Adipocyte-like signature in ovarian cancer minimal residual disease identifies metabolic vulnerabilities of tumor-initiating cells.

Similar to tumor-initiating cells (TICs), minimal residual disease (MRD) is capable of reinitiating tumors and causing recurrence. However, the molecular characteristics of solid tumor MRD cells and drivers of their survival have remained elusive. Here we performed dense multiregion transcriptomics analysis of paired biopsies from 17 ovarian cancer patients before and after chemotherapy. We reveal that while MRD cells share important molecular signatures with TICs, they are also characterized by an adipocyte-like gene expression signature and a portion of them had undergone epithelial-mesenchymal transition (EMT). In a cell culture MRD model, MRD-mimic cells showed the same phenotype and were dependent on fatty acid oxidation (FAO) for survival and resistance to cytotoxic agents. These findings identify EMT and FAO as attractive targets to eradicate MRD in ovarian cancer and make a compelling case for the further testing of FAO inhibitors in treating MRD.

A highly accurate platform for clone-specific mutation discovery enables the study of active mutational processes.

Bulk whole genome sequencing (WGS) enables the analysis of tumor evolution but, because of depth limitations, can only identify old mutational events. The discovery of current mutational processes for predicting the tumor's evolutionary trajectory requires dense sequencing of individual clones or single cells. Such studies, however, are inherently problematic because of the discovery of excessive false positive (FP) mutations when sequencing picogram quantities of DNA. Data pooling to increase the confidence in the discovered mutations, moves the discovery back in the past to a common ancestor. Here we report a robust WGS and analysis pipeline (DigiPico/MutLX) that virtually eliminates all F results while retaining an excellent proportion of true positives. Using our method, we identified, for the first time, a hyper-mutation (kataegis) event in a group of ∼30 cancer cells from a recurrent ovarian carcinoma. This was unidentifiable from the bulk WGS data. Overall, we propose DigiPico/MutLX method as a powerful framework for the identification of clone-specific variants at an unprecedented accuracy.

Hyperactive piggyBac transposase improves transformation efficiency in diverse insect species.

Even in times of advanced site-specific genome editing tools, the improvement of DNA transposases is still on high demand in the field of transgenesis: especially in emerging model systems where evaluated integrase landing sites have not yet been created and more importantly in non-model organisms such as agricultural pests and disease vectors, in which reliable sequence information and genome annotations are still pending. In fact, random insertional mutagenesis is essential to identify new genomic locations that are not influenced by position effects and thus can serve as future stable transgene integration sites. In this respect, a hyperactive version of the most widely used piggyBac transposase (PBase) has been engineered. The hyperactive version (hyPBase) is currently available with the original insect codon-based coding sequence (ihyPBase) as well as in a mammalian codon-optimized (mhyPBase) version. Both facilitate significantly higher rates of transposition when expressed in mammalian in vitro and in vivo systems compared to the classical PBase at similar protein levels. Here we demonstrate that the usage of helper plasmids encoding the hyPBase - irrespective of the codon-usage - also strikingly increases the rate of successful germline transformation in the Mediterranean fruit fly (Medfly) Ceratitis capitata, the red flour beetle Tribolium castaneum, and the vinegar fly Drosophila melanogaster. hyPBase-encoding helpers are therefore highly suitable for the generation of transgenic strains of diverse insect orders. Depending on the species, we achieved up to 15-fold higher germline transformation rates compared to PBase and generated hard to obtain transgenic T. castaneum strains that express constructs affecting fitness and viability. Moreover, previously reported high sterility rates supposedly caused by hyPBase (iPB7), encoded by ihyPBase, could not be confirmed by our study. Therefore, we value hyPBase as an effective genetic engineering tool that we highly recommend for insect transgenesis.

Loss of PFKFB4 induces cell death in mitotically arrested ovarian cancer cells.

Taxanes represent some of the most commonly used chemotherapeutic agents for ovarian cancer treatment. However, they are only effective in approximately 40% of patients. Novel therapeutic strategies are required to potentiate their effect and improve patient outcome. A hallmark of many cancers is the constitutive activation of the PI3K/AKT pathway, which drives cell survival and metabolism. We discovered a striking decrease in AKT activity coupled with a significant reduction in glucose 6-phosphate and ATP levels during mitotic arrest in the majority of ovarian cancer cell lines tested, indicating a potential metabolic vulnerability. A high-content siRNA screen to detect novel metabolic targets in mitotically arrested ovarian cancer cells identified the glycolytic enzyme PFKFB4. PFKFB4 depletion increased caspase 3/7 activity, and levels of reactive oxygen species only in mitotically arrested cells, and significantly enhanced mitotic cell death after paclitaxel treatment. Depletion of PFKFB3 demonstrated a similar phenotype. The observation that some ovarian cancer cells lose AKT activity during mitotic arrest and become vulnerable to metabolic targeting is a new concept in cancer therapy. Thus, combining mitotic-targeted therapies with glycolytic inhibitors may act to potentiate the effects of antimitotics in ovarian cancer through mitosis-specific cell death.