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Abstract MYCN oncogene amplification is frequently observed in aggressive childhood neuroblastoma. Using an unbiased large-scale mutagenesis screen in neuroblastoma-prone transgenic mice, we identify a single germline point mutation in the transcriptional corepressor Runx1t1, which abolishes MYCN-driven tumorigenesis. This loss-of-function mutation disrupts a highly conserved zinc finger domain within Runx1t1. Deletion of one Runx1t1 allele in an independent Runx1t1 knockout mouse model is also sufficient to prevent MYCN-driven neuroblastoma development, and reverse ganglia hyperplasia, a known pre-requisite for tumorigenesis. Silencing RUNX1T1 in human neuroblastoma cells decreases colony formation in vitro, and inhibits tumor growth in vivo. Moreover, RUNX1T1 knockdown inhibits the viability of PAX3-FOXO1 fusion-driven rhabdomyosarcoma and MYC-driven small cell lung cancer cells. Despite the role of Runx1t1 in MYCN-driven tumorigenesis neither gene directly regulates the other. We show RUNX1T1 forms part of a transcriptional LSD1-CoREST3-HDAC repressive complex recruited by HAND2 to enhancer regions to regulate chromatin accessibility and cell-fate pathway genes.

Abstract Background Despite the advent of neoadjuvant chemoradiotherapy (CRT), overall survival rates of esophageal adenocarcinoma (EAC) remain low. A readily induced mesenchymal transition of EAC cells contributes to resistance to CRT. Methods In this study, we aimed to chart the heterogeneity in cell state transition after CRT and to identify its underpinnings. A panel of 12 esophageal cultures were treated with CRT and ranked by their relative epithelial-mesenchymal plasticity. RNA-sequencing was performed on 100 pre-treatment biopsies. After RNA-sequencing, Ridge regression analysis was applied to correlate gene expression to ranked plasticity, and models were developed to predict mesenchymal transitions in patients. Plasticity score predictions of the three highest significant predictive models were projected on the pre-treatment biopsies and related to clinical outcome data. Motif enrichment analysis of the genes associated with all three models was performed. Results This study reveals NANOG as the key associated transcription factor predicting mesenchymal plasticity in EAC. Expression of NANOG in pre-treatment biopsies is highly associated with poor response to neoadjuvant chemoradiation, the occurrence of recurrences, and median overall survival difference in EAC patients (>48 months). Perturbation of NANOG reduces plasticity and resensitizes cell lines, organoid cultures, and patient-derived in vivo grafts. Conclusions In conclusion, NANOG is a key transcription factor in mesenchymal plasticity in EAC and a promising predictive marker for outcome.

Epithelial-mesenchymal transition (EMT) has been identified as a driver of therapy resistance, particularly in esophageal adenocarcinoma (EAC), where transforming growth factor beta (TGF-β) can induce this process. Inhibitors of TGF-β may counteract the occurrence of mesenchymal, resistant tumor cell populations following chemo(radio)therapy and improve treatment outcomes in EAC. Here, we aimed to identify predictive biomarkers for the response to TGF-β targeting. In vitro approximations of neoadjuvant treatment were applied to publicly available primary EAC cell lines. TGF-β inhibitors fresolimumab and A83–01 were employed to inhibit EMT, and mesenchymal markers were quantified via flow cytometry to assess efficacy. Our results demonstrated a robust induction of mesenchymal cell states following chemoradiation, with TGF-β inhibition leading to variable reductions in mesenchymal markers. The cell lines were clustered into responders and non-responders. Genomic expression profiles were obtained through RNA-seq analysis. Differentially expressed gene (DEG) analysis identified 10 positively- and 23 negatively-associated hub genes, which were bioinformatically identified. Furthermore, the correlation of DEGs with response to TGF-β inhibition was examined using public pharmacogenomic databases, revealing 9 positively associated and 11 negatively associated DEGs. Among these, ERBB2, EFNB1, and TNS4 were the most promising candidates. Our findings reveal a distinct gene expression pattern associated with the response to TGF-β inhibition in chemo(radiated) EAC. The identified DEGs and predictive markers may assist patient selection in clinical studies investigating TGF-β targeting.

