Medulloblastoma (MB) is the most common malignant primary brain tumor of childhood. High risk patients still have a poor outcome, and especially young patients suffer from standard therapy induced ...sequelae. Therefore, other therapeutic options need to be explored. In glioblastoma (GBM), application of 5-aminolaevulinic acid (5-ALA) results in selective accumulation of protoporphyrin IX (PPIX) in the tumor cells, which can be exploited during fluorescence-guided surgery to increase the extent of resection or for photodynamic therapy (PDT) induced phototoxicity. It is not entirely clear, whether MB cells accumulate PPIX and are sensitive to PDT.
Human MYC-amplified (Med8A and D283) and non-amplified (UW228–2 and ONS76) MB cell lines were incubated for 2, 4 or 6 h with increasing doses (0–100 μg/ml) of 5-ALA, and PPIX accumulation was determined by flow cytometry. To assess sensitivity to 5-ALA/PDT, cells were incubated with 5-ALA and subsequently exposed to laser light of 635 nm wavelength (18.75 J/cm2). After an additional 24 h culture period, viability of cells was quantified using the WST-1 assay. Expression of ferrochelatase was detected by reverse transcription and quantitative polymerase chain reaction. Ferrochelatase activity was quantified by measuring the enzymatic conversion of PPIX to zinc-protoporphyrin. Expression of the ABCG2 transporter protein CD338 was detected by flow cytometry.
All MB cell lines showed a time- and dose-dependent accumulation of PPIX after exposure to exogenous 5-ALA and became sensitive to 5-ALA/PDT-induced phototoxicity. PPIX accumulation was reduced compared to U373 GBM cells at shorter incubation periods and limiting 5-ALA doses. Moreover, not all MB cells became PPIX positive and overall phototoxicity was lower in the MB cell lines. Notably, the MYC-amplified MB cells demonstrated a more pronounced photosensitivity compared to their non-amplified counterparts. There was no difference in expression of ferrochelatase, but enzymatic activity appeared to be reduced in the MB cells compared to U373 GBM cells, whereas CD338 was expressed on the MB cells only.
Medulloblastoma cell lines accumulate PPIX after application of 5-ALA and become sensitive to PDT, associated with low ferrochelatase expression and activity. Photosensitivity is more pronounced in MYC-amplified cell lines. In contrast to GBM cells, however, PPIX accumulation appears to be reduced, restricted to a subset of cells and associated with lower photosensitivity of the MB cell lines, possibly due to expression of the ABCG2 transporter protein CD338 on MB cells.
Abstract
INTRODUCTION
Pilocytic astrocytomas (PA) are the most common pediatric brain tumors. They are characterized by MAPK pathway alterations, leading to its constitutive activation and modulating ...the balance between proliferation and oncogene-induced senescence (OIS) sustained by senescence-associated secretory phenotype (SASP). Little is known about the molecular implications of MAPK pathway inhibition in the proliferating and senescent tumor compartments.
METHODS
DKFZ-BT66 cells derived from a primary KIAA:BRAF-fusion positive PA cell line and BT40 cells derived from pleomorphic xanthoastrocytoma with a BRAFV600E mutation and CDKN2A/B deletion, were used as model systems. RNA-sequencing and phospho-/proteomic datasets were generated in both the proliferative and senescent cells, and treated with the MEKi trametinib for different time-spans. A multi-omics factor analysis tool (MEFISTO) was used to identify key OIS/proliferation effectors.
RESULTS
Differential gene expression analysis revealed that MEK inhibition leads to the inhibition of the OIS/SASP gene program in senescent DKFZ-BT66. In addition, the protein level of several SASP factors was decreased. This translated in reduced sensitivity towards senolytics drugs, indicating inhibition of senescence features upon MEKi. MEFISTO analysis allowed to identify key transcription factors, genes and proteins involved in MAPK-induced OIS in the senescent PA cells, that were mapped using a prior knowledge network approach. Finally, single sample geneset enrichment analysis showed that most MAPK-related signatures were downregulated upon MEKi treatment, while pathways related to upstream MAPK activators (including several RTK pathways) were predicted to be upregulated, in both proliferating and senescent cells.
CONCLUSION
This data suggests that MAPKi reverses OIS in senescent PA cells, while inducing the activation of MAPK upstream regulators, identifying putative co-targets for the treatment of PA. Further validation of the targetability of these pathways is pending. Furthermore, the identification of the MAPK-related OIS/SASP genes provide insight about the regulation of OIS/SASP by the MAPK pathway.
