Multiple receptor tyrosine kinases regulate dengue infection of hepatocytes.

Introduction: Dengue is an arboviral disease causing severe illness in over 500,000 people each year. Currently, there is no way to constrain dengue in the clinic. Host kinase regulators of dengue virus (DENV) infection have the potential to be disrupted by existing therapeutics to prevent infection and/or disease progression. Methods: To evaluate kinase regulation of DENV infection, we performed kinase regression (KiR), a machine learning approach that predicts kinase regulators of infection using existing drug-target information and a small drug screen. We infected hepatocytes with DENV in vitro in the presence of a panel of 38 kinase inhibitors then quantified the effect of each inhibitor on infection rate. We employed elastic net regularization on these data to obtain predictions of which of 291 kinases are regulating DENV infection. Results: Thirty-six kinases were predicted to have a functional role. Intriguingly, seven of the predicted kinases - EPH receptor A4 (EPHA4), EPH receptor B3 (EPHB3), EPH receptor B4 (EPHB4), erb-b2 receptor tyrosine kinase 2 (ERBB2), fibroblast growth factor receptor 2 (FGFR2), Insulin like growth factor 1 receptor (IGF1R), and ret proto-oncogene (RET) - belong to the receptor tyrosine kinase (RTK) family, which are already therapeutic targets in the clinic. We demonstrate that predicted RTKs are expressed at higher levels in DENV infected cells. Knockdown of EPHB4, ERBB2, FGFR2, or IGF1R reduces DENV infection in hepatocytes. Finally, we observe differential temporal induction of ERBB2 and IGF1R following DENV infection, highlighting their unique roles in regulating DENV. Discussion: Collectively, our findings underscore the significance of multiple RTKs in DENV infection and advocate further exploration of RTK-oriented interventions against dengue.

Viral protein engagement of GBF1 induces host cell vulnerability through synthetic lethality.

Viruses co-opt host proteins to carry out their lifecycle. Repurposed host proteins may thus become functionally compromised; a situation analogous to a loss-of-function mutation. We term such host proteins as viral-induced hypomorphs. Cells bearing cancer driver loss-of-function mutations have successfully been targeted with drugs perturbing proteins encoded by the synthetic lethal (SL) partners of cancer-specific mutations. Similarly, SL interactions of viral-induced hypomorphs can potentially be targeted as host-based antiviral therapeutics. Here, we use GBF1, which supports the infection of many RNA viruses, as a proof-of-concept. GBF1 becomes a hypomorph upon interaction with the poliovirus protein 3A. Screening for SL partners of GBF1 revealed ARF1 as the top hit, disruption of which selectively killed cells that synthesize 3A alone or in the context of a poliovirus replicon. Thus, viral protein interactions can induce hypomorphs that render host cells selectively vulnerable to perturbations that leave uninfected cells otherwise unscathed. Exploiting viral-induced vulnerabilities could lead to broad-spectrum antivirals for many viruses, including SARS-CoV-2.

Exploiting polypharmacology to dissect host kinases and kinase inhibitors that modulate endothelial barrier integrity.

Kinase inhibitors are promising drugs to stabilize the endothelial barrier following inflammatory damage. However, our limited knowledge of how kinase signaling activates barrier-restorative pathways and the complexity of multi-target drugs have hindered drug discovery and repurposing efforts. Here, we apply a kinase regression approach that exploits drug polypharmacology to investigate endothelial barrier regulation. A screen of 28 kinase inhibitors identified multiple inhibitors that promote endothelial barrier integrity and revealed divergent barrier phenotypes for BCR-ABL drugs. Target deconvolution predicted 50 barrier-regulating kinases from diverse kinase families. Using gene knockdowns, we identified kinases with a role in endothelial barrier regulation and dissected different mechanisms of action of barrier-protective kinase inhibitors. These results demonstrate the importance of polypharmacology in the endothelial barrier phenotype of kinase inhibitors and provide promising new leads for barrier-strengthening therapies.

Viral protein engagement of GBF1 induces host cell vulnerability through synthetic lethality.

