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1.
Elife ; 102021 03 01.
Article in English | MEDLINE | ID: mdl-33647232

ABSTRACT

BLM (Bloom syndrome protein) is a RECQ-family helicase involved in the dissolution of complex DNA structures and repair intermediates. Synthetic lethality analysis implicates BLM as a promising target in a range of cancers with defects in the DNA damage response; however, selective small molecule inhibitors of defined mechanism are currently lacking. Here, we identify and characterise a specific inhibitor of BLM's ATPase-coupled DNA helicase activity, by allosteric trapping of a DNA-bound translocation intermediate. Crystallographic structures of BLM-DNA-ADP-inhibitor complexes identify a hitherto unknown interdomain interface, whose opening and closing are integral to translocation of ssDNA, and which provides a highly selective pocket for drug discovery. Comparison with structures of other RECQ helicases provides a model for branch migration of Holliday junctions by BLM.


Subject(s)
RecQ Helicases/antagonists & inhibitors , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology , DNA/metabolism , DNA, Cruciform , DNA, Single-Stranded , Drug Discovery , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Escherichia coli , High-Throughput Screening Assays , Humans , RecQ Helicases/metabolism
2.
J Clin Invest ; 130(6): 3188-3204, 2020 06 01.
Article in English | MEDLINE | ID: mdl-32125284

ABSTRACT

As there is growing evidence for the tumor microenvironment's role in tumorigenesis, we investigated the role of fibroblast-expressed kinases in triple-negative breast cancer (TNBC). Using a high-throughput kinome screen combined with 3D invasion assays, we identified fibroblast-expressed PIK3Cδ (f-PIK3Cδ) as a key regulator of cancer progression. Although PIK3Cδ was expressed in primary fibroblasts derived from TNBC patients, it was barely detectable in breast cancer (BC) cell lines. Genetic and pharmacological gain- and loss-of-function experiments verified the contribution of f-PIK3Cδ in TNBC cell invasion. Integrated secretomics and transcriptomics analyses revealed a paracrine mechanism via which f-PIK3Cδ confers its protumorigenic effects. Inhibition of f-PIK3Cδ promoted the secretion of factors, including PLGF and BDNF, that led to upregulation of NR4A1 in TNBC cells, where it acts as a tumor suppressor. Inhibition of PIK3Cδ in an orthotopic BC mouse model reduced tumor growth only after inoculation with fibroblasts, indicating a role of f-PIK3Cδ in cancer progression. Similar results were observed in the MMTV-PyMT transgenic BC mouse model, along with a decrease in tumor metastasis, emphasizing the potential immune-independent effects of PIK3Cδ inhibition. Finally, analysis of BC patient cohorts and TCGA data sets identified f-PIK3Cδ (protein and mRNA levels) as an independent prognostic factor for overall and disease-free survival, highlighting it as a therapeutic target for TNBC.


Subject(s)
Class I Phosphatidylinositol 3-Kinases/biosynthesis , Fibroblasts/enzymology , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Neoplastic , Neoplasm Proteins/biosynthesis , Triple Negative Breast Neoplasms/enzymology , Animals , Class I Phosphatidylinositol 3-Kinases/genetics , Female , Fibroblasts/pathology , Heterografts , Humans , Mice , Mice, Transgenic , Neoplasm Invasiveness , Neoplasm Metastasis , Neoplasm Proteins/genetics , Neoplasm Transplantation , Triple Negative Breast Neoplasms/genetics , Triple Negative Breast Neoplasms/pathology
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