumorigenicity in vitro and in vivo. Mechanistically, HHIP-AS1 binds directly to the mRNA of cytoplasmic dynein 1 intermediate chain 2 (DYNC1I2) and attenuates its degradation by hsa-miR-425-5p. We uncover that neither HHIP-AS1 nor the corresponding regulatory element in DYNC1I2 are evolutionary conserved in mice. Taken together, we discover an lncRNA-mediated mechanism that enables the pro-mitotic effects of SHH pathway activation in human tumors.

EIF4EBP1 is transcriptionally upregulated by MYCN and associates with poor prognosis in neuroblastoma.

Neuroblastoma (NB) accounts for 15% of cancer-related deaths in childhood despite considerable therapeutic improvements. While several risk factors, including MYCN amplification and alterations in RAS and p53 pathway genes, have been defined in NB, the clinical outcome is very variable and difficult to predict. Since genes of the mechanistic target of rapamycin (mTOR) pathway are upregulated in MYCN-amplified NB, we aimed to define the predictive value of the mTOR substrate-encoding gene eukaryotic translation initiation factor 4E-binding protein 1 (EIF4EBP1) expression in NB patients. Using publicly available data sets, we found that EIF4EBP1 mRNA expression is positively correlated with MYCN expression and elevated in stage 4 and high-risk NB patients. In addition, high EIF4EBP1 mRNA expression is associated with reduced overall and event-free survival in the entire group of NB patients in three cohorts, as well as in stage 4 and high-risk patients. This was confirmed by monitoring the clinical value of 4EBP1 protein expression, which revealed that high levels of 4EBP1 are significantly associated with prognostically unfavorable NB histology. Finally, functional analyses revealed that EIF4EBP1 expression is transcriptionally controlled by MYCN binding to the EIF4EBP1 promoter in NB cells. Our data highlight that EIF4EBP1 is a direct transcriptional target of MYCN whose high expression is associated with poor prognosis in NB patients. Therefore, EIF4EBP1 may serve to better stratify patients with NB.

Eukaryotic translation initiation factor 4E binding protein 1 (EIF4EBP1) expression in glioblastoma is driven by ETS1- and MYBL2-dependent transcriptional activation.

Eukaryotic translation initiation factor 4E binding protein 1 (EIF4EBP1) encodes the 4EBP1 protein, a negative regulator of mRNA translation and a substrate of the mechanistic target of rapamycin (mTOR), whose function and relevance in cancer is still under debate. Here, we analyzed EIF4EBP1 expression in different glioma patient cohorts and investigated its mode of transcriptional regulation in glioblastoma cells. We verified that EIF4EBP1 mRNA is overexpressed in malignant gliomas, including isocitrate dehydrogenase (IDH)-wildtype glioblastomas, relative to non-neoplastic brain tissue in multiple publically available datasets. Our analyses revealed that EIF4EBP1 overexpression in malignant gliomas is neither due to gene amplification nor to altered DNA methylation, but rather results from aberrant transcriptional activation by distinct transcription factors. We found seven transcription factor candidates co-expressed with EIF4EBP1 in gliomas and bound to the EIF4EBP1 promoter, as revealed by chromatin immunoprecipitation (ChIP)-sequencing data. We investigated the ability of these candidates to activate the EIF4EBP1 promoter using luciferase reporter assays, which supported four transcription factors as candidate EIF4EBP1 regulators, namely MYBL2, ETS1, HIF-1A, and E2F6. Finally, by employing transient knock-down experiments to repress either of these transcription factors, we identified MYBL2 and ETS1 as the relevant transcriptional drivers of enhanced EIF4EBP1 expression in malignant glioma cells. Taken together, our findings confirm enhanced expression of EIF4EBP1 in malignant gliomas relative to non-neoplastic brain tissue and characterize the underlying molecular pathomechanisms.

The long non-coding RNA HOTAIRM1 promotes tumor aggressiveness and radiotherapy resistance in glioblastoma.

