Glioblastoma evolution and heterogeneity from a 3D whole-tumor perspective.

Treatment failure for the lethal brain tumor glioblastoma (GBM) is attributed to intratumoral heterogeneity and tumor evolution. We utilized 3D neuronavigation during surgical resection to acquire samples representing the whole tumor mapped by 3D spatial coordinates. Integrative tissue and single-cell analysis revealed sources of genomic, epigenomic, and microenvironmental intratumoral heterogeneity and their spatial patterning. By distinguishing tumor-wide molecular features from those with regional specificity, we inferred GBM evolutionary trajectories from neurodevelopmental lineage origins and initiating events such as chromothripsis to emergence of genetic subclones and spatially restricted activation of differential tumor and microenvironmental programs in the core, periphery, and contrast-enhancing regions. Our work depicts GBM evolution and heterogeneity from a 3D whole-tumor perspective, highlights potential therapeutic targets that might circumvent heterogeneity-related failures, and establishes an interactive platform enabling 360° visualization and analysis of 3D spatial patterns for user-selected genes, programs, and other features across whole GBM tumors.

Mammalian RNAi represses pericentromeric lncRNAs to maintain genome stability.

Mammalian pericentromeric tandem repeats produce long noncoding RNAs (lncRNAs) that are dysregulated in cancer and linked to genomic instability. Identifying the basic molecular characteristics of these lncRNAs and their regulation is important to understanding their biological function. Here, we determine that the Argonaute (Ago) proteins of the RNA interference (RNAi) pathway directly and uniformly repress bidirectional pericentromeric lncRNAs in a Dicer-dependent manner in mouse embryonic and adult stem cells. Ago-dependent and Dicer-dependent autoregulatory small RNAs were identified within pericentromeric lncRNA degradation intermediates. We develop an RNase H cleavage assay to determine the relative proportions and lengths of the pericentromeric lncRNA targets. We find that 5'-phosphate and non-polyadenylated bidirectional pericentromeric lncRNAs are expressed at similar proportions. These lncRNAs can span up to 9 repeats, with transcription from the reverse strand template yielding the longer products. Using pericentromeric repeat RNA reporters, we determine that Ago represses pericentromeric lncRNAs after S phase transcription. Upon loss of Ago, pericentromeric lncRNA dysregulation results in delayed cell cycle progression, a defective mitotic spindle assembly checkpoint (SAC) and genomic instability. These results show that an evolutionarily conserved Ago activity at pericentromeres contributes to mammalian genome stability.

Tumor-wide RNA splicing aberrations generate immunogenic public neoantigens.

T-cell-mediated immunotherapies are limited by the extent to which cancer-specific antigens are homogenously expressed throughout a tumor. We reasoned that recurrent splicing aberrations in cancer represent a potential source of tumor-wide and public neoantigens, and to test this possibility, we developed a novel pipeline for identifying neojunctions expressed uniformly within a tumor across diverse cancer types. Our analyses revealed multiple neojunctions that recur across patients and either exhibited intratumor heterogeneity or, in some cases, were tumor-wide. We identified CD8+ T-cell clones specific for neoantigens derived from tumor-wide and conserved neojunctions in GNAS and RPL22 , respectively. TCR-engineered CD8 + T-cells targeting these mutations conferred neoantigen-specific tumor cell eradication. Furthermore, we revealed that cancer-specific dysregulation in splicing factor expression leads to recurrent neojunction expression. Together, these data reveal that a subset of neojunctions are both intratumorally conserved and public, providing the molecular basis for novel T-cell-based immunotherapies that address intratumoral heterogeneity.