Viruses co-opt host proteins to carry out their lifecycle. Repurposed host proteins may thus become functionally compromised; a situation analogous to a loss-of-function mutation. We term such host proteins viral-induced hypomorphs. Cells bearing cancer driver loss-of-function mutations have successfully been targeted with drugs perturbing proteins encoded by the synthetic lethal partners of cancer-specific mutations. Synthetic lethal interactions of viral-induced hypomorphs have the potential to be similarly targeted for the development of host-based antiviral therapeutics. Here, we use GBF1, which supports the infection of many RNA viruses, as a proof-of-concept. GBF1 becomes a hypomorph upon interaction with the poliovirus protein 3A. Screening for synthetic lethal partners of GBF1 revealed ARF1 as the top hit, disruption of which, selectively killed cells that synthesize poliovirus 3A. Thus, viral protein interactions can induce hypomorphs that render host cells vulnerable to perturbations that leave uninfected cells intact. Exploiting viral-induced vulnerabilities could lead to broad-spectrum antivirals for many viruses, including SARS-CoV-2. SUMMARY: Using a viral-induced hypomorph of GBF1, Navare et al., demonstrate that the principle of synthetic lethality is a mechanism to selectively kill virus-infected cells.

Liver stage malaria infection is controlled by host regulators of lipid peroxidation.

The facets of host control during Plasmodium liver infection remain largely unknown. We find that the SLC7a11-GPX4 pathway, which has been associated with the production of reactive oxygen species, lipid peroxidation, and a form of cell death called ferroptosis, plays a critical role in control of Plasmodium liver stage infection. Specifically, blocking GPX4 or SLC7a11 dramatically reduces Plasmodium liver stage parasite infection. In contrast, blocking negative regulators of this pathway, NOX1 and TFR1, leads to an increase in liver stage infection. We have shown previously that increased levels of P53 reduces Plasmodium LS burden in an apoptosis-independent manner. Here, we demonstrate that increased P53 is unable to control parasite burden during NOX1 or TFR1 knockdown, or in the presence of ROS scavenging or when lipid peroxidation is blocked. Additionally, SLC7a11 inhibitors Erastin and Sorafenib reduce infection. Thus, blocking the host SLC7a11-GPX4 pathway serves to selectively elevate lipid peroxides in infected cells, which localize within the parasite and lead to the elimination of liver stage parasites.

A Molecular Signature in Blood Reveals a Role for p53 in Regulating Malaria-Induced Inflammation.

Immunity that controls parasitemia and inflammation during Plasmodium falciparum (Pf) malaria can be acquired with repeated infections. A limited understanding of this complex immune response impedes the development of vaccines and adjunctive therapies. We conducted a prospective systems biology study of children who differed in their ability to control parasitemia and fever following Pf infection. By integrating whole-blood transcriptomics, flow-cytometric analysis, and plasma cytokine and antibody profiles, we demonstrate that a pre-infection signature of B cell enrichment, upregulation of T helper type 1 (Th1) and Th2 cell-associated pathways, including interferon responses, and p53 activation associated with control of malarial fever and coordinated with Pf-specific immunoglobulin G (IgG) and Fc receptor activation to control parasitemia. Our hypothesis-generating approach identified host molecules that may contribute to differential clinical outcomes during Pf infection. As a proof of concept, we have shown that enhanced p53 expression in monocytes attenuated Plasmodium-induced inflammation and predicted protection from fever.

Spatial presentation of biological molecules to cells by localized diffusive transfer.

Cellular decisions in human development, homeostasis, regeneration, and disease are coordinated in large part by signals that are spatially localized in tissues. These signals are often soluble, such that biomolecules produced by one cell diffuse to receiving cells. To recapitulate soluble factor patterning in vitro, several microscale strategies have been developed. However, these techniques often introduce new variables into cell culture experiments (e.g., fluid flow) or are limited in their ability to pattern diverse solutes in a user-defined manner. To address these challenges, we developed an adaptable method that facilitates spatial presentation of biomolecules across cells in traditional open cultures in vitro. This technique employs device inserts that are placed in standard culture wells, which support localized diffusive pattern transmission through microscale spaces between device features and adherent cells. Devices can be removed and cultures can be returned to standard media following patterning. We use this method to spatially control cell labeling with pattern features ranging in scale from several hundred microns to millimeters and with sequential application of multiple patterns. To better understand the method we investigate relationships between pattern fidelity, device geometry, and consumption and diffusion kinetics using finite element modeling. We then apply the method to spatially defining reporter cell heterogeneity by patterning a small molecule modulator of genetic recombination with the requisite sustained exposure. Finally, we demonstrate use of this method for patterning larger and more slowly diffusing particles by creating focal sites of gene delivery and infection with adenoviral, lentiviral, and Zika virus particles. Thus, our method leverages devices that interface with standard culture vessels to pattern diverse diffusible factors, geometries, exposure dynamics, and recipient cell types, making it well poised for adoption by researchers across various fields of biological research.

