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Neural development requires the orchestration of dynamic changes in gene expression to regulate cell fate decisions. This regulation is heavily influenced by epigenetics, heritable changes in gene expression not directly explained by genomic information alone. An understanding of the complexity of epigenetic regulation is rapidly emerging through the development of novel technologies that can assay various features of epigenetics and gene regulation. Here, we provide a broad overview of several commonly investigated modes of epigenetic regulation, including DNA methylation, histone modifications, noncoding RNAs, as well as epitranscriptomics that describe modifications of RNA, in neurodevelopment and diseases. Rather than functioning in isolation, it is being increasingly appreciated that these various modes of gene regulation are dynamically interactive and coordinate the complex nature of neurodevelopment along multiple axes. Future work investigating these interactions will likely utilize 'multi-omic' strategies that assay cell fate dynamics in a high-dimensional and high-throughput fashion. Novel human neurodevelopmental models including iPSC and cerebral organoid systems may provide further insight into human-specific features of neurodevelopment and diseases.

Glioblastoma (GBM) is the most prevalent and lethal primary brain tumor, which carries a poor prognosis due to a high level of therapeutic resistance and rate of tumor recurrence. Underlying this aggressive behavior is extraordinary genomic and cellular heterogeneity, conferring numerous avenues for treatment escape and refractory growth. This work leverages single cell genomic analyses to link somatic mutations to transcriptomic signatures within the native tumor tissue. We implemented a three-stage pipeline to perform the following tasks: variant calling, machine learning (ML) classification, and biological pathway analysis. Somatic variants were called from smart-seq based scRNA-seq data and filtered based on predicted lethality, reference in cancer mutation databases e.g. COSMIC, and population prevalence. In the second stage, the genotyped cells and normalized gene expression profiles were pre-processed using differential gene expression, and input into ML classifiers trained on the task of predicting mutation status. The utilized ML models included a Support Vector Machine and Ridge Classifier and were selected based on performance and interpretability. The efficacy of the models was evaluated using the AUC-ROC metric across multiple dataset partitions. The output of this stage was a set of “important genes” identified using the learned ML model weights. In the third stage, the sets of identified genes were subsampled and analyzed using the g:Profiler API to identify significant biological pathways. The analysis was performed on 2 datasets containing 677 samples from 5 patients and 1091 samples from 4 patients. The EGFRvIII mutation was used to validate the performance of the methodology due to its prevalence in primary GBM tumors. The mutation was detected in 17 single-cell samples. The ML models both produced a mean AUC-ROC of 0.93 [0.92, 094], and the classifiers identified NPM1, TCEA1, PPIA, SUMO2, among other genes, to be of significance. The individual genes have functions involving cell cycle regulation and protein folding, while the identified biological pathways associated with gene subsets were linked to ribonuclease activity and transcription factor post-transcriptional modifications. The ML component was applied to an additional dataset containing cell samples from 4 patients and correctly identified the 2 patients positive for EGFRvIII. The larger analysis identified point mutations in EGFR, TP53, and ATRX, and the classifiers exhibited AUC-ROC metrics ranging from 0.94 [0.93, 0.94] to 0.99 [0.99, 1.0]. Overall, we developed and employed a pipeline to link somatic variants with their gene expression signatures at the single cell level, identified biological pathways for further examination and established a ML classifier that can be applied to high-throughput scRNA-seq methods that lack the transcript coverage of the variant loci. Citation Format: Christopher Streiffer, Daniel Zhang, Ryan Salinas, Steven Brem, Donald O'Rourke, Zev Binder, Han-Chiao Isaac Chen, Hongjun Song. Identifying the transcriptomic signatures of mutational heterogeneity in GBM using single cell genomics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2203.