Glioblastoma is the most common malignant primary brain tumor. To date, clinically relevant biomarkers are restricted to isocitrate dehydrogenase (IDH) gene 1 or 2 mutations and O6-methylguanine DNA methyltransferase (MGMT) promoter methylation. Long non-coding RNAs (lncRNAs) have been shown to contribute to glioblastoma pathogenesis and could potentially serve as novel biomarkers. The clinical significance of HOXA Transcript Antisense RNA, Myeloid-Specific 1 (HOTAIRM1) was determined by analyzing HOTAIRM1 in multiple glioblastoma gene expression data sets for associations with prognosis, as well as, IDH mutation and MGMT promoter methylation status. Finally, the role of HOTAIRM1 in glioblastoma biology and radiotherapy resistance was characterized in vitro and in vivo. We identified HOTAIRM1 as a candidate lncRNA whose up-regulation is significantly associated with shorter survival of glioblastoma patients, independent from IDH mutation and MGMT promoter methylation. Glioblastoma cell line models uniformly showed reduced cell viability, decreased invasive growth and diminished colony formation capacity upon HOTAIRM1 down-regulation. Integrated proteogenomic analyses revealed impaired mitochondrial function and determination of reactive oxygen species (ROS) levels confirmed increased ROS levels upon HOTAIRM1 knock-down. HOTAIRM1 knock-down decreased expression of transglutaminase 2 (TGM2), a candidate protein implicated in mitochondrial function, and knock-down of TGM2 mimicked the phenotype of HOTAIRM1 down-regulation in glioblastoma cells. Moreover, HOTAIRM1 modulates radiosensitivity of glioblastoma cells both in vitro and in vivo. Our data support a role for HOTAIRM1 as a driver of biological aggressiveness, radioresistance and poor outcome in glioblastoma. Targeting HOTAIRM1 may be a promising new therapeutic approach.

Rapalink-1 Targets Glioblastoma Stem Cells and Acts Synergistically with Tumor Treating Fields to Reduce Resistance against Temozolomide.

Glioblastoma (GBM) is a lethal disease with limited clinical treatment options available. Recently, a new inhibitor targeting the prominent cancer signaling pathway mTOR was discovered (Rapalink-1), but its therapeutic potential on stem cell populations of GBM is unknown. We applied a collection of physiological relevant organoid-like stem cell models of GBM and studied the effect of RL1 exposure on various cellular features as well as on the expression of mTOR signaling targets and stem cell molecules. We also undertook combination treatments with this agent and clinical GBM treatments tumor treating fields (TTFields) and the standard-of-care drug temozolomide, TMZ. Low nanomolar (nM) RL1 treatment significantly reduced cell growth, proliferation, migration, and clonogenic potential of our stem cell models. It acted synergistically to reduce cell growth when applied in combination with TMZ and TTFields. We performed an in silico analysis from the molecular data of diverse patient samples to probe for a relationship between the expression of mTOR genes, and mesenchymal markers in different GBM cohorts. We supported the in silico results with correlative protein data retrieved from tumor specimens. Our study further validates mTOR signaling as a druggable target in GBM and supports RL1, representing a promising therapeutic target in brain oncology.

How does mTOR sense glucose starvation? AMPK is the usual suspect.

Glucose is a major requirement for biological life. Its concentration is constantly sensed at the cellular level, allowing for adequate responses to any changes of glucose availability. Such responses are mediated by key sensors and signaling pathway components that adapt cellular metabolism to glucose levels. One of the major hubs of these responses is mechanistic target of rapamycin (mTOR) kinase, which forms the mTORC1 and mTORC2 protein complexes. Under physiological glucose concentrations, mTORC1 is activated and stimulates a number of proteins and enzymes involved in anabolic processes, while restricting the autophagic process. Conversely, when glucose levels are low, mTORC1 is inhibited, in turn leading to the repression of numerous anabolic processes, sparing ATP and antioxidants. Understanding how mTORC1 activity is regulated by glucose is not only important to better delineate the biological function of mTOR, but also to highlight potential therapeutic strategies for treating diseases characterized by deregulated glucose availability, as is the case of cancer. In this perspective, we depict the different sensors and upstream proteins responsible of controlling mTORC1 activity in response to changes in glucose concentration. This includes the major energy sensor AMP-activated protein kinase (AMPK), as well as other independent players. The impact of such modes of regulation of mTORC1 on cellular processes is also discussed.