Abstract Over the past 10 years, institutional and national molecular tumor boards have been implemented for relapsed or refractory pediatric cancer to prioritize targeted drugs for individualized treatment based on actionable oncogenic lesions, including the Dutch iTHER platform. Hematological malignancies form a minority in precision medicine studies. Here, we report on 56 iTHER leukemia/lymphoma patients for which we considered cell surface markers and oncogenic aberrations as actionable events, supplemented with ex vivo drug sensitivity for six patients. Prior to iTHER registration, 34% of the patients had received allogeneic hematopoietic cell transplantation (HCT) and 18% CAR‐T therapy. For 51 patients (91%), a sample with sufficient tumor percentage (≥20%) required for comprehensive diagnostic testing was obtained. Up to 10 oncogenic actionable events were prioritized in 49/51 patients, and immunotherapy targets were identified in all profiled patients. Targeted treatment(s) based on the iTHER advice was given to 24 of 51 patients (47%), including immunotherapy in 17 patients, a targeted drug matching an oncogenic aberration in 12 patients, and a drug based on ex vivo drug sensitivity in one patient, resulting in objective responses and a bridge to HCT in the majority of the patients. In conclusion, comprehensive profiling of relapsed/refractory hematological malignancies showed multiple oncogenic and immunotherapy targets for a precision medicine approach, which requires multidisciplinary expertise to prioritize the best treatment options for this rare, heavily pretreated pediatric population.

Abstract In principle, germline cells possess the capability to transmit a nearly unaltered set of genetic material to infinite future generations, whereas somatic cells are limited by strict growth constraints necessary to assure an organism’s physical structure and eventual mortality. As the potential to replicate indefinitely is a key feature of cancer, we hypothesized that the activation of a “germline program” in somatic cells can contribute to oncogenesis. Our group recently described over one thousand germline specific genes that can be ectopically expressed in cancer, yet how germline specific processes contribute to the malignant properties of cancer is poorly understood. We here show that the expression of germ cell/cancer (GC) genes correlates with malignancy in lung adenocarcinoma (LUAD). We found that LUAD cells expressing more GC genes can repair DNA double strand breaks more rapidly, show higher rates of proliferation and are more resistant to ionizing radiation, compared to LUAD cells that express fewer GC genes. In particular, we identified the HORMA domain protein regulator TRIP13 to be predominantly responsible for this malignant phenotype, and that TRIP13 inhibition or expression levels affect the response to ionizing radiation and subsequent DNA repair. Our results demonstrate that GC genes are viable targets in oncology, as they induce increased radiation resistance and increased propagation in cancer cells. Because their expression is normally restricted to germline cells, we anticipate that GC gene directed therapeutic options will effectively target cancer, with limited side effects besides (temporary) infertility.

Summary: Small intestine (SI) maturation during early life is pivotal in preventing the onset of gut diseases. In this study we interrogated the milestones of SI development by gene expression profiling and ingenuity pathway analyses. We identified a set of cytokines as main regulators of changes observed across different developmental stages. Upon cytokines stimulation, with IFNγ as the most contributing factor, human fetal organoids (HFOs) increase brush border gene expression and enzyme activity as well as trans-epithelial electrical resistance. Electron microscopy revealed developed brush border and loss of fetal cell characteristics in HFOs upon cytokine stimulation. We identified T cells as major source of IFNγ production in the fetal SI lamina propria. Co-culture of HFOs with T cells recapitulated the major effects of cytokine stimulation. Our findings underline pro-inflammatory cytokines derived from T cells as pivotal factors inducing functional SI maturation in vivo and capable of modulating the barrier maturation of HFOs in vitro.

Pancreatic ductal adenocarcinoma (PDAC) is a dismal disease with a low 5-year survival rate of only 13%. Despite intense research efforts, PDAC remains insufficiently understood. In part, this is attributed to opposing effects of key players being unraveled, including the stroma but also molecules that act in a context-dependent manner. One such molecule is the transcription factor C/EBPδ, where we recently showed that C/EBPδ exerts tumor-suppressive effects in PDAC cells in vitro. To better understand the role of C/EBPδ in different contexts and the development of PDAC, we here build on these findings and assess the effect of C/EBPδ in a PDAC model in mice. We establish that the lack of oxygen in vivo—hypoxia—counteracts the tumor-suppressive effects of C/EBPδ, and identify a reciprocal feedback loop between C/EBPδ and HIF-1α. RNA sequencing of C/EBPδ-induced cells under hypoxia also suggests that the growth-limiting effects of C/EBPδ decrease with oxygen tension. Consequently, in vitro proliferation assays reveal that the tumor-suppressive activities of C/EBPδ are abrogated due to hypoxia. This study demonstrates the importance of considering major physiological parameters in preclinical approaches.

The development of neuroblastoma (NB) is regulated by multiple core transcription factors. Here, SOX11 is identified as a potential epigenetic master regulator upstream of the core regulatory circuitry in adrenergic high-risk neuroblastoma.

