Showing papers by "Jason W. H. Wong published in 2020"

Organoid cultures of early-onset colorectal cancers reveal distinct and rare genetic profiles

Abstract: Objective Sporadic early-onset colorectal cancer (EOCRC) has bad prognosis, yet is poorly represented by cell line models. We examine the key mutational and transcriptomic alterations in an organoid biobank enriched in EOCRCs. Design We established paired cancer (n=32) and normal organoids (n=18) from 20 patients enriched in microsatellite-stable EOCRC. Exome and transcriptome analysis was performed. Results We observed a striking diversity of molecular phenotypes, including PTPRK-RSPO3 fusions. Transcriptionally, RSPO fusion organoids resembled normal colon organoids and were distinct from APC mutant organoids, with high BMP2 and low PTK7 expression. Single cell transcriptome analysis confirmed the similarity between RSPO fusion organoids and normal organoids, with a propensity for maturation on Wnt withdrawal, whereas the APC mutant organoids were locked in progenitor stages. CRISPR/Cas9 engineered mutation of APC in normal human colon organoids led to upregulation of PTK7 protein and suppression of BMP2, but less so with an engineered RNF43 mutation. The frequent co-occurrence of RSPO fusions with SMAD4 or BMPR1A mutation was confirmed in TCGA database searches. RNF43 mutation was found in organoid from a leukaemia survivor with a novel mutational signature; and organoids with POLE proofreading mutation displayed ultramutation. The cancer organoid genomes were stable over long culture periods, while normal human colon organoids tended to be subject to clonal dominance over time. Conclusions These organoid models enriched in EOCRCs with linked genomic data fill a gap in existing CRC models and reveal distinct genetic profiles and novel pathway cooperativity.

RNA Splicing Alterations Induce a Cellular Stress Response Associated with Poor Prognosis in Acute Myeloid Leukemia

Abstract: Purpose: RNA splicing is a fundamental biological process that generates protein diversity from a finite set of genes. Recurrent somatic mutations of splicing factor genes are common in some hematological cancers but are relatively uncommon in Acute Myeloid Leukemia (AML,

Mutational processes of distinct POLE exonuclease domain mutants drive an enrichment of a specific TP53 mutation in colorectal cancer.

Abstract: Cancer genomes with mutations in the exonuclease domain of Polymerase Epsilon (POLE) present with an extraordinarily high somatic mutation burden. In vitro studies have shown that distinct POLE mutants exhibit different polymerase activity. Yet, genome-wide mutation patterns and driver mutation formation arising from different POLE mutants remains unclear. Here, we curated somatic mutation calls from 7,345 colorectal cancer samples from published studies and publicly available databases. These include 44 POLE mutant samples including 9 with whole genome sequencing data available. The POLE mutant samples were categorized based on the specific POLE mutation present. Mutation spectrum, associations of somatic mutations with epigenomics features and co-occurrence with specific driver mutations were examined across different POLE mutants. We found that different POLE mutants exhibit distinct mutation spectrum with significantly higher relative frequency of C>T mutations in POLE V411L mutants. Our analysis showed that this increase frequency in C>T mutations is not dependent on DNA methylation and not associated with other genomic features and is thus specifically due to DNA sequence context alone. Notably, we found strong association of the TP53 R213* mutation specifically with POLE P286R mutants. This truncation mutation occurs within the TT[C>T]GA context. For C>T mutations, this sequence context is significantly more likely to be mutated in POLE P286R mutants compared with other POLE exonuclease domain mutants. This study refines our understanding of DNA polymerase fidelity and underscores genome-wide mutation spectrum and specific cancer driver mutation formation observed in POLE mutant cancers.

Disruption of a GATA2, TAL1, ERG regulatory circuit promotes erythroid transition in healthy and leukemic stem cells

Abstract: Changes in gene regulation and expression govern orderly transitions from hematopoietic stem cells to terminally differentiated blood cell types. These transitions are disrupted during leukemic transformation but knowledge of the gene regulatory changes underpinning this process is elusive. We hypothesised that identifying core gene regulatory networks in healthy hematopoietic and leukemic cells could provide insights into network alterations that perturb cell state transitions. A heptad of transcription factors (LYL1, TAL1, LMO2, FLI1, ERG, GATA2, RUNX1) bind key hematopoietic genes in human CD34+ haematopoietic stem and progenitor cells (HSPCs) and have prognostic significance in acute myeloid leukemia (AML). These factors also form a densely interconnected circuit by binding combinatorially at their own, and each other′s, regulatory elements. However, their mutual regulation during normal haematopoiesis and in AML cells, and how perturbation of their expression levels influences cell fate decisions remains unclear. Here, we integrated bulk and single cell data and found that the fully connected heptad circuit identified in healthy HSPCs persists with only minor alterations in AML, and that chromatin accessibility at key heptad regulatory elements was predictive of cell identity in both healthy progenitors and in leukemic cells. The heptad factors GATA2, TAL1 and ERG formed an integrated sub-circuit that regulates stem cell to erythroid transition in both healthy and leukemic cells. Components of this triad could be manipulated to facilitate erythroid transition providing a proof of concept that such regulatory circuits could be harnessed to promote specific cell type transitions and overcome dysregulated haematopoiesis.

