A kinase to cytokine explorer to identify molecular regulators and potential therapeutic opportunities.

Cytokines and chemokines are secreted proteins that regulate various biological processes, such as inflammation, immune response, and cell differentiation. Therefore, disruption of signaling pathways involving these proteins has been linked to a range of diseases, including cancer. However, targeting individual cytokines, chemokines, or their receptors is challenging due to their regulatory redundancy and incomplete understanding of their signaling networks. To transform these difficult-to-drug targets into a pharmacologically manageable class, we developed a web-based platform called KinCytE. This platform was designed to link the effects of kinase inhibitors, a well-established class of drugs, with cytokine and chemokine release and signaling networks. The resulting KinCytE platform enables users to investigate protein kinases that regulate specific cytokines or chemokines, generate a ranked list of FDA-approved kinase inhibitors that affect cytokine/chemokine activity, and explore and visualize cytokine signaling network thus facilitating drugging this challenging target class. KinCytE is freely accessible via https://atlas.fredhutch.org/kincyte.

A test combining multiplex-PCR with pooled antibiotic susceptibility testing has high correlation with expanded urine culture for detection of live bacteria in urine samples of suspected UTI patients.

We evaluated whether multiplex polymerase chain reaction (M-PCR) detects viable micro-organisms by comparing micro-organism identification with standard urine culture (SUC) and expanded quantitative urine culture (EQUC). Of the 395 organisms detected by M-PCR, EQUC detected 89.1% (p = 0.10), whereas SUC detected 27.3% (p < 0.0001 vs. M-PCR and p < 0.0001 vs EQUC). M-PCR identified 260 nonfastidious bacteria, EQUC detected 96.5% (p = 0.68), whereas SUC detected 41.5% (p < 0.0001). Common nonfastidious bacteria missed by SUC included Escherichia coli (72.5% detected), Klebsiella pneumoniae (66.7% detected), Enterococcus faecalis (34.6% detected) and Enterococcus faecium (0% detected). M-PCR identified 135 fastidious bacteria and EQUC 101 (74.8%, p = 0.01), whereas SUC failed to detect any (0%, p < 0.0001). Clinical samples evaluated using EQUC and M-PCR yielded very similar findings, indicating that most microbes identified by M-PCR represented viable organisms, and validating M-PCR as a diagnostic tool for UTIs.

Blockade of interleukin 10 potentiates antitumour immune function in human colorectal cancer liver metastases.

OBJECTIVE: Programmed cell death protein 1 (PD-1) checkpoint inhibition and adoptive cellular therapy have had limited success in patients with microsatellite stable colorectal cancer liver metastases (CRLM). We sought to evaluate the effect of interleukin 10 (IL-10) blockade on endogenous T cell and chimeric antigen receptor T (CAR-T) cell antitumour function in CRLM slice cultures. DESIGN: We created organotypic slice cultures from human CRLM (n=38 patients' tumours) and tested the antitumour effects of a neutralising antibody against IL-10 (αIL-10) both alone as treatment and in combination with exogenously administered carcinoembryonic antigen (CEA)-specific CAR-T cells. We evaluated slice cultures with single and multiplex immunohistochemistry, in situ hybridisation, single-cell RNA sequencing, reverse-phase protein arrays and time-lapse fluorescent microscopy. RESULTS: αIL-10 generated a 1.8-fold increase in T cell-mediated carcinoma cell death in human CRLM slice cultures. αIL-10 significantly increased proportions of CD8+ T cells without exhaustion transcription changes, and increased human leukocyte antigen - DR isotype (HLA-DR) expression of macrophages. The antitumour effects of αIL-10 were reversed by major histocompatibility complex class I or II (MHC-I or MHC-II) blockade, confirming the essential role of antigen presenting cells. Interrupting IL-10 signalling also rescued murine CAR-T cell proliferation and cytotoxicity from myeloid cell-mediated immunosuppression. In human CRLM slices, αIL-10 increased CEA-specific CAR-T cell activation and CAR-T cell-mediated cytotoxicity, with nearly 70% carcinoma cell apoptosis across multiple human tumours. Pretreatment with an IL-10 receptor blocking antibody also potentiated CAR-T function. CONCLUSION: Neutralising the effects of IL-10 in human CRLM has therapeutic potential as a stand-alone treatment and to augment the function of adoptively transferred CAR-T cells.

