Alluri, R. | bioRxiv Science

bioRxiv2021

Lymphocyte-specific Protein Tyrosine Kinase Regulates Homologous Recombination (HR) DNA Repair and Targeting Enhances PARPi Utility in HR Proficient Ovarian Cancer

Poly-ADP Ribose Polymerase (PARP) targeted therapy is clinically approved for the treatment of homologous recombination (HR) repair deficient tumors. The remarkable success in treatment of HR repair deficient cancers has not translated to HR-proficient cancer. Our studies identify the mechanism of non-receptor lymphocyte-specific protein tyrosine kinase (LCK) in HR repair in endometrioid epithelial ovarian cancer (eEOC). LCK expression is induced and activation in the nucleus in response to DNA damage insult. LCK inhibition attenuates expression of RAD51, BRCA1, and BRCA2 proteins necessary for HR-mediated DNA repair, sufficient to suppress RAD51 foci formation, and augments {gamma}H2AX foci formation. Mechanistically, DNA damage leads to direct interaction of LCK with RAD51 and BRCA1 in a kinase dependent manner. Attenuation of LCK sensitized HR-proficient eEOC cells to PARP inhibitor in cell culture and pre-clinical mouse studies. These findings identify a new mechanism for expanding utility of PARP inhibitors in HR proficient ovarian cancer. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=120 SRC="FIGDIR/small/433791v2_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@1715c60org.highwire.dtl.DTLVardef@167cb5forg.highwire.dtl.DTLVardef@158d807org.highwire.dtl.DTLVardef@1ced201_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2020

X-ray and solution structures of human beta-2 glycoprotein I reveal a new mechanism of autoantibody recognition

Venous and arterial thromboses in patients suffering from the autoimmune disorder Antiphospholipid Syndrome (APS) are caused by the presence of antiphospholipid antibodies (aPL). Emerging evidence indicates that autoantibodies targeting the epitