Biology subjects

Reifenberger, G.

Publications and source records attributed to Reifenberger, G..

14 recordsLinked to original sources

bioRxiv2026

GALE-dependent glycoproteome remodelling is a determinant of oncogenic RAS transformation

Oncogenic transformation is accompanied by extensive remodelling of the cellular glycosylation landscape, yet the mechanisms linking oncogenic signalling to glycoproteome reprogramming remain poorly defined. We used site-resolved intact glycoproteomics to systematically map oncogenic RAS-dependent changes across the N- and O-linked glycoproteome. Integrating multiomics profiling with functional approaches, we identify UDP-glucose 4-epimerase (GALE) as a transcriptional target of oncogenic RAS that is induced through MAPK signalling and MYC-dependent transcription. GALE depletion selectively disrupted RAS-dependent glycoproteome remodelling, preferentially impairing sialylation of N-linked glycans and O-linked GalNAc-type glycosylation. Functionally, these glycosylation defects were accompanied by suppressed anchorage-independent growth and reduced in vivo tumorigenicity of KRAS-mutant cells, establishing GALE-dependent nucleotide-sugar interconversion as a requirement for malignant growth. Clinically, GALE expression was elevated in pancreatic ductal adenocarcinoma, wherein KRAS mutations are highly prevalent, and across a broad spectrum of human cancers. Collectively, our findings define a regulatory axis that connects oncogenic signalling to nucleotide-sugar precursor availability and diversification of the cellular glycan repertoire, revealing GALE as a metabolic dependency in RAS-driven cancer.

Cancer Biology↗

bioRxiv2024

Identification and characterization of tertiary lymphoid structures in brain metastases

Brain metastases (BrM) are the most common cancers in the brain. We performed transcriptome-wide gene expression profiling combined with spatial immune cell profiling to characterize the tumor immune microenvironment in BrM from different primary tumors. We found that BrM from lung carcinoma and malignant melanoma showed overall higher immune cell infiltration as compared to BrM from breast carcinoma. RNA sequencing-based immune cell deconvolution revealed gene expression signatures indicative of tertiary lymphoid structures (TLS) in subsets of BrM, mostly from lung cancer and melanoma. This finding was corroborated by multiplex immunofluorescence staining of immune cells in BrM tissue sections. Detection of TLS signatures was more common in treatment-naive BrM and associated with prolonged survival after BrM diagnosis in lung cancer patients. Our findings highlight the cellular diversity of the tumor immune microenvironment in BrM of different cancer types and suggest a role of TLS formation for BrM patient outcome.

cancer biology↗

bioRxiv2024

The EIF4EBP1 gene encoding 4EBP1 is transcriptionally upregulated by MYC and linked to shorter survival in medulloblastoma

Medulloblastoma (MB) is the most common malignant brain tumor in childhood and is stratified into four molecular groups - WNT, SHH, Group 3 and Group 4. Group 3 MB patients exhibit the poorest prognosis, with a 5-year overall survival of <60%, followed by Group 4 MB patients. Apart from MYC amplification in a subset of Group 3 MBs, the molecular pathomechanisms driving aggressiveness of these tumors remain incompletely characterized. The gene encoding the mTOR substrate and mRNA translation inhibitor eukaryotic translation initiation factor 4E-binding protein 1 (EIF4EBP1) represents a possible MYC target gene whose corresponding protein, 4EBP1, was shown to be more active in Group 3 versus Group 4 MBs. However, the prognostic role of 4EBP1 in MB and the mechanisms supporting 4EBP1 overexpression in Group 3 MB are still elusive. We analyzed EIF4EBP1 mRNA expression in publicly available data sets and found an upregulation in MB as compared to non-neoblastic brain. EIF4EBP1 mRNA expression levels were higher in Group 3 compared to Group 4 MBs. EIF4EBP1 mRNA expression was correlated with MYC expression, most prominently in Group 3 MBs. Survival analyses highlighted that high EIF4EBP1 mRNA expression was associated with reduced overall and event-free survival across all MB patients and in Group 3/Group 4 MB patients. Immunohistochemical evaluation of 4EBP1 protein expression in MB tissues confirmed that high levels of 4EBP1 are associated with poor outcome. Functional analyses revealed that MYC directly regulates EIF4EBP1 promoter activity, providing a mechanism for increased EIF4EBP1 mRNA levels in Group 3 MBs. Finally, we observed that 4EBP1 may support colony formation of in vitro cultured MB cells. Our data highlight that transcriptional upregulation of EIF4EBP1 by MYC promotes in vitro tumorigenicity of MB cells and associates with shorter survival of MB patients.

