Abstract BACKGROUND The topologically associated domain (TAD) on 8q24 surrounding MYC is important for many types of cancer. The region is also important in the formation of IDH-mutant glioma; the risk allele rs55705857 being associated with the development of these tumors. The rs55705857 risk allele significantly increased the formation of tumors in an IDH-mutant mouse model. Cerebral organoids have been found to recapitulate the early development of the brain. We hypothesized that the rs55705857 risk allele may alter the phenotype of cerebral organoids. METHODS Isogenic induced pluripotent stem cells (iPSCs) were developed with (n=3) and without (n=2) the rs55705857 risk allele from the parental cell line PGP-1 (GM23338). These cells were cultured and differentiated into unpatterned cerebral organoids and observed throughout differentiation. Organoids were fixed, sectioned, and stained by H&E and immunofluorescence. RESULTS Organoids containing the risk allele were smaller throughout development, starting at the embryoid body stage and continuing up to over 75 days in culture, compared to two isogenic non-risk lines. At 4 weeks the risk allele containing organoids also contained a larger number of small rosette-like neuroepithelia, instead of the more continuous neuroepithelia observed in the non-risk allele organoids. At 75 days, the organoids with the risk allele maintained a larger proportion of Sox2 positive cells, which are enriched in the tight clusters of small cells with round nuclei. CONCLUSIONS The risk allele rs55705857 in the MYC TAD at 8q24 is altering the development of unpatterned cerebral organoids. This difference is visible as early as embryoid body development and persists through neuroepithelial development. Further studies of these differences may help our understanding of how rs55705857 accelerates IDH-mutant glioma development.

Establishing causal links between inherited polymorphisms and cancer risk is challenging. Here, we focus on the single-nucleotide polymorphism rs55705857, which confers a sixfold greater risk of isocitrate dehydrogenase ( IDH) –mutant low-grade glioma (LGG). We reveal that rs55705857 itself is the causal variant and is associated with molecular pathways that drive LGG. Mechanistically, we show that rs55705857 resides within a brain-specific enhancer, where the risk allele disrupts OCT2/4 binding, allowing increased interaction with the Myc promoter and increased Myc expression. Mutating the orthologous mouse rs55705857 locus accelerated tumor development in an Idh1 R132H -driven LGG mouse model from 472 to 172 days and increased penetrance from 30% to 75%. Our work reveals mechanisms of the heritable predisposition to lethal glioma in ~40% of LGG patients.

Abstract Establishing causal links between genetic polymorphisms and increased heritable risk of developing brain cancer is a major challenge. The non-coding single nucleotide polymorphism rs55705857 (A >G) is associated with a ~6-fold increased risk to develop IDH-mutant low-grade glioma (LGG). The rs55705857 G allele has a minor allele frequency of only ~5% in the general population but is found in ~40% of patients with IDH-mutant LGG and patients carrying risk-alleles are diagnosed on average 7-12 years earlier than those carrying non-risk A alleles. This makes rs55705857 one of the highest reported genetic associations with cancer, comparable with inherited BRCA1 gene mutations and the risk of developing breast cancer or other familial glioma genes such as NF1/2, CDKN2A or p53. To generate a LGG mouse model, we combined clonal activation of IDH1R132H with mutations of Trp53 and Atrx, which resulted in the development of LGG-like brain tumors in 25% of mice. Mutating the highly conserved, orthologous mouse rs55705857 locus to mimic the human risk allele dramatically accelerated tumor development from 463 to 172 days and increased penetrance to 75%. The resulting tumors exhibit elevated R-2-hydroxyglutarate levels, well-differentiated fibrillary neoplastic histology and metabolic rewiring, recapitulating histopathological and molecular hallmarks of human LGG. Mechanistically, we show that the rs55705857 locus resides within a brain-specific enhancer, which shows enhanced activity in IDH-mutant tumors. In addition, we found that the risk allele disrupts OCT2/4 binding, allowing increased interaction with the Myc promoter and increased Myc expression. The hyperactive chromatin status combined with the tissue specificity of this enhancer explains the cooperativity between mutant IDH and rs55705857 and why rs55705857 is associated specifically with IDH-mutant glioma, but not other cancers. Overall, we generated new LGG mouse models, which provide insights into the pathophysiology of this deadly disease and shed light into the heritable predisposition to LGG development.

Low-grade glioma (LGG) are generally slowly growing brain cancers, that frequently undergo malignant progression to aggressive, secondary glioblastoma with a dismal prognosis. By combining genetically engineered Idh1-mutant mice with in vivo CRISPR gene editing we generated a mouse model faithfully recapitulating the founder mutations of LGG. Clonal activation of the neomorphic Idh1 R132H mutation cooperates with Trp53 and Atrx mutations to trigger development of brain tumors but only with ~30% penetrance and very long latency. To elucidate the molecular mechanisms underlying the malignant progression of IDH1-mutant LGG, we devised and deployed a direct in vivo CRISPR screen targeting genes commonly mutated in human IDH-mutant secondary glioblastoma. Stereotaxic delivery of a lentiviral sgRNA library targeting the mouse orthologs of these genes into the brain of Idh1 R132H ;Trp53;Atrx;Cas9 and control Idh1 wt ;Trp53;Atrx;Cas9 compound mutant mice resulted in rapid formation of tumors that recapitulate human Idh1-mutant glioblastoma. Deconvoluting the screen showed that PI3K pathway members Pten and Pik3ca as well as Notch1, Smarca4 and Fat1 are preferentially enriched in Idh1 R132H-tumors, while Rb1 and NF2 were enriched in Idh1 wt tumors. Co-mutation analysis further identified additional co-occurring driver combinations such as Bcor-Met, Olig2-Met, Olig2-Med12 or Bcor-Olig2. We validated the tumor suppressive function of Notch1 and Pten using conventional floxed knock-out alleles and found that Notch1 functions in a haploinsufficient manner. Interestingly, Idh1 R132H did not alter tumor latency or pathology in a high grade p53;Pten;Rb1 mutant background, indicating that the neomorphic IDH-mutations can drive low but not high grade glioma development. Our study provides a functional landscape of gliomagenesis suppressors in vivo.

