Dissecting signaling regulators driving AXL-mediated bypass resistance and associated phenotypes by phosphosite perturbations.

Receptor tyrosine kinase (RTK)-targeted therapies are often effective but invariably limited by drug resistance. A major mechanism of acquired resistance involves "bypass" switching to alternative pathways driven by non-targeted RTKs that restore proliferation. One such RTK is AXL whose overexpression, frequently observed in bypass resistant tumors, drives both cell survival and associated malignant phenotypes such as epithelial-to-mesenchymal (EMT) transition and migration. However, the signaling molecules and pathways eliciting these responses have remained elusive. To explore these coordinated effects, we generated a panel of mutant lung adenocarcinoma PC9 cell lines in which each AXL intracellular tyrosine residue was mutated to phenylalanine. By integrating measurements of phosphorylation signaling and other phenotypic changes associated with resistance through multivariate modeling, we mapped signaling perturbations to specific resistant phenotypes. Our results suggest that AXL signaling can be summarized into two clusters associated with progressive disease and poor clinical outcomes in lung cancer patients. These clusters displayed favorable Abl1 and SFK motifs and their phosphorylation was consistently decreased by dasatinib. High-throughput kinase specificity profiling showed that AXL likely activates the SFK cluster through FAK1 which is known to complex with Src. Moreover, the SFK cluster overlapped with a previously established focal adhesion kinase (FAK1) signature conferring EMT-mediated erlotinib resistance in lung cancer cells. Finally, we show that downstream of this kinase signaling, AXL and YAP form a positive feedback loop that sustains drug tolerant persister cells. Altogether, this work demonstrates an approach for dissecting signaling regulators by which AXL drives erlotinib resistance-associated phenotypic changes.

Systems approaches to uncovering the contribution of environment-mediated drug resistance.

Cancer drug response is heavily influenced by the extracellular matrix (ECM) environment. Despite a clear appreciation that the ECM influences cancer drug response and progression, a unified view of how, where, and when environment-mediated drug resistance contributes to cancer progression has not coalesced. Here, we survey some specific ways in which the ECM contributes to cancer resistance with a focus on how materials development can coincide with systems biology approaches to better understand and perturb this contribution. We argue that part of the reason that environment-mediated resistance remains a perplexing problem is our lack of a wholistic view of the entire range of environments and their impacts on cell behavior. We cover a series of recent experimental and computational tools that will aid exploration of ECM reactions space, and how they might be synergistically integrated.

Dual data and motif clustering improves the modeling and interpretation of phosphoproteomic data.

Cell signaling is orchestrated in part through a network of protein kinases and phosphatases. Dysregulation of kinase signaling is widespread in diseases such as cancer and is readily targetable through inhibitors. Mass spectrometry-based analysis can provide a global view of kinase regulation, but mining these data is complicated by its stochastic coverage of the proteome, measurement of substrates rather than kinases, and the scale of the data. Here, we implement a dual data and motif clustering (DDMC) strategy that simultaneously clusters peptides into similarly regulated groups based on their variation and their sequence profile. We show that this can help to identify putative upstream kinases and supply more robust clustering. We apply this clustering to clinical proteomic profiling of lung cancer and identify conserved proteomic signatures of tumorigenicity, genetic mutations, and immune infiltration. We propose that DDMC provides a general and flexible clustering strategy for the analysis of phosphoproteomic data.

Integrated Proteomics-Based Physical and Functional Mapping of AXL Kinase Signaling Pathways and Inhibitors Define Its Role in Cell Migration.

To better understand the signaling complexity of AXL, a member of the tumor-associated macrophage (TAM) receptor tyrosine kinase family, we created a physical and functional map of AXL signaling interactions, phosphorylation events, and target-engagement of three AXL tyrosine kinase inhibitors (TKI). We assessed AXL protein complexes using proximity-dependent biotinylation (BioID), effects of AXL TKI on global phosphoproteins using mass spectrometry, and target engagement of AXL TKI using activity-based protein profiling. BioID identifies AXL-interacting proteins that are mostly involved in cell adhesion/migration. Global phosphoproteomics show that AXL inhibition decreases phosphorylation of peptides involved in phosphatidylinositol-mediated signaling and cell adhesion/migration. Comparison of three AXL inhibitors reveals that TKI RXDX-106 inhibits pAXL, pAKT, and migration/invasion of these cells without reducing their viability, while bemcentinib exerts AXL-independent phenotypic effects on viability. Proteomic characterization of these TKIs demonstrates that they inhibit diverse targets in addition to AXL, with bemcentinib having the most off-targets. AXL and EGFR TKI cotreatment did not reverse resistance in cell line models of erlotinib resistance. However, a unique vulnerability was identified in one resistant clone, wherein combination of bemcentinib and erlotinib inhibited cell viability and signaling. We also show that AXL is overexpressed in approximately 30% to 40% of nonsmall but rarely in small cell lung cancer. Cell lines have a wide range of AXL expression, with basal activation detected rarely. IMPLICATIONS: Our study defines mechanisms of action of AXL in lung cancers which can be used to establish assays to measure drug targetable active AXL complexes in patient tissues and inform the strategy for targeting it's signaling as an anticancer therapy.

