Targeting oncogenic pathways holds promise for brain tumor treatment, but inhibition of Sonic Hedgehog (SHH) signaling has failed in SHH-driven medulloblastoma. Cellular diversity within tumors and reduced lineage commitment can undermine targeted therapy by increasing the probability of treatment-resistant populations. Using single-cell RNA-seq and lineage tracing, we analyzed cellular diversity in medulloblastomas in transgenic, medulloblastoma-prone mice, and responses to the SHH-pathway inhibitor vismodegib. In untreated tumors, we find expected stromal cells and tumor-derived cells showing either a spectrum of neural progenitor-differentiation states or glial and stem cell markers. Vismodegib reduces the proliferative population and increases differentiation. However, specific cell types in vismodegib-treated tumors remain proliferative, showing either persistent SHH-pathway activation or stem cell characteristics. Our data show that even in tumors with a single pathway-activating mutation, diverse mechanisms drive tumor growth. This diversity confers early resistance to targeted inhibitor therapy, demonstrating the need to target multiple pathways simultaneously.
Background Medulloblastoma (MB) is a heterogeneous disease in which neoplastic cells and associated immune cells contribute to disease progression. We aimed to determine the influence of neoplastic and immune cell diversity on MB biology in patient samples and animal models. Methods To better characterize cellular heterogeneity in MB we used single-cell RNA sequencing, immunohistochemistry and deconvolution of transcriptomic data to profile neoplastic and immune populations in patient samples and animal models across childhood MB subgroups. Results Neoplastic cells cluster primarily according to individual sample of origin which is influenced by chromosomal copy number variance. Harmony alignment reveals novel MB subgroup/subtype-associated subpopulations that recapitulate neurodevelopmental processes, including photoreceptor and glutamatergic neuron-like cells in molecular subgroups GP3 and GP4, and a specific nodule-associated neuronally-differentiated subpopulation in subgroup molecular SHH. We definitively chart the spectrum of MB immune cell infiltrates, which include subpopulations that recapitulate developmentally-related neuron-pruning and antigen presenting myeloid cells. MB cellular diversity matching human samples is mirrored in subgroup-specific mouse models of MB. Conclusions These findings provide a clearer understanding of the diverse neoplastic and immune cell subpopulations that constitute the MB microenvironment.
Cancer-derived loss-of-function mutations in the KEAP1 tumor suppressor gene stabilize the NRF2 transcription factor, resulting in a pro-survival gene expression program that alters cellular metabolism and neutralizes oxidative stress. In a recent genotype-phenotype study, we classified 40% of KEAP1 mutations as ANCHOR mutants. By immunoprecipitation, these mutants bind more NRF2 than wild-type KEAP1 and ubiquitylate NRF2, but are incapable of promoting NRF2 degradation. BioID-based protein interaction studies confirmed increased abundance of NRF2 within the KEAP1 ANCHOR mutant complexes with no other statistically significant changes to the complexes. Discrete molecular dynamic simulation modeling and limited proteolysis suggest that the ANCHOR mutations stabilize residues in KEAP1 that contact NRF2. The modeling supports an intramolecular salt bridge between the R470C ANCHOR mutation and E493; mutation of the E493 residue confirmed the model, resulting in the ANCHOR phenotype. In live cells, the KEAP1 R320Q and R470C ANCHOR mutants co-localize with NRF2, p62/SQSTM1, and polyubiquitin in structured spherical droplets that rapidly fuse and dissolve. Transmission electron microscopy coupled with confocal fluorescent imaging revealed membraneless phase-separated biomolecular condensates. We present a model wherein ANCHOR mutations form p62-dependent biomolecular condensates that may represent a transitional state between impaired proteasomal degradation and autophagy.
Medulloblastoma (MB) is a heterogeneous disease in which neoplastic cells and associated immune cells contribute to disease progression. To better understand cellular heterogeneity in MB we profile neoplastic and immune populations in childhood MB samples using single-cell RNA sequencing, immunohistochemistry and deconvolution of transcriptomic data. Neoplastic cells cluster primarily according to individual sample of origin which is in part due to the effect of chromosomal copy number gains and losses. Harmony alignment reveals novel MB subgroup/subtype-associated subpopulations that recapitulate neurodevelopmental processes and are associated with clinical outcomes. We identify discrete photoreceptor-like cells in MB subgroups GP3 and GP4 and nodule-associated neuronally-differentiated cells in subgroup SHH. MB immune infiltrates consist of both developmentally-related neuron-pruning and antigen presenting myeloid cells. We show that this MB cellular diversity is recapitulated in genetically engineered mouse subgroup-specific models of MB. These findings advance our understanding of both the neoplastic and immune landscape of MB.
It is unclear why medulloblastoma patients receiving similar treatments experience different outcomes. Transcriptomic profiling identified subgroups with different prognoses, but in each subgroup, individuals remain at risk of incurable recurrence. To investigate why similar-appearing tumors produce variable outcomes, we analyzed medulloblastomas triggered in transgenic mice by a common driver mutation expressed at different points in brain development. We genetically engineered mice to express oncogenic SmoM2, starting in multipotent glio-neuronal stem cells, or committed neural progenitors. Both groups developed medulloblastomas with similar transcriptomic profiles. We compared medulloblastoma progression, radiosensitivity, and cellular heterogeneity, determined by single-cell transcriptomic analysis (scRNA-seq). Stem cell-triggered medulloblastomas progressed faster, contained more OLIG2-expressing stem-like cells, and consistently showed radioresistance. In contrast, progenitor-triggered MBs progressed slower, down-regulated stem-like cells and were curable with radiation. Progenitor-triggered medulloblastomas also contained more diverse stromal populations, with more Ccr2+ macrophages and fewer Igf1+ microglia, indicating that developmental events affected the subsequent tumor microenvironment. Reduced mTORC1 activity in M-Smo tumors suggests that differential Igf1 contributed to differences in phenotype. Developmental events in tumorigenesis that were obscure in transcriptomic profiles thus remained cryptic determinants of tumor composition and outcome. Precise understanding of medulloblastoma pathogenesis and prognosis requires supplementing transcriptomic/methylomic studies with analyses that resolve cellular heterogeneity.