Glioblastoma multiforme (GBM) is the most common primary and aggressive brain tumors in adults with extremely poor prognosis and limited treatment options. A major hallmark of GBM is the rapid and diffused infiltration of tumor cells into the surrounding healthy tissue that contribute to tumor recurrence and therapeutic resistance. However, existing in vitro cell culture or in vivo xenograft models inadequately recapitulate the inter-tumoral and intra-tumoral heterogeneity which are key features of GBM. For example, common oncogenic drivers of GBM such as epidermal growth factor receptor (EGFR) amplification and EGFRvIII mutation do not persist in traditional in vitro models due to selection pressures, thus requires exogenous overexpression. Alternatively, EGFR statuses can be maintained in xenografted mice, but implantation of the primary GBM cells into the flank is required to first establish the tumor prior to secondary injection into the brains. Recently, we have established a novel protocol for culturing GBM tissue as organoids (GBOs) directly from patient tumor resection that retain many distinct cell populations in vitro with high fidelity evidenced by histological, whole-exome, bulk and single cell RNA analyses. Compared to prolonged generation time of previously established in vitro and xenograft models, our methodology is robust for generating GBOs within 1–2 weeks from initial resection. In addition, these GBOs can be readily xenografted into the adult mouse brains as an intact organoid, exhibit rapid and aggressive infiltration phenotypes, and maintains driver mutation EGFRviii within as little as one month. Consequently, they can be used to test in vivo treatment efficacies in a timely fashion. The presence of diverse cell types in this GBO model offers a promising platform for not only understanding of tumor biology, but also more strategic development of new therapies.

Immunotherapy, including chimeric antigen receptor (CAR) T cell therapy, has yielded major advancements for a number of hard-to-treat cancers; however, its transformative potential has yet to be realized in glioblastoma (GBM). Clinical studies of EGFRvIII targeted CAR T cell therapy have indicated that tumor heterogeneity, immune microenvironment, and adaptive responses to treatment may play important roles in limiting overall efficacy. We sought to examine comprehensively the dynamic molecular landscape of CAR T cell therapy in GBM using patient-derived GBM organoids (GBOs), a newly established laboratory model of inter- and intra- tumoral heterogeneity. Especially advantageous in these studies, GBOs preserve the intrinsic composition of somatic mutations, transcriptomic states, and non-neoplastic cells that contribute to the tumor microenvironment. Complementing the complexity of this biological system, we constructed an integrated single-cell multi-omics platform to interrogate gene expression, cell surface protein expression, somatic variants, and TCR sequences all from the same cell. Co-culture of GBOs and EGFRvIII targeted CAR T cells led to rapid T cell activation with concomitant upregulation of cytokine response gene programs in both antigen-positive and antigen-negative tumor cells. The adaptive tumor response also included expression of immune checkpoint receptor (ICR) ligands, such as PD-L1. At later time points, T cells transitioned into a dysfunctional or exhausted state, as characterized by increased cell surface expression of inhibitory receptors, such as PD-1, and decreased markers of effector activity despite the presence of antigen. Interestingly, CAR T cell therapy not only led to changes in immune-related pathways and tumor microenvironment, but it also induced shifts in tumor cell states with the depletion of an oligodendrocyte precursor cell-like state and corresponding enrichment in an astrocyte-like state. This finding suggests that EGFRvIII targeted CAR T cell therapy may leverage intrinsic cellular plasticity to induce differentiation-like effects in the surviving tumor cells.

Summary Glioblastomas exhibit vast inter- and intra-tumoral heterogeneity, complicating the development of effective therapeutic strategies. Current in vitro models are limited in preserving the cellular and mutational diversity of parental tumors and require a prolonged generation time. Here, we report methods for generating and biobanking patient-derived glioblastoma organoids (GBOs) that recapitulate the histological features, cellular diversity, gene expression, and mutational profiles of their corresponding parental tumors. GBOs can be generated quickly with high reliability and exhibit rapid, aggressive infiltration when transplanted into adult rodent brains. We further demonstrate the utility of GBOs to test personalized therapies by correlating GBO mutational profiles with responses to specific drugs and by modeling chimeric antigen receptor T cell immunotherapy. Our studies show that GBOs maintain many key features of glioblastomas and can be rapidly deployed to investigate patient-specific treatment strategies. Additionally, our live biobank establishes a rich resource for basic and translational glioblastoma research.

SummaryWhile thousands of long noncoding RNAs (lncRNAs) have been identified, few lncRNAs that control neural stem cell (NSC) behavior are known. Here, we identify Pinky (Pnky) as a neural-specific lncRNA that regulates neurogenesis from NSCs in the embryonic and postnatal brain. In postnatal NSCs, Pnky knockdown potentiates neuronal lineage commitment and expands the transit-amplifying cell population, increasing neuron production several-fold. Pnky is evolutionarily conserved and expressed in NSCs of the developing human brain. In the embryonic mouse cortex, Pnky knockdown increases neuronal differentiation and depletes the NSC population. Pnky interacts with the splicing regulator PTBP1, and PTBP1 knockdown also enhances neurogenesis. In NSCs, Pnky and PTBP1 regulate the expression and alternative splicing of a core set of transcripts that relates to the cellular phenotype. These data thus unveil Pnky as a conserved lncRNA that interacts with a key RNA processing factor and regulates neurogenesis from embryonic and postnatal NSC populations.