cancer biology↗

bioRxiv2024

Oncogenic RAS signaling suppresses ferroptosis via transcriptional upregulation of GCH1

Ferroptosis is an iron-dependent form of regulated cell death arising from excessive lipid peroxidation. The role of oncogenic RAS signaling in modulating the cellular response to ferroptosis is controversial. While seminal studies described that oncogenic RAS transformation drives a synthetic lethal vulnerability to archetypal ferroptosis inducers including erastin (eradicator of RAS and ST-expressing cells) and RSL3 (Ras selective lethal 3), more recent work suggest that oncogenic RAS signaling may confer ferroptosis resistance. Thus, the impact of oncogenic RAS expression on ferroptosis sensitivity is still poorly understood. Here, using orthogonal cellular systems across multiple classes of ferroptosis- inducing agents, as well as in silico therapeutic drug-response analyses, we provide unifying evidence that oncogenic RAS signaling suppresses ferroptosis. Integrated proteo- and transcriptomic analyses in oncogenic RAS-transformed cells further uncovered that RAS signaling upregulates the ferroptosis suppressor GTP cyclohydrolase I (GCH1) via transcriptional induction by the transcription factor ETS1 downstream of the RAS-MAPK signaling cascade. Targeted repression of Gch1 or of Gch1-controlled tetrahydrobiopterin (BH4) synthesis pathway is sufficient to sensitize RAS-mutant cell lines to ferroptosis in 2D and 3D cell models, as well as in tumor organoids and tumor xenografts, highlighting a mechanism through which RAS promotes resistance to ferroptosis induction. Furthermore, we found that GCH1 expression is clinically relevant and correlates with RAS signaling activation in human cancers. Overall, this study redefines oncogenic RAS signaling to be a ferroptosis suppressor, and identifies GCH1 as a mediator of this effect and a potential clinical target for the sensitization of RAS-driven cancers to ferroptosis-inducing agents. Significance StatementAlthough it is commonly accepted that ferroptosis induction is a mutant RAS-selective lethality, accumulating evidence suggests that oncogenic RAS protects cells against this form of cell death. However, a systematic survey establishing the relationship between RAS and ferroptosis sensitivity is lacking, and the molecular mechanisms this entails are still poorly understood. Here, we report across RAS-mutant isoforms, in diverse cellular models, and using multiple ferroptosis-inducing compounds that oncogenic RAS consistently suppresses ferroptosis. Further, we show that oncogenic RAS-mediated ferroptosis suppression is attributed to the upregulation of GCH1 and its downstream metabolite, tetrahydrobiopterin. Our study delivers a shift towards a new paradigm in which oncogenic RAS confers ferroptosis resistance, and a potential clinical strategy to re-engage ferroptosis sensitivity in RAS-driven cancers.

cancer biology↗

bioRxiv2024

Direct excitatory synapses between neurons and tumor cells drive brain metastatic seeding of breast cancer and melanoma

Interactions between neurons and cancer cells are found in many malignancies, but their relevance for metastatic organ colonization remain largely unknown. It is also unclear whether any direct synaptic communication between neurons and cancer cells of non-neural tumor types exists, and if so, whether this can support metastasis and thus cancer progression. Here we show that excitatory synapses are formed between neurons and brain-metastatic melanoma and breast cancer cells. This starts at an early microscopic stage after extravasation into the brain parenchyma, during residence of cancer cells in the perivascular niche, a critical step for their survival. These neuron-cancer synapses showed a bona fide synaptic ultrastructure, and generated excitatory postsynaptic currents mediated by glutamate receptors of the AMPA subtype in cancer cells. In accordance, AMPA receptor signatures were consistently detected in preclinical and patient samples of melanoma and breast cancer brain metastases. Genetic perturbation and pharmacological inhibition of AMPA receptors with the approved antiepileptic drug perampanel in models of breast and melanoma cancer reduced the number of brain metastases and overall brain metastatic burden. These findings demonstrate for the first time that neurons can form biologically relevant direct synapses with non-neural cancer cells. In brain metastasis, a particularly challenging complication of many common malignancies, this non-canonical stimulatory synaptic interaction offers novel therapeutic opportunities.