Deficiency in DNA mismatch repair of methylation damage is a major mutational process in cancer

Abstract: DNA mismatch repair (MMR) is essential for maintaining genome integrity with its deficiency predisposing to cancer1. MMR is well known for its role in the post-replicative repair of mismatched base pairs that escape proofreading by DNA polymerases following cell division2. Yet, cancer genome sequencing has revealed that MMR deficient cancers not only have high mutation burden but also harbour multiple mutational signatures3, suggesting that MMR has pleotropic effects on DNA repair. The mechanisms underlying these mutational signatures have remained unclear despite studies using a range of in vitro4,5 and in vivo6 models of MMR deficiency. Here, using mutation data from cancer genomes, we identify a previously unknown function of MMR, showing that the loss of non-canonical replication-independent MMR activity is a major mutational process in human cancers. MMR is comprised of the MutSα (MSH2/MSH6) and MutLα (MLH1/PMS2) complexes7. Cancers with deficiency of MutSα exhibit mutational signature contributions distinct from those deficient of MutLα. This disparity is attributed to mutations arising from the unrepaired deamination of 5-methylcytosine (5mC), i.e. methylation damage, as opposed to replicative errors by DNA polymerases induced mismatches. Repair of methylation damage is strongly associated with H3K36me3 chromatin but independent of binding of MBD4, a DNA glycosylase that recognise 5mC and can repair methylation damage. As H3K36me3 recruits MutSα, our results suggest that MutSα is the essential factor in mediating the repair of methylation damage. Cell line models of MMR deficiency display little evidence of 5mC deamination-induced mutations as their rapid rate of proliferation limits for the opportunity for methylation damage. We thus uncover a non-canonical role of MMR in the protection against methylation damage in non-dividing cells.

Induction of Muscle Regenerative Multipotent Stem Cells from Human Adipocytes by PDGF-AB and 5-Azacytidine

Abstract: Terminally differentiated murine osteocytes and adipocytes can be reprogrammed using platelet-derived growth factor–AB and 5-Azacytidine into multipotent stem cells with stromal cell characteristics. To generate a product that is amenable for therapeutic application, we have modified and optimised culture conditions to reprogram human adipocytes into induced multipotent stem cells (iMS) and expand them in vitro. The basal transcriptomes of adipocyte-derived iMS cells and matched adipose-tissue-derived mesenchymal stem cells were remarkably similar. However, there were distinct changes in histone modifications and CpG methylation at cis-regulatory regions consistent with an epigenetic landscape that was primed for tissue development and differentiation. In a non-specific tissue injury xenograft model, iMS cells contributed directly to new muscle, bone, cartilage and blood vessels with no evidence of teratogenic potential. In a cardiotoxin muscle injury model, iMS cells contributed specifically to satellite cells and myofibres without ectopic tissue formation. Taken together, human adipocyte derived iMS cells regenerate tissues in a context dependent manner without ectopic or neoplastic growth.

Overexpression of transposable elements is associated with immune overdrive and poor clinical outcome in colorectal cancer patients

Abstract: Objective The immune system plays a key role in protecting against cancer. Increased immune infiltration in tumor tissue is usually associated with improved clinical outcome, but in colorectal cancer (CRC), excessive immune infiltration has also been shown to lead to worst prognosis. The factors underlying this immune overdrive phenotype remains unknown. Design Using RNA sequencing data from The Cancer Genome Atlas, the expression of over 1,000 transposable element (TE) subfamilies were quantified using the “REdiscoverTE” pipeline. Candidate prognostic and immunogenic TEs were screened by survival and correlation analysis, respectively. Based on these candidates, a TE expression score was developed and CRC patients were clustered using the “kaps” algorithm. Results In CRC, we found that the TE expression score stratified patients into four clusters each with distinctive prognosis. Those with the highest TE expression were associated with immune overdrive and had the poorest outcomes. Importantly, this association was independent of microsatellite instability status and tumor mutation burden. To link TE overexpression to the immune overdrive phenotype, we showed that cell lines treated with DNA methyltransferase inhibitors also had a high TE expression score and activation of cellular innate immune response pathways. Finally, a pan-cancer survey of TE expression identified a subset of kidney renal clear cell carcinoma with a similar adverse immune overdrive phenotype with poor prognosis. Conclusion Our findings reveal that TE expression is associated with immune overdrive in cancer and is an independent predictor of immune infiltration and prognosis in CRC patients. 1. What is already known about this subject? Cancers with high immune infiltration generally have better prognosis, but it is unknown why a subset of colorectal cancers (CRC) with high immune infiltration have the poorest outcomes. Transposable element (TE) expression has been shown to be strongly associated with immune infiltration in cancers but its role in patient prognosis is unclear. TEs can be reactivated by DNA hypomethylation in cancers, resulting in immune response via viral mimicry. 2. What are the new findings? A TE expression score has been developed that is predicative of prognosis in CRC patients where those who have the highest TE score show an immune overdrive phenotype and have the worst prognosis. The TE expression score predicts prognostic and immune infiltration independent of microsatellite instability and tumor mutation burden (TMB). Immune response pathways and infiltrate profiles of high TE expression CRC recapitulates those of DNA methyltransferase inhibitor treated cells where TEs are reactivated, suggesting that TE overexpression may drive immune infiltration in CRC. A pan-cancer analysis found that kidney renal clear cell carcinoma shares are a similar TE expression associated immune overdrive phenotype with adverse prognosis. 3. How might it impact on clinical practice in the foreseeable future? Our work highlights the importance of TE expression in evaluating CRC patient prognosis. The association of TE expression with the immune overdrive phenotype independent of MSI and TMB status suggests that by considering TE expression, there may be new opportunities to identify MSS CRC patients for immunotherapy and develop new strategies to harness TE driven immune response.

RNA splicing alterations induce a cellular stress response associated with poor prognosis in AML

Abstract: RNA splicing is a fundamental biological process that generates protein diversity from a finite set of genes. Recurrent somatic mutations of splicing factor genes are relatively uncommon in Acute Myeloid Leukemia (AML,