Year: 2020
Type: article
Abstract: Relevant patient-derived models may help to interrogate clinical and molecular data from patients and models and to work on clinically relevant questions of the contribution of glioblastoma heterogeneity to treatment resistance as well as the role of evolutionary programs during spontaneous tumor progression or development under treatment pressure. Patient-derived xenografts (PDX) with variable sources are heavily used models requiring host mice. The tissue source (full pretreatment tumor, post-treatment tissue, tissue from metastatic sites or minimal residual disease or circulating tumor cells) is variable and chosen according to the hypothesis examined; the mouse model is usually immune compromised, but examples of challenging humanized PDX models with a lengthy humanization process are available. In vitro models might be easier but are less complex, lacking the microenvironment, and are subject to genetic and post-translational changes due to the cultivation processes and the media growth factors that are not favorable to maintaining various cellular subtypes and key driver gene expression of parental tumors. In a recent issue of Cell, Jacob et al present a workable, fast (1–2 weeks), and effective workflow and paradigm of a patient-derived glioblastoma organoid model that allows for valid recapitulation of mutations, gene expression, various cellular states, and also histological features mimicking the parental glioblastoma.1 An important difference to established glioblastoma organoid (GBO) model is the generation directly from fresh tumor specimens without single-cell dissociation, a process that allows the maintenance of potentially critical cell-cell contacts and communication 2 and also avoids clonal selection by the avoidance of matrix compounds or growth factors. Instead, the protocol used H+GPSA medium containing Hibernate A, L-glutamine, penicillin/streptomycin, and amphotericin B.1 GBO demonstrated not only qualitatively but also quantitatively a similar cellular composition, i.e. level of OLIG2- or SOX2-positive cells, but also bona fide precursor cells, diversely proliferating cell progenies and non-neoplastic cells. As a limitation, over time GBO showed a decreased abundance of vasculature and macrophage/microglia populations and lower expression of immune-related genes. The success rate of GBO growth was 91.4% from a total of 53 patients, with 66.7% for isocitrate dehydrogenase (IDH)1 mutant tumors, 75% for recurrent tumors and 96.4% for primary IDH1-wildtype tumors, differential also at a level of subregional clones. As a critical prerequisite to use GBO for targeted analyses, GBOs also maintain inter- and intra-tumoral transcriptomic and genomic heterogeneity.1 At the single-cell transcriptomic level there was patient-specific clustering of GBO and parental tumor cells, whereas non-neoplastic cells of several patients clustered together. Looking at levels of recently described transcription cell types,3 again, similarities of the paired GBO-primary tumor distribution remained stable. Engraftment into female 4–8-week-old athymic nude (NU/J) mice was very effective and tumors prone to formation of distant satellites in patients are described to display similar features in the intracranial model. The work did not suffice in establishing a correlation between patient and GBO treatment responses to radiation, temozolomide in vitro, or targeted experimental treatments in vivo. A reason might be the low sample number, although molecular signatures were predictive of pathway-directed therapies. Also successful was an anti-EGFRvIII+ GBO-specific CAR-T cell treatment with CAR-T cell expansion and specific toxicity sparing EGFR+ GBO, all-in-all showing the utility of GBO for rapid testing of responses with this endogenous target in culture. Oncogene amplification is a common event in cancer and glioblastoma (GBM). The presence of large circular extrachromosomal DNA (ecDNA) in human cancers has been known for many years, but its importance was previously underestimated. Until recently, ecDNA was thought to occur in only 1.4% of tumors. Two seminal papers published by Mischel and colleagues in 2014 and 2017 as well as others changed this perspective by showing that resistance to epidermal growth factor receptor (EGFR) inhibitors in GBM was conferred by reversible loss of the gain of function mutation in EGFRvIII on ecDNA1 and that ecDNA was found in nearly half of human cancers.2 In a third recent publication in the journal Nature, the same group demonstrated that ecDNA allows for high copy number and high chromatin accessibility of oncogenes, which in turn leads to their high transcription in GBM and other cancers.3 Using multiple advanced sequencing and microscopy techniques, the research team set out to address three questions: 1) verify the circular nature of ecDNA; 2) determine the effects of ecDNA on oncogene expression; and 3) assess chromatin