cancer biology↗

bioRxiv2023

Epigenetic modification and characterization of the MGMT promoter region using CRISPRoff in glioblastoma cells

The methylation status of the O6-methylguanine DNA methyltransferase (MGMT) promoter region is a critical predictor of response to alkylating agents in glioblastoma. However, current approaches to study the MGMT status focus on analyzing models with non-identical backgrounds. Here, we present an epigenetic editing approach using CRISPRoff to introduce site-specific CpG methylation in the MGMT promoter region of glioma cell lines. Sanger sequencing revealed successful introduction of methylation, effectively generating differently methylated glioma cell lines with an isogenic background. The introduced methylation resulted in reduced MGMT mRNA and protein levels. Furthermore, the cell lines with MGMT promoter region methylation exhibited increased sensitivity to temozolomide, consistent with the impact of methylation on treatment outcomes in patients with glioblastoma. This precise epigenome-editing approach provides valuable insights into the functional relevance of MGMT promoter regional methylation and its potential for prognostic and predictive assessments, as well as epigenetic-targeted therapies.

cancer biology↗

bioRxiv2023

Functional synapses between small cell lung cancer and glutamatergic neurons

Small cell lung cancer (SCLC) is a highly aggressive type of lung cancer, characterized by rapid proliferation, early metastatic spread, clinical recurrence and high rate of mortality. Using in vivo insertional mutagenesis screening in conjunction with cross-species genomic and transcriptomic validation, we identified a strong and consistent signal for neuronal, synaptic, and glutamatergic signaling gene sets in murine and human SCLC. We show that SCLC cells have the ability to develop intimate contacts with neuronal glutamatergic terminals in vitro, in autochthonous primary lung tumors and in brain-engrafted tumors. These contacts can develop into bona fide synapses, allowing SCLC cells to receive glutamatergic inputs. Fitting with a potential oncogenic role of neuron-SCLC interactions, we show that SCLC cells derive a robust proliferation advantage when co-cultured with neurons. Moreover, the repression of glutamate release and the stimulation of the inhibitory glutamate receptor GRM8 displayed therapeutic efficacy in an autochthonous mouse model of SCLC. Therefore, following malignant transformation, SCLC cells appear to hijack glutamatergic signaling to sustain tumor growth, thereby exposing a novel entry route for therapeutic intervention.

cancer biology↗

bioRxiv2022

4EBP1/2 support tumorigenicity and cell survival during energetic stress by translationally regulating fatty acid synthesis

Energetic stress compels cells to evolve adaptive mechanisms to maintain homeostasis. Here, we report that the negative regulators of mRNA translation initiation eukaryotic initiation factor 4E binding proteins 1/2 (4EBP1/2) are essential to promote the survival of mammalian cells and budding yeast under glucose starvation. Functionally, 4EBP1/2 inhibit fatty acid synthesis upon energetic stress via repression of Acetyl-CoA Carboxylase Alpha (ACACA) mRNA translation, sparing NADPH, to maintain intracellular redox balance. This has important relevance in cancers, as we uncovered that oncogene-transformed cells and glioma cells exploit the 4EBP1/2 regulation of ACACA expression and redox balance to combat energetic stress, thereby supporting transformation and tumorigenicity in vitro and in vivo. Clinically, high EIF4EBP1 (encoding 4EBP1) expression is associated with poor outcomes in several cancer types, including glioma. Our data reveal that 4EBP1/2 are conserved mediators of the survival response to energetic stress which are exploited by cancer cells for metabolic adaptation.

