Lily Cabrera Cosme (Kent Lab) & Diogo Candeias (Hamilton Lab) - University of York

Lily Cabrera Cosme (Kent Lab) & Diogo Candeias (Hamilton Lab) - University of York

Talk 1: Preleukaemic disorders are primarily a cause of the accumulation of somatic mutations in haematopoeietic stem cell (HSCs). Mutations in the epigenetic enzymes, ten-eleven translocase 2 (TET2) and DNA methyl transferase 3 (DNMT3A) are amongst the most frequent in myeloid malignancies. Although these have been extensively studied at the bulk population level, clonal characterisation studies are still lacking. We have developed a clonal competition method that has allowed the identification of rare competitive clone phenotypes of TET2- and DNMT3A-mutant models. Likewise, since our method enables splitting of single HSC-derived clones, we have also been able to perform cross-treatment of clones with different molecules. Together, these experimental applications of our method allow for scrutinisation of clonal dynamics of cell competition at the single-cell level.

Talk 2: Patients with biallelic mutations in the gene SUPV3L1 present with demyelination, neuroinflammation, and progressive loss of cognitive and motor function. SUPV3L1 is a double-stranded RNA helicase that localises to the mitochondria, where it prevents the accumulation of dsRNA, a known immunogenic molecule, generated during mitochondrial genome transcription.
Genetic targeting of supv3l1 in a zebrafish model resulted in impaired locomotion, increased lethality, accumulation of dsRNA, and a 2000-fold increase in type 1 interferon-controlled genes.  Activation of type 1 interferon was also found in blood samples of 4 tested patients. How mitochondrial dsRNA accumulation can trigger an antiviral immune response, leading to the pathophysiology, remains unclear. 
Using a type I interferon-deficient zebrafish mutant, we showed that blocking antiviral immunity is sufficient to rescue the pathology. To establish which cells are promoting neuroinflammation, we combined locomotion assays, live imaging, flow cytometry and cell depletion assays to identify the microglia as the key drivers of neuroinflammation in supv3l1-deficient zebrafish. Next, we performed a targeted genetic screen against known dsRNA receptors, which found the endolysosomal dsRNA receptor TLR3 as a driver of the pathology. To understand how mitochondrial dsRNA reaches the endolysosome, we are investigating microglial cell-autonomous mitochondria fission and efferocytosis. Our data so far points to a dual mechanism by which dsRNA is trafficked to the lysosome by the phagocytosis of dying cells in the brain and cell-autonomous mitochondrial recycling.
Overall, our work has established the first in vivo models of a novel white matter disorder, which have allowed us to identify the role of type 1 interferon in disease progression, TLR3 as a possible drug target and microglia as the drivers of the pathology. To our knowledge, this is the first report of TLR3 detecting mitochondrial dsRNA through mitophagy. We are therefore developing SUPV3L1 iPSC-derived microglia to validate our findings in human models.