Hierarchical Organization Endows the Kinase Domain with Regulatory Plasticity.

The functional diversity of kinases enables specificity in cellular signal transduction. Yet how more than 500 members of the human kinome specifically receive regulatory inputs and convey information to appropriate substrates-all while using the common signaling output of phosphorylation-remains enigmatic. Here, we perform statistical co-evolution analysis, mutational scanning, and quantitative live-cell assays to reveal a hierarchical organization of the kinase domain that facilitates the orthogonal evolution of regulatory inputs and substrate outputs while maintaining catalytic function. We find that three quasi-independent "sectors"-groups of evolutionarily coupled residues-represent functional units in the kinase domain that encode for catalytic activity, substrate specificity, and regulation. Sector positions impact both disease and pharmacology: the catalytic sector is significantly enriched for somatic cancer mutations, and residues in the regulatory sector interact with allosteric kinase inhibitors. We propose that this functional architecture endows the kinase domain with inherent regulatory plasticity.

The Feasibility of Encapsulated Embryonic Medullary Reticular Cells to Grow and Differentiate Into Neurons in Functionalized Gelatin-Based Hydrogels.

The study of the behavior of embryonic neurons in controlled in vitro conditions require methodologies that take advantage of advanced tissue engineering approaches to replicate elements of the developing brain extracellular matrix. We report here a series of experiments that explore the potential of photo-polymerized gelatin hydrogels to culture primary embryonic neurons. We employed large medullary reticular neurons whose activity is essential for brain arousal as well as a library of gelatin hydrogels that span a range of mechanical properties, inclusion of brain-mimetic hyaluronic acid, and adhesion peptides. These hydrogel platforms showed inherent capabilities to sustain neuronal viability and were permissive for neuronal differentiation, resulting in the development of neurite outgrowth under specific conditions. The maturation of embryonic medullary reticular cells took place in the absence of growth factors or other exogenous bioactive molecules. Immunocytochemistry labeling of neuron-specific tubulin confirmed the initiation of neural differentiation. Thus, this methodology provides an important validation for future studies of nerve cell growth and maintenance.

Macroscopic DNA helices.

Presently there is great interest in the construction of nanostructures from DNA fragments. Here we report the preparation of much larger helical textures with different shapes from pure DNA fragments. We have observed them while preparing crystals of the dodecamer d(AAAAAATTTTTT), which only contains adenine and thymine. Noncoding regions of the genome are always rich in these two bases. We have found a strong influence of ions either monovalent or divalent, with which different crystalline structures are found. All of them contain long helical stacks of duplexes in the unit cell. The most remarkable structures are macroscopic helices with diameters and pitch in the range of 20-40 microm. Thus, pure DNA oligonucleotides may form a hierarchy of helical structures, going from the B-form double helix (pitch, p = 33 A) to helical stacks of duplexes (p approximately 900 A), and to macroscopic helices (p approximately 300,000 A). These different levels of organization are reminiscent of the different levels of organization of DNA in eukaryotic chromosomes.

Overview of the structure of all-AT oligonucleotides: organization in helices and packing interactions.

We present the crystalline organization of 33 all-AT deoxyoligonucleotide duplexes, studied by x-ray diffraction. Most of them have very similar structures, with Watson-Crick basepairs and a standard average twist close to 36 degrees. The molecules are organized as parallel columns of stacked duplexes in a helical arrangement. Such organization of duplexes is very regular and repetitive: all sequences show the same pattern. It is mainly determined by the stacking of the terminal basepairs, so that the twist in the virtual TA base step between neighbor duplexes is always negative, approximately -22 degrees. The distance between the axes of parallel columns is practically identical in all cases, approximately 26 A. Interestingly, it coincides with that found in DNA viruses and fibers in their hexagonal phase. It appears to be a characteristic distance for ordered parallel DNA molecules. This feature is due to the absence of short range intermolecular forces, which are usually due to the presence of CG basepairs at the end of the oligonucleotide sequence. The duplexes apparently interact only through their diffuse ionic atmospheres. The results obtained can thus be considered as intermediate between liquid crystals, fibers, and standard crystal structures. They provide new information on medium range DNA-DNA interactions.