No abstract

Assays to study cancer cell responses to pharmacologic or genetic perturbations are typically restricted to using simple phenotypic readouts such as proliferation rate. Information-rich assays, such as gene-expression profiling, have generally not permitted efficient profiling of a given perturbation across multiple cellular contexts. Here, we develop MIX-Seq, a method for multiplexed transcriptional profiling of post-perturbation responses across a mixture of samples with single-cell resolution, using SNP-based computational demultiplexing of singlecell RNA-sequencing data. We show that MIX-Seq can be used to profile responses to chemical or genetic perturbations across pools of 100 or more cancer cell lines. We combine it with Cell Hashing to further multiplex additional experimental conditions, such as posttreatment time points or drug doses. Analyzing the high-content readout of scRNA-seq reveals both shared and context-specific transcriptional response components that can identify drug mechanism of action and enable prediction of long-term cell viability from shortterm transcriptional responses to treatment.

Despite advances in precision medicine, the clinical prospects for patients with ovarian and uterine cancers have not substantially improved. Here, we analyzed genome-scale CRISPR/Cas9 loss-of-function screens across 851 human cancer cell lines and found that frequent overexpression of SLC34A2 – encoding a phosphate importer – is correlated to sensitivity to loss of the phosphate exporter XPR1 in vitro and in vivo. In patient-derived tumor samples, we observed frequent PAX8-dependent overexpression of SLC34A2 , XPR1 copy number amplifications, and XPR1 mRNA overexpression. Mechanistically, in SLC34A2 -high cancer cell lines, genetic or pharmacologic inhibition of XPR1-dependent phosphate efflux leads to the toxic accumulation of intracellular phosphate. Finally, we show that XPR1 requires the novel partner protein KIDINS220 for proper cellular localization and activity, and that disruption of this protein complex results in acidic vacuolar structures preceding cell death. These data point to the XPR1:KIDINS220 complex and phosphate dysregulation as a therapeutic vulnerability in ovarian cancer.

Abstract Tunicates, the closest living relatives of vertebrates, have served as a foundational model of early embryonic development for decades. Comparative studies of tunicate phylogeny and genome evolution provide a critical framework for analyzing chordate diversification and the emergence of vertebrates. Toward this goal, we sequenced the genome of Corella inflata (Ascidiacea, Phlebobranchia), so named for the capacity to brood self-fertilized embryos in a modified, “inflated” atrial chamber. Combining the new genome sequence for Co. inflata with publicly available tunicate data, we estimated a tunicate species phylogeny, reconstructed the ancestral Hox gene cluster at important nodes in the tunicate tree, and compared patterns of gene loss between Co. inflata and Ciona robusta, the prevailing tunicate model species. Our maximum-likelihood and Bayesian trees estimated from a concatenated 210-gene matrix were largely concordant and showed that Aplousobranchia was nested within a paraphyletic Phlebobranchia. We demonstrated that this relationship is not an artifact due to compositional heterogeneity, as had been suggested by previous studies. In addition, within Thaliacea, we recovered Doliolida as sister to the clade containing Salpida and Pyrosomatida. The Co. inflata genome provides increased resolution of the ancestral Hox clusters of key tunicate nodes, therefore expanding our understanding of the evolution of this cluster and its potential impact on tunicate morphological diversity. Our analyses of other gene families revealed that several cardiovascular associated genes (e.g., BMP10, SCL2A12, and PDE2a) absent from Ci. robusta, are present in Co. inflata. Taken together, our results help clarify tunicate relationships and the genomic content of key ancestral nodes within this phylogeny, providing critical insights into tunicate evolution.

Endosomal trafficking of receptors and associated proteins plays a critical role in signal processing. Until recently, it was thought that trafficking was shut down during cell division. Thus, remarkably, the regulation of trafficking during division remains poorly characterized. Here we delineate the role of mitotic kinases in receptor trafficking during asymmetric division. Targeted perturbations reveal that Cyclin-dependent Kinase 1 (CDK1) and Aurora Kinase promote storage of Fibroblast Growth Factor Receptors (FGFRs) by suppressing endosomal degradation and recycling pathways. As cells progress through metaphase, loss of CDK1 activity permits differential degradation and targeted recycling of stored receptors, leading to asymmetric induction. Mitotic receptor storage, as delineated in this study, may facilitate rapid reestablishment of signaling competence in nascent daughter cells. However, mutations that limit or enhance the release of stored signaling components could alter daughter cell fate or behavior thereby promoting oncogenesis.