SummaryThe epigenetic mechanisms that enable lifelong neurogenesis from neural stem cells (NSCs) in the adult mammalian brain are poorly understood. Here, we show that JMJD3, a histone H3 lysine 27 (H3K27) demethylase, acts as a critical activator of neurogenesis from adult subventricular zone (SVZ) NSCs. JMJD3 is upregulated in neuroblasts, and Jmjd3 deletion targeted to SVZ NSCs in both developing and adult mice impairs neuronal differentiation. JMJD3 regulates neurogenic gene expression via interaction at not only promoter regions but also neurogenic enhancer elements. JMJD3 localizes at neural enhancers genome-wide in embryonic brain, and in SVZ NSCs, JMJD3 regulates the I12b enhancer of Dlx2. In Jmjd3-deleted SVZ cells, I12b remains enriched with H3K27me3 and Dlx2-dependent neurogenesis fails. These findings support a model in which JMJD3 and the poised state of key transcriptional regulatory elements comprise an epigenetic mechanism that enables the activation of neurogenic gene expression in adult NSCs throughout life.

Non-neurogenic cell types, such as cortical astroglia and fibroblasts, can be directly converted into neurons by the overexpression of defined transcription factors. Normally, the cellular phenotype of such differentiated cells is remarkably stable and resists direct cell transdifferentiation. Here we show that theInk4a/Arf(also known asCdkn2a) locus is a developmental barrier to direct neuronal transdifferentiation induced by transcription factor overexpression. With serial passagein vitro, wild-type postnatal cortical astroglia become progressively resistant toDlx2-induced neuronal transdifferentiation. In contrast, the neurogenic competence ofInk4a/Arf-deficient astroglia is both greatly increased and does not diminish through serial cell culture passage. Electrophysiological analysis further demonstrates the neuronal identity of cells induced fromInk4a/Arf-null astroglia, and short hairpin RNA-mediated acute knockdown of p16Ink4a and p19Arf p16Ink4aand p19Arfindicates that these gene products function postnatally as a barrier to cellular transdifferentiation. Finally, we found that mouse fibroblasts deficient forInk4a/Arfalso exhibit greatly enhanced transcription factor-induced neuronal induction. These data indicate thatInk4a/Arfis a potent barrier to direct neuronal transdifferentiation and further suggest that this locus functions normally in the progressive developmental restriction of postnatal astrocytes.

Author(s): Potts, Matthew B; Siu, Jason J; Price, James D; Salinas, Ryan D; Cho, Mathew J; Ramos, Alexander D; Hahn, Junghyun; Margeta, Marta; Oldham, Michael C; Lim, Daniel A | Abstract: BackgroundMixed lineage leukemia-1 (Mll1) epigenetically regulates gene expression patterns that specify cellular identity in both embryonic development and adult stem cell populations. In the adult mouse brain, multipotent neural stem cells (NSCs) in the subventricular zone generate new neurons throughout life, and Mll1 is required for this postnatal neurogenesis but not for glial cell differentiation. Analysis of Mll1-dependent transcription may identify neurogenic genes useful for the direct reprogramming of astrocytes into neurons.ObjectiveTo identify Mll1-dependent transcriptional modules and to determine whether genes in the neurogenic modules can be used to directly reprogram astrocytes into neurons.MethodsWe performed gene coexpression module analysis on microarray data from differentiating wild-type and Mll1-deleted subventricular zone NSCs. Key developmental regulators belonging to the neurogenic modules were overexpressed in Mll1-deleted cells and cultured cortical astrocytes, and cell phenotypes were analyzed by immunocytochemistry and electrophysiology.ResultsTranscriptional modules that correspond to neurogenesis were identified in wild-type NSCs. Modules related to astrocytes and oligodendrocytes were enriched in Mll1-deleted NSCs, consistent with their gliogenic potential. Overexpression of genes selected from the neurogenic modules enhanced the production of neurons from Mll1-deleted cells, and overexpression of Brn4 (Pou3f4) in nonneurogenic cortical astroglia induced their transdifferentiation into electrophysiologically active neurons.ConclusionOur results demonstrate that Mll1 is required for the expression of neurogenic but not gliogenic transcriptional modules in a multipotent NSC population and further indicate that specific Mll1-dependent genes may be useful for direct reprogramming strategies.