Embryonal tumors with multilayered rosettes (ETMRs) are rare, deadly pediatric brain tumors characterized by high-level amplification of the microRNA cluster C19MC. We performed integrated genetic ...and epigenetic analyses of 12 ETMR samples and identified, in all cases, C19MC fusions to TTYH1 driving expression of the microRNAs. ETMR tumors, cell lines and xenografts showed a specific DNA methylation pattern distinct from those of other tumors and normal tissues. We detected extreme overexpression of a previously uncharacterized isoform of DNMT3B originating at an alternative promoter that is active only in the first weeks of neural tube development. Transcriptional and immunohistochemical analyses suggest that C19MC-dependent DNMT3B deregulation is mediated by RBL2, a known repressor of DNMT3B. Transfection with individual C19MC microRNAs resulted in DNMT3B upregulation and RBL2 downregulation in cultured cells. Our data suggest a potential oncogenic re-engagement of an early developmental program in ETMR via epigenetic alteration mediated by an embryonic, brain-specific DNMT3B isoform.
Postoperative residual tumor can occur for intentional or unintentional reasons. Decision-making regarding second-look surgery has to weigh molecular biology, probability of total resection and ...prognostic relevance against potential additional morbidity. In interdisciplinary tumor boards the neurosurgeon has to estimate risk and efficacy of second-look surgery in individual cases, based on precise data.
Aim of this study was to provide such data by analyzing morbidity and volumetric efficacy of second-look surgery at a designated pediatric neuro-oncology unit.
Children who received second-look surgery in 2007–2018 after incomplete resections were analyzed retrospectively. Measurements were performed on early postoperative magnetic resonance imaging, comparing axial diameter-based measurement as well as computer-assisted volumetric analysis.
59 patients (37% of the overall cohort; 21 female; mean age: 8 ± 5 years) received a subtotal (n = 35) or near total (n = 24) resection. After interdisciplinary case review, 12 of these patients received second-look surgery mainly for residual ependymoma. This led to further tumor volume reduction in all cases (new degrees of resection: subtotal = 2, near total = 6, gross total = 4). No new permanent morbidity or perioperative mortality was observed.
Second-look surgery did not increase mortality and permanent morbidity, had an 8% rate of transient morbidity and achieved tumor volume reduction above 95% in 75% of selected cases, with 4 additional gross total resections. Second-look surgery is safe and effective with regard to volumetric outcome parameters even in cases with good initial resections, although the role of second-look surgery regarding oncological outcome has to be further investigated in times of personalized molecular medicine.
•Second-look surgery after incomplete initial resection of a pediatric brain tumor does not increase mortality and permanent surgical morbidity.•It achieves a reduction of tumor volume above 95% in 75% of selected cases, with 4 additional gross total resections per 12 patients undergoing second-look surgery.•Irrespective of two-dimensional or three-dimensional measurement methodology, criteria for near total resection correspond well and consistently showed an extent of resection above 95%.•In the era of molecular and personalized medicine, children with specific tumors and molecular biology (e.g. PF-EPN-A ependymoma or group 4 medulloblastoma) might be candidates for second-look surgery after interdisciplinary review. This study gives the neurosurgeon accessible information to precisely characterize the neurosurgical implications of second-look surgery in such tumor board discussions.
