SUMMARY Apicomplexan parasites are leading causes of human and livestock diseases—like malaria and toxoplasmosis—yet most of their genes remain uncharacterized. Here, we present the first genome-wide genetic screen of an apicomplexan. We adapted CRISPR/Cas9 to assess the contribution of each gene from the human parasite Toxoplasma gondii during infection of fibroblasts. Our analysis defines ~200 previously uncharacterized, fitness-conferring genes unique to the phylum, from which 16 were investigated, revealing essential functions during infection of human cells. Secondary screens identify as an invasion factor the claudin-like apicomplexan microneme protein (CLAMP), which resembles mammalian tight-junction proteins and localizes to secretory organelles, making it critical to the initiation of infection. CLAMP is present throughout sequenced apicomplexan genomes, and is essential during the asexual stages of the malaria parasite Plasmodium falciparum. These results provide broad-based functional information on T. gondii genes and will facilitate future approaches to expand the horizon of antiparasitic interventions.

As the concentrations of highly consumed nutrients, particularly glucose, are generally lower in tumours than in normal tissues1,2, cancer cells must adapt their metabolism to the tumour microenvironment. A better understanding of these adaptations might reveal cancer cell liabilities that can be exploited for therapeutic benefit. Here, we developed a continuous flow culture apparatus (Nutrostat) for maintaining proliferating cells in low nutrient media for long periods of time and used it to undertake competitive proliferation assays on a pooled collection of barcoded cancer cell lines cultured in low glucose conditions. Sensitivity to low glucose varies amongst cell lines, and an RNAi screen pinpointed mitochondrial oxidative phosphorylation (OXPHOS) as the major pathway required for optimal proliferation in low glucose. We found that cell lines most sensitive to low glucose are defective in the upregulation of OXPHOS normally caused by glucose limitation as a result of either mtDNA mutations in Complex I genes or impaired glucose utilization. These defects predict sensitivity to biguanides, anti-diabetic drugs that inhibit OXPHOS3,4, when cancer cells are grown in low glucose or as tumour xenografts. Remarkably, the biguanide sensitivity of cancer cells with mtDNA mutations was reversed by ectopic expression of yeast NDI1, a ubiquinone oxidoreductase that allows bypass of Complex I function5. Thus, we conclude that mtDNA mutations and impaired glucose utilization are potential biomarkers for identifying tumours with increased sensitivity to OXPHOS inhibitors.

Carcinoma cells residing in an intermediate phenotypic state along the epithelial–mesenchymal (E–M) spectrum are associated with malignant phenotypes, such as invasiveness, tumor-initiating ability, and metastatic dissemination. Using the recently described CD104+/CD44hi antigen marker combination, we isolated highly tumorigenic breast cancer cells residing stably—both in vitro and in vivo—in an intermediate phenotypic state and coexpressing both epithelial (E) and mesenchymal (M) markers. We demonstrate that tumorigenicity depends on individual cells residing in this E/M hybrid state and cannot be phenocopied by mixing two cell populations that reside stably at the two ends of the spectrum, i.e., in the E and in the M state. Hence, residence in a specific intermediate state along the E–M spectrum rather than phenotypic plasticity appears critical to the expression of tumor-initiating capacity. Acquisition of this E/M hybrid state is facilitated by the differential expression of EMT-inducing transcription factors (EMT-TFs) and is accompanied by the expression of adult stem cell programs, notably, active canonical Wnt signaling. Furthermore, transition from the highly tumorigenic E/M state to a fully mesenchymal phenotype, achieved by constitutive ectopic expression of Zeb1, is sufficient to drive cells out of the E/M hybrid state into a highly mesenchymal state, which is accompanied by a substantial loss of tumorigenicity and a switch from canonical to noncanonical Wnt signaling. Identifying the gatekeepers of the various phenotypic states arrayed along the E–M spectrum is likely to prove useful in developing therapeutic approaches that operate by shifting cancer cells between distinct states along this spectrum.