RAS-driven oncogenesis is supported by downstream antioxidant programs.

In our recent study, we demonstrated that oncogenic RAS (rat sarcoma)-mediated transformation and tumorigenesis are supported by transcriptional induction of a crucial antioxidant component, SLC7A11 (solute carrier family 7 member 11), otherwise known as XCT, a gene encoding the cystine/glutamate transporter. Our data highlight that this promotes the biosynthesis of glutathione, in turn allowing RAS transformed cells to mitigate tumorigenesis-linked oxidative stress.

The impact of oncogenic RAS on redox balance and implications for cancer development.

The RAS family of proto-oncogenes comprises HRAS, KRAS, and NRAS, which are among the most mutated genes in human cancers. The RAS family genes encode small GTPases that coordinate key signaling pathways in response to growth factors. Mutations in RAS result in a constitutively active form of the protein that supports cellular transformation and tumorigenesis. The mechanisms of oncogenic RAS-mediated transformation encompass uncontrolled proliferation and inhibition of cell death through overactivation of the RAF-MEK-ERK and the PI3K-AKT pathways, respectively. In addition, the control of redox balance by RAS has also been proposed to play a role in its oncogenic properties. However, the exact role of redox balance in mediating mutant RAS transformation is still under debate. Here, we present, on one hand, the involvement of pro-oxidant components in oncogenic RAS transformation, such as NADPH oxidases and mitochondrial reactive oxygen species, and how these promote transformation. On the other hand, we describe the contribution of antioxidant components to mutant RAS transformation, including Nrf2, glutathione biosynthesis and xCT, as well as the mechanisms by which antioxidant programs drive transformation. Finally, we aim to reconcile the seemingly opposite effects of oncogenic RAS on redox balance and discuss a model for the complementary role of both pro-oxidant and antioxidant pathways in mutant RAS-driven tumor progression.

The long noncoding RNA TP73-AS1 promotes tumorigenicity of medulloblastoma cells.

Medulloblastoma is the most common malignant brain cancer in children. Since previous studies have mainly focused on alterations in the coding genome, our understanding of the contribution of long noncoding RNAs (lncRNAs) to medulloblastoma biology is just emerging. Using patient-derived data, we show that the promoter of lncRNA TP73-AS1 is hypomethylated and that the transcript is highly expressed in the SHH subgroup. Furthermore, high expression of TP73-AS1 is correlated with poor outcome in patients with TP53 wild-type SHH tumors. Silencing TP73-AS1 in medulloblastoma tumor cells induced apoptosis, while proliferation and migration were inhibited in culture. In vivo, silencing TP73-AS1 in medulloblastoma tumor cells resulted in reduced tumor growth, reduced proliferation of tumor cells, increased apoptosis and led to prolonged survival of tumor-bearing mice. Together, our study suggests that the lncRNA TP73-AS1 is a prognostic marker and therapeutic target in medulloblastoma tumors and serves as a proof of concept that lncRNAs are important factors in the disease.

Cystine/glutamate antiporter xCT (SLC7A11) facilitates oncogenic RAS transformation by preserving intracellular redox balance.

The RAS family of proto-oncogenes are among the most commonly mutated genes in human cancers and predict poor clinical outcome. Several mechanisms underlying oncogenic RAS transformation are well documented, including constitutive signaling through the RAF-MEK-ERK proproliferative pathway as well as the PI3K-AKT prosurvival pathway. Notably, control of redox balance has also been proposed to contribute to RAS transformation. However, how homeostasis between reactive oxygen species (ROS) and antioxidants, which have opposing effects in the cell, ultimately influence RAS-mediated transformation and tumor progression is still a matter of debate and the mechanisms involved have not been fully elucidated. Here, we show that oncogenic KRAS protects fibroblasts from oxidative stress by enhancing intracellular GSH levels. Using a whole transcriptome approach, we discovered that this is attributable to transcriptional up-regulation of xCT, the gene encoding the cystine/glutamate antiporter. This is in line with the function of xCT, which mediates the uptake of cystine, a precursor for GSH biosynthesis. Moreover, our results reveal that the ETS-1 transcription factor downstream of the RAS-RAF-MEK-ERK signaling cascade directly transactivates the xCT promoter in synergy with the ATF4 endoplasmic reticulum stress-associated transcription factor. Strikingly, xCT was found to be essential for oncogenic KRAS-mediated transformation in vitro and in vivo by mitigating oxidative stress, as knockdown of xCT strongly impaired growth of tumor xenografts established from KRAS-transformed cells. Overall, this study uncovers a mechanism by which oncogenic RAS preserves intracellular redox balance and identifies an unexpected role for xCT in supporting RAS-induced transformation and tumorigenicity.

The lncRNA TP73-AS1 is linked to aggressiveness in glioblastoma and promotes temozolomide resistance in glioblastoma cancer stem cells.

Glioblastoma multiform (GBM) is the most common brain tumor characterized by a dismal prognosis. GBM cancer stem cells (gCSC) or tumor-initiating cells are the cell population within the tumor-driving therapy resistance and recurrence. While temozolomide (TMZ), an alkylating agent, constitutes the first-line chemotherapeutic significantly improving survival in GBM patients, resistance against this compound commonly leads to GBM recurrence and treatment failure. Although the roles of protein-coding transcripts, proteins and microRNA in gCSC, and therapy resistance have been comprehensively investigated, very little is known about the role of long noncoding RNAs (lncRNAs) in this context. Using nonoverlapping, independent RNA sequencing and gene expression profiling datasets, we reveal that TP73-AS1 constitutes a clinically relevant lncRNA in GBM. Specifically, we demonstrate significant overexpression of TP73-AS1 in primary GBM samples, which is particularly increased in the gCSC. More importantly, we demonstrate that TP73-AS1 comprises a prognostic biomarker in glioma and in GBM with high expression identifying patients with particularly poor prognosis. Using CRISPRi to downregulate our candidate lncRNA in gCSC, we demonstrate that TP73-AS1 promotes TMZ resistance in gCSC and is linked to regulation of the expression of metabolism- related genes and ALDH1A1, a protein known to be expressed in cancer stem cell markers and protects gCSC from TMZ treatment. Taken together, our results reveal that high TP73-AS1 predicts poor prognosis in primary GBM cohorts and that this lncRNA promotes tumor aggressiveness and TMZ resistance in gCSC.

High-Throughput Screening Identified Compounds Sensitizing Tumor Cells to Glucose Starvation in Culture and VEGF Inhibitors In Vivo.

Tumor cells utilize glucose to fuel their anabolic needs, including rapid proliferation. However, due to defective vasculature and increased glucose uptake, tumor cells must overcome glucose deprivation. Accordingly, tumor cells depend on cellular pathways promoting survival under such conditions. Targeting these survival mechanisms can thus serve as a new therapeutic strategy in oncology. As such, we sought to identify small-molecule inhibitors which sensitize tumor cells to glucose starvation by high-throughput drug screening in vitro. Specifically, we searched for inhibitors that selectively killed tumor cells growing in glucose-free but not in normal medium. This phenotypic drug screen of 7000 agents with MCF7 cells led to the identification of 67 potential candidates, 31 of which were validated individually. Among the identified compounds, we found a high number of compounds known to target mitochondria. The efficacies of two of the identified compounds, QNZ (EVP4593) and papaverine, were validated in four different tumor cell lines. We found that these agents inhibited the mTOR(Mechamistic\Mammilian Target of Rapamycin) pathway in tumor cells growing under glucose starvation, but not under normal conditions. The results were validated and confirmed in vivo, with QNZ and papaverine exhibiting superior antitumor activity in a tumor xenograft model when combined with the VEGF inhibitor bevacizumab (avastin). Administering these drug combinations (i.e., avastin and papaverine, and avastin and QNZ) led to significant reductions in proliferation and mTOR activity of the aggressive DLD1 colon cell line in mice. Given our findings, we propose that compounds targeting metabolically challenged tumors, such as inhibitors of mitochondrial activity, be considered as a therapeutic strategy in cancer.