Brain metastases represent the most common intracranial tumors in adults and are associated with a poor prognosis. We used a personalized in vitro drug screening approach to characterize individual ...therapeutic vulnerabilities in brain metastases.PURPOSEBrain metastases represent the most common intracranial tumors in adults and are associated with a poor prognosis. We used a personalized in vitro drug screening approach to characterize individual therapeutic vulnerabilities in brain metastases.Short-term cultures of cancer cells isolated from brain metastasis patients were molecularly characterized using next-generation sequencing and functionally evaluated using high-throughput in vitro drug screening to characterize pharmacological treatment sensitivities.METHODSShort-term cultures of cancer cells isolated from brain metastasis patients were molecularly characterized using next-generation sequencing and functionally evaluated using high-throughput in vitro drug screening to characterize pharmacological treatment sensitivities.Next-generation sequencing identified matched genetic alterations in brain metastasis tissue samples and corresponding short-term cultures, suggesting that short-term cultures of brain metastases are suitable models for recapitulating the genetic profile of brain metastases that may determine their sensitivity to anti-cancer drugs. Employing a high-throughput in vitro drug screening platform, we successfully screened the cultures of five brain metastases for response to 267 anticancer compounds and related drug response to genetic data. Among others, we found that targeted treatment with JAK3, HER2, or FGFR3 inhibitors showed anti-cancer effects in individual brain metastasis cultures.RESULTSNext-generation sequencing identified matched genetic alterations in brain metastasis tissue samples and corresponding short-term cultures, suggesting that short-term cultures of brain metastases are suitable models for recapitulating the genetic profile of brain metastases that may determine their sensitivity to anti-cancer drugs. Employing a high-throughput in vitro drug screening platform, we successfully screened the cultures of five brain metastases for response to 267 anticancer compounds and related drug response to genetic data. Among others, we found that targeted treatment with JAK3, HER2, or FGFR3 inhibitors showed anti-cancer effects in individual brain metastasis cultures.Our preclinical study provides a proof-of-concept for combining molecular profiling with in vitro drug screening for predictive evaluation of therapeutic vulnerabilities in brain metastasis patients. This approach could advance the use of patient-derived cancer cells in clinical practice and might eventually facilitate decision-making for personalized drug treatment.CONCLUSIONOur preclinical study provides a proof-of-concept for combining molecular profiling with in vitro drug screening for predictive evaluation of therapeutic vulnerabilities in brain metastasis patients. This approach could advance the use of patient-derived cancer cells in clinical practice and might eventually facilitate decision-making for personalized drug treatment.
While major advances have been made in improving the quality of life and survival of children with most forms of medulloblastoma (MB), those with MYC-driven tumors (Grp3-MB) still suffer significant ...morbidity and mortality. There is an urgent need to explore multimodal therapeutic regimens which are effective and safe for children. Large-scale studies have revealed abnormal cancer epigenomes caused by mutations and structural alterations of chromatin modifiers, aberrant DNA methylation, and histone modification signatures. Therefore, targeting epigenetic modifiers for cancer treatment has gained increasing interest, and inhibitors for various epigenetic modulators have been intensively studied in clinical trials. Here, we report a cross-entity, epigenetic drug screen to evaluate therapeutic vulnerabilities in MYC amplified MB, which sensitizes them to macrophage-mediated phagocytosis by targeting the CD47-signal regulatory protein α (SIRPα) innate checkpoint pathway.
We performed a primary screen including 78 epigenetic inhibitors and a secondary screen including 20 histone deacetylase inhibitors (HDACi) to compare response profiles in atypical teratoid/rhabdoid tumor (AT/RT, n=11), MB (n=14), and glioblastoma (n=14). This unbiased approach revealed the preferential activity of HDACi in MYC-driven MB. Importantly, the class I selective HDACi, CI-994, showed significant cell viability reduction mediated by induction of apoptosis in MYC-driven MB, with little-to-no activity in non-MYC-driven MB, AT/RT, and glioblastoma in vitro. We tested the combinatorial effect of targeting class I HDACs and the CD47-SIRPa phagocytosis checkpoint pathway using in vitro phagocytosis assays and in vivo orthotopic xenograft models.
CI-994 displayed antitumoral effects at the primary site and the metastatic compartment in two orthotopic mouse models of MYC-driven MB. Furthermore, RNA sequencing revealed nuclear factor-kB (NF-κB) pathway induction as a response to CI-994 treatment, followed by transglutaminase 2 (TGM2) expression, which enhanced inflammatory cytokine secretion. We further show interferon-γ release and cell surface expression of engulfment ('eat-me') signals (such as calreticulin). Finally, combining CI-994 treatment with an anti-CD47 mAb targeting the CD47-SIRPα phagocytosis checkpoint enhanced in vitro phagocytosis and survival in tumor-bearing mice.
Together, these findings suggest a dynamic relationship between MYC amplification and innate immune suppression in MYC amplified MB and support further investigation of phagocytosis modulation as a strategy to enhance cancer immunotherapy responses.