A call to arms: Unifying the fight against resistance.

This Editorial discusses the state of research on drug resistance in the fields of cancer, infectious disease, and agriculture. Reaching across the aisle for a more cross-collaborative approach may lead to exciting breakthroughs toward tackling the challenges of drug resistance in each field.

Opportunities for Host-targeted Therapies for Malaria.

Despite the recent successes of artemisinin-based antimalarial drugs, many still die from severe malaria, and eradication efforts are hindered by the limited drugs currently available to target transmissible gametocyte parasites and liver-resident dormant Plasmodium vivax hypnozoites. Host-targeted therapy is a new direction for infectious disease drug development and aims to interfere with host molecules, pathways, or networks that are required for infection or that contribute to disease. Recent advances in our understanding of host pathways involved in parasite development and pathogenic mechanisms in severe malaria could facilitate the development of host-targeted interventions against Plasmodium infection and malaria disease. This review discusses new opportunities for host-targeted therapeutics for malaria and the potential to harness drug polypharmacology to simultaneously target multiple host pathways using a single drug intervention.

Identifying host regulators and inhibitors of liver stage malaria infection using kinase activity profiles.

Plasmodium parasites have extensive needs from their host hepatocytes during the obligate liver stage of infection, yet there remains sparse knowledge of specific host regulators. Here we assess 34 host-targeted kinase inhibitors for their capacity to eliminate Plasmodium yoelii-infected hepatocytes. Using pre-existing activity profiles of each inhibitor, we generate a predictive computational model that identifies host kinases, which facilitate Plasmodium yoelii liver stage infection. We predict 47 kinases, including novel and previously described kinases that impact infection. The impact of a subset of kinases is experimentally validated, including Receptor Tyrosine Kinases, members of the MAP Kinase cascade, and WEE1. Our approach also predicts host-targeted kinase inhibitors of infection, including compounds already used in humans. Three of these compounds, VX-680, Roscovitine and Sunitinib, each eliminate >85% of infection. Our approach is well-suited to uncover key host determinants of infection in difficult model systems, including field-isolated parasites and/or emerging pathogens.

Malaria parasites target the hepatocyte receptor EphA2 for successful host infection.

The invasion of a suitable host hepatocyte by mosquito-transmitted Plasmodium sporozoites is an essential early step in successful malaria parasite infection. Yet precisely how sporozoites target their host cell and facilitate productive infection remains largely unknown. We found that the hepatocyte EphA2 receptor was critical for establishing a permissive intracellular replication compartment, the parasitophorous vacuole. Sporozoites productively infected hepatocytes with high EphA2 expression, and the deletion of EphA2 protected mice from liver infection. Lack of host EphA2 phenocopied the lack of the sporozoite proteins P52 and P36. Our data suggest that P36 engages EphA2, which is likely to be a key step in establishing the permissive replication compartment.

Plasmodium vivax liver stage development and hypnozoite persistence in human liver-chimeric mice.

Plasmodium vivax malaria is characterized by periodic relapses of symptomatic blood stage parasite infections likely initiated by activation of dormant liver stage parasites-hypnozoites. The lack of tractable P. vivax animal models constitutes an obstacle in examining P. vivax liver stage infection and drug efficacy. To overcome this obstacle, we have used human liver-chimeric (huHep) FRG KO mice as a model for P. vivax infection. FRG KO huHep mice support P. vivax sporozoite infection, liver stage development, and hypnozoite formation. We show complete P. vivax liver stage development, including maturation into infectious exo-erythrocytic merozoites as well as the formation and persistence of hypnozoites. Prophylaxis or treatment with the antimalarial primaquine can prevent and eliminate liver stage infection, respectively. Thus, P. vivax-infected FRG KO huHep mice are a model to investigate liver stage development and dormancy and may facilitate the discovery of drugs targeting relapsing malaria.

Host-based Prophylaxis Successfully Targets Liver Stage Malaria Parasites.