Olverembatinib inhibits SARS-CoV-2-Omicron variant-mediated cytokine release in human peripheral blood mononuclear cells.

The N-terminus domain (NTD) of the SARS-CoV-2 Omicron variant spike protien strongly induces multiple inflammatory molecules in human peripheral blood mononuclear cells, unaffected by the mutations observed in the NTD. Olverembatinib, a clinical-stage multi-kinase inhibitor, potently inhibits Omicron NTD-mediated cytokine release.

Machine learning identifies molecular regulators and therapeutics for targeting SARS-CoV2-induced cytokine release.

Although 15-20% of COVID-19 patients experience hyper-inflammation induced by massive cytokine production, cellular triggers of this process and strategies to target them remain poorly understood. Here, we show that the N-terminal domain (NTD) of the SARS-CoV-2 spike protein substantially induces multiple inflammatory molecules in myeloid cells and human PBMCs. Using a combination of phenotypic screening with machine learning-based modeling, we identified and experimentally validated several protein kinases, including JAK1, EPHA7, IRAK1, MAPK12, and MAP3K8, as essential downstream mediators of NTD-induced cytokine production, implicating the role of multiple signaling pathways in cytokine release. Further, we found several FDA-approved drugs, including ponatinib, and cobimetinib as potent inhibitors of the NTD-mediated cytokine release. Treatment with ponatinib outperforms other drugs, including dexamethasone and baricitinib, inhibiting all cytokines in response to the NTD from SARS-CoV-2 and emerging variants. Finally, ponatinib treatment inhibits lipopolysaccharide-mediated cytokine release in myeloid cells in vitro and lung inflammation mouse model. Together, we propose that agents targeting multiple kinases required for SARS-CoV-2-mediated cytokine release, such as ponatinib, may represent an attractive therapeutic option for treating moderate to severe COVID-19.

KiRNet: Kinase-centered network propagation of pharmacological screen results.

The ever-increasing size and scale of biological information have popularized network-based approaches as a means to interpret these data. We develop a network propagation method that integrates kinase-inhibitor-focused functional screens with known protein-protein interactions (PPIs). This method, dubbed KiRNet, uses an a priori edge-weighting strategy based on node degree to establish a pipeline from a kinase inhibitor screen to the generation of a predictive PPI subnetwork. We apply KiRNet to uncover molecular regulators of mesenchymal cancer cells driven by overexpression of Frizzled 2 (FZD2). KiRNet produces a network model consisting of 166 high-value proteins. These proteins exhibit FZD2-dependent differential phosphorylation, and genetic knockdown studies validate their role in maintaining a mesenchymal cell state. Finally, analysis of clinical data shows that mesenchymal tumors exhibit significantly higher average expression of the 166 corresponding genes than epithelial tumors for nine different cancer types.

Pharmacoproteomics Identifies Kinase Pathways that Drive the Epithelial-Mesenchymal Transition and Drug Resistance in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a complex and deadly disease lacking druggable genetic mutations. The limited efficacy of systemic treatments for advanced HCC implies that predictive biomarkers and drug targets are urgently needed. Most HCC drugs target protein kinases, indicating that kinase-dependent signaling networks drive HCC progression. To identify HCC signaling networks that determine responses to kinase inhibitors (KIs), we apply a pharmacoproteomics approach integrating kinome activity in 17 HCC cell lines with their responses to 299 KIs, resulting in a comprehensive dataset of pathway-based drug response signatures. By profiling patient HCC samples, we identify signatures of clinical HCC drug responses in individual tumors. Our analyses reveal kinase networks promoting the epithelial-mesenchymal transition (EMT) and drug resistance, including a FZD2-AXL-NUAK1/2 signaling module, whose inhibition reverses the EMT and sensitizes HCC cells to drugs. Our approach identifies cancer drug targets and molecular signatures of drug response for personalized oncology.

Comparison of tumor-associated YAP1 fusions identifies a recurrent set of functions critical for oncogenesis.