Abstract Nowadays medulloblastoma (MB) tumors can be treated with risk-stratified approaches with up to 80% success rate. However, disease relapses occur in approximately 30% of patients and successful salvage treatment strategies at relapse remain scarce. Acquired copy number changes or TP53 mutations are known to occur frequently in relapses, while methylation profiles usually remain highly similar to those of the matching primary tumors, indicating that in general molecular subgrouping does not change during the course of the disease. In the current study, we have used RNA sequencing data to analyze the transcriptome profiles of 43 primary-relapse MB pairs in order to identify specific molecular features of relapses within various tumor groups. Gene variance analysis between primary and relapse samples demonstrated the impact of age in SHH-MB: the changes in gene expression relapse profiles were more pronounced in the younger patients (

Colorectal cancer can lead to the development of peritoneal metastases, which are associated with worse disease outcome. Here, the authors characterize peritoneal metastases from 52 patients using RNA-seq and mutational sequencing and show a distinct molecular subtype.

Abstract Background SP140 is a bromodomain-containing protein expressed predominantly in immune cells. Genetic polymorphisms and epigenetic modifications in the SP140 locus have been linked to Crohn’s disease (CD), suggesting a role in inflammation. Results We report the development of the first small molecule SP140 inhibitor (GSK761) and utilize this to elucidate SP140 function in macrophages. We show that SP140 is highly expressed in CD mucosal macrophages and in in vitro-generated inflammatory macrophages. SP140 inhibition through GSK761 reduced monocyte-to-inflammatory macrophage differentiation and lipopolysaccharide (LPS)-induced inflammatory activation, while inducing the generation of CD206+ regulatory macrophages that were shown to associate with a therapeutic response to anti-TNF in CD patients. SP140 preferentially occupies transcriptional start sites in inflammatory macrophages, with enrichment at gene loci encoding pro-inflammatory cytokines/chemokines and inflammatory pathways. GSK761 specifically reduces SP140 chromatin binding and thereby expression of SP140-regulated genes. GSK761 inhibits the expression of cytokines, including TNF, by CD14+ macrophages isolated from CD intestinal mucosa. Conclusions This study identifies SP140 as a druggable epigenetic therapeutic target for CD.

Summary: Memory CD8 T cells play an important role in the protection against breakthrough infections with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Whether the route of antigen exposure impacts these cells at a functional level is incompletely characterized. Here, we compare the memory CD8 T cell response against a common SARS-CoV-2 epitope after vaccination, infection, or both. CD8 T cells demonstrate comparable functional capacity when restimulated directly ex vivo, independent of the antigenic history. However, analysis of T cell receptor usage shows that vaccination results in a narrower scope than infection alone or in combination with vaccination. Importantly, in an in vivo recall model, memory CD8 T cells from infected individuals show equal proliferation but secrete less tumor necrosis factor (TNF) compared with those from vaccinated people. This difference is negated when infected individuals have also been vaccinated. Our findings shed more light on the differences in susceptibility to re-infection after different routes of SARS-CoV-2 antigen exposure.

BackgroundStudies conducted in the last decades have revealed a role for the non-coding microRNAs (miRNAs) in cancer development and progression. Several miRNAs within the chromosome region 14q32, a region commonly deleted in cancers, are associated with poor clinical outcome in the childhood cancer neuroblastoma. We have previously identified miR-323a-3p from this region to be downregulated in chemotherapy treated neuroblastoma cells compared to pre-treatment cells from the same patients. Furthermore, in neuroblastoma tumors, this miRNA is downregulated in advanced stage 4 disease compared to stage 1–2. In this study, we attempt to delineate the unknown functional roles of miR-323a-3p in neuroblastoma.MethodsSynthetic miRNA mimics were used to overexpress miR-323a-3p in neuroblastoma cell lines. To investigate the functional roles of miR-323a-3p, cell viability assay, flow cytometry, reverse transcription-quantitative polymerase chain reaction, luciferase reporter assay and western blot were conducted on the neuroblastoma cell lines Kelly, SH-SY5Y and SK-N-BE(2)-C.ResultsEctopic expression of miR-323a-3p resulted in marked reduction of cell viability in Kelly, SH-SY5Y and SK-N-BE(2)-C by causing G1-cell cycle arrest in Kelly and SH-SY5Y and apoptosis in all the cell lines tested. Furthermore, mRNA and protein levels of signal transducer and activator of transcription 3 (STAT3) were reduced upon miR-323a-3p overexpression. A direct binding of the miR-323a-3p to the 3′UTR of STAT3 was experimentally validated by luciferase reporter assay, where miR-323a-3p reduced luminescent signal from full length STAT3 3′UTR luciferase reporter, but not from a reporter with mutation in the predicted seed sequence.ConclusionsmiR-323a-3p inhibits growth of neuroblastoma cell lines through G1-cell cycle arrest and apoptosis, and the well-known oncogene STAT3 is a direct target of this miRNA.

