Tumor progression involves the ability of cancer cells to communicate with each other and with neighboring normal cells in their microenvironment. Microvesicles (MV) derived from human cancer cells have received a good deal of attention because of their ability to participate in the horizontal transfer of signaling proteins between cancer cells and to contribute to their invasive activity. Here we show that MV may play another important role in oncogenesis. In particular, we demonstrate that MV shed by two different human cancer cells, MDAMB231 breast carcinoma cells and U87 glioma cells, are capable of conferring onto normal fibroblasts and epithelial cells the transformed characteristics of cancer cells (e.g., anchorage-independent growth and enhanced survival capability) and that this effect requires the transfer of the protein cross-linking enzyme tissue transglutaminase (tTG). We further demonstrate that tTG is not sufficient to transform fibroblasts but rather that it must collaborate with another protein to mediate the transforming actions of the cancer cell-derived MV. Proteomic analyses of the MV derived from MDAMB231 and U87 cells indicated that both these vesicle preparations contained the tTG-binding partner and cross-inking substrate fibronectin (FN). Moreover, we found that tTG cross-links FN in MV from cancer cells and that the ensuing MV-mediated transfers of cross-linked FN and tTG to recipient fibroblasts function cooperatively to activate mitogenic signaling activities and to induce their transformation. These findings highlight a role for MV in the induction of cellular transformation and identify tTG and FN as essential participants in this process.

There is an urgent need to create novel models using human disease-relevant cells to study SARS-CoV-2 biology and to facilitate drug screening. As SARS-CoV-2 primarily infects the respiratory tract, we developed a lung organoid model using human pluripotent stem cells (hPSC-LOs). The hPSC-LOs, particularly alveolar type II-like cells, are permissive to SARS-CoV-2 infection, and showed robust induction of chemokines upon SARS-CoV-2 infection, similar to what is seen in COVID-19 patients. Nearly 25% of these patients also have gastrointestinal manifestations, which are associated with worse COVID-19 outcomes 1. We therefore also generated complementary hPSC-derived colonic organoids (hPSC-COs) to explore the response of colonic cells to SARS-CoV-2 infection. We found that multiple colonic cell types, especially enterocytes, express ACE2 and are permissive to SARS-CoV-2 infection. Using hPSC-LOs, we performed a high throughput screen of FDA-approved drugs and identified entry inhibitors of SARS-CoV-2, including imatinib, mycophenolic acid (MPA), and quinacrine dihydrochloride (QNHC). Treatment at physiologically relevant levels of these drugs significantly inhibited SARS-CoV-2 infection of both hPSC-LOs and hPSC-COs. Together, these data demonstrate that hPSC-LOs and hPSC-COs infected by SARS-CoV-2 can serve as disease models to study SARS-CoV-2 infection and provide a valuable resource for drug screening to identify candidate COVID-19 therapeutics. The development of anti-SARS-CoV-2 drugs could change the scope of the ongoing COVID-19 pandemic. While this strategy is being pursued, high throughput screens are typically performed in transformed cell lines which fail to capture the physiologically relevant dynamics of human SARS-CoV-2 infection. To overcome limitations of these cell lines, several adult organoid models have been developed to study SARS-CoV-2 2-4. Here, we developed human pluripotent stem cell-derived lung and colonic organoids (hPSC-LOs and hPSC-COs) optimized as in vitro platforms for high throughput drug screening. hPSC-LOs are permissive to SARS-CoV-2 We differentiated hPSCs to lung organoids (hPSC-LOs) based on previously reported stepwise strategies 5-13 (Extended Data Fig. 1a-1c). qRT-PCR and RNA-seq profiling validates the expression of alveolar type II (AT2) cell markers in the hPSC-LOs (Extended Data Fig. 1d, 1e). Intra-cellular flow cytometry further confirmed the presence of Pro-SP-C + cells in hPSC-LOs (Extended Data Fig. 1f). Single cell transcriptomic profiles of hPSC-LOs identified AT2-like cells, which were enriched for adult human lung AT2 cell markers (Fig. 1a-1c and Extended Data Fig. 2a-2c).

Activating mutations in PIK3CA are frequent in human breast cancer, and phosphoinositide 3-kinase alpha (PI3Kα) inhibitors have been approved for therapy. To characterize determinants of sensitivity to these agents, we analyzed PIK3CA-mutant cancer genomes and observed the presence of multiple PIK3CA mutations in 12 to 15% of breast cancers and other tumor types, most of which (95%) are double mutations. Double PIK3CA mutations are in cis on the same allele and result in increased PI3K activity, enhanced downstream signaling, increased cell proliferation, and tumor growth. The biochemical mechanisms of dual mutations include increased disruption of p110α binding to the inhibitory subunit p85α, which relieves its catalytic inhibition, and increased p110α membrane lipid binding. Double PIK3CA mutations predict increased sensitivity to PI3Kα inhibitors compared with single-hotspot mutations.

Establishment of cell polarity—or symmetry breaking—relies on local accumulation of polarity regulators. Although simple positive feedback is sufficient to drive symmetry breaking, it is highly sensitive to stochastic fluctuations typical for living cells. Here, by integrating mathematical modelling with quantitative experimental validations, we show that in the yeast Saccharomyces cerevisiae a combination of actin- and guanine nucleotide dissociation inhibitor-dependent recycling of the central polarity regulator Cdc42 is needed to establish robust cell polarity at a single site during yeast budding. The guanine nucleotide dissociation inhibitor pathway consistently generates a single-polarization site, but requires Cdc42 to cycle rapidly between its active and inactive form, and is therefore sensitive to perturbations of the GTPase cycle. Conversely, actin-mediated recycling of Cdc42 induces robust symmetry breaking but cannot restrict polarization to a single site. Our results demonstrate how cells optimize symmetry breaking through coupling between multiple feedback loops.