Eliminating malaria parasites during the asymptomatic but obligate liver stages (LSs) of infection would stop disease and subsequent transmission. Unfortunately, only a single licensed drug that targets all LSs, Primaquine, is available. Targeting host proteins might significantly expand the repertoire of prophylactic drugs against malaria. Here, we demonstrate that both Bcl-2 inhibitors and P53 agonists dramatically reduce LS burden in a mouse malaria model in vitro and in vivo by altering the activity of key hepatocyte factors on which the parasite relies. Bcl-2 inhibitors act primarily by inducing apoptosis in infected hepatocytes, whereas P53 agonists eliminate parasites in an apoptosis-independent fashion. In combination, Bcl-2 inhibitors and P53 agonists act synergistically to delay, and in some cases completely prevent, the onset of blood stage disease. Both families of drugs are highly effective at doses that do not cause substantial hepatocyte cell death in vitro or liver damage in vivo. P53 agonists and Bcl-2 inhibitors were also effective when administered to humanized mice infected with Plasmodium falciparum. Our data demonstrate that host-based prophylaxis could be developed into an effective intervention strategy that eliminates LS parasites before the onset of clinical disease and thus opens a new avenue to prevent malaria.

Susceptibility to Plasmodium yoelii preerythrocytic infection in BALB/c substrains is determined at the point of hepatocyte invasion.

After transmission by Anopheles mosquitoes, Plasmodium sporozoites travel to the liver, infect hepatocytes, and rapidly develop as intrahepatocytic liver stages (LS). Rodent models of malaria exhibit large differences in the magnitude of liver infection, both between parasite species and between strains of mice. This has been mainly attributed to differences in innate immune responses and parasite infectivity. Here, we report that BALB/cByJ mice are more susceptible to Plasmodium yoelii preerythrocytic infection than BALB/cJ mice. This difference occurs at the level of early hepatocyte infection, but expression levels of reported host factors that are involved in infection do not correlate with susceptibility. Interestingly, BALB/cByJ hepatocytes are more frequently polyploid; thus, their susceptibility converges on the previously observed preference of sporozoites to infect polyploid hepatocytes. Gene expression analysis demonstrates hepatocyte-specific differences in mRNA abundance for numerous genes between BALB/cByJ and BALB/cJ mice, some of which encode hepatocyte surface molecules. These data suggest that a yet-unknown receptor for sporozoite infection, present at elevated levels on BALB/cByJ hepatocytes and also polyploid hepatocytes, might facilitate Plasmodium liver infection.

Systems biology of megakaryocytes.

The molecular pathways that regulate megakaryocyte production have historically been identified through multiple candidate gene approaches. Several transcription factors critical for generating megakaryocytes were identified by promoter analysis of megakaryocyte-specific genes, and their biological roles then verified by gene knockout studies; for example, GATA-1, NF-E2, and RUNX1 were identified in this way. In contrast, other transcription factors important for megakaryopoiesis were discovered through a systems approach; for example, c-Myb was found to be critical for the erythroid versus megakaryocyte lineage decision by genome-wide loss-of-function studies. The regulation of the levels of these transcription factors is, for the most part, cell intrinsic, although that assumption has recently been challenged. Epigenetics also impacts megakaryocyte gene expression, mediated by histone acetylation and methylation. Several cytokines have been identified to regulate megakaryocyte survival, proliferation, and differentiation, most prominent of which is thrombopoietin. Upon binding to its receptor, the product of the c-Mpl proto-oncogene, thrombopoietin induces a conformational change that activates a number of secondary messengers that promote cell survival, proliferation, and differentiation, and down-modulate receptor signaling. Among the best studied are the signal transducers and activators of transcription (STAT) proteins; phosphoinositol-3-kinase; mitogen-activated protein kinases; the phosphatases PTEN, SHP1, SHP2, and SHIP1; and the suppressors of cytokine signaling (SOCS) proteins. Additional signals activated by these secondary mediators include mammalian target of rapamycin; ß(beta)-catenin; the G proteins Rac1, Rho, and CDC42; several transcription factors, including hypoxia-inducible factor 1α(alpha), the homeobox-containing proteins HOXB4 and HOXA9, and a number of signaling mediators that are reduced, including glycogen synthase kinase 3α(alpha) and the FOXO3 family of forkhead proteins. More recently, systematic interrogation of several aspects of megakaryocyte formation have been conducted, employing genomics, proteomics, and chromatin immunoprecipitation (ChIP) analyses, among others, and have yielded many previously unappreciated signaling mechanisms that regulate megakaryocyte lineage determination, proliferation, and differentiation. This chapter focuses on these pathways in normal and neoplastic megakaryopoiesis, and suggests areas that are ripe for further study.

Phosphorylated c-Mpl tyrosine 591 regulates thrombopoietin-induced signaling.