cell biology↗

bioRxiv2021

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

BackgroundNeuroblastoma (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 mTOR pathway are up-regulated 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. MethodsSeveral independent NB patient cohorts with corresponding mRNA expression data were analyzed for EIF4EBP1 expression. An institutional NB cohort consisting of 69 prospectively collected tumors was employed to immunohistochemically analyze expression of EIF4EBP1-encoded protein (4EBP1). In addition, we performed an in vitro luciferase reporter gene assay with an episomal EIF4EBP1 promoter and genetically modulated MYCN expression in NB cells. FindingsEIF4EBP1 mRNA expression was positively correlated with MYCN expression and elevated in stage 4 and high-risk NB patients. High EIF4EBP1 mRNA expression was 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. High levels of 4EBP1 were significantly associated with prognostically unfavorable NB histology. Functional analyses in vitro revealed that EIF4EBP1 expression is transcriptionally controlled by MYCN binding to the EIF4EBP1 promoter. InterpretationHigh EIF4EBP1 expression is associated with poor prognosis in NB patients and may serve to stratify patients with high-risk NB. FundingG.L. was supported by funding from the Elterninitiative Dusseldorf e.V., the Research Commission of the Medical Faculty of Heinrich Heine University, the Deutsche Forschungsgemeinschaft (Grant LE 3751/2-1), and the German Cancer Aid (Grant 70112624). The laboratory of T.G.P.G. is supported by the Barbara und Wilfried Mohr Foundation. BR is supported by the Israel Science Foundation (grant No. 1436/19). RESEARCH IN CONTEXT Evidence before this studyNB represents a particularly heterogeneous cancer entity, with 5-year event-free survival rate ranging from 50% to 98% depending on the patients risk group. While genes of the nutrient-sensing mTOR pathway were found to be up-regulated in MYCN-amplified NB tumors, their clinical relevance and prognostic value in NB patients remain unclear. In particular, the mTOR substrate-encoding gene EIF4EBP1 was studied in NB by three different groups and high EIF4EBP1 mRNA expression was observed in MYCN-amplified or contradictorily in more favorable stages 1 and 2 patients. Also, EIF4EBP1 was included in a prognostic gene signature for poor overall survival in NB. However, the prognostic value of EIF4EBP1 alone was not determined in NB and the expression of EIF4EBP1 encoded protein, 4EBP1, was not analyzed in NB tumor tissues and not correlated with clinicopathological features such as histological subtypes. Additionally, the transcriptional regulation of the EIF4EBP1 promoter by MYCN was not characterized. Added value of this studyThis study uncovers the prognostic potential of EIF4EBP1 at the mRNA and protein levels in NB patients. We report that high EIF4EBP1 expression is correlated with poor survival in three independent cohorts and that high 4EBP1 levels is associated with a prognostically unfavorable histological subtype. High EIF4EBP1 expression is also a factor of poor prognosis in stage 4 and high-risk patient groups. Finally, we found that MYCN activates the human EIF4EBP1 promoter through binding at three binding motifs. Implications of all the available evidenceEIF4EBP1 mRNA and 4EBP1 protein expression have prognostic value in NB, especially to stratify patients with advanced and more aggressive NB, such as patients with stage 4 disease and high-risk patients including those with unfavorable histological subtype NB. Enhanced EIF4EBP1 mRNA and 4EBP1 protein expression in NB are driven by direct transcriptional activation of EIF4EBP1 by MYCN.

cancer biology↗

bioRxiv2021

The epigenetic evolution of gliomas is determined by their IDH1 mutation status and treatment regimen