Patients with medulloblastoma are typically treated with a narrow range of therapies, but may experience widely divergent outcomes; 80-90% become long-term survivors while 20% develop incurable recurrence. Transcriptomic profiling has identified four subgroups with different recurrence risks, but outcomes remain variable for individual patients within each subgroup. To gain new insight into why patients with similar-appearing tumors have variable outcomes, we examined how the timing of tumor initiation effects medulloblastomas triggered by a single, common driver mutation. We genetically-engineered mice to express an oncogenic Smo allele starting early in development in the broad lineage of neural stem cells, or later, in the more committed lineage of cerebellar granule neuron progenitors. Both groups developed medulloblastomas and no other tumors. We compared medulloblastoma progression, response to therapy, gene expression profile and cellular heterogeneity, determined by single cell transcriptomic analysis (scRNA-seq). The average transcriptomic profiles of the tumors were similar. However, stem cell-triggered medulloblastomas progressed faster, contained more OLIG2-expressing tumor stem cells, and consistently showed radioresistance. In contrast, progenitor-triggered MBs progressed slower, lost stem cell character over time and were radiosensitive. Progenitor-triggered medulloblastomas also contained more diverse stromal populations, including tumor-associated macrophages, indicating that the timing of oncogenesis affected the subsequent interactions between the tumor and microenvironment. Our findings show that developmental events in tumorigenesis may be impossible to infer from transcriptomic profile, but while remaining cryptic can nevertheless influence tumor composition and the outcome of therapy. Precise understanding of medulloblastoma pathogenesis and prognosis requires supplementing transcriptomic data with biomarkers of cellular heterogeneity.
Activation of the nuclear factor (erythroid‐derived 2)‐like 2 (NFE2L2 or NRF2) transcription factor is a critical and evolutionarily conserved cellular response to oxidative stress, metabolic stress, and xenobiotic insult. Deficiency of NRF2 results in hypersensitivity to a variety of stressors, whereas its aberrant activation contributes to several cancer types, most commonly squamous cell carcinomas of the esophagus, oral cavity, bladder, and lung. Between 10% and 35% of patients with squamous cell carcinomas display hyperactive NRF2 signaling, harboring activating mutations and copy number amplifications of the NFE2L2 oncogene or inactivating mutations or deletions of KEAP1 or CUL3, the proteins of which co‐complex to ubiquitylate and degrade NRF2 protein. To better understand the role of NRF2 in tumorigenesis and more broadly in development, we engineered the endogenous Nfe2l2 genomic locus to create a conditional mutant LSL‐Nrf2E79Q mouse model. The E79Q mutation, one of the most commonly observed NRF2‐activating mutations in human squamous cancers, codes for a mutant protein that does not undergo KEAP1/CUL3‐dependent degradation, resulting in its constitutive activity. Expression of NRF2 E79Q protein in keratin 14 (KRT14)‐positive murine tissues resulted in hyperplasia of squamous cell tissues of the tongue, forestomach, and esophagus, a stunted body axis, decreased weight, and decreased visceral adipose depots. RNA‐seq profiling and follow‐up validation studies of cultured NRF2E79Q murine esophageal epithelial cells revealed known and novel NRF2‐regulated transcriptional programs, including genes associated with squamous cell carcinoma (e.g. Myc), lipid and cellular metabolism (Hk2, Ppard), and growth factors (Areg, Bmp6, Vegfa). These data suggest that in addition to decreasing adipogenesis, KRT14‐restricted NRF2 activation drives hyperplasia of the esophagus, forestomach, and tongue, but not formation of squamous cell carcinoma. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Nuclear factor erythroid 2-related factor 2 (NFE2L2, also known as NRF2) is a transcription factor and master regulator of cellular antioxidant response. Aberrantly high NRF2-dependent transcription is recurrent in human cancer, but conversely NRF2 activity diminishes with age and in neurodegenerative and metabolic disorders. Although NRF2-activating drugs are clinically beneficial, NRF2 inhibitors do not yet exist. Here, we describe use of a gain-of-function genetic screen of the kinome to identify new druggable regulators of NRF2 signaling. We found that the under-studied protein kinase brain-specific kinase 2 (BRSK2) and the related BRSK1 kinases suppress NRF2-dependent transcription and NRF2 protein levels in an activity-dependent manner. Integrated phosphoproteomics and RNAseq studies revealed that BRSK2 drives 5′-AMP-activated protein kinase α2 (AMPK) signaling and suppresses the mTOR pathway. As a result, BRSK2 kinase activation suppresses ribosome-RNA complexes, global protein synthesis and NRF2 protein levels. Collectively, our data illuminate the BRSK2 and BRSK1 kinases, in part by functionally connecting them to NRF2 signaling and mTOR. This signaling axis might prove useful for therapeutically targeting NRF2 in human disease.This article has an associated First Person interview with the first author of the paper.
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