Diffuse infiltrating tumour with the molecular profile of an atypical teratoid rhabdoid tumour (AT/RT SHH-1B) in an adult patient.

We describe a 46-year-old patient with an IDH-wildtype diffusely infiltrating atypical teratoid/rhabdoid tumour (AT/RT), SHH-1B molecular subtype. The unusual histology and subsequent diagnosis in an adult patient will be discussed.

Low-grade diffusely infiltrative tumour (LGDIT), SMARCB1-mutant: A clinical and histopathological distinct entity showing epigenetic similarity with ATRT-MYC.

Low-grade diffusely infiltrative tumour (LGDIT), SMARCB1-mutant, is a histopathological distinct low-grade lesion encountered in older children and young adults that shows epigenetic similarity with ATRT-MYC and has the potential for malignant progression.

ATRT-SHH comprises three molecular subgroups with characteristic clinical and histopathological features and prognostic significance.

Atypical teratoid/rhabdoid tumor (ATRT) is an aggressive central nervous system tumor characterized by loss of SMARCB1/INI1 protein expression and comprises three distinct molecular groups, ATRT-TYR, ATRT-MYC and ATRT-SHH. ATRT-SHH represents the largest molecular group and is heterogeneous with regard to age, tumor location and epigenetic profile. We, therefore, aimed to investigate if heterogeneity within ATRT-SHH might also have biological and clinical importance. Consensus clustering of DNA methylation profiles and confirmatory t-SNE analysis of 65 ATRT-SHH yielded three robust molecular subgroups, i.e., SHH-1A, SHH-1B and SHH-2. These subgroups differed by median age of onset (SHH-1A: 18 months, SHH-1B: 107 months, SHH-2: 13 months) and tumor location (SHH-1A: 88% supratentorial; SHH-1B: 85% supratentorial; SHH-2: 93% infratentorial, often extending to the pineal region). Subgroups showed comparable SMARCB1 mutational profiles, but pathogenic/likely pathogenic SMARCB1 germline variants were over-represented in SHH-2 (63%) as compared to SHH-1A (20%) and SHH-1B (0%). Protein expression of proneural marker ASCL1 (enriched in SHH-1B) and glial markers OLIG2 and GFAP (absent in SHH-2) as well as global mRNA expression patterns differed, but all subgroups were characterized by overexpression of SHH as well as Notch pathway members. In a Drosophila model, knockdown of Snr1 (the fly homologue of SMARCB1) in hedgehog activated cells not only altered hedgehog signaling, but also caused aberrant Notch signaling and formation of tumor-like structures. Finally, on survival analysis, molecular subgroup and age of onset (but not ASCL1 staining status) were independently associated with overall survival, older patients (> 3 years) harboring SHH-1B experiencing relatively favorable outcome. In conclusion, ATRT-SHH comprises three subgroups characterized by SHH and Notch pathway activation, but divergent molecular and clinical features. Our data suggest that molecular subgrouping of ATRT-SHH has prognostic relevance and might aid to stratify patients within future clinical trials.

FOXD1 promotes dedifferentiation and targeted therapy resistance in melanoma by regulating the expression of connective tissue growth factor.

Metastatic melanoma is an aggressive skin cancer and associated with a poor prognosis. In clinical terms, targeted therapy is one of the most important treatments for patients with BRAFV600E -mutated advanced melanoma. However, the development of resistance to this treatment compromises its therapeutic success. We previously demonstrated that forkhead box D1 (FOXD1) regulates melanoma migration and invasion. Here, we found that FOXD1 was highly expressed in melanoma cells and was associated with a poor survival of patients with metastatic melanoma. Upregulation of FOXD1 expression enhanced melanoma cells' resistance to vemurafenib (BRAF inhibitor [BRAFi]) or vemurafenib and cobimetinib (MEK inhibitor) combination treatment whereas loss of FOXD1 increased the sensitivity to treatment. By comparing gene expression levels between FOXD1 knockdown (KD) and overexpressing (OE) cells, we identified the connective tissue growth factor (CTGF) as a downstream factor of FOXD1. Chromatin immunoprecipitation and luciferase assay demonstrated the direct binding of FOXD1 to the CTGF promoter. Similar to FOXD1, knockdown of CTGF increased the sensitivity of BRAFi-resistant cells to vemurafenib. FOXD1 KD cells treated with recombinant CTGF protein were less sensitive towards vemurafenib compared to untreated FOXD1 KD cells. Based on these findings, we conclude that FOXD1 might be a promising new diagnostic marker and a therapeutic target for the treatment of targeted therapy resistant melanoma.

