An oncogenic phenoscape of colonic stem cell polarization.

Cancer cells are regulated by oncogenic mutations and microenvironmental signals, yet these processes are often studied separately. To functionally map how cell-intrinsic and cell-extrinsic cues co-regulate cell fate, we performed a systematic single-cell analysis of 1,107 colonic organoid cultures regulated by (1) colorectal cancer (CRC) oncogenic mutations, (2) microenvironmental fibroblasts and macrophages, (3) stromal ligands, and (4) signaling inhibitors. Multiplexed single-cell analysis revealed a stepwise epithelial differentiation phenoscape dictated by combinations of oncogenes and stromal ligands, spanning from fibroblast-induced Clusterin (CLU)+ revival colonic stem cells (revCSCs) to oncogene-driven LRIG1+ hyper-proliferative CSCs (proCSCs). The transition from revCSCs to proCSCs is regulated by decreasing WNT3A and TGF-ß-driven YAP signaling and increasing KRASG12D or stromal EGF/Epiregulin-activated MAPK/PI3K flux. We find that APC loss and KRASG12D collaboratively limit access to revCSCs and disrupt stromal-epithelial communication-trapping epithelia in the proCSC fate. These results reveal that oncogenic mutations dominate homeostatic differentiation by obstructing cell-extrinsic regulation of cell-fate plasticity.

Trellis tree-based analysis reveals stromal regulation of patient-derived organoid drug responses.

Patient-derived organoids (PDOs) can model personalized therapy responses; however, current screening technologies cannot reveal drug response mechanisms or how tumor microenvironment cells alter therapeutic performance. To address this, we developed a highly multiplexed mass cytometry platform to measure post-translational modification (PTM) signaling, DNA damage, cell-cycle activity, and apoptosis in >2,500 colorectal cancer (CRC) PDOs and cancer-associated fibroblasts (CAFs) in response to clinical therapies at single-cell resolution. To compare patient- and microenvironment-specific drug responses in thousands of single-cell datasets, we developed "Trellis"-a highly scalable, tree-based treatment effect analysis method. Trellis single-cell screening revealed that on-target cell-cycle blockage and DNA-damage drug effects are common, even in chemorefractory PDOs. However, drug-induced apoptosis is rarer, patient-specific, and aligns with cancer cell PTM signaling. We find that CAFs can regulate PDO plasticity-shifting proliferative colonic stem cells (proCSCs) to slow-cycling revival colonic stem cells (revCSCs) to protect cancer cells from chemotherapy.

Targeting the HER3 pseudokinase domain with small molecule inhibitors.

HER3 is a potent oncogenic growth factor receptor belonging to the human epidermal growth factor (HER/EGFR) family of receptor tyrosine kinases. In contrast to other EGFR family members, HER3 is a pseudokinase, lacking functional kinase activity. As such, efforts to develop small molecule tyrosine kinase inhibitors against this family member have been limited. In response to HER3-specific growth factors such as neuregulin (NRG, also known as heregulin or HRG), HER3 must couple with catalytically active family members, including its preferred partner HER2. Dimerization of the intracellular HER2:HER3 kinase domains is a critical part of the activation mechanism and HER3 plays a specialized role as an allosteric activator of the active HER2 kinase partner. Intriguingly, many pseudokinases retain functionally important nucleotide binding capacity, despite loss of kinase activity. We demonstrated that occupation of the nucleotide pocket of the pseudokinase HER3 retains functional importance for growth factor signaling through oncogenic HER2:HER3 heterodimers. Mutation of the HER3 nucleotide pocket both disrupts signaling and disrupts HER2:HER3 dimerization. Conversely, ATP competitive drugs which bind to HER3, but not HER2, can stabilize HER2:HER3 dimers, induce signaling and promote cell growth in breast cancer models. This indicates a nucleotide-dependent conformational role for the HER3 kinase domain. Critically, our recent proof-of-concept work demonstrated that HER3-directed small molecule inhibitors can also disrupt HER2:HER3 dimerization and signaling, supporting the prospect that HER3 can be a direct drug target despite its lack of intrinsic activity. In this chapter we will describe methods for identifying and validating small molecule inhibitors against the HER3 pseudokinase.

A small molecule inhibitor of HER3: a proof-of-concept study.