YAP1 is a transcriptional coactivator and the principal effector of the Hippo signaling pathway, which is causally implicated in human cancer. Several YAP1 gene fusions have been identified in various human cancers and identifying the essential components of this family of gene fusions has significant therapeutic value. Here, we show that the YAP1 gene fusions YAP1-MAMLD1, YAP1-FAM118B, YAP1-TFE3, and YAP1-SS18 are oncogenic in mice. Using reporter assays, RNA-seq, ChIP-seq, and loss-of-function mutations, we can show that all of these YAP1 fusion proteins exert TEAD-dependent YAP activity, while some also exert activity of the C'-terminal fusion partner. The YAP activity of the different YAP1 fusions is resistant to negative Hippo pathway regulation due to constitutive nuclear localization and resistance to degradation of the YAP1 fusion proteins. Genetic disruption of the TEAD-binding domain of these oncogenic YAP1 fusions is sufficient to inhibit tumor formation in vivo, while pharmacological inhibition of the YAP1-TEAD interaction inhibits the growth of YAP1 fusion-expressing cell lines in vitro. These results highlight TEAD-dependent YAP activity found in these gene fusions as critical for oncogenesis and implicate these YAP functions as potential therapeutic targets in YAP1 fusion-positive tumors.

Pan-cancer analysis of the developmental pathways reveals non-canonical wnt signaling as a driver of mesenchymal-type tumors.

The processes of angiogenesis, cell proliferation, invasion, and migration, and the signaling pathways that drive these events, are activated in both cancer and during embryonic development. Here, we systematically assessed how the activity of major developmental signaling pathways, represented by the expression of genes encoding components of the pathways, correlated with patient survival in ∼8000 patients across 17 cancer types. We also compared the expressed genes enriched in developmental pathways with those associated with epithelial-mesenchymal transition (EMT) both in a cancer cohort and in mice during embryonic development. We found that EMT and gene expression profiles consistent with high activity of several developmental pathways, including the TGFß, Notch, and non-canonical Wnt pathways, significantly correlated with poor patient survival in multiple cancer types. We investigated individual components of these pathways and found that expression of the gene encoding the non-canonical Wnt receptor, frizzled 2 (FZD2), is highly correlated with both poor patient survival and gene expression indicating EMT in the tumors. Further mechanistic studies and pathway analyses revealed that FZD2-regulated genes in cancer cells in culture or FZD2-regulated gene sets from the TCGA data or FZD2-regulated genes involved in mouse organogenesis converged in EMT-associated biological processes, suggesting that FZD2 is a key driver of mesenchymal-like cell state and thus, a contributor to cancer progression and metastasis.

Organotypic tumor slice cultures provide a versatile platform for immuno-oncology and drug discovery.

Organotypic tumor slices represent a physiologically-relevant culture system for studying the tumor microenvironment. Systematic characterization of the tumor slice culture system will enable its effective application for translational research. Here, using flow cytometry-based immunophenotyping, we performed a comprehensive characterization of the immune cell composition in organotypic tumor slices prepared from four syngeneic mouse tumor models and a human liver tumor. We found that the immune cell compositions of organotypic tumor slices prepared on the same day as the tumor cores were harvested are similar. Differences were primarily observed in the lymphocyte population of a clinical hepatocellular carcinoma case. Viable populations of immune cells persisted in the tumor slices for 7 days. Despite some changes in the immune cell populations, we showed the utility of mouse tumor slices for assessing responses to immune-modulatory agents. Further, we demonstrated the ability to use patient-derived xenograft tumor slices for assessing responses to targeted and cytotoxic drugs. Overall, tumor slices provide a broadly useful platform for studying the tumor microenvironment and evaluating the preclinical efficacy of cancer therapeutics.

Evaluations of Conflicts Between Latino Values and Autonomy Desires Among Puerto Rican Adolescents.

Puerto Rican adolescents (N = 105; Mage  = 15.97 years, SD = 1.40) evaluated hypothetical situations describing conflicts between Latino values (family obligations and respeto) and autonomy desires regarding personal, friendship, and dating activities. Adolescents judged that peers should prioritize Latino values over autonomy, which led to greater feelings of pride than happiness. However, they believed that teens would prioritize autonomy over Latino values, which led to greater feelings of happiness than pride. Adolescents reasoned about autonomy desires as personal issues, whereas reasoning about Latino values was multifaceted, including references to conventions and concerns for others. Furthermore, judgments and reasoning depended on the type of autonomy desire and Latino value and sometimes, by participants' age and sex.