T-type calcium channel inhibition restores sensitivity to MAPK inhibitors in de-differentiated and adaptive melanoma cells.

BACKGROUND: Drug resistance remains as one of the major challenges in melanoma therapy. It is well known that tumour cells undergo phenotypic switching during melanoma progression, increasing melanoma plasticity and resistance to mitogen-activated protein kinase inhibitors (MAPKi). METHODS: We investigated the melanoma phenotype switching using a partial reprogramming model to de-differentiate murine melanoma cells and target melanoma therapy adaptation against MAPKi. RESULTS: Here, we show that partially reprogrammed cells are a less proliferative and more de-differentiated cell population, expressing a gene signature for stemness and suppressing melanocyte-specific markers. To investigate adaptation to MAPKi, cells were exposed to B-Raf Proto-Oncogene (BRAF) and mitogen-activated protein kinase kinase (MEK) inhibitors. De-differentiated cells became less sensitive to MAPKi, showed increased cell viability and decreased apoptosis. Furthermore, T-type calcium channels expression increased in adaptive murine cells and in human adaptive melanoma cells. Treatment with the calcium channel blocker mibefradil induced cell death, differentiation and susceptibility to MAPKi in vitro and in vivo. CONCLUSION: In summary, we show that partial reprogramming of melanoma cells induces de-differentiation and adaptation to MAPKi. Moreover, we postulated a calcium channel blocker such as mibefradil, as a potential candidate to restore sensitivity to MAPKi in adaptive melanoma cells.

Histone methyltransferase SETDB1 contributes to melanoma tumorigenesis and serves as a new potential therapeutic target.

Alterations in histone modifications play a crucial role in the progression of various types of cancer. The histone methyltransferase SETDB1 catalyzes the addition of methyl groups to histone H3 at lysine 9. Here, we describe how overexpression of SETDB1 contributes to melanoma tumorigenesis. SETDB1 is highly amplified in melanoma cells and in the patient tumors. Increased expression of SETDB1, which correlates with SETDB1 amplification, is associated with a more aggressive phenotype in in vitro and in vivo studies. Mechanistically, SETDB1 implements its effects via regulation of thrombospondin 1, and the SET-domain of SETDB1 is essential for the maintenance of its tumorigenic activity. Inhibition of SETDB1 reduces cell growth in melanomas resistant to targeted treatments. Our results indicate that SETDB1 is a major driver of melanoma development and may serve as a potential future target for the treatment of this disease.

SOX2-mediated upregulation of CD24 promotes adaptive resistance toward targeted therapy in melanoma.

Melanoma is often characterized by a constitutively active RAS-RAF-MEK-ERK pathway. For targeted therapy, BRAF inhibitors are available that are powerful in the beginning but resistance occurs rather fast. A better understanding of the mechanisms of resistance is urgently needed to increase the success of the treatment. Here, we observed that SOX2 and CD24 are upregulated upon BRAF inhibitor treatment. A similar upregulation was seen in targeted therapy-resistant, melanoma-derived induced pluripotent cancer cells (iPCCs). SOX2 and CD24 are known to promote an undifferentiated and cancer stem cell-like phenotype associated with resistance. We, therefore, elucidated the role of SOX2 and CD24 in targeted therapy resistance in more detail. We found that the upregulation of SOX2 and CD24 required activation of STAT3 and that SOX2 induced the expression of CD24 by binding to its promoter. We find that the overexpression of SOX2 or CD24 significantly increases the resistance toward BRAF inhibitors, while SOX2 knock-down rendered cells more sensitivity toward treatment. The overexpression of CD24 or SOX2 induced Src and STAT3 activity. Importantly, by either CD24 knock-down or Src/STAT3 inhibition in resistant SOX2-overexpressing cells, the sensitivity toward BRAF inhibitors was re-established. Hence, we suggest a novel mechanism of adaptive resistance whereby BRAF inhibition is circumvented via the activation of STAT3, SOX2 and CD24. Thus, to prevent adaptive resistance, it might be beneficial to combine Src/STAT3 inhibitors together with MAPK pathway inhibitors.

Comparison of transcriptome profiles between medulloblastoma primary and recurrent tumors uncovers novel variance effects in relapses.