Despite being catalytically defective, pseudokinases are typically essential players of cellular signalling, acting as allosteric regulators of their active counterparts. Deregulation of a growing number of pseudokinases has been linked to human diseases, making pseudokinases therapeutic targets of interest. Pseudokinases can be dynamic, adopting specific conformations critical for their allosteric function. Interfering with their allosteric role, with small molecules that would lock pseudokinases in a conformation preventing their productive partner interactions, is an attractive therapeutic strategy to explore. As a well-known allosteric activator of epidermal growth factor receptor family members, and playing a major part in cancer progression, the pseudokinase HER3 is a relevant context in which to address the potential of pseudokinases as drug targets for the development of allosteric inhibitors. In this proof-of-concept study, we developed a multiplex, medium-throughput thermal shift assay screening strategy to assess over 100 000 compounds and identify selective small molecule inhibitors that would trap HER3 in a conformation which is unfavourable for the formation of an active HER2-HER3 heterodimer. As a proof-of-concept compound, AC3573 bound with some specificity to HER3 and abrogated HER2-HER3 complex formation and downstream signalling in cells. Our study highlights the opportunity to identify new molecular mechanisms of action interfering with the biological function of pseudokinases.

Inhibitor-induced HER2-HER3 heterodimerisation promotes proliferation through a novel dimer interface.

While targeted therapy against HER2 is an effective first-line treatment in HER2+ breast cancer, acquired resistance remains a clinical challenge. The pseudokinase HER3, heterodimerisation partner of HER2, is widely implicated in the resistance to HER2-mediated therapy. Here, we show that lapatinib, an ATP-competitive inhibitor of HER2, is able to induce proliferation cooperatively with the HER3 ligand neuregulin. This counterintuitive synergy between inhibitor and growth factor depends on their ability to promote atypical HER2-HER3 heterodimerisation. By stabilising a particular HER2 conformer, lapatinib drives HER2-HER3 kinase domain heterocomplex formation. This dimer exists in a head-to-head orientation distinct from the canonical asymmetric active dimer. The associated clustering observed for these dimers predisposes to neuregulin responses, affording a proliferative outcome. Our findings provide mechanistic insights into the liabilities involved in targeting kinases with ATP-competitive inhibitors and highlight the complex role of protein conformation in acquired resistance.

Cluster Analysis of Endogenous HER2 and HER3 Receptors in SKBR3 Cells.

The Human Epidermal Growth Factor Receptor (HER) family of receptor tyrosine kinases consists of four, single pass, transmembrane receptor homologs (HER1-4) that act to regulate many critical processes in normal and tumor cells. HER2 is overexpressed in many tumors, and the deregulated proliferation of cancerous cells is driven by cooperation with its preferred receptor partner, HER3. The assessment of the in-situ organization of tagged HER2 and HER3 using super-resolution microscopy reveals quantitative Single Molecule Localization Microscopy (SMLM) as an ideal bioanalytical tool to characterize receptor clusters. Clustering of receptors is an important regulatory mechanism to prime cells to respond to stimuli so, to understand these processes, it is necessary to measure parameters such as numbers of clusters, cluster radii and the number of localizations per cluster for different perturbations. Previously, Fluorescence Localization Imaging with Photobleaching (FLImP), another nanoscale, single-molecule technique, characterized the oligomerization state of HER1 [or Epidermal Growth Factor Receptors (EGFR)] in cell membranes. To achieve an unprecedented resolution (< 5 nm) for inter-molecular separations in EGFR oligomers using FLImP, very few receptors are tagged, and so this method is unsuitable for measurements of whole receptor populations in cancer cells where receptors are frequently upregulated. Here, in order to detect all receptors involved in cluster formation, we saturate endogenous HER2 and HER3 membrane receptors with ligands at a 1:1 dye to protein ratio, in the presence or absence of therapeutic drugs (lapatinib or bosutinib). This is performed in the commonly used breast cancer cell line model SKBR3 cells, where there are ~1.6 million HER2 receptors/cell and 10,000-40,000 HER3 receptors/cell. The basal state of these receptors is studied using HER2- or HER3-specific Affibodies, and likewise, the active state is probed using the natural HER3 ligand, Neuregulin-beta1 (NRGß1). Stochastic Optical Reconstruction Microscopy (STORM), one form of SMLM, was used here to image cells, which were chemically fixed to minimize image blurring and provide data (x and y coordinates and standard deviation of the measured localizations) for cluster analysis. Further analysis can also determine proportions of receptor colocalizations. Our findings show that lapatinib-bound HER2, complexed with HER3 via a non-canonical kinase dimer structure, induces higher order oligomers. We hypothesized that nucleation of receptors creates signaling platforms that explain the counterintuitive, increase in cell proliferation upon ligand binding, in the presence of the HER2-inhibitor lapatinib.

Regulation of the cytokinesis cleavage furrow by PKCε.

