Summary StatementThe generation and transcriptome analysis of the first induced pluripotent stem cells from the platypus reveals SOX2 has been a key driver of the expanded pluripotency regulatory network in placental mammals.ABSTRACTThe mechanisms by which pluripotency has evolved remain unclear. To gain insight into the evolution of mammalian pluripotency we have generated induced pluripotent stem cells (piPSCs) from the platypus. Deep sequencing of the piPSC transcriptome revealed that piPSCs robustly express the core eutherian pluripotency factors OCT4, SOX2 and NANOG. Given the more extensive role of SOX3 over SOX2 in avian pluripotency, our data indicate that between 315 million years and 166 million years ago primitive mammals replaced the role of SOX3 in the vertebrate pluripotency network with SOX2. DAX1/NR0B1 is not expressed in piPSCs and an analysis of the platypus DAX1 promoter revealed the absence of a proximal SOX2-binding DNA motif known to be critical for DAX1 expression in eutherian pluripotent stem cells, suggesting that the acquisition of SOX2 responsiveness by DAX1 has facilitated its recruitment into the pluripotency network of eutherians. We further show that the expression ratio of X chromosomes to autosomes (X1-5 X1-5:AA) is approximately equal to 1 indicating that there is no upregulation of X-linked genes and that there is no preference for silencing of maternal or paternal alleles (ie imprinting).

The Australian Zero Childhood Cancer (ZERO) program aims to assess the feasibility of a comprehensive precision medicine approach to improve outcomes for patients with an expected survival <30%. ZERO combines molecular profiling (whole genome sequencing, whole transcriptome sequencing, DNA methylation profiling) with in vitro high-throughput drug screening (HTS) and patient-derived xenograft drug efficacy testing. We report on the cohort of patients with midline high-grade glioma (HGG), including H3-K27M DMG, enrolled on the pilot study (TARGET) and on the ongoing ZERO clinical trial (PRISM). We identified 48 patients with midline HGG. Fresh or cryopreserved samples were submitted in 37 cases and cell culture was attempted in 30/37 cases with 45% success rate. The most commonly mutated genes/pathways identified by molecular profiling include H3-K27M mutations, DNA repair pathway, and PI3K/mTOR pathway. Two targetable fusions (NTRK and FGFR1) were reported. Five patients with germline alterations were identified. Thirty-five (72%) patients received a therapeutic recommendation from the ZERO molecular tumour board and the main recommended therapies were mTOR inhibitors, PARP inhibitors or tyrosine kinase inhibitors. HTS added evidence for the recommended therapy (n=3) or identified novel potential therapy (n=1). Out of the 35 patients, 16 received a recommended drug. Response to treatment was complete response for five months (n=1), partial response for nine months (n=1), stable disease (n=4), and progressive disease (n=10). These results highlight the feasibility of the ZERO platform and the value of fresh biopsy, necessary for pre-clinical drug testing. Targetable alterations were identified leading to clinical benefit in six patients.