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 (< 10 years old), which were also associated with increased DNA aberrations and somatic mutations at relapse probably driving this effect. For Group 3/4 MB transcriptome data analysis uncovered clear sets of genes either active or decreased at relapse that are significantly associated with survival, thus could be potential predictive markers. In addition, deconvolution analysis of bulk transcriptome data identified progression-associated differences in cell type enrichment. The proportion of undifferentiated progenitors increased in SHH-MB relapses with a concomitant decrease of differentiated neuron-like cells, while in Group 3/4 MB relapses cell cycle activity increases and differentiated neuron-like cells proportion decreases as well. Thus, our findings uncovered significant transcriptome changes in the molecular signatures of relapsed MB and could be potentially useful for further clinical purposes.

Secretogranin II influences the assembly and function of MHC class I in melanoma.

Melanoma is the deadliest form of skin cancer showing rising incidence over the past years. New insights into the mechanisms of melanoma progression contributed to the development of novel treatment options, such as immunotherapies. However, acquiring resistance to treatment poses a big problem to therapy success. Therefore, understanding the mechanisms underlying resistance could improve therapy efficacy. Correlating expression levels in tissue samples of primary melanoma and metastases revealed that secretogranin 2 (SCG2) is highly expressed in advanced melanoma patients with poor overall survival (OS) rates. By conducting transcriptional analysis between SCG2-overexpressing (OE) and control melanoma cells, we detected a downregulation of components of the antigen presenting machinery (APM), which is important for the assembly of the MHC class I complex. Flow cytometry analysis revealed a downregulation of surface MHC class I expression on melanoma cells that showed resistance towards the cytotoxic activity of melanoma-specific T cells. IFNγ treatment partially reversed these effects. Based on our findings, we suggest that SCG2 might stimulate mechanisms of immune evasion and therefore be associated with resistance to checkpoint blockade and adoptive immunotherapy.

Atypical teratoid/rhabdoid tumoroids reveal subgroup-specific drug vulnerabilities.

Atypical teratoid/rhabdoid tumors (ATRTs) represent a rare, but aggressive pediatric brain tumor entity. They are genetically defined by alterations in the SWI/SNF chromatin remodeling complex members SMARCB1 or SMARCA4. ATRTs can be further classified in different molecular subgroups based on their epigenetic profiles. Although recent studies suggest that the different subgroups have distinct clinical features, subgroup-specific treatment regimens have not been developed thus far. This is hampered by the lack of pre-clinical in vitro models representative of the different molecular subgroups. Here, we describe the establishment of ATRT tumoroid models from the ATRT-MYC and ATRT-SHH subgroups. We demonstrate that ATRT tumoroids retain subgroup-specific epigenetic and gene expression profiles. High throughput drug screens on our ATRT tumoroids revealed distinct drug sensitivities between and within ATRT-MYC and ATRT-SHH subgroups. Whereas ATRT-MYC universally displayed high sensitivity to multi-targeted tyrosine kinase inhibitors, ATRT-SHH showed a more heterogeneous response with a subset showing high sensitivity to NOTCH inhibitors, which corresponded to high expression of NOTCH receptors. Our ATRT tumoroids represent the first pediatric brain tumor organoid model, providing a representative pre-clinical model which enables the development of subgroup-specific therapies.

Patient- and xenograft-derived organoids recapitulate pediatric brain tumor features and patient treatments.

Brain tumors are the leading cause of cancer-related death in children. Experimental in vitro models that faithfully capture the hallmarks and tumor heterogeneity of pediatric brain cancers are limited and hard to establish. We present a protocol that enables efficient generation, expansion, and biobanking of pediatric brain cancer organoids. Utilizing our protocol, we have established patient-derived organoids (PDOs) from ependymomas, medulloblastomas, low-grade glial tumors, and patient-derived xenograft organoids (PDXOs) from medulloblastoma xenografts. PDOs and PDXOs recapitulate histological features, DNA methylation profiles, and intratumor heterogeneity of the tumors from which they were derived. We also showed that PDOs can be xenografted. Most interestingly, when subjected to the same routinely applied therapeutic regimens, PDOs respond similarly to the patients. Taken together, our study highlights the potential of PDOs and PDXOs for research and translational applications for personalized medicine.

SMARCB1-deficient and SMARCA4-deficient Malignant Brain Tumors With Complex Copy Number Alterations and TP53 Mutations May Represent the First Clinical Manifestation of Li-Fraumeni Syndrome.