IntroductionMutations affecting the RAS-MAPK pathway occur frequently in relapsed neuroblastoma tumors and are associated with response to MEK inhibition in vitro. However, these inhibitors alone do not lead to tumor regression in vivo, indicating the need for combination therapy.Methods and resultsVia high-throughput combination screening, we identified that the MEK inhibitor trametinib can be combined with BCL-2 family member inhibitors, to efficiently inhibit growth of neuroblastoma cell lines with RAS-MAPK mutations. Suppressing the RAS-MAPK pathway with trametinib led to an increase in pro-apoptotic BIM, resulting in more BIM binding to anti-apoptotic BCL-2 family members. By favoring the formation of these complexes, trametinib treatment enhances sensitivity to compounds targeting anti-apoptotic BCL-2 family members. In vitro validation studies confirmed that this sensitizing effect is dependent on an active RAS-MAPK pathway. In vivo combination of trametinib with BCL-2 inhibitors led to tumor inhibition in NRAS-mutant and NF1-deleted xenografts.ConclusionTogether, these results show that combining MEK inhibition with BCL-2 family member inhibition could potentially improve therapeutic outcomes for RAS-MAPK-mutated neuroblastoma patients.

Measurement(s) mRNA Sequencing • MicroRNA Sequencing Technology Type(s) sequencer Sample Characteristic - Organism Homo sapiens Sample Characteristic - Location Belgium

Abstract The epithelial signaling pathways involved in damage and regeneration, and neoplastic transformation are known to be similar. We noted upregulation of argininosuccinate synthetase (ASS1) in hyperproliferative intestinal epithelium. Since ASS1 leads to de novo synthesis of arginine, an important amino acid for the growth of intestinal epithelial cells, its upregulation can contribute to epithelial proliferation necessary to be sustained during oncogenic transformation and regeneration. Here we investigated the function of ASS1 in the gut epithelium during tissue regeneration and tumorigenesis, using intestinal epithelial conditional Ass1 knockout mice and organoids, and tissue specimens from colorectal cancer patients. We demonstrate that ASS1 is strongly expressed in the regenerating and Apc-mutated intestinal epithelium. Furthermore, we observe an arrest in amino acid flux of the urea cycle, which leads to an accumulation of intracellular arginine. However, loss of epithelial Ass1 does not lead to a reduction in proliferation or increase in apoptosis in vivo, also in mice fed an arginine-free diet. Epithelial loss of Ass1 seems to be compensated by altered arginine metabolism in other cell types and the liver.

Despite intensive therapy, children with high-risk neuroblastoma are at risk of treatment failure. We applied a multiomic system approach to evaluate metabolic vulnerabilities in human neuroblastoma. We combined metabolomics, CRISPR screening, and transcriptomic data across more than 700 solid tumor cell lines and identified dihydroorotate dehydrogenase (DHODH), a critical enzyme in pyrimidine synthesis, as a potential treatment target. Of note, DHODH inhibition is currently under clinical investigation in patients with hematologic malignancies. In neuroblastoma, DHODH expression was identified as an independent risk factor for aggressive disease, and high DHODH levels correlated to worse overall and event-free survival. A subset of tumors with the highest DHODH expression was associated with a dismal prognosis, with a 5-year survival of less than 10%. In xenograft and transgenic neuroblastoma mouse models treated with the DHODH inhibitor brequinar, tumor growth was dramatically reduced, and survival was extended. Furthermore, brequinar treatment was shown to reduce the expression of MYC targets in 3 neuroblastoma models in vivo. A combination of brequinar and temozolomide was curative in the majority of transgenic TH-MYCN neuroblastoma mice, indicating a highly active clinical combination therapy. Overall, DHODH inhibition combined with temozolomide has therapeutic potential in neuroblastoma, and we propose this combination for clinical testing.