A noncanonical Frizzled2 pathway regulates epithelial-mesenchymal transition and metastasis.

Wnt signaling plays a critical role in embryonic development, and genetic aberrations in this network have been broadly implicated in colorectal cancer. We find that the Wnt receptor Frizzled2 (Fzd2) and its ligands Wnt5a/b are elevated in metastatic liver, lung, colon, and breast cancer cell lines and in high-grade tumors and that their expression correlates with markers of epithelial-mesenchymal transition (EMT). Pharmacologic and genetic perturbations reveal that Fzd2 drives EMT and cell migration through a previously unrecognized, noncanonical pathway that includes Fyn and Stat3. A gene signature regulated by this pathway predicts metastasis and overall survival in patients. We have developed an antibody to Fzd2 that reduces cell migration and invasion and inhibits tumor growth and metastasis in xenografts. We propose that targeting this pathway could provide benefit for patients with tumors expressing high levels of Fzd2 and Wnt5a/b.

Family-wide investigation of PDZ domain-mediated protein-protein interactions implicates ß-catenin in maintaining the integrity of tight junctions.

ß-catenin is a multifunctional protein that plays a critical role in cell-cell contacts and signal transduction. ß-catenin has previously been shown to interact with PDZ-domain-containing proteins through its C terminus. Using protein microarrays comprising 206 mouse PDZ domains, we identified 26 PDZ-domain-mediated interactions with ß-catenin and confirmed them biochemically and in cellular lysates. Many of the previously unreported interactions involved proteins with annotated roles in tight junctions. We found that four tight-junction-associated PDZ proteins-Scrib, Magi-1, Pard3, and ZO-3-colocalize with ß-catenin at the plasma membrane. Disrupting these interactions by RNA interference, overexpression of PDZ domains, or overexpression of the ß-catenin C terminus altered localization of the full-length proteins, weakened tight junctions, and decreased cellular adhesion. These results suggest that ß-catenin serves as a scaffold to establish the location and function of tight-junction-associated proteins.

A dual array-based approach to assess the abundance and posttranslational modification state of signaling proteins.

A system-wide analysis of cell signaling requires detecting and quantifying many different proteins and their posttranslational modification states in the same cellular sample. Here, we present Protocols for two miniaturized, array-based methods, one of which provides detailed information on a central signaling protein and the other of which provides a broad characterization of the surrounding signaling network. We describe a bead-based array and its use in characterizing the different forms and functions of ß-catenin, as well as lysate microarrays (reverse-phase protein arrays) and their use in detecting and quantifying proteins involved in the canonical and noncanonical Wnt signaling pathways. As an application of this dual approach, we characterized the state of ß-catenin signaling in cell lysates and linked these molecule-specific data with pathway-wide changes in signaling. The Protocols described here provide detailed instructions for cell culture methods, bead arrays, and lysate microarrays and outline how to use these complementary approaches to obtain insight into a complex network at a systems level.

Developmental changes and individual differences in young children's moral judgments.

Developmental trajectories and individual differences in 70 American middle-income 2½- to 4-year olds' moral judgments were examined 3 times across 1 year using latent growth modeling. At Wave 1, children distinguished hypothetical moral from conventional transgressions on all criteria, but only older preschoolers did so when rating deserved punishment. Children's understanding of moral transgressions as wrong independent of authority grew over time. Greater surgency and effortful control were both associated with a better understanding of moral generalizability. Children higher in effortful control also grew more slowly in understanding that moral rules are not alterable and that moral transgressions are wrong independent of rules. Girls demonstrated sharper increases across time than boys in understanding the nonalterability of moral rules.

Keeping secrets from parents: daily variations among poor, urban adolescents.