Atypical teratoid/rhabdoid tumor (AT/RT) is a malignant central nervous system tumor predominantly affecting infants. Mutations of SMARCB1 or (rarely) SMARCA4 causing loss of nuclear SMARCB1 or SMARCA4 protein expression are characteristic features, but further recurrent genetic alterations are lacking. Most AT/RTs occur de novo, but secondary AT/RTs arising from other central nervous system tumors have been reported. Malignant gliomas, IDH wild-type, arising in patients with Li-Fraumeni syndrome typically show somatic mutations of TP53 as well as complex copy number alterations, but little is known about the loss of SMARCB1 or SMARCA4 protein expression in this context. Here, we report 2 children in whom malignant supratentorial brain tumors with SMARCB1 deficiency, complex copy number alterations, and somatic TP53 mutations lead to the discovery of pathogenic/likely pathogenic TP53 variants in the germline. Screening of the molecularneuropathology.org dataset for cases with similar genetic and epigenetic alterations yielded another case with SMARCA4 deficiency in a young adult with Li-Fraumeni syndrome. In conclusion, SMARCB1-deficient or SMARCA4-deficient malignant brain tumors with complex copy number alterations and somatic TP53 mutations in children and young adults may represent the first clinical manifestation of Li-Fraumeni syndrome and should prompt genetic counseling and investigation for TP53 germline status.

The Effect of Weaning with Adult Food Typical of the Mediterranean Diet on Taste Development and Eating Habits of Children: A Randomized Trial.

Mediterranean Diet (Med Diet) is one of the healthiest dietary patterns. We aimed to verify the effects of weaning (i.e., the introduction of solid foods in infants previously fed only with milk) using adult foods typical of Med Diet on children eating habits, and on the microbiota composition. A randomized controlled clinical trial on 394 healthy infants randomized in a 1:1 ratio in a Med Diet group weaned with fresh; seasonal and tasty foods of Med Diet and control group predominantly weaned with industrial baby foods. The primary end point was the percentage of children showing a good adherence to Med Diet at 36 months. Secondary end points were mother's changes in adherence to Med Diet and differences in children gut microbiota. At 36 months, children showing a good adherence to Med Diet were 59.3% in the Med Diet group and 34.3% in the control group (p < 0.001). An increase in adherence to the Med Diet was observed in the mothers of the Med Diet group children (p < 0.001). At 4 years of age children in the Med Diet group had a higher gut microbial diversity and a higher abundance of beneficial taxa. A Mediterranean weaning with adult food may become a strategy for early nutritional education, to develop a healthy microbiota, to prevent inflammatory chronic diseases and to ameliorate eating habits in children and their families.

Defining the molecular features of radiation-induced glioma: A systematic review and meta-analysis.

BACKGROUND: Cranial radiation therapy is essential in treating many pediatric cancers, especially brain tumors; however, its use comes with the risk of developing second malignancies. Cranial radiation-induced gliomas (RIGs) are aggressive high-grade tumors with a dismal prognosis, for which no standard therapy exists. A definitive molecular signature for RIGs has not yet been established. We sought to address this gap by performing a systematic review and meta-analysis of the molecular features of cranial RIGs. METHODS: A systematic review of the literature was performed according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. Articles and case reports that described molecular analyses of cranial radiation-induced high-grade gliomas were identified and evaluated, and data extracted for collation. RESULTS: Of 1727 records identified, 31 were eligible, containing 102 unique RIGs with molecular data. The most frequent genetic alterations in RIGs included PDGFRA or TP53 mutations, PDGFRA or CDK4 amplifications, and CDKN2A deletion, along with 1q gain, 1p loss and 13q loss. Of note, mutations in ACVR1, EGFR, H3F3A, HIST1H3B, HIST1H3C, IDH2, SMARCB1 or the TERT promoter were not observed. A comparative analysis revealed that RIGs are molecularly distinct from most other astrocytomas and gliomas and instead align most closely with the pedGBM_RTK1 subgroup of pediatric glioblastoma. CONCLUSIONS: This comprehensive analysis highlights the major molecular features of RIGs, demonstrates their molecular distinction from many other astrocytomas and gliomas, and reveals potential genetic drivers and therapeutic targets for this currently fatal disease.

Atypical Teratoid/Rhabdoid Tumor (AT/RT) With Molecular Features of Pleomorphic Xanthoastrocytoma.