Fibrodysplasia Ossificans Progressiva (FOP) is a very rare genetic disease characterized by progressive heterotopic ossification (HO) of soft tissues, leading to immobility and premature death. FOP is caused by a mutation in the Activin receptor Type 1 (ACVR1) gene, resulting in altered responsiveness to Activin-A. We recently revealed that Activin-A induces fewer, but larger and more active, osteoclasts regardless of the presence of the mutated ACVR1 receptor. The underlying mechanism of Activin-A-induced changes in osteoclastogenesis at the gene expression level remains unknown. Transcriptomic changes induced by Activin-A during osteoclast formation from healthy controls and patient-derived CD14-positive monocytes were studied using RNA sequencing. CD14-positive monocytes from six FOP patients and six age- and sex-matched healthy controls were differentiated into osteoclasts in the absence or presence of Activin-A. RNA samples were isolated after 14 days of culturing and analyzed by RNA sequencing. Non-supervised principal component analysis (PCA) showed that samples from the same culture conditions (e.g., without or with Activin-A) tended to cluster, indicating that the variability induced by Activin-A treatment was larger than the variability between the control and FOP samples. RNA sequencing analysis revealed 1480 differentially expressed genes induced by Activin-A in healthy control and FOP osteoclasts with p(adj) < 0.01 and a Log2 fold change of ≥±2. Pathway and gene ontology enrichment analysis revealed several significantly enriched pathways for genes upregulated by Activin-A that could be linked to the differentiation or function of osteoclasts, cell fusion or inflammation. Our data showed that Activin-A has a substantial effect on gene expression during osteoclast formation and that this effect occurred regardless of the presence of the mutated ACVR1 receptor causing FOP.

Summary: Diffuse midline gliomas (DMG) are highly malignant incurable pediatric brain tumors. In this study, we show that Aurora kinase A (AURKA) is overexpressed in DMG and can be used as a therapeutic target. Additionally, AURKA inhibition combined with CRISPR/Cas9 screening in DMG cells, revealed polo-like kinase 1 (PLK1) as a synergistic target with AURKA. Using a panel of patient-derived DMG culture models, we demonstrate that treatment with volasertib, a clinically relevant and selective PLK1 inhibitor, synergizes with different AURKA inhibitors, supporting the CRISPR screen results. Mechanistically, our results show that combined loss of PLK1 and AURKA causes a G2/M cell cycle arrest which blocks vital parts of DNA-damage repair and induces apoptosis, solely in DMG cells. Altogether, our findings highlight the importance of AURKA and PLK1 for DMG propagation and demonstrate the potential of concurrently targeting these proteins as a therapeutic strategy for these devastating pediatric brain tumors.

Abstract Up to 10% of pediatric cancer patients harbor pathogenic germline variants in one or more cancer susceptibility genes. A recent study from the US reported pathogenic variants in 22 out of 60 analyzed autosomal dominant cancer susceptibility genes, implicating 8.5% of pediatric cancer patients. Here we aimed to assess the prevalence of germline pathogenic variants in these 22 genes in a population-based Swedish cohort and to compare the results to those described in other populations. We found pathogenic variants in 10 of the 22 genes covering 3.8% of these patients. The prevalence of TP53 mutations was significantly lower than described in previous studies, which can largely be attributed to differences in tumor diagnosis distributions across the three cohorts. Matched family history for relatives allowed assessment of familial cancer incidence, however, no significant difference in cancer incidence was found in families of children carrying pathogenic variants compared to those who did not.

Alternative lengthening of telomeres (ALT) is associated with a poor outcome in neuroblastoma. Here, the authors find that ALT is associated with mutated ATRX and/or reduced protein abundance, frequent telomeric repeat loci and heterochromatic telomeric chromatin.

Background & Aims: The use of antibiotics (ABs) is a common practice during the first months of life. ABs can perturb the intestinal microbiota, indirectly influencing the intestinal epithelial cells (IECs), but can also directly affect IECs independent of the microbiota. Previous studies have focused mostly on the impact of AB treatment during adulthood. However, the difference between the adult and neonatal intestine warrants careful investigation of AB effects in early life. Methods: Neonatal mice were treated with a combination of amoxicillin, vancomycin, and metronidazole from postnatal day 10 to 20. Intestinal permeability and whole-intestine gene and protein expression were analyzed. IECs were sorted by a fluorescence-activated cell sorter and their genome-wide gene expression was analyzed. Mouse fetal intestinal organoids were treated with the same AB combination and their gene and protein expression and metabolic capacity were determined. Results: We found that in vivo treatment of neonatal mice led to decreased intestinal permeability and a reduced number of specialized vacuolated cells, characteristic of the neonatal period and necessary for absorption of milk macromolecules. In addition, the expression of genes typically present in the neonatal intestinal epithelium was lower, whereas the adult gene expression signature was higher. Moreover, we found altered epithelial defense and transepithelial-sensing capacity. In vitro treatment of intestinal fetal organoids with AB showed that part of the consequences observed in vivo is a result of the direct action of the ABs on IECs. Lastly, ABs reduced the metabolic capacity of intestinal fetal organoids. Conclusions: Our results show that early life AB treatment induces direct and indirect effects on IECs, influencing their maturation and functioning.