Ribosomal protein haploinsufficiency occurs in diverse human diseases including Diamond-Blackfan anemia (DBA),1,2 congenital asplenia,3 and T-cell leukemia.4 Yet how mutations in such ubiquitously expressed proteins result in cell-type and tissue specific defects remains a mystery.5 Here, we show that GATA1 mutations that reduce full-length protein levels of this critical hematopoietic transcription factor can cause DBA in rare instances. We show that ribosomal protein haploinsufficiency, the more common cause of DBA, can similarly reduce translation of GATA1 mRNA - a phenomenon that appears to result from this mRNA having a higher threshold for initiation of translation. In primary hematopoietic cells from patients with RPS19 mutations, a transcriptional signature of GATA1 target genes is globally and specifically reduced, confirming that the activity, but not the mRNA level, of GATA1 is reduced in DBA patients with ribosomal protein mutations. The defective hematopoiesis observed in DBA patients with ribosomal protein haploinsufficiency can be at least partially overcome by increasing GATA1 protein levels. Our results provide a paradigm by which selective defects in translation due to mutations in ubiquitous ribosomal proteins can result in human disease.

A list of authors and their affiliations appears at the end of the paperWe present a draft genome sequence of the platypus, Ornithorhynchus anatinus. This monotreme exhibits a fascinating combination of reptilian and mammalian characters. For example, platypuses have a coat of fur adapted to an aquatic lifestyle; platypus females lactate, yet lay eggs; and males are equipped with venom similar to that of reptiles. Analysis of the first monotreme genome aligned these features with genetic innovations. We find that reptile and platypus venom proteins have been co-opted independently from the same gene families; milk protein genes are conserved despite platypuses laying eggs; and immune gene family expansions are directly related to platypus biology. Expansions of protein, non-protein-coding RNA and microRNA families, as well as repeat elements, are identified. Sequencing of this genome now provides a valuable resource for deep mammalian comparative analyses, as well as for monotreme biology and conservation.

Diamond-Blackfan anemia (DBA) is a hypoplastic anemia characterized by impaired production of red blood cells, with approximately half of all cases attributed to ribosomal protein gene mutations. We performed exome sequencing on two siblings who had no known pathogenic mutations for DBA and identified a mutation in the gene encoding the hematopoietic transcription factor GATA1. This mutation, which occurred at a splice site of the GATA1 gene, impaired production of the full-length form of the protein. We further identified an additional patient carrying a distinct mutation at the same splice site of the GATA1 gene. These findings provide insight into the pathogenesis of DBA, showing that the reduction in erythropoiesis associated with the disease can arise from causes other than defects in ribosomal protein genes. These results also illustrate the multifactorial role of GATA1 in human hematopoiesis.

The mechanisms by which cells adapt to proteotoxic stress are largely unknown, but key to understanding how tumor cells, particularly in vivo, are largely resistant to proteasome inhibitors. Analysis of cancer cell lines, mouse xenografts and patient-derived tumor samples all showed an association between mitochondrial metabolism and proteasome inhibitor sensitivity. When cells were forced to use oxidative phosphorylation rather than glycolysis, they became proteasome inhibitor-resistant. This mitochondrial state, however, creates a unique vulnerability: sensitivity to the small-molecule compound elesclomol. Genome-wide CRISPR/Cas9 screening showed that a single gene, encoding the mitochondrial reductase FDX1, could rescue elesclomol-induced cell death. Enzymatic function and NMR-based analyses further showed that FDX1 is the direct target of elesclomol, which promotes a unique form of copper-dependent cell death. These studies elucidate a fundamental mechanism by which cells adapt to proteotoxic stress and suggests strategies to mitigate proteasome inhibitor-resistance.

The epithelial-to-mesenchymal transition (EMT) enables carcinoma cells to acquire malignancy-associated traits and properties of tumor-initiating cells (TICs). TICs have emerged in recent years as important targets for cancer therapy owing to their ability to drive clinical relapse and enable metastasis. Here we propose a strategy to eliminate mesenchymal TICs by inducing their conversion to more epithelial counterparts that have lost tumor-initiating ability. We report that increases in intracellular levels of the second messenger, cAMP, and the subsequent activation of protein kinase A (PKA) induce a mesenchymal-to-epithelial transition (MET) in mesenchymal human mammary epithelial cells. Activation of PKA triggers PHF2-mediated epigenetic reprogramming of TICs, promoting their differentiation that leads to loss of tumor-initiating ability. This study provides proof-of-principle for inducing an MET as differentiation therapy for TICs and uncovers a novel role for PKA in enforcing and maintaining the epithelial state.