Thrombopoietin (TPO) is the primary regulator of platelet production, affecting cell survival, proliferation, and differentiation through binding to and stimulation of the cell surface receptor the cellular myeloproliferative leukemia virus oncogene (c-Mpl). Activating mutations in c-Mpl constitutively stimulate downstream signaling pathways, leading to aberrant hematopoiesis, and contribute to development of myeloproliferative neoplasms. Several studies have mapped the tyrosine residues within the cytoplasmic domain of c-Mpl that mediate these cellular signals; however, secondary signaling pathways are incompletely understood. In this study, we focused on c-Mpl tyrosine 591 (Y591). We found Y591 of wild-type c-Mpl to be phosphorylated in the presence of TPO. Additionally, eliminating Y591 phosphorylation by mutation to Phe resulted in decreased total receptor phosphorylation. Using a Src homology 2/phosphotyrosine-binding (SH2/PTB) domain binding microarray, we identified novel c-Mpl binding partners for phosphorylated Y591, including Src homology region 2 domain-containing phosphatase-1 (SHP-1), spleen tyrosine kinase (SYK) and Bruton's tyrosine kinase (BTK). The functional significance of binding partners was determined through small interfering RNA treatment of Ba/F3-Mpl cells, confirming that the increase in pERK1/2 resulting from removal of Y591 may be mediated by spleen tyrosine kinase. These findings identify a novel negative regulatory pathway that controls TPO-mediated signaling, advancing our understanding of the mechanisms required for successful maintenance of hematopoietic stem cells and megakaryocyte development.

Susceptibility to Plasmodium liver stage infection is altered by hepatocyte polyploidy.

Plasmodium parasites infect hepatocytes of their mammalian hosts and undergo obligate liver stage development. The specific host cell attributes that are important for liver infection remain largely unknown. Several host signalling pathways are perturbed in infected hepatocytes, some of which are important in the generation of hepatocyte polyploidy. To test the functional consequence of polyploidy on liver infection, we infected hepatocytes with the rodent malaria parasite Plasmodium yoelii both in vitro and in vivo and examined the ploidy of infected and uninfected hepatocytes by flow cytometry. In both hepatoma cell lines and in the mouse liver, the fraction of polyploid cells was higher in the infected cell population than in the uninfected cell population. When the data were reanalysed by comparing the extent of Plasmodium infection within each ploidy subset, we found that infection rates were elevated in more highly polyploid cells and lower in diploid cells. Furthermore, we found that the parasite's preference for host cells with high ploidy is conserved among rodent malaria species and the human malaria parasite Plasmodium falciparum. This parasite preference for host cells of high ploidy cannot be explained by differences in hepatocyte size or DNA replication. We conclude that Plasmodium preferentially infects and develops in polyploid hepatocytes.

Suppression of host p53 is critical for Plasmodium liver-stage infection.

Plasmodium parasites infect the liver and replicate inside hepatocytes before they invade erythrocytes and trigger clinical malaria. Analysis of host signaling pathways affected by liver-stage infection could provide critical insights into host-pathogen interactions and reveal targets for intervention. Using protein lysate microarrays, we found that Plasmodium yoelii rodent malaria parasites perturb hepatocyte regulatory pathways involved in cell survival, proliferation, and autophagy. Notably, the prodeath protein p53 was substantially decreased in infected hepatocytes, suggesting that it could be targeted by the parasite to foster survival. Indeed, mice that express increased levels of p53 showed reduced liver-stage parasite burden, whereas p53 knockout mice suffered increased liver-stage burden. Furthermore, boosting p53 levels with the use of the small molecule Nutlin-3 dramatically reduced liver-stage burden in vitro and in vivo. We conclude that perturbation of the hepatocyte p53 pathway critically impacts parasite survival. Thus, host pathways might constitute potential targets for host-based antimalarial prophylaxis.

Phosphotyrosine signaling proteins that drive oncogenesis tend to be highly interconnected.

Mutation and overexpression of receptor tyrosine kinases or the proteins they regulate serve as oncogenic drivers in diverse cancers. To better understand receptor tyrosine kinase signaling and its link to oncogenesis, we used protein microarrays to systematically and quantitatively measure interactions between virtually every SH2 or PTB domain encoded in the human genome and all known sites of tyrosine phosphorylation on 40 receptor tyrosine kinases and on most of the SH2 and PTB domain-containing adaptor proteins. We found that adaptor proteins, like RTKs, have many high affinity bindings sites for other adaptor proteins. In addition, proteins that drive cancer, including both receptors and adaptor proteins, tend to be much more highly interconnected via networks of SH2 and PTB domain-mediated interactions than nononcogenic proteins. Our results suggest that network topological properties such as connectivity can be used to prioritize new drug targets in this well-studied family of signaling proteins.