Drug synergies impact the efficacy of combination therapies but are difficult to identify. Here Narayan et al. describe the drug atlas, a method to predict effective drug combinations from common exclusive drug effects providing a resource for exploring and understanding effective drug combinations.

Background & Aims: Recent evidence has suggested that the intact intestinal epithelial barrier protects our body from a range of immune-mediated diseases. The epithelial layer has an impressive ability to reconstitute and repair upon damage and this process of repair increasingly is seen as a therapeutic target. In vitro models to study this process in primary intestinal cells are lacking. Methods: We established and characterized an in vitro model of intestinal damage and repair by applying γ-radiation on small-intestinal organoids. We then used this model to identify novel regulators of intestinal regeneration. Results: We identified hepatocyte nuclear factor 4α (HNF4α) as a pivotal upstream regulator of the intestinal regenerative response. Organoids lacking Hnf4a were not able to propagate in vitro. Importantly, intestinal Hnf4a knock-out mice showed impaired regeneration after whole-body irradiation, confirming intestinal organoids as a valuable alternative to in vivo studies. Conclusions: In conclusion, we established and validated an in vitro damage–repair model and identified HNF4α as a crucial regulator of intestinal regeneration. Transcript profiling: GSE141515 and GSE141518.

Abstract Background Breast cancer (BC) immune infiltrates play a critical role in tumor progression and response to treatment. Besides stromal tumor infiltrating lymphocytes (sTILs) which have recently reached level 1B evidence as a prognostic marker in triple negative BC, a plethora of methods to assess immune infiltration exists, and it is unclear how these compare to each other and if they can be used interchangeably. Methods Two experienced pathologists scored sTIL, intra-tumoral TIL (itTIL), and 6 immune cell types (CD3+, CD4+, CD8+, CD20+, CD68+, FOXP3+) in the International Cancer Genomics Consortium breast cancer cohort using hematoxylin and eosin-stained (n = 243) and immunohistochemistry-stained tissue microarrays (n = 254) and whole slides (n = 82). The same traits were evaluated using transcriptomic- and methylomic-based deconvolution methods or signatures. Results The concordance correlation coefficient (CCC) between pathologists for sTIL was very good (0.84) and for cell-specific immune infiltrates slightly lower (0.63–0.66). Comparison between tissue microarray and whole slide pathology scores revealed systematically higher values in whole slides (ratio 2.60–5.98). The Spearman correlations between microscopic sTIL and transcriptomic- or methylomic-based assessment of immune infiltrates were highly variable (r = 0.01–0.56). Similar observations were made for cell type-specific quantifications (r = 0.001–0.54). We observed a strong inter-method variability between the omics-derived estimations, which is further cell type dependent. Finally, we demonstrated that most methods more accurately identify highly infiltrated (sTIL ≥ 60%; area under the curve, AUC, 0.64–0.99) as compared to lowly infiltrated tumors (sTIL ≤ 10%; AUC 0.52–0.82). Conclusions There is a lower inter-pathologist concordance for cell-specific quantification as compared to overall infiltration quantification. Microscopic assessments are underestimated when considering small cores (tissue microarray) instead of whole slides. Results further highlight considerable differences between the microscopic-, transcriptomic-, and methylomic-based methods in the assessment of overall and cell-specific immune infiltration in BC. We therefore call for extreme caution when assessing immune infiltrates using current methods and emphasize the need for standardized immune characterization beyond TIL.

Background: Pediatric high-grade gliomas (pHGG) are the leading cause of cancer-related death during childhood. Due to their diffuse growth characteristics, chemoresistance and location behind the blood-brain barrier (BBB), the prognosis of pHGG has barely improved in the past decades. As such, there is a dire need for new therapies that circumvent those difficulties. Since aberrant expression of DNA damage-response associated Fanconi anemia proteins play a central role in the onset and therapy resistance of many cancers, we here investigated if FANCD2 depletion could sensitize pHGG to additional DNA damage. Methods: We determined the capacity of celastrol, a BBB-penetrable compound that degrades FANCD2, to sensitize glioma cells to the archetypical DNA-crosslinking agent carboplatin in vitro in seven patient-derived pHGG models. In addition, we tested this drug combination in vivo in a patient-derived orthotopic pHGG xenograft model. Underlying mechanisms to drug response were investigated using mRNA expression profiling, western blotting, immunofluorescence, FANCD2 knockdown and DNA fiber assays. Findings: FANCD2 is overexpressed in HGGs and depletion of FANCD2 by celastrol synergises with carboplatin to induce cytotoxicity. Combination therapy prolongs survival of pHGG-bearing mice over monotherapy and control groups in vivo (P