Cytokinesis is the final act of the cell cycle where the replicated DNA and cellular contents are finally split into two daughter cells. This process is very tightly controlled as DNA segregation errors and cytokinesis failure is commonly associated with aneuploidy and aggressive tumours. Protein kinase Cε (PKCε) is a lipid-activated serine/threonine kinase that is part of the PKC superfamily. PKCε plays a complex role in the regulation of migration, adhesion and cytokinesis and in the present article we discuss the interplay between these processes. Integrin-mediated interaction with the actin cytoskeleton is a known regulator of cell adhesion and migration and there is emerging evidence that this pathway may also be essential for cytokinesis. We discuss evidence that a known actin-binding region in PKCε is involved in PKCε-mediated regulation of cytokinesis, providing a link between integrin-mediated stabilization of the cytokinesis furrow and PKCε recruitment.

A role for the pseudokinase HER3 in the acquired resistance against EGFR- and HER2-directed targeted therapy.

Specific inhibition of members of the EGFR (epidermal growth factor receptor) family, particularly EGFR and HER2 (human epidermal growth factor receptor 2), are an important therapeutic strategy in many human cancers. Compared with classical chemotherapy, these targeted therapeutics are very specific and initially effective, but acquired resistance against these targeted therapies is a recurring threat. A growing body of recent work has highlighted a pseudokinase in the EGFR family, HER3, and its ligand, NRG (neuregulin ß1), to be of importance in models of resistant cancers, as well as in patients. In the present article, we describe some of the roles in which HER3 can mediate acquired resistance and discuss the current efforts to target HER3 itself in cancer.

Functional implications of assigned, assumed and assembled PKC structures.

The empirical derivation of PKC (protein kinase C) domain structures and those modelled by homology or imputed from protein behaviour have been extraordinarily valuable both in the elucidation of PKC pathway mechanisms and in the general lessons that extrapolate to other signalling pathways. For PKC family members, there are many domain/subdomain structures and models, covering all of the known domains, variably present in this family of protein serine/threonine kinases (C1, C2, PB1, HR1, kinase domains). In addition to these structures, there are a limited number of complexes defined, including the structure of the PKCε V3-14-3-3 complex. In the context of structure-driven insights into PKC pathways, there are several broadly applicable principles and mechanisms relevant to the operation of and intervention in signalling pathways. These principles have an impact in unexpected ways, from the regulation of membrane targeting, through strategies for pharmacological intervention, to biomarkers.

Update on imatinib for gastrointestinal stromal tumors: duration of treatment.

Gastrointestinal stromal tumors (GISTs) are the most common sarcoma of the gastrointestinal tract, with transformation typically driven by activating mutations of c-KIT and less commonly platelet-derived growth factor receptor alpha (PDGFRA). Successful targeting of c-KIT and PDGFRA with imatinib, a tyrosine kinase inhibitor (TKI), has had a major impact in advanced GIST and as an adjuvant and neoadjuvant treatment. If treatment with imatinib fails, further lines of TKI therapy have a role, but disease response is usually only measured in months, so strategies to maximize the benefit from imatinib are paramount. Here, we provide an overview of the structure and signaling of c-KIT coupled with a review of the clinical trials of imatinib in GIST. In doing so, we make recommendations about the duration of imatinib therapy and suggest how best to utilize imatinib in order to improve patient outcomes in the future.

Pseudokinase drug intervention: a potentially poisoned chalice.

Pseudokinases, the catalytically impaired component of the kinome, have recently been found to share more properties with active kinases than previously thought. In many pseudokinases, ATP binding and even some activity is preserved, highlighting these proteins as potential drug targets. In both active kinases and pseudokinases, binding of ATP or drugs in the nucleotide-binding pocket can stabilize specific conformations required for activity and protein-protein interactions. We discuss the implications of locking particular conformations in a selection of (pseudo)kinases and the dual potential impact on the druggability of these proteins.

Distribution of resting and ligand-bound ErbB1 and ErbB2 receptor tyrosine kinases in living cells using number and brightness analysis.

Ligand-driven dimerizations of ErbB receptor subunits fulfill a fundamental role in their activation. We have used the number and brightness analysis technique to investigate the existence of preformed ligand-independent dimers and clusters and to characterize the initial steps in the activation of ErbB1 and ErbB2. In cells expressing 50,000-200,000 receptors, ErbB1 was monomeric in the absence of ligand stimulation, whereas in CHO cells with receptor levels >500,000 as much as 30% of ErbB1 was present as preformed dimers. EGF induced the formation of ErbB1 dimers as well as larger clusters (up to pentamers) that colocalized with clathrin-coated pits. The distribution of unstimulated ErbB2 in cells expressing 3·10(5)-10(6) receptors was fundamentally different, in that this receptor was present in preformed homoassociated aggregates containing 5-10 molecules. These constitutive ErbB2 homoclusters colocalized with caveolae, increased in size at subphysiological temperatures, but decreased in size upon EGF stimulation. We conclude that these ErbB2 clusters are promoted primarily by membrane-mediated interactions and are dispersed upon ligand stimulation.