Tomoyuki Koga

Genome instability, including chromothripsis, is a hallmark of cancer. Cancer cells frequently contain micronuclei—small, nucleus-like structures formed by chromosome missegregation—that are susceptible to rupture, exposing chromatin to cytoplasmic nucleases. Through an unbiased, imaging-based small interfering RNA screen that targeted all 204 known and putative human nucleases, we identified a previously uncharacterized cytoplasmic endonuclease, NEDD4-binding protein 2 (N4BP2), that enters ruptured micronuclei and initiates DNA damage, leading to chromosome fragmentation. N4BP2 promoted genome rearrangements (including chromothripsis), formation of extrachromosomal DNA (ecDNA) in drug-induced gene amplification, tumorigenesis, and tumor cell proliferation in an induced model of human high-grade glioma. Analysis of more than 10,000 human cancer genomes revealed elevated N4BP2 expression to be predictive of chromothripsis and copy number amplifications, including ecDNA.

Widespread alterations in RNA alternative splicing (AS) have been identified in adult gliomas. However, their regulatory mechanism, biological significance, and therapeutic potential remain largely elusive. Here, using a computational approach with both bulk and single-cell RNA-Seq, we uncover a prognostic AS signature linked with neural developmental hierarchies. Using advanced iPSC glioma models driven by glioma driver mutations, we show that this AS signature could be enhanced by EGFRvIII and inhibited by in situ IDH1 mutation. Functional validations of 2 isoform switching events in CERS5 and MPZL1 show regulations of sphingolipid metabolism and SHP2 signaling, respectively. Analysis of upstream RNA binding proteins reveals PTBP1 as a key regulator of the AS signature where targeting of PTBP1 suppresses tumor growth and promotes the expression of a neuron marker TUJ1 in glioma stem-like cells. Overall, our data highlights the role of AS in affecting glioma malignancy and heterogeneity and its potential as a therapeutic vulnerability for treating adult gliomas.

Nervous system tumors, particularly brain tumors, represent the most common tumors in children and one of the most lethal tumors in adults. Despite decades of research, there are few effective therapies for these cancers. Although human nervous system tumor cells and genetically engineered mouse models have served as excellent platforms for drug discovery and preclinical testing, they have limitations with respect to accurately recapitulating important aspects of the pathobiology of spontaneously arising human tumors. For this reason, attention has turned to the deployment of human stem cell engineering involving human embryonic or induced pluripotent stem cells, in which genetic alterations associated with nervous system cancers can be introduced. These stem cells can be used to create self-assembling three-dimensional cerebral organoids that preserve key features of the developing human brain. Moreover, stem cell-engineered lines are amenable to xenotransplantation into mice as a platform to investigate the tumor cell of origin, discover cancer evolutionary trajectories and identify therapeutic vulnerabilities. In this article, we review the current state of human stem cell models of nervous system tumors, discuss their advantages and disadvantages, and provide consensus recommendations for future research.

In 2021, the World Health Organization reclassified glioblastoma, the most common form of adult brain cancer, into isocitrate dehydrogenase (IDH)-wild-type glioblastomas and grade IV IDH mutant (G4 IDHm) astrocytomas. For both tumor types, intratumoral heterogeneity is a key contributor to therapeutic failure. To better define this heterogeneity, genome-wide chromatin accessibility and transcription profiles of clinical samples of glioblastomas and G4 IDHm astrocytomas were analyzed at single-cell resolution. These profiles afforded resolution of intratumoral genetic heterogeneity, including delineation of cell-to-cell variations in distinct cell states, focal gene amplifications, as well as extrachromosomal circular DNAs. Despite differences in IDH mutation status and significant intratumoral heterogeneity, the profiled tumor cells shared a common chromatin structure defined by open regions enriched for nuclear factor 1 transcription factors (NFIA and NFIB). Silencing of NFIA or NFIB suppressed in vitro and in vivo growths of patient-derived glioblastomas and G4 IDHm astrocytoma models. These findings suggest that despite distinct genotypes and cell states, glioblastoma/G4 astrocytoma cells share dependency on core transcriptional programs, yielding an attractive platform for addressing therapeutic challenges associated with intratumoral heterogeneity.

Diffuse midline glioma (DMG) is a leading cause of brain tumor death in children. In addition to hallmark H3.3K27M mutations, significant subsets also harbor alterations of other genes, such as TP53 and PDGFRA . Despite the prevalence of H3.3K27M, the results of clinical trials in DMG have been mixed, possibly due to the lack of models recapitulating its genetic heterogeneity. To address this gap, we developed human iPSC-derived tumor models harboring TP53 ^R248Q with or without heterozygous H3.3K27M and/or PDGFRA ^D842V overexpression. The combination of H3.3K27M and PDGFRA ^D842V resulted in more proliferative tumors when gene-edited neural progenitor (NP) cells were implanted into mouse brains compared to NP with either mutation alone. Transcriptomic comparison of tumors and their NP cells of origin identified conserved JAK/STAT pathway activation across genotypes as characteristic of malignant transformation. Conversely, integrated genome-wide epigenomic and transcriptomic analyses, as well as rational pharmacologic inhibition, revealed targetable vulnerabilities unique to the TP53 ^R248Q ; H3.3K27M; PDGFRA ^D842V tumors and related to their aggressive growth phenotype. These include AREG -mediated cell cycle control, altered metabolism, and vulnerability to combination ONC201/trametinib treatment. Taken together, these data suggest that cooperation between H3.3K27M and PDGFRA influences tumor biology, underscoring the need for better molecular stratification in DMG clinical trials.