Summary: Autotaxin (ATX; ENPP2) produces lysophosphatidic acid (LPA) that regulates multiple biological functions via cognate G protein-coupled receptors LPAR1-6. ATX/LPA promotes tumor cell migration and metastasis via LPAR1 and T cell motility via LPAR2, yet its actions in the tumor immune microenvironment remain unclear. Here, we show that ATX secreted by melanoma cells is chemorepulsive for tumor-infiltrating lymphocytes (TILs) and circulating CD8+ T cells ex vivo, with ATX functioning as an LPA-producing chaperone. Mechanistically, T cell repulsion predominantly involves Gα12/13-coupled LPAR6. Upon anti-cancer vaccination of tumor-bearing mice, ATX does not affect the induction of systemic T cell responses but, importantly, suppresses tumor infiltration of cytotoxic CD8+ T cells and thereby impairs tumor regression. Moreover, single-cell data from melanoma tumors are consistent with intratumoral ATX acting as a T cell repellent. These findings highlight an unexpected role for the pro-metastatic ATX-LPAR axis in suppressing CD8+ T cell infiltration to impede anti-tumor immunity, suggesting new therapeutic opportunities.

Esophageal adenocarcinoma (EAC) is a highly aggressive cancer and its response to chemo- and radiotherapy is unpredictable. EACs are highly heterogeneous at the molecular level. The aim of this study was to perform gene expression analysis of EACs to identify distinct molecular subgroups and to investigate expression signatures in relation to treatment response. In this prospective observational study, RNA sequencing was performed on pre-treatment endoscopic EAC biopsies from a discovery cohort included between 2012 and 2017 in one Dutch Academic Center. Four additional cohorts were analyzed for validation purposes. Unsupervised clustering was performed on 107 patients to identify biological EAC subgroups. Specific cell signaling profiles were identified and evaluated with respect to predicting response to neo-adjuvant chemo(radio)therapy. We identified and validated three distinct biological EAC subgroups, characterized by (1) p38 MAPK/Toll-like receptor signaling; (2) activated immune system; and (3) impaired cell adhesion. Subgroup 1 was associated with poor response to chemo-radiotherapy. Moreover, an immune signature with activated T-cell signaling, and increased number of activated CD4 T memory cells, neutrophils and dendritic cells, and decreased M1 and M2 macrophages and plasma cells, was associated with complete histopathological response. This study provides a novel molecular classification for EACs. EAC subgroup 1 proved to be more therapy-resistant, while immune signaling was increased in patients with complete response to chemo-radiotherapy. Our findings give insight into the biology of EACs and in cellular signaling mechanisms underlying response to neo-adjuvant treatment. Future implementation of this classification will improve patient stratification and enhance the development of targeted therapies.

Abstract Background The clinical behavior of prostate cancer (PCa) is variable, and while the majority of cases remain indolent, 10% of patients progress to deadly forms of the disease. Current clinical predictors used at the time of diagnosis have limitations to accurately establish progression risk. Here we describe the development of a tumor suppressor regulated, cell-cycle gene expression based prognostic signature for PCa, and validate its independent contribution to risk stratification in several radical prostatectomy (RP) patient cohorts. Methods We used RNA interference experiments in PCa cell lines to identify a gene expression based gene signature associated with Tmeff2, an androgen regulated, tumor suppressor gene whose expression shows remarkable heterogeneity in PCa. Gene expression was confirmed by qRT-PCR. Correlation of the signature with disease outcome (time to recurrence) was retrospectively evaluated in four geographically different cohorts of patients that underwent RP (834 samples), using multivariate logistical regression analysis. Multivariate analyses were adjusted for standard clinicopathological variables. Performance of the signature was compared to previously described gene expression based signatures using the SigCheck software. Results Low levels of TMEFF2 mRNA significantly (p

Neuroblastoma includes adrenergic and mesenchymal cell types that can interconvert. Here, the authors show that this transdifferentiation is driven by a NOTCH feedforward loop that allows a swift transition between two semi-stable cellular states.