accessibility on ecDNA. The authors first showed that the structure of ecDNA is indeed circular. Using super-resolution, three-dimensional structured illumination microscopy, scanning electron microscopy, and transmission electron microscopy, they were able to visualize individual, fixed ecDNA circular structures within the nuclei of different cancer cells. The circular nature of ecDNA was further verified with computational reconstruction tools that the authors developed. They then went on to show that the oncogenes located on ecDNA are among the highest expressed genes in GBM and other cancer genomes and exhibited higher expression than oncogenes that were only located on chromosomal DNA (chrDNA). The authors showed that one reason for this increase is that DNA copy numbers on ecDNA are greatly increased. The second reason for increased expression was enhanced chromatin accessibility of genes located on ecDNA. In fact, using ChIP-seq, ATAC-seq, and MNase-seq, the authors determined that chromatins are more accessible when located on ecDNA than on chrDNA because nucleosomes are less condensed in the former as compared with the latter. Finally, using PLAC-seq and circular chromosome conformation capture combined with 4C-seq, they showed that ecDNA has a significantly greater number of ultra-long-range interactions with active chromatin. Altogether, the data showed that ecDNA is indeed circular and can markedly increase oncogene expression owing to increased DNA copy numbers and enhanced chromatin accessibility. The intra-tumoral cellular heterogeneity of glioblastoma (GBM) is notorious and is thought to be a significant contributor to therapeutic inefficiency. Our understanding of the mechanisms giving rise to this heterogeneity is now becoming clearer with the advent of single-cell RNA sequencing technologies. In a recent report by Bhaduri and colleagues,1 the authors analyzed freshly excised GBMs by scRNA-seq and, similar to other studies, found that each tumor includes proliferating and differentiated cells in addition to various immune cell populations, an exact mixture that creates a patient-specific tumor composition. To gain a better understanding of the provenance of the non-immune cell composition, the authors created an atlas of GBM cell types and states by mapping their data onto a reference framework of single-cell data from the developing and adult human brain. They found that a variety of cell types expressing marker genes of glioma stem cells (GSCs) could exist within a single tumor, with some tumors having multiple clusters of GSCs. These results are somewhat iconoclastic to the prevailing models of cancer stem cell biology that purports that a single stem cell can initiate and sustain the tumor and that this stem cell emerges from a single cell or origin. In their analysis, the authors observed representation of OPCs, astrocytes, and neuronal cell types expressing GSC markers. Furthermore, outer radial glia (oRG), a stem cell critical in the developing brain and not believed to be present in adult normal brain, appeared as a distinct cell type that strongly expressed GSC marker genes in GBM. The authors then demonstrated that oRG-like GSCs express the protein tyrosine phosphatase receptor zeta 1 (PTPRZ1), a known marker of oRG. PTPRZ1-positive oRG-like GSCs are more migratory in vitro and in a brain organoid model system, and PTPRZ1-positive GBM cells are more invasive in vivo. Knockdown of PTPRZ1 resulted in downregulation of other key markers of radial glia identity. Taken together, these results suggest that PTPRZ1 plays a role in defining a highly invasive, tumorigenic signaling axis in oRG-like GBM GSCs. While the authors recognize the unique features of the oRG-like GSCs, they also point out the possibility of spatial and temporal roles for each of the GSC populations or perhaps their acting in parallel to promote GBM growth and maintenance and resistance to standard-of-care therapy. Recognizing and characterizing multiple GSC cell types is a first step towards identifying targets to pursue drug discovery efforts against GBM. Embryonal tumors with multilayered rosettes (ETMRs) are a histologically diverse and molecularly defined group of aggressive brain tumors primarily occurring in infants and very young children with an extremely poor prognosis. Up to 90% of ETMRs are characterized by amplification of the chromosome 19q13.41 microRNA cluster (C19MC) and fusion with the promoter of the brain-specific expressed gene TTYH1.1 Despite knowledge of the most frequent genetic lesion in this disease, our understanding of the cellular, genomic, and transcriptional basis has been limited. Three important recent advances illuminate the cellular and molecular underpinnings of ETMR, expand future research opportunities, and offer potential therapeutic strategies. Possible Origins - Several pediatric brain tumors, including ETMRs, are thought to arise from cell types normally expressed