Daily variations in secrecy with mothers and fathers were examined in 108 poor, urban, diverse middle adolescents (M=15.16 years, SD=0.89). Adolescents completed online diaries over 14 days assessing secrecy from parents about school, personal, and multifaceted activities (e.g., staying out late), and bad behavior. Three-level hierarchical linear models indicated that there were significant daily fluctuations in adolescents' secrecy with mothers and that adolescents kept more secrets from mothers about personal than other activities. Secrecy with mothers also was associated with greater involvement in problem behavior. For both mothers and fathers, secrecy on the current day was associated with greater secrecy on the previous day and with poorer overall relationships (as aggregated across study days) with that parent. Thus, for mothers, secrecy appeared to be associated with both stable factors and daily variations, whereas for fathers, secrecy was associated primarily with stable factors. The results provide a detailed picture of secrecy in diverse adolescents' everyday lives.

Disclosure to parents about everyday activities among american adolescents from mexican, chinese, and European backgrounds.

Disclosure to parents and reasons for not disclosing different activities were examined in 489 Chinese, Mexican, and European American adolescents (M = 16.37 years, SD = 0.77). With generational status controlled, Chinese American adolescents disclosed less to mothers about personal and multifaceted activities than European Americans and less about personal feelings than other youth, primarily because these acts were considered personal, not harmful, or because parents would not listen or understand. Disclosure regarding prudential behavior was lower among Mexican American than among European American adolescents, primarily due to concerns with parental disapproval. Multigroup path analyses indicated that greater closeness to parents is associated with more disclosure for all youth and activities; associations between family obligation and disclosure varied by domain and ethnicity.

How much do I tell thee? Strategies for managing information to parents among American adolescents from Chinese, Mexican, and European backgrounds.

Strategies for managing information about activities to parents, including partial disclosure, avoidance, lying, and full disclosure, were examined in 479 American adolescents (M = 16.38 years, SD = 0.77) varying in generational status and from Mexican, Chinese, and European backgrounds. Information management strategies for personal, prudential, and overlapping (multifaceted) activities as defined within social domain theory were examined. With age, parental education, and generational status controlled, Chinese American adolescents partially disclosed more to mothers about personal and multifaceted activities than did Mexican American adolescents and more to fathers about personal activities than did European American teens. In contrast, European and Mexican American adolescents fully disclosed more to mothers about personal activities than did Chinese-origin adolescents. Strategies varied by generation among Chinese American youth; second-generation adolescents avoided discussing activities with parents more than did immigrants. Adolescents who fully disclosed about all activities and lied less about multifaceted and personal activities reported stronger endorsement of obligations to assist their families, more trust in parents, and less problem behavior. More depressed mood was associated with more lying about personal activities.

Adolescents' and parents' evaluations of helping versus fulfilling personal desires in family situations.

A sample of 118 predominantly European American families with early and middle adolescents (M(ages)= 12.32 and 15.18 years) and 1 parent evaluated hypothetical conflicts between adolescents' and parents' requests for assistance versus the other's personal desires. Evaluations differed by level of need, but in low-need situations, adolescents viewed teens as more obligated to help parents than did parents, whereas parents rated it as more permissible for teens to satisfy personal desires than did teenagers. Justifications for helping focused on concern for others, role responsibilities, and among parents, psychological reasons. Middle adolescents reasoned about role responsibilities more and viewed satisfying personal desires as less selfish than did early adolescents, but satisfying personal desires was seen as more selfish by parents of middle than early adolescents. Implications for adolescent-parent relationships are discussed.

Early and middle adolescents' disclosure to parents about activities in different domains.

Disclosure, disclosure strategies, and justifications for nondisclosure for prudential, peer, multifaceted, and personal acts were assessed using a sorting task with 118 lower-middle class early and middle adolescents (Ms=12.77 and 15.68 years). Adolescents were less involved in prudential than other behaviors, although prudential behavior was greater among middle than early adolescents; adolescents disclosed more about prudential and personal than multifaceted and peer behaviors. Nondisclosure was primarily due to concerns about parental disapproval (for prudential acts), claims that acts were personal or not harmful (for personal acts), and their mixture (for peer and multifaceted acts). When concerned about parental disapproval, older adolescents fully disclosed less (and lied somewhat more) than younger adolescents, whereas adolescents primarily avoided discussing the issue when they viewed acts as personal. Full disclosure was associated with better relationships with parents and less depressed mood; lying was associated with more parental behavioral control over personal issues and poorer relationships with fathers.