The association between prolonged antibiotic (AB) use in neonates and increased incidence of later life diseases is not yet fully understood. AB treatment in early life alters intestinal epithelial cell composition, functioning, and maturation, which could be the basis for later life health effects. Here, we investigated whether AB-induced changes in the neonatal gut persisted up to adulthood and whether early life AB had additional long-term consequences for gut functioning. Mice received AB orally from postnatal day 10 to 20. Intestinal morphology, permeability, and gene and protein expression at 8 weeks were analyzed. Our data showed that the majority of the early life AB-induced gut effects did not persist into adulthood, yet early life AB did impact later life gut functioning. Specifically, the proximal small intestine (SI) of adult mice treated with AB in early life was characterized by hyperproliferative crypts, increased number of Paneth cells, and alterations in enteroendocrine cell-specific gene expression profiles. The distal SI of adult mice displayed a reduced expression of antibacterial defense markers. Together, our results suggest that early life AB leads to structural and physiological changes in the adult gut, which may contribute to disease development when homeostatic conditions are under challenge.

The memory CD8 T-cell pool must select for clones that bind immunodominant epitopes with high affinity to efficiently counter reinfection. At the same time, it must retain a level of clonal diversity to allow recognition of pathogens with mutated epitopes. How the level of diversity within the memory pool is controlled is unclear, especially in the context of a selective drive for antigen affinity. We find that preservation of clones that bind the activating antigen with low affinity depends on expression of the transcription factor Eomes in the first days after antigen encounter. Eomes is induced at low activating signal strength and directly drives transcription of the prosurvival protein Bcl-2. At higher signal intensity, T-bet is induced which suppresses Bcl-2 and causes a relative survival advantage for cells of low affinity. Clones activated with high-affinity antigen form memory largely independent of Eomes and have a proliferative advantage over clones that bind the same antigen with low affinity. This causes high-affinity clones to prevail in the memory pool, despite their relative survival deficit. Genetic or therapeutic targeting of the Eomes/Bcl-2 axis reduces the clonal diversity of the memory pool, which diminishes its ability to respond to pathogens carrying mutations in immunodominant epitopes. Thus, we demonstrate on a molecular level how sufficient diversity of the memory pool is established in an environment of affinity-based selection.

Abstract Background To better predict the likelihood of response to chemotherapy, we have conducted a study comparing the gene expression patterns of primary tumours with their corresponding response to systemic chemotherapy in the metastatic setting. Methods mRNA expression profiles of breast carcinomas of patients that later developed distant metastases were analyzed using supervised and non-supervised classification techniques to identify predictors of response to chemotherapy. The top differentially expressed genes between the responders and non-responders were identified and further explored. An independent dataset which was generated to predict response to neo-adjuvant CT was utilized for the purpose of validation. Response to chemotherapy was also correlated to the clinicopathologic characteristics, molecular subtypes, metastatic behavior and survival outcomes. Results Anthracycline containing regimens were the most common first line treatment (58.4%), followed by non-anthracycline/non-taxane containing (25.8%) and taxane containing (15.7%) regimens. Response was achieved in 41.6% of the patients to the first line CT and in 21.8% to second line CT. Response was not found to be significantly correlated to tumour type, grade, lymph node status, ER and PR status. Patients with HER2+ tumours showed better response to anthracycline containing therapy (p: 0.002). Response to first and second line chemotherapy did not differ among gene expression based molecular subtypes (p: 0.236 and p: 0.20). Using supervised classification, a 14 gene response classifier was identified. This 14-gene predictor could successfully predict the likelihood of better response to first and second line CT (p:

Ornithine decarboxylase (ODC1), a critical regulatory enzyme in polyamine biosynthesis, is a direct transcriptional target of MYCN, amplification of which is a powerful marker of aggressive neuroblastoma. A single nucleotide polymorphism (SNP), G316A, within the first intron of ODC1, results in genotypes wildtype GG, and variants AG/AA. CRISPR-cas9 technology was used to investigate the effects of AG clones from wildtype MYCN-amplified SK-N-BE(2)-C cells and the effect of the SNP on MYCN binding, and promoter activity was investigated using EMSA and luciferase assays. AG clones exhibited decreased ODC1 expression, growth rates, and histone acetylation and increased sensitivity to ODC1 inhibition. MYCN was a stronger transcriptional regulator of the ODC1 promoter containing the G allele, and preferentially bound the G allele over the A. Two neuroblastoma cohorts were used to investigate the clinical impact of the SNP. In the study cohort, the minor AA genotype was associated with improved survival, while poor prognosis was associated with the GG genotype and AG/GG genotypes in MYCN-amplified and non-amplified patients, respectively. These effects were lost in the GWAS cohort. We have demonstrated that the ODC1 G316A polymorphism has functional significance in neuroblastoma and is subject to allele-specific regulation by the MYCN oncoprotein.