Advances in genome and tissue engineering have spurred significant progress and opportunity for innovation in cancer modeling. Human induced pluripotent stem cells (iPSCs) are an established and powerful tool to study cellular processes in the context of disease-specific genetic backgrounds; however, their application to cancer has been limited by the resistance of many transformed cells to undergo successful reprogramming. Here, we review the status of human iPSC modeling of solid tumors in the context of genetic engineering, including how base and prime editing can be incorporated into “bottom-up” cancer modeling, a term we coined for iPSC-based cancer models using genetic engineering to induce transformation. This approach circumvents the need to reprogram cancer cells while allowing for dissection of the genetic mechanisms underlying transformation, progression, and metastasis with a high degree of precision and control. We also discuss the strengths and limitations of respective engineering approaches and outline experimental considerations for establishing future models.

Diffuse intrinsic pontine glioma (DIPG) is a subset of high-grade glioma that occurs predominantly in children and has no cure. Up to 80% of DIPG harbor a heterozygous point mutation that results in a lysine 27 to methionine substitution in histone variant H3.3 (H3.3K27M). However, H3.3K27M alone is insufficient for tumorigenesis in existing DIPG models, suggesting that it interacts with co-occurring oncogenic mutations. NF1 deletion co-occurs with TP53 mutation and H3.3K27M in DIPG, but the impact of these mutations individually and together are understudied. To address this gap, we designed a model of DIPG based on human induced pluripotent stem cells (iPSC) edited via CRISPR to express either wild-type or deleted NF1 in conjunction with heterozygous H3.3K27M and a loss of function mutation in TP53. Edited iPSC were chemically differentiated into neural progenitor cells (iNPC), which upon implantation into the brainstems of immunodeficient mice formed diffusely invasive tumors that were histologically consistent with high-grade glioma. Mice with TP53^mut;H3.3K27M tumors survived significantly (pTP53^mut;NF1^-/-;H3.3K27M tumors. In vitro proliferation assays of neurospheres generated from these tumors (termed iDIPG) confirmed this result, suggesting that NF1 deletion synergizes with the other two mutations to accelerate tumor growth. To determine which transcriptional pathways could be involved in this faster tumorigenesis, we performed RNA-sequencing TP53^mut;H3.3K27M (TK) and TP53^mut;NF1^-/-;H3.3WT (TNK) iDIPG neurospheres as well as their iNPC precursors. iDIPG and iNPC clustered by NF1 status. Differential expression analysis comparing TNK and TK iDIPG neurospheres revealed 7226 differentially expressed genes (pNF1 deletion significantly impacts expression of the kinome. We found 141 kinases upregulated in TNK iDIPG neurospheres, including several members of the MAP kinase family and EGFR, which is amplified or activated in a large number of high-grade gliomas and therefore represents an attractive therapeutic target. Taken together, these data show that NF1 deletion is associated with a significant alteration of kinase expression in H3.3K27M iDIPG, potentially opening up a new therapeutic avenue in these devastating tumors. Further work using this model will focus on screening for kinases necessary for TNK iDIPG neurospheres survival in culture and investigating synergy between targeted kinase inhibition and HDAC inhibitors, which have shown promise in H3.3K27M DIPG. Citation Format: Kasey Skinner, Tomoyuki Koga, Shunichiro Miki, Robert F. Gruener, R. Stephanie Huang, Frank Furnari, Ryan Miller. NF1 deletion potentiates tumorigenesis and activates expression of cancer-related kinases in an iPSC-based model of H3.3K27M diffuse intrinsic pontine glioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB067.

Heterozygous TERT (telomerase reverse transcriptase) promoter mutations (TPMs) facilitate TERT expression and are the most frequent mutation in glioblastoma (GBM). A recent analysis revealed this mutation is one of the earliest events in gliomagenesis. However, no appropriate human models have been engineered to study the role of this mutation in the initiation of these tumors. We established GBM models by introducing the heterozygous TPM in human induced pluripotent stem cells (hiPSCs) using a two-step targeting approach in the context of GBM genetic alterations, CDKN2A/B and PTEN deletion, and EGFRvIII overexpression. The impact of the mutation was evaluated through the in vivo passage and in vitro experiment and analysis. Orthotopic injection of neuronal precursor cells (NPCs) derived from hiPSCs with the TPM into immunodeficient mice did not enhance tumorigenesis compared to TERT promoter wild type NPCs at initial in vivo passage presumably due to relatively long telomeres. However, the mutation recruited GA-Binding Protein and engendered low-level TERT expression resulting in enhanced tumorigenesis and maintenance of short telomeres upon secondary passage as observed in human GBM. These results provide the first insights regarding increased tumorigenesis upon introducing a TPM compared to isogenic controls without TPMs. Our novel GBM models presented the growth advantage of heterozygous TPMs for the first time in the context of GBM driver mutations relative to isogenic controls, thereby allowing for the identification and validation of TERT promoter-specific vulnerabilities in a genetically accurate background.