Focal high-level amplifications of
MYC
(
or MYCC
) define a subset of high-risk medulloblastoma patients. However, the prognostic role of
MYCN
oncogene amplification remains unresolved. We aimed to ...evaluate the prognostic value of this alteration alone and in combination with biological modifiers in 67 pediatric medulloblastomas with
MYCN
amplification (
MYCN
-MB). Twenty-one
MYCN
-MB were examined using gene expression profiling and array-CGH, whereas for 46 tumors immunohistochemical analysis and FISH were performed. All 67 tumors were further subjected to mutational analyses. We compared molecular, clinical, and prognostic characteristics both within biological
MYCN
-MB groups and with non-amplified tumors. Transcriptomic analysis revealed SHH-driven tumorigenesis in a subset of
MYCN
-MBs indicating a biological dichotomy of
MYCN
-MB. Activation of SHH was accompanied by variant-specific cytogenetic aberrations including deletion of 9q in SHH tumors. Non-SHH MB were associated with gain of 7q and isochromosome 17q/17q gain. Among clinically relevant variables, SHH subtype and 10q loss for non-SHH tumors comprised the most powerful markers of favorable prognosis in
MYCN
-MB. In conclusion, we demonstrate considerable heterogeneity within
MYCN
-MB in terms of genetics, tumor biology, and clinical outcome. Thus, assessment of disease group and 10q copy-number status may improve risk stratification of this group and may delineate
MYCN
-MB with the same dismal prognosis as
MYC
amplified tumors. Furthermore, based on the enrichment of
MYCN
and
GLI2
amplifications in SHH-driven medulloblastoma, amplification of these downstream signaling intermediates should be taken into account before a patient is enrolled into a clinical trial using a smoothened inhibitor.
The synthesis and biological evaluation of potent hydroxamate-based dual HDAC1/6 inhibitors with modest HDAC6 preference and a novel alkoxyurea connecting unit linker region are described. The ...biological studies included the evaluation of antiproliferative effects and HDAC inhibitory activity in the human ovarian cancer cell line A2780, the human squamous carcinoma cell line Cal27, and their cisplatin resistant sublines A2780CisR and Cal27CisR. The three most potent compounds 1g–i showed IC50 values in the low μM and sub-μM range. 1g–i revealed low nM IC50 values for HDAC6 with up to 15-fold preference over HDAC1, >3500-fold selectivity over HDAC4, and >100-fold selectivity over HDAC8. Furthermore, their ability to enhance cisplatin sensitivity was analyzed in Cal27 and Cal27CisR cells. Notably, a 48 h preincubation of 1g–i significantly enhanced the antiproliferative effects of cisplatin in Cal27 and Cal27CisR. 1g–i interacted synergistically with cisplatin. These effects were more pronounced for the cisplatin resistant subline Cal27CisR.
Abstract
Pilocytic astrocytomas (PA) are the most common pediatric brain tumors. They are characterized by driving alterations in the mitogen-activated protein kinase (MAPK) pathway, leading to its ...constitutive activation and modulating the balance between cell proliferation and oncogene-induced senescence (OIS) sustained by senescence-associated secretory phenotype (SASP) factors. This makes PA susceptible to MAPK inhibitor (MAPKi) therapies, which show encouraging results in phase 1/2 clinical trials. However, little is known about the molecular implications of MAPK inhibition in PA. The DKFZ-BT66 cell line, derived from a primary KIAA:BRAF-fusion positive PA, was used as a model system. DKFZ-BT66 were treated with the MEKi trametinib for different durations in both proliferative and senescent states. Gene expression was analyzed by gene expression profiling and protein expression/phospho-regulation by data-dependent mass spectrometry followed by label-free quantitative analysis. A time course analysis based on differentially expressed genes and phosphorylated proteins was performed, followed by a single-sample gene set enrichment analysis (ssGSEA) and kinase substrate enrichment analysis, respectively. Differential gene expression analysis revealed that MEK inhibition led to the inhibition of the OIS/SASP gene programs in senescent DKFZ-BT66, with downregulation of key OIS/SASP partners such as IL1B on the protein level. This functionally translated into a de-sensitization of these cells towards the senolytic agent navitoclax. ssGSEA showed that most MAPK-related signatures were downregulated upon MEKi treatment, while pathways related to upstream MAPK activators (including FGFR, NTRK and TGFB pathways) were upregulated, in both proliferating and senescent DKFZ-BT66. This data indicates that MAPKi reverses OIS in senescent PA cells, while inducing the activation of MAPK upstream regulators in proliferating and senescent PA cells, identifying putative co-targets that could help increase treatment’s efficacy. Validation of these targets by post-translational modification enrichment analysis of the phospho-proteomics dataset is ongoing.
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