Metastases account for 90% of cancer-related deaths; thus, it is vital to understand the biology of tumour dissemination. Here, we collected and monitored >50 patient specimens ex vivo to investigate the cell biology of colorectal cancer (CRC) metastatic spread to the peritoneum. This reveals an unpredicted mode of dissemination. Large clusters of cancer epithelial cells displaying a robust outward apical pole, which we termed tumour spheres with inverted polarity (TSIPs), were observed throughout the process of dissemination. TSIPs form and propagate through the collective apical budding of hypermethylated CRCs downstream of canonical and non-canonical transforming growth factor-β signalling. TSIPs maintain their apical-out topology and use actomyosin contractility to collectively invade three-dimensional extracellular matrices. TSIPs invade paired patient peritoneum explants, initiate metastases in mice xenograft models and correlate with adverse patient prognosis. Thus, despite their epithelial architecture and inverted topology TSIPs seem to drive the metastatic spread of hypermethylated CRCs.

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Large-scale sequencing studies of hematologic malignancies have revealed notable epistasis among high-frequency mutations. One of the most striking examples of epistasis occurs for mutations in RNA splicing factors. These lesions are amongst the most common alterations in myeloid neoplasms and generally occur in a mutually exclusive manner, a finding attributed to their synthetic lethal interactions and/or convergent effects. Curiously, however, patients with multiple concomitant splicing factor mutations have been observed, challenging our understanding of one of the most common examples of epistasis in hematologic malignancies. Here we performed bulk and single cell analyses of myeloid malignancy patients harboring >2 splicing factor mutations to understand the frequency and basis for the co-existence of these mutations. Although mutations in splicing factors were strongly mutually exclusive across 4,231 patients (q<0.001), 0.85% harbored two concomitant bona fide splicing factor mutations, ~50% of which were present in the same individual cells. However, the distribution of mutations in double mutants deviated from those in single mutants with selection against the most common alleles, SF3B1K700E and SRSF2P95H/L/R, and selection for less common alleles, such as SF3B1 non-K700E mutations, rare amino acid substitutions at SRSF2P95, and combined U2AF1S34/Q157 mutations. SF3B1 and SRSF2 alleles enriched in double mutants had reduced effects on RNA splicing and/or binding compared to the most common alleles. Moreover, dual U2AF1 mutations occurred in cis with preservation of the wild-type allele. These data highlight allele-specific differences as critical in regulating molecular effects of splicing factor mutations as well as their co-occurrences/exclusivities with one another.

Mutations in genes encoding RNA splicing factors were discovered nearly ten years ago and are now understood to be amongst the most recurrent genetic abnormalities in patients with all forms of myeloid neoplasms and several types of lymphoproliferative disorders as well as subjects with clonal hematopoiesis. These discoveries implicate aberrant RNA splicing, the process by which precursor RNA is converted into mature messenger RNA, in the development of clonal hematopoietic conditions. Both the protein as well as the RNA components of the splicing machinery are affected by mutations at highly specific residues and a number of these mutations alter splicing in a manner distinct from loss of function. Importantly, cells bearing these mutations have now been shown to generate mRNA species with novel aberrant sequences, some of which may be critical to disease pathogenesis and/or novel targets for therapy. These findings have opened new avenues of research to understand biological pathways disrupted by altered splicing. In parallel, multiple studies have revealed that cells bearing change-of-function mutation in splicing factors are preferentially sensitized to any further genetic or chemical perturbations of the splicing machinery. These discoveries are now being pursued in several early phase clinical trials using molecules with diverse mechanisms of action. Here we review the molecular effects of splicing factor mutations on splicing, mechanisms by which these mutations drive clonal transformation of hematopoietic cells, and the development of new therapeutics targeting these genetic subsets of hematopoietic malignancies.

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