Abstract BACKGROUND Determination of the causation of germline single nucleotide polymorphisms (SNPs) located in non-coding regions of the genome is challenging. The genomic region of 8q24 has been identified as important in many kinds of cancer, linked to a topologically associated domain (TAD) encompassing MYC; this TAD contains a GWAS SNP (rs55705857) associated with IDH-mutant glioma. METHODS Germline genotyping data from 622 IDH-mutant glioma and 668 controls were used to fine map the rs55705857 locus by detailed haplotype analysis. Chromatin immunoprecipitation sequencing (ChIP-seq) of histone markers H3K4me1, H3K4me3, H3K27ac and H3K36me3 was performed on normal brain samples (n=8) and human glioma samples (n=11 IDH-wt and 52 IDH-mut). RNAseq from 9 normal and 83 brain tumors (n=26 IDH-wt and 55 IDH-mut) were used to assess differential gene expression. RESULTS Fine-mapping identified rs55705857 SNP as the most likely causative allele (OR=8.69; p<0.001) within 8q24 for the development of IDH-mutant glioma. At rs55705857, both H3K27ac and H3K4me1 in IDH-mutant vs IDH-wt tumors were increased 3.05- and 1.58-fold, respectively (DiffBind; p=5.81×10-7 and p=2.31×10-3). ChromHMM analysis of the marks indicated that promoter and enhancer functions were significantly increased, and the activity broadened at rs55705857 in IDH-mut gliomas compared to IDH-wt tumors and normal brain samples. This enhancement correlated with significant increased MYC expression in IDH-mut gliomas (p=3.1×10-13), as well as alterations of Myc signaling targets. Publicly available ATACseq, ChIPseq and long-range DNA interaction data demonstrated that the rs55705857 locus is open and interacts with the MYC promoter. CONCLUSIONS Fine-mapping of the 8q24 locus provided strong evidence that rs55705857 is the causative 8q24 locus associated with IDH-mut glioma. Functional experiments suggest that IDH mutation facilitates rs55705857 interaction with MYC to alter downstream MYC targets.

CRISPR-Cas9 is an efficient and versatile tool for genome engineering in many species. However, inducible CRISPR-Cas9 editing systems that regulate Cas9 activity or sgRNA expression often suffer from significant limitations, including reduced editing capacity, off-target effects, or leaky expression. Here, we develop a precisely controlled sgRNA expression cassette that can be combined with widely-used Cre systems, termed CRISPR-Switch (SgRNA With Induction/Termination by Cre Homologous recombination). Switch-ON facilitates controlled, rapid induction of sgRNA activity. In turn, Switch-OFF-mediated termination of editing improves generation of heterozygous genotypes and can limit off-target effects. Furthermore, we design sequential CRISPR-Switch-based editing of two loci in a strictly programmable manner and determined the order of mutagenic events that leads to development of glioblastoma in mice. Thus, CRISPR-Switch substantially increases the versatility of gene editing through precise and rapid switching ON or OFF sgRNA activity, as well as switching OVER to secondary sgRNAs.

The repositioning or “repurposing” of existing therapies for alternative disease indications is an attractive approach that can save significant investments of time and money during drug development. For cancer indications, the primary goal of repurposed therapies is on efficacy, with less restriction on safety due to the immediate need to treat this patient population. This report provides a high-level overview of how drug developers pursuing repurposed assets have previously navigated funding efforts, regulatory affairs, and intellectual property laws to commercialize these “new” medicines in oncology. This article provides insight into funding programs (e.g., government grants and philanthropic organizations) that academic and corporate initiatives can leverage to repurpose drugs for cancer. In addition, we highlight previous examples where secondary uses of existing, Food and Drug Administration- or European Medicines Agency-approved therapies have been predicted in silico and successfully validated in vitro and/or in vivo (i.e., animal models and human clinical trials) for certain oncology indications. Finally, we describe the strategies that the pharmaceutical industry has previously employed to navigate regulatory considerations and successfully commercialize their drug products. These factors must be carefully considered when repurposing existing drugs for cancer to best benefit patients and drug developers alike.

A leading hypothesis for the evolutionary maintenance of sexual reproduction proposes that sex is advantageous because it facilitates adaptation. Changes in the environment stimulate adaptation but not all changes are equivalent; a change may occur along one or multiple environmental dimensions. In two evolution experiments with the facultatively sexual rotifer Brachionus calyciflorus, we test how environmental complexity affects the evolution of sex by adapting replicate populations to various environments that differ from the original along one, two, or three environmental dimensions. Three different estimates of fitness (growth, lifetime reproduction, and population density) confirmed that populations adapted to their new environment. Growth measures revealed an intriguing cost of complex adaptations: populations that adapted to more complex environments lost greater amounts of fitness in the original environment. Furthermore, both experiments showed that B. calyciflorus became more sexual when adapting to a greater number of environmental dimensions. Common garden experiments confirmed that observed changes in sex were heritable. As environments in nature are inherently complex these findings help explain why sex is maintained in natural populations.