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Discovery of Potent Small-Molecule Inhibitors of WDR5-MYC Interaction
No full textWD repeat domain 5 (WDR5) is a member of the WD40-repeat protein family that plays a critical role in multiple processes and is also a prominent target for pharmacological inhibition in multiple disease such as cancer, aging and neurodegenerative disorders. Interaction between WDR5 and its various partners are essential for sustaining its function. Most drug-discovery efforts center on the WIN site of WDR5 that is responsible for the recruitment of WDR5 to chromatin. Here, we describe the discovery of novel WDR5 inhibitors for the other WBM pocket on this scaffold protein, to disrupt WDR5 interaction with its binding partner MYC by high-throughput biochemical screening, subsequent molecule optimization and biological assessment. These new WDR5 inhibitors provide useful probes for future investigations of WDR5 and an avenue for targeting WDR5 as a therapeutic strategy
Two peak elution behavior of a monoclonal antibody in cation exchange chromatography as a screening tool for excipients
No full textAggregation of proteins is a critical quality attribute and a major concern during the purification of therapeutic proteins, like monoclonal antibodies. In-solution experiments applying different stress scenarios, e.g., mechanical, or physical stresses, can determine the overall conformational stability of the protein to enhance drug product shelf-life. Several groups have reported surface-induced unfolding and aggregation of monoclonal antibodies and their derivatives during cation exchange chromatography, which results in a two-peak elution behavior of the protein and its species. We have investigated universal influencing factors, like temperature and hold time, on this phenomenon. The formation of the second peak is a kinetic process, which is strongly influenced by temperature during the hold time. However, our main focus was the application of excipients and their influence on the two-peak elution behavior. We compared the on-column screening results with results obtained through a “traditional” in-solution screening using nanoDSF. Mostly, stabilizing excipients, like Sucrose, show their stabilizing abilities in both systems, but some discrepancies, e.g., using Arginine, between the two orthogonal techniques show the potential of the on-column screening system to lead to unexpected results, which would not necessarily be visible in in-solution experiments
Loss of Wdr5 attenuates MLL-rearranged leukemogenesis by suppressing Myc targets.
No full textWD repeat domain 5 (WDR5) is a prominent target for pharmacological inhibition in cancer through its scaffolding role with various oncogenic partners such as MLL and MYC. WDR5-related drug discovery efforts center on blocking these binding interfaces or degradation have been devoted to developing small-molecule inhibitors or degraders of WDR5 for cancer treatment. Nevertheless, the precise role of WDR5 in these cancer cells has not been well elucidated genetically. Here, by using an MLL-AF9 murine leukemia model, we found that genetically deletion of Wdr5 impairs cell growth and colony forming ability of MLL-AF9 leukemia cells in vitro or ex vivo and attenuates the leukemogenesis in vivo as well, which acts through direct regulation of ribosomal genes. Pharmacological inhibition of Wdr5 recapitulates genetic study results in the same model. In conclusion, our current study demonstrated the first genetic evidence for the indispensable role of Wdr5 in MLL-r leukemogenesis in vivo, which supports therapeutically targeting WDR5 in MLL-rearranged leukemia by strengthening its disease linkage genetically and deepening insights into its mechanism of action
Intrinsic BMP inhibitor Gremlin regulates alveolar epithelial type II cell proliferation and differentiation.
No full textType 1 alveolar epithelial cells (AT1s) and type 2 alveolar epithelial cells (AT2s) regulate the structural integrity and function of alveoli. AT1s mediate gas exchange, whereas AT2s serve multiple functions, including surfactant secretion and alveolar repair through proliferation and differentiation into AT1s as progenitors. However, mechanisms regulating AT2 proliferation and differentiation remain unclear. Here we demonstrate that Gremlin, an intrinsic inhibitor of bone morphogenetic protein (BMP), induces AT2 proliferation and differentiation. Transient overexpression of Gremlin in rat lungs by adenovirus vector delivery suppressed BMP signaling, induced proliferation of AT2s and the production of Bmp2, which in turn led to the recovery of BMP signaling and induced AT2 differentiation into AT1s. Bleomycin-induced lung injury upregulated Gremlin and showed a similar time course of biomarker expression comparable to the adenovirus model. TGF-β and IL-1β induced Gremlin expression in fibroblasts. Taken toge
Vascular-Parenchymal Cross-Talk Promotes Lung Fibrosis through BMPR2 Signaling.