Atypical teratoid/rhabdoid tumor (AT/RT) is a highly malignant central nervous system tumor predominantly occurring in infants that may also arise in older children and adults. Rare secondary AT/RT developing from other tumors such as pleomorphic xanthoastrocytoma (PXA) are on record, but AT/RT presenting with molecular features of PXA have not been described. Here, we report 3 malignant central nervous system tumors in children (10, 13, and 18 y old). All tumors were located in the temporal lobe. In 2 cases, there was no history of a low-grade precursor lesion; in 1 case anaplastic PXA had been diagnosed 3 months earlier. Histopathologically, all tumors were composed of RT cells and showed frank signs of malignancy as well as loss of nuclear SMARCB1/INI1 protein expression. Two cases displayed homozygous deletions of the SMARCB1 region while the third case showed an exon 7 mutation (c.849_850delGT; p.Met283Ilefs*77). Of note, DNA methylation profiles did not group with AT/RT or other tumor entities using the Heidelberg Brain Tumor Classifier (version v11b4). By unsupervised t-distributed stochastic neighbor embedding analysis and hierarchical clustering analysis, however, all tumors clearly grouped with PXA. Genome-wide copy number analysis revealed homozygous CDNK2A/B deletions and gains of whole chromosome 7. BRAF V600E mutations could be demonstrated in all cases. In conclusion, the possibility of AT/RT with molecular features of PXA needs to be taken into account and warrants molecular characterization of AT/RT especially in older children. Since treatments targeting mutated BRAF are available, identification of such cases may also have therapeutic consequences.

Mithramycin A and Mithralog EC-8042 Inhibit SETDB1 Expression and Its Oncogenic Activity in Malignant Melanoma.

Malignant melanoma is the most deadly skin cancer, associated with rising incidence and mortality rates. Most of the patients with melanoma, treated with current targeted therapies, develop a drug resistance, causing tumor relapse. The attainment of a better understanding of novel cancer-promoting molecular mechanisms driving melanoma progression is essential for the development of more effective targeted therapeutic approaches. Recent studies, including the research previously conducted in our laboratory, reported that the histone methyltransferase SETDB1 contributes to melanoma pathogenesis. In this follow-up study, we further elucidated the role of SETDB1 in melanoma, showing that SETDB1 modulated relevant transcriptomic effects in melanoma, in particular, as activator of cancer-related secreted (CRS) factors and as repressor of melanocyte-lineage differentiation (MLD) and metabolic enzymes. Next, we investigated the effects of SETDB1 inhibition via compounds belonging to the mithramycin family, mithramycin A and mithramycin analog (mithralog) EC-8042: melanoma cells showed strong sensitivity to these drugs, which effectively suppressed the expression of SETDB1 and induced changes at the transcriptomic, morphological, and functional level. Moreover, SETDB1 inhibitors enhanced the efficacy of mitogen-activated protein kinase (MAPK) inhibitor-based therapies against melanoma. Taken together, this work highlights the key regulatory role of SETDB1 in melanoma and supports the development of SETDB1-targeting therapeutic strategies for the treatment of melanoma patients.

HER3-Receptor-Mediated STAT3 Activation Plays a Central Role in Adaptive Resistance toward Vemurafenib in Melanoma.

Melanoma is an aggressive form of skin cancer that is often characterized by activating mutations in the Mitogen-Activated Protein (MAP) kinase pathway, causing hyperproliferation of the cancer cells. Thus, inhibitors targeting this pathway were developed. These inhibitors are initially very effective, but the occurrence of resistance eventually leads to a failure of the therapy and is the major obstacle for clinical success. Therefore, investigating the mechanisms causing resistance and discovering ways to overcome them is essential for the success of therapy. Here, we observed that treatment of melanoma cells with the B-Raf Proto-Oncogene, Serine/Threonine Kinase (BRAF) inhibitor vemurafenib caused an increased cell surface expression and activation of human epidermal growth factor receptor 3 (HER3) by shed ligands. HER3 promoted the activation of signal transducer and activator of transcription 3 (STAT3) resulting in upregulation of the STAT3 target gene SRY-Box Transcription Factor 2 (SOX2) and survival of the cancer cells. Pharmacological blocking of HER led to a diminished STAT3 activation and increased sensitivity toward vemurafenib. Moreover, HER blocking sensitized vemurafenib-resistant cells to drug treatment. We conclude that the inhibition of the STAT3 upstream regulator HER might help to overcome melanoma therapy resistance toward targeted therapies.