Tumor adaptation or selection is thought to underlie therapy resistance of gliomas. To investigate the longitudinal epigenetic evolution of gliomas in response to therapeutic pressure, we performed an epigenomic analysis of 143 matched initial and recurrent patients with IDH-wildtype (IDHwt) and IDH-mutant (IDHmut) gliomas. IDHwt gliomas showed a longitudinally stable epigenome with relatively low levels of global methylation, whereas the epigenome of IDHmut gliomas showed initial high levels genome-wide of DNA methylation that was progressively reduced to levels similar to those of IDHwt tumors. By integrating DNA methylation and gene expression data, adaptive changes of putative master regulators of the cell cycle and of differentiation were seen in IDHmut recurrent tumors. Furthermore, relapses of IDHmut tumors were accompanied by histological progression which in turn influenced survival, as validated in an independent cohort. Finally, the initial cell composition of the tumor microenvironment differed between IDHwt and IDHmut tumors and changed differentially following treatment, suggesting increased neo-angiogenesis and T-cell infiltration upon treatment for IDHmut gliomas. Our study provides one of the largest cohorts of paired glioma samples profiled with epigenomics, transcriptomics and genomics; and our results demonstrate that the treatment of IDHmut gliomas reshapes the epigenome towards an IDHwt-like phenotype. Accordingly, the prevalent practice of early genotoxic treatment in this patient population may need to be revisited.

genomics↗

bioRxiv2021

Integrated phospho-proteogenomic and single-cell transcriptomic analysis of meningiomas establishes robust subtyping and reveals subtype-specific immune invasion

Meningiomas are the most frequent primary intracranial tumors. They can follow a wide clinical spectrum from benign to highly aggressive clinical course. No specific therapy exists for refractory cases or cases not amenable to resection and radiotherapy. Identification of risk of recurrence and malignant transformation for the individual patients is challenging. However, promising molecular markers and prognostic subgrouping by DNA methylation are emerging. Still, the biological underpinnings of these diagnostic subgroups are elusive, and, consequently, no novel therapeutic options arise thereof. Here we establish robust subgroups across the full landscape of meningiomas, consistent through DNA methylation, mutations, the transcriptomic, proteomic and phospho-proteomic level. Pronounced proliferative stress and DNA damage repair signals in malignant cells and in clusters exclusive to recurrent tumors are in line with their higher mitotic activity, but also provide an explanation for the accumulation of genomic instability in anaplastic meningiomas. Although homozygous deletion of CDKN2A/B is a diagnostic marker of high-grade meningioma, the expression of its gene product increased from low to non-deleted high-grade cases. Differences between subgroups in lymphocyte and myeloid cell infiltration, representing a majority of tumor mass in low-grade NF2 tumors, could be assigned to cluster-specific interaction with tumor cells. Activation to a more proinflammatory phenotype and decreased infiltration of myeloid cells in high-grade cases correlated with lower expression of CSF1, located on chromosome arm 1p, whose deletion is known as prognostic marker, with no proposed mechanism before. Our results demonstrate a robust molecular subclassification of a tumor type across multiple layers, provide insight into heterogeneous growth dynamics despite shared pathognomonic mutations, and highlight immune infiltration modulation as a novel target for meningioma therapy.

genomics↗

bioRxiv2021

Distribution of GOPC:ROS1 and other ROS1 fusions in glioma types

The ROS proto-oncogene 1 (ROS1) gene is rearranged in various cancers. The translated fusion protein presents an attractive therapeutic target, since specific inhibitors have been approved for several tumor types. In glioma, ROS1 fusions are frequent within infantile hemispheric glioma, and single case reports on occurrences in other glioma types exist. However, a comprehensive analysis spanning the full width of glioma types and subtypes is lacking. We here assessed the spectrum and distribution of ROS1 fusions by screening >20,000 glioma cases for typical chromosomal alterations, with subsequent RNA-sequencing for confirmation of candidate cases. ROS1 fusions were identified in 16 cases, from low grade pilocytic astrocytoma WHO grade 1 to glioblastoma, IDH wildtype WHO grade 4. Thus, despite being enriched in some tumor types, ROS1 fusions are not pathognomonic for specific glioma types and may consitute a relevant target in a variety of cases.

genomics↗

bioRxiv2021

Recurrent fusions in PLAGL1 define a distinct subset of pediatric-type supratentorial ependymoma