No full textIdiopathic pulmonary fibrosis (IPF) is a fatal lung disease characterized by progressive lung scarring. IPF-related pulmonary vascular remodeling and pulmonary hypertension (PH) result in a particularly poor prognosis. To study the pathogenesis of vascular remodeling in fibrotic lungs and its contribution to progression of fibrosis. We used an experimental model of lung fibrosis associated with PH by transient overexpression of active TGF-β1 (transforming growth factor-β1). Samples from patients with fibrotic lung diseases were analyzed in depth using immunostaining, gene expression, and gene mutations. We found a reduction in endothelial cells (ECs) and activation of vascular smooth muscle cells (VSMCs) in fibrotic lungs. Coculturing fibroblasts with VSMCs or ECs from fibrotic lungs induced fibrotic phenotypes in fibroblasts. IPF fibroblasts induced EC death and activation of VSMCs in coculture systems. Decreased concentrations of BMPR2 (bone morphogenic protein receptor 2) and its signaling were observe
Cancer lineage-specific regulation of YAP responsive elements revealed through large-scale functional epigenomic screens.
No full textYAP is a key transcriptional co-activator of TEADs, it regulates cell growth and is frequently activated in cancer. In Malignant Pleural Mesothelioma (MPM), YAP is activated by loss-of-function mutations in upstream components of the Hippo pathway, while, in Uveal Melanoma (UM), YAP is activated in a Hippo-independent manner. To date, it is unclear if and how the different oncogenic lesions activating YAP impact its oncogenic program, which is particularly relevant for designing selective anti-cancer therapies. Here we show that, despite YAP being essential in both MPM and UM, its interaction with TEAD is unexpectedly dispensable in UM, limiting the applicability of TEAD inhibitors in this cancer type. Systematic functional interrogation of YAP regulatory elements in both cancer types reveals convergent regulation of broad oncogenic drivers in both MPM and UM, but also strikingly selective programs. Our work reveals unanticipated lineage-specific features of the YAP regulatory network that provide important insights to guide the design of tailored therapeutic strategies to inhibit YAP signaling across different cancer types
In vitro flow cytometry assay to assess human and mouse macrophage phagocytosis of tumor cells
No full textAlthough tumor-associated macrophages are generally immunosuppressive, macrophages may also promote tumor clearance via phagocytosis of live tumor cells. Here, we describe a protocol for assessing macrophage engulfment of tumor cells in vitro using flow cytometry. The method is applicable to both mouse bone marrow-derived macrophages and human monocyte-derived macrophages. SMAC mimetics and recombinant IFN- or lymphotoxin are used as positive controls. This assay can be used to identify compounds affecting phagocytosis or to mechanistically interrogate pathways involved in phagocytic uptake
Discovery of new binders for DCAF1, an emerging ligase target in the Targeted Protein Degradation field
No full textIn this study, we describe the rapid identification of potent binders for the WD40 repeat domain (WDR) of DCAF1. This was achieved by two rounds of iterative focused screening of a small set of compounds selected based on internal WDR domain knowledge followed by hit expansion. Subsequent structure-based design led to nanomolar potency binders with a clear exit vector enabling DCAF1-based bifunctional degrader exploration
A consensus protocol for functional connectivity analysis in the rat brain
No full textTask-free functional connectivity in animal models provides an experimental framework to examine connectivity phenomena under controlled conditions and allows for comparisons with data modalities collected under invasive or terminal procedures. Currently, animal acquisitions are performed with varying protocols and analyses that hamper result comparison and integration. Here we introduce StandardRat, a consensus rat functional magnetic resonance imaging acquisition protocol tested across 20 centers. To develop this protocol with optimized acquisition and processing parameters, we initially aggregated 65 functional imaging datasets acquired from rats across 46 centers. We developed a reproducible pipeline for analyzing rat data acquired with diverse protocols and determined experimental and processing parameters associated with the robust detection of functional connectivity across centers. We show that the standardized protocol enhances biologically plausible functional connectivity patterns relative to previous acquisitions. The protocol and processing pipeline described here is openly shared with the neuroimaging community to promote interoperability and cooperation toward tackling the most important challenges in neuroscience
Human PIK3R1 mutations disrupt lymphocyte differentiation to cause activated PI3Kδ syndrome 2.
No full textHeterozygous loss-of-function (LOF) mutations in PIK3R1 (encoding phosphatidylinositol 3-kinase [PI3K] regulatory subunits) cause activated PI3Kδ syndrome 2 (APDS2), which has a similar clinical profile to APDS1, caused by heterozygous gain-of-function (GOF) mutations in PIK3CD (encoding the PI3K p110δ catalytic subunit). While several studies have established how PIK3CD GOF leads to immune dysregulation, less is known about how PIK3R1 LOF mutations alter cellular function. By studying a novel CRISPR/Cas9 mouse model and patients' immune cells, we determined how PIK3R1 LOF alters cellular function. We observed some overlap in cellular defects in APDS1 and APDS2, including decreased intrinsic B cell class switching and defective Tfh cell function. However, we also identified unique APDS2 phenotypes including defective expansion and affinity maturation of Pik3r1 LOF B cells following immunization, and decreased survival of Pik3r1 LOF pups. Further, we observed clear differences in the way Pik3r1 LOF and Pik3cd GOF altered signaling. Together these results demonstrate crucial differences between these two genetic etiologies