The epigenetic mechanisms that enable specialized astrocytes to retain neurogenic competence throughout adult life are still poorly understood. Here we show that astrocytes that serve as neural stem cells (NSCs) in the adult mouse subventricular zone (SVZ) express the histone methyltransferase EZH2. This Polycomb repressive factor is required for neurogenesis independent of its role in SVZ NSC proliferation, as Ink4a/Arf-deficiency in Ezh2-deleted SVZ NSCs rescues cell proliferation, but neurogenesis remains defective. Olig2 is a direct target of EZH2, and repression of this bHLH transcription factor is critical for neuronal differentiation. Furthermore, Ezh2 prevents the inappropriate activation of genes associated with non-SVZ neuronal subtypes. In the human brain, SVZ cells including local astroglia also express EZH2, correlating with postnatal neurogenesis. Thus, EZH2 is an epigenetic regulator that distinguishes neurogenic SVZ astrocytes, orchestrating distinct and separable aspects of adult stem cell biology, which has important implications for regenerative medicine and oncogenesis. DOI: http://dx.doi.org/10.7554/eLife.02439.001, eLife digest In addition to the billions of nerve cells called neurons, the brain and spinal cord also contain star-shaped cells called astrocytes. At first it was thought that all astrocytes were the same, but it later became clear that there are several different types of astrocytes that perform different functions. Most neurons are formed in the embryo, but some astrocytes that are found deep within the brain can act as ‘neurogenic stem cells’ and continue to produce new neurons during adult life. However, it was not clear how these neurogenic astrocytes were different from other astrocytes. Now Hwang et al. have found that neurogenic astrocytes contain a protein called EZH2 that is not found in other types of astrocyte in the adult brain. Researchers already knew that this protein, which acts to help keep DNA tightly packed inside the nucleus and to keep genes switched off, was important for brain development. EZH2 was also known to prevent stem cells from prematurely turning into specialized cell types. But, surprisingly, Hwang et al. found that EZH2 has two distinct roles in neurogenic astrocytes: it allows them to multiply to make more astrocytes, and it also helps guide astrocytes into becoming neurons. Hwang et al. showed that different sets of genes were involved in these two roles. DOI: http://dx.doi.org/10.7554/eLife.02439.002

It is increasingly clear that while genetic alterations drive glioblastoma development, epigenetic dysregulation can also contribute to oncogenesis. Although there is clear evidence of tumors driven by specific mutations to histones or their modifiers, genomic studies have demonstrated that the majority of brain tumors are not molecularly classified by mutations in key epigenetic regulators. We hypothesized that some of the most common genetic mutations in glioblastoma can lead to chromatin-based transcriptional dysregulation. First, to investigate the transcriptional changes from specific genetic mutations, we performed RNA-sequencing analysis of a mouse tumor progression model with Ink4a/arf−/- cells derived from the subventricular zone (SVZ) which do not form tumors when intracranially injected into SCID mice, "primary" tumors produced by overexpression of EGFRviii in Ink4a/Arf −/- cells, and "secondary" tumors that models tumor recurrence via reinjection of primary tumor cells into the brains of other mice. Compared to Ink4a/Arf-/-, both primary and secondary tumors downregulated genes related to neuron differentiation and cell adhesion, while overexpressing homeobox genes related to cellular morphogenesis, including the HoxA locus. Secondary tumors, which carry worse survival, upregulate invasion-associated extracellular matrix and basement membrane genes. Intriguingly, ChIP-seq analysis revealed a clear loss of the repressive H3K27me3 mark at the HoxA locus and other homeodomain containing genes upon Ink4a/Arf inactivation. Subsequently, a subset of the genes became overexpressed upon tumorigenesis with a concomitant and progressive increase of the promoter activating H3K4me3 mark. Furthermore, IHC in mouse and a subset of human samples demonstrated heterogeneity of H3K27me3 staining compared to H3, while Western blot analysis also demonstrated a relative decrease in HK27me3 staining in mouse tumor samples compared to Ink4a/Arf−/-. These results support a model whereby Ink4a/Arf deficiency is sufficient to induce epigenetic changes via loss of H3K27me3, generating heterogeneity within a given tumor sample and allowing for EGFRviii driven oncogenic selection.