Ependymomas encompass a heterogeneous group of central nervous system (CNS) neoplasms that occur along the entire neuroaxis. In recent years, extensive (epi-)genomic profiling efforts have identified several molecular groups of ependymoma that are characterized by distinct molecular alterations and/or patterns. Based on unsupervised visualization of a large cohort of genome-wide DNA methylation data, we identified a highly distinct group of pediatric-type tumors (n = 40) forming a cluster separate from all established CNS tumor types, of which a high proportion were histopathologically diagnosed as ependymoma. RNA sequencing revealed recurrent fusions involving the pleomorphic adenoma gene-like 1 (PLAGL1) gene in 19 of 20 of the samples analyzed, with the most common fusion being EWSR1:PLAGL1 (n = 13). Five tumors showed a PLAGL1:FOXO1 fusion and one a PLAGL1:EP300 fusion. High transcript levels of PLAGL1 were noted in these tumors, with concurrent overexpression of the imprinted genes H19 and IGF2, which are regulated by PLAGL1. Histopathological review of cases with sufficient material (n = 16) demonstrated a broad morphological spectrum of largely ependymoma-like tumors. Immunohistochemically, tumors were GFAP-positive and OLIG2- and SOX10-negative. In 3/16 of the cases, a dot-like positivity for EMA was detected. Consistent with other fusion-positive ependymal groups, all tumors in our series were located in the supratentorial compartment. Median age of the patients at the time of diagnosis was 6.2 years. Analysis of time to progression or recurrence revealed survival times comparable to those of patients with ZFTA:RELA-fused ependymoma. In summary, our findings suggest the existence of a novel group of supratentorial ependymomas that are characterized by recurrent PLAGL1 fusions and enriched for pediatric patients.

cancer biology↗

bioRxiv2020

A subset of pediatric thalamic gliomas share a distinct DNA methylation profile, H3K27me3 loss and frequent alteration of EGFR

BackgroundMalignant astrocytic gliomas in children show a remarkable biological and clinical diversity. Small in-frame insertions or missense mutations in the EGFR gene have recently been identified in a distinct subset of pediatric bithalamic gliomas with a unique DNA methylation pattern. MethodsHere, we investigated an epigenetically homogeneous cohort of malignant gliomas (n=58) distinct from other subtypes and enriched for pediatric cases and thalamic location, in order to elucidate the overlap with this recently identified subtype of pediatric bithalamic gliomas. ResultsEGFR gene amplification was detected in 16/58 (27%) tumors, and missense mutations or small in-frame insertions in EGFR were found in 20/30 tumors with available sequencing data (67%; five of them co-occurring with EGFR amplification). Additionally, eight of the 30 tumors (27%) harbored an H3.1 or H3.3 K27M mutation (six of them with a concomitant EGFR alteration). All tumors tested showed loss of H3K27me3 staining, with evidence of EZHIP overexpression in the H3 wildtype cases. Although some tumors indeed showed a bithalamic growth pattern, a significant proportion of tumors occurred in the unilateral thalamus or in other (predominantly midline) locations. ConclusionsOur findings present a distinct molecular class of pediatric malignant gliomas largely overlapping with the recently reported bithalamic gliomas characterized by EGFR alteration, but additionally showing a broader spectrum of EGFR alterations and tumor localization. Global H3K27me3 loss in this group appears to be mediated by either H3 K27 mutation or EZHIP overexpression. EGFR inhibition may represent a potential therapeutic strategy in these highly aggressive gliomas. Key pointsO_LIThis study confirms a distinct new subset of pediatric diffuse midline glioma with H3K27me3 loss, with or without H3 K27 mutation C_LIO_LIThe poor outcome of these tumors is in line with the broader family of pediatric diffuse midline gliomas with H3 K27 mutation or EZHIP overexpression C_LIO_LIFrequent EGFR alterations in these tumors may represent a therapeutic target in this subset C_LI Importance of the StudyMalignant astrocytic gliomas in children show a remarkable biological and clinical diversity. Here, we highlight a distinct molecular class of pediatric malignant gliomas characterized by EGFR alteration and global H3K27me3 loss that appears to be mediated by either H3 K27 mutation or EZHIP overexpression. EGFR inhibition may represent a potential therapeutic strategy in these highly aggressive gliomas.

cancer biology↗