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Background Intratumoral heterogeneity is crucially involved in metastasis, resistance to therapy, and cancer relapse. Amplifications of the proto-oncogene MYC display notable heterogeneity at the single-cell level and are associated with a particularly dismal prognosis in high-risk medulloblastomas (MBs). The aim of this study was to establish the relevance of interclonal cross-talk between MYC-driven and non-MYC-driven MB cells. Methods We used fluorescence in situ hybridization, single-cell transcriptomics, and immunohistochemistry, in vitro isogenic cell models, non-targeted proteomics, mass spectrometry-based metabolite quantification, HUVECs tube formation assay, and orthotopic in vivo experiments to investigate interclonal cross-talk in MB. Results We found that the release of lactate dehydrogenase A (LDHA) from MYC-driven cells facilitates metastatic seeding and outgrowth, while secretion of dickkopf WNT signaling pathway inhibitor 3 from non-MYC-driven cells promotes tumor angiogenesis. This tumor-supporting interaction between both subclones was abrogated by targeting the secretome through pharmacological and genetic inhibition of LDHA, which significantly suppressed tumor cell migration. Conclusion Our study reveals the functional relevance of clonal diversity and highlights the therapeutic potential of targeting the secretome to interrupt interclonal communication and progression in high-risk MB.

BackgroundWhile 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.MethodsWe 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.ResultsCI-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.ConclusionTogether, 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.

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Introduction Resistance to chemotherapy is a common cause of treatment failure in cancer patients and a major problem facing current cancer research. Targeted modulation of oncogenic signaling pathways may be used to systematically characterize drug resistance mechanisms across tumor entities and may help to identify new therapeutic strategies. Since the transcription factor MYC is aberrantly activated in many cancers including pediatric malignant brain tumors, like medulloblastoma (MB), our study focused on MYC-related drug resistance. Methods and Results We performed high-throughput drug screening using our in-house semi-automated platform and identified the HDAC inhibitor Entinostat as a drug that shows promising effects in MYC-driven MB. Investigating genome-wide dCas9-based transcriptional activation screening, potential drug response modulators, mainly TGFB1/Erk/MKNK1 signaling including neural EGFL like 2 (NELL2), were discovered. For further validation, we stably overexpressed NELL2 in MYC-driven MB cells and treated overexpressing cells and the corresponding control cells with Entinostat. Using PI staining, cell cycle status was tracked. Entinostat treatment led to modest induction of cell death in MYC-driven MB control cells but only slightly increased cell death rate in MYC-driven MB cells with NELL2-overexpression. Conclusion We report that the combination of genetic and pharmacological approaches is a powerful approach to study drug resistance. Our data suggest that activation of the TGFB1/Erk/MKNK1 signaling pathway desensitizes MYC-driven MB cells to Entinostat. Synergistic targeting of TGFB1/Erk/MKNK1 signaling and MYC could therefore provide a novel therapeutic option in this aggressive MB subtype.

Atypical teratoid/rhabdoid tumors (AT/RT) are among the most common malignant brain tumors in infants. Comprehensive genomic studies revealed three distinct molecular subgroups (AT/RT-TYR, -MYC and -SHH). As primary cilia (PC) have already been shown to play a pivotal role in other tumor entities, we aimed to characterize the distribution of PC across AT/RT subgroups and to target primary ciliogenesis in these tumors with dismal prognosis. We performed immunofluorescence to detect PC in AT/RT primary tumor sections and cell lines. The functional role of PC was investigated in vitro by knockdown of KIF3A. The relevance of primary ciliogenesis in AT/RT biology was further elucidated in vivo using a fly model of SMARCB1 deficiency and an orthotopic mouse model. We detected PC in all AT/RT primary tumor sections and cell lines investigated in this study. Notably, we observed a significant subgroup-specific difference in the proportion of PC-positive cells. Specifically, AT/RT-TYR demonstrated the highest percentage of ciliated cells. Knockdown of KIF3A significantly reduced cell proliferation and stem cell properties. Additionally, apoptosis was significantly increased via upregulation of JAK1/STAT1 signaling. In a fly model of SMARCB1 deficiency, concomitant knockdown of several cilia-associated genes resulted in a substantial shift of the lethal phenotype with >20% of flies reaching adulthood. Finally, we demonstrated a significantly prolonged survival in an orthotopic xenograft model upon knockdown of KIF3A. In conclusion, our results implicate PC as a key feature of AT/RT biology and suggest primary ciliogenesis as a potential therapeutic target, especially in AT/RT-TYR.

Atypical teratoid/rhabdoid tumors (AT/RT) are among the most common malignant brain tumors in infants. Comprehensive genomic studies revealed three distinct molecular subgroups (AT/RT-TYR, -MYC and -SHH). As primary cilia (PC) have already been shown to play a central role in other tumor entities, we aimed to characterize the distribution of PC across AT/RT subgroups and to target primary ciliogenesis in these tumors with dismal prognosis. We performed immunofluorescence to detect PC in AT/RT cell lines (n=2) and primary tumor sections (n=13). The functional role of PC was investigated by knockdown of KIF3A and pharmacological inhibition with Ciliobrevin D in vitro. The relevance of primary ciliogenesis in AT/RT biology was further elucidated using a fly model of SMARCB1 deficiency. We detected PC in both AT/RT cell lines and all primary tumors investigated in this study. Notably, we observed a significant subgroup-specific difference in the proportion of PC-positive cells. Specifically, AT/RT-TYR demonstrated the highest percentage of ciliated cells. Knockdown of KIF3A significantly reduced cell proliferation and stem cell properties. Additionally, death receptor 5 signaling was significantly up-regulated. Pharmacological inhibition by Ciliobrevin D phenocopied these results, specifically after induction of primary ciliogenesis through serum starvation. In a fly model of SMARCB1 deficiency, concomitant knockdown of several cilia-associated genes resulted in a substantial shift of the lethal phenotype with >20% of flies reaching adulthood. In conclusion, these results implicate PC as a key feature of AT/RT biology and suggest primary ciliogenesis as a potential therapeutic target, especially in AT/RT-TYR.