Targeting cancer hallmarks is a cardinal strategy to improve antineoplastic treatment. However, cross-talk between signaling pathways and key oncogenic processes frequently convey resistance to targeted therapies. The p53 and Wnt pathway play vital roles for the biology of many tumors, as they are critically involved in cancer onset and progression. Over recent decades, a high level of interaction between the two pathways has been revealed. Here, we provide a comprehensive overview of molecular interactions between the p53 and Wnt pathway discovered in cancer, including complex feedback loops and reciprocal transactivation. The mutational landscape of genes associated with p53 and Wnt signaling is described, including mutual exclusive and co-occurring genetic alterations. Finally, we summarize the functional consequences of this cross-talk for cancer phenotypes, such as invasiveness, metastasis or drug resistance, and discuss potential strategies to pharmacologically target the p53-Wnt interaction.

Plasmodium falciparum (Pf) parasite development in liver represents the initial step of the life-cycle in the human host after a Pf-infected mosquito bite. While an attractive stage for life-cycle interruption, understanding of parasite-hepatocyte interaction is inadequate due to limitations of existing in vitro models. We explore the suitability of hepatocyte organoids (HepOrgs) for Pf-development and show that these cells permitted parasite invasion, differentiation and maturation of different Pf strains. Single-cell messenger RNA sequencing (scRNAseq) of Pf-infected HepOrg cells has identified 80 Pf-transcripts upregulated on day 5 post-infection. Transcriptional profile changes are found involving distinct metabolic pathways in hepatocytes with Scavenger Receptor B1 (SR-B1) transcripts highly upregulated. A novel functional involvement in schizont maturation is confirmed in fresh primary hepatocytes. Thus, HepOrgs provide a strong foundation for a versatile in vitro model for Pf liver-stages accommodating basic biological studies and accelerated clinical development of novel tools for malaria control.

Canonical Wnt signaling plays a key role during organ development, homeostasis and regeneration and these processes are conserved between invertebrates and vertebrates. Mutations in Wnt pathway components are commonly found in various types of cancer. Upon activation of canonical Wnt signaling, β-catenin binds in the nucleus to members of the TCF-LEF family and activates the transcription of target genes. Multiple Wnt target genes, including Lgr5/LGR5 and Axin2/AXIN2, have been identified in mouse models and human cancer cell lines. Here we set out to identify the transcriptional targets of Wnt signaling in five human tissues using organoid technology. Organoids are derived from adult stem cells and recapitulate the functionality as well as the structure of the original tissue. Since the Wnt pathway is critical to maintain the organoids from the human intestine, colon, liver, pancreas and stomach, organoid technology allows us to assess Wnt target gene expression in a human wildtype situation. We performed bulk mRNA sequencing of organoids immediately after inhibition of Wnt pathway and identified 41 genes as commonly regulated genes in these tissues. We also identified large numbers of target genes specific to each tissue. One of the shared target genes is TEAD4, a transcription factor driving expression of YAP/TAZ signaling target genes. In addition to TEAD4, we identified a variety of genes which encode for proteins that are involved in Wnt-independent pathways, implicating the possibility of direct crosstalk between Wnt signaling and other pathways. Collectively, this study identified tissue-specific and common Wnt target gene signatures and provides evidence for a conserved role for these Wnt targets in different tissues.

Wnt ligands are secreted signaling proteins that display a wide range of biological effects. They play key roles in stimulating Wnt signaling pathways to facilitate processes such as tissue homeostasis and regeneration. Dysregulation of Wnt signaling is a hallmark of many cancers and genetic alterations in various Wnt signaling components, which result in ligand-independent or ligand-dependent hyperactivation of the pathway that have been identified. Recently, research is focusing on the impact of Wnt signaling on the interaction between tumor cells and their micro-environment. This Wnt-mediated crosstalk can act either in a tumor promoting or suppressing fashion. In this review, we comprehensively outline the function of Wnt ligands in different tumor entities and their impact on key phenotypes, including cancer stemness, drug resistance, metastasis, and immune evasion. Lastly, we elaborate approaches to target Wnt ligands in cancer therapy.

Background: Colorectal carcinoma exhibits high heterogeneity, comprising subtypes that show poor efficacy of T cell-based immunotherapies, such as programmed cell death protein 1 (PD-1) checkpoint inhibitors. Although natural killer (NK) cells are considered a promising approach for cancer immunotherapy, it remains unclear what molecular mechanisms drive NK cell activation or suppression within the tumor microenvironment. Moreover, limitations in human tumor models that reflect the diversity of individual patient tumors hinder the ability to effectively select patients who would benefit most from NK cell-based therapies. Methods: Here, we established a co-culture platform of genetically diverse colorectal cancer (CRC) patient-derived organoids (PDOs) with primary allogeneic NK cells. We performed bulk RNA sequencing analysis of sorted NK cells after exposure to PDOs and aligned gene expression signatures derived from our findings with publicly available single-cell RNA sequencing data of NK cells from peripheral blood and CRC tissues of patients. Moreover, we evaluated identified pathways using flow cytometry and IncuCyte live-cell imaging analysis to quantify phenotypic alterations and NK cell-mediated killing of PDOs over time, respectively. Ultimately, we tested CRISPR-Cas9-edited NK cells and PDOs, small molecule compounds, and clinically relevant monoclonal antibodies (mAbs) to increase NK cell potency. Results: On co-culture, NK cells acquired common transcriptional signatures related to hypoxia and transforming growth factor-beta (TGF-β), similar to NK cells infiltrating CRC tissues of patients. In addition, we observed patient-specific differential PDO susceptibilities to NK cell-mediated lysis. Major histocompatibility complex class I deficiency and natural killer group 2, member D (NKG2D)-ligand expression on PDOs facilitated NK cell-mediated cytotoxicity, and induced phenotypic NK cell diversification related to activation and the acquisition of inflammation and tissue-residency-related transcriptional signatures. Genetic or pharmaceutical targeting of hypoxia-inducible factors HIF1A / EPAS1 or TGF-βR1, or the addition of anti-CEACAM1 mAbs, enhanced NK cell-mediated PDO killing or activation, respectively. Conclusions: The NK cell/PDO co-culture platform allows the identification of both common and patient-specific impacts of the tumor microenvironment on NK cell function and can aid the development of patient-tailored immunotherapies. The majority of CRC (CMS2/CMS3) PDOs from our cohort were susceptible to NK cell-mediated killing and induced NK cell activation, highlighting the potential of NK cells for CRC immunotherapies.

Protein kinases control most cellular processes and aberrant kinase activity is involved in numerous diseases. Here we introduce molecular recorders of kinase activities for later analysis to investigate the link between specific kinase activities and cellular phenotypes in heterogeneous cell populations and in vivo. Based on split-HaloTag and a phosphorylation-dependent molecular switch, our recorders become rapidly labeled in the presence of a specific kinase activity and a fluorescent HaloTag substrate. The kinase activity in a given cell controls the degree of fluorescent labeling, whereas the recording window is set by the presence of the fluorescent substrate. We designed specific recorders for four protein kinases, including protein kinase A. We apply our protein kinase A recorder to sort heterogeneous cell populations for subsequent transcriptome analysis, in genome-wide CRISPR screens to discover regulators of PKA activity and to track neuromodulation in freely moving mice.