primarily during embryonic development. To explore this concept, in a recent paper in Nature Genetics Jessa et al generated a single-cell transcriptome atlas of human and murine embryonal pons and forebrain by profiling >65,000 cells, which allowed identification of 191 cell populations.2 Gene signatures from these developmental cell populations were then projected across 240 human bulk RNA-sequencing samples, including 186 brain tumor patient samples. ETMRs were able to be segregated from other tumor pathologies based on their distinctive developmental cell population, which clustered with early fetal brain (13–18 post-conception weeks). Assessment of temporal TTYH1 expression suggested prenatal neurogenic radial glia cells as possible ETMR cells of origin. In line with other pediatric brain tumor entities, the authors propose that ETMR tumor cells remain developmentally trapped as early neural progenitors and are unable to complete cellular differentiation, propagating oncogenesis. These findings provide possible insight into cell types relevant for mouse modeling of ETMRs and underscore relevant developmental programs necessary for neoplastic transformation to ETMR. Collusion and Partners in Crime—Another detailed genomic investigation of ETMRs published in Cancer Cell investigated the mechanisms by which C19MC downstream signaling contributes to ETMR tumor cell growth.3 The authors first confirmed by both methylation and transcriptional profiling that ETMRs are distinct from other embryonal brain tumors of infancy and that they are characterized by enrichment of embryonal neural developmental pathways; these findings highlight the early developmental cellular origins of these tumors. C19MC miRNAs upregulated genes involved in neural stem cell maintenance and decreased expression of the tumor suppressor proteins p21, p27, and RBL2. Most importantly, C19MC miRNAs were found to inhibit the tumor suppressor protein TTP and consequently upregulate both LIN28A and MYCN. This establishes a C19MC-LIN28A-MYCN oncogenic circuit that is hypothesized by the authors to be the driver of ETMR formation. The C19MC-LIN28A-MYCN loop is amplified by a hybrid super-enhancer formed by the TTYH1-C19MC fusion bringing together their gene enhancers as well as by long-range interactions between the MYCN promoter and early embryonic enhancers. Other Candidates Suggest Unifying Pathways—What defines the 10% of non-C19MC-amplified ETMRs? Can we learn from other recurrent mutations across patients and over the course of treatment to understand common/distinct pathways in ETMRS? In a study authored by Lambo et al, published in Nature in 2019, the molecular characteristics of 193 primary ETMR samples as well as 23 matched relapsed samples were described.4 In the approximately 10% of ETMRs without C19MC amplification, the authors identified either germline mutations in DICER1 or other microRNA-related aberrations, including amplification of the miR-17-92miRNA cluster, C19MC-MYO9B fusions, and clustered breakpoints affecting the C19MC locus. Importantly, however, all ETMRs, regardless of location, histology, or the presence or absence of C19MC amplification, clustered together on cluster analysis of ETMR DNA methylation profiles. Additionally, all ETMRs harbored similar copy-number aberrations, genomic structural instability, and patterns of microRNA expression, suggesting a common cell of origin. When the genomics of primary and recurrent ETMR were compared, structural variants were maintained; in contrast, somatic single-nucleotide variants were increased in the recurrent samples, likely secondary to platinum-based treatment. Recurrent samples additionally demonstrated enrichment of expression of stem cell-like genes with less differentiation than primary ETMR cells. Consistent with the observed genomic instability of ETMRs, these tumors were also found to have high levels of R-loops in the region surrounding C19MC, structures formed by stalled RNA polymerase leading to increased DNA breakpoints. Combination treatment of ETMR cells with topotecan, a topoisomerase I inhibitor, and the PARP inhibitor pamiparib synergistically increased R-loop formation and consequently increased DNA damage. Overall, these three manuscripts collectively provide important clues related to the genomic instability of the C19MC region and enrichment of developmental regulatory elements resulting in synthesis of an oncogenic program tied to developmental gene expression programs (Fig. 1). Future investigations will likely capitalize on these molecular discoveries by developing pre-clinical therapeutic strategies for ETMR. Recent scientific advances in ETMR biology refine our understanding of the tumor’s cellular origins, oncogenic mechanisms, and potential therapeutic vulnerabilities.
Source: Neuro-Oncology
Language: English
Subfield: Genetics (clinical)
Field: Medicine
Domain: Health Sciences
Open Access status: bronze
OpenAlex ID: W4256166205