Protein phosphorylation is one of the most widespread post-translational modifications in biology1,2. With advances in mass-spectrometry-based phosphoproteomics, 90,000 sites of serine and threonine phosphorylation have so far been identified, and several thousand have been associated with human diseases and biological processes3,4. For the vast majority of phosphorylation events, it is not yet known which of the more than 300 protein serine/threonine (Ser/Thr) kinases encoded in the human genome are responsible3. Here we used synthetic peptide libraries to profile the substrate sequence specificity of 303 Ser/Thr kinases, comprising more than 84% of those predicted to be active in humans. Viewed in its entirety, the substrate specificity of the kinome was substantially more diverse than expected and was driven extensively by negative selectivity. We used our kinome-wide dataset to computationally annotate and identify the kinases capable of phosphorylating every reported phosphorylation site in the human Ser/Thr phosphoproteome. For the small minority of phosphosites for which the putative protein kinases involved have been previously reported, our predictions were in excellent agreement. When this approach was applied to examine the signalling response of tissues and cell lines to hormones, growth factors, targeted inhibitors and environmental or genetic perturbations, it revealed unexpected insights into pathway complexity and compensation. Overall, these studies reveal the intrinsic substrate specificity of the human Ser/Thr kinome, illuminate cellular signalling responses and provide a resource to link phosphorylation events to biological pathways.

The subcellular localization of the Rho family GTPases is of fundamental importance to their proper functioning in cells. The Rho guanine nucleotide dissociation inhibitor (RhoGDI) plays a key regulatory role by influencing the cellular localization of Rho GTPases and is essential for the transforming activity of oncogenic forms of Cdc42. However, the mechanism by which RhoGDI helps Cdc42 to undergo the transition between a membrane-associated protein and a soluble (cytosolic) species has been poorly understood. Here, we examine how RhoGDI influences the binding of Cdc42 to lipid bilayers. Despite having similar affinities for the signaling-inactive (GDP-bound) and signaling-active (GTP-bound) forms of Cdc42 in solution, we show that when RhoGDI interacts with Cdc42 along the membrane surface, it has a much higher affinity for GDP-bound Cdc42 compared with its GTP-bound counterpart. Interestingly, the rate for the dissociation of Cdc42⅐RhoGDI complexes from membranes is unaffected by the nucleotide-bound state of Cdc42. Moreover, the membrane release of Cdc42⅐RhoGDI complexes occurs at a similar rate as the release of Cdc42 alone, with the major effect of RhoGDI being to impede the re-association of Cdc42 with membranes. These findings lead us to propose a new model for how RhoGDI influences the ability of Cdc42 to move between membranes and the cytosol, which highlights the role of the membrane in helping RhoGDI to distinguish between the GDP-and GTP-bound forms of Cdc42 and holds important implications for how it functions as a key regulator of the cellular localization and signaling activities of this GTPase.

While the majority of phosphatidylinositol-4, 5-bisphosphate (PI-4, 5-P) in mammalian cells is generated by the conversion of phosphatidylinositol-4-phosphate (PI-4-P) to PI-4, 5-P, a small fraction can be made by phosphorylating phosphatidylinositol-5-phosphate (PI-5-P). The physiological relevance of this second pathway is not clear. Here, we show that deletion of the genes encoding the two most active enzymes in this pathway, Pip4k2a and Pip4k2b, in the liver of mice causes a large enrichment in lipid droplets and in autophagic vesicles during fasting. These changes are due to a defect in the clearance of autophagosomes that halts autophagy and reduces the supply of nutrients salvaged through this pathway. Similar defects in autophagy are seen in nutrient-starved Pip4k2aPip4k2b mouse embryonic fibroblasts and in C. elegans lacking the PI5P4K ortholog. These results suggest that this alternative pathway for PI-4, 5-P synthesis evolved, in part, to enhance the ability of multicellular organisms to survive starvation.

ParagraphThe SARS-CoV-2 virus has caused already over 3.5 million COVID-19 cases and 250,000 deaths globally. There is an urgent need to create novel models to study SARS-CoV-2 using human disease-relevant cells to understand key features of virus biology and facilitate drug screening. As primary SARS-CoV-2 infection is respiratory-based, we developed a lung organoid model using human pluripotent stem cells (hPSCs) that could be adapted for drug screens. The lung organoids, particularly aveolar type II cells, express ACE2 and are permissive to SARS-CoV-2 infection.Transcriptomic analysis following SARS-CoV-2 infection revealed a robust induction of chemokines and cytokines with little type I/III interferon signaling, similar to that observed amongst human COVID-19 pulmonary infections. We performed a high throughput screen using hPSC-derived lung organoids and identified FDA-approved drug candidates, including imatinib and mycophenolic acid, as inhibitors of SARS-CoV-2 entry. Pre-or post-treatment with these drugs at physiologically relevant levels decreased SARS-CoV-2 infection of hPSC-derived lung organoids. Together, these data demonstrate that hPSC-derived lung cells infected by SARS-CoV-2 can model human COVID-19 disease and provide a valuable resource to screen for FDAapproved drugs that might be repurposed and should be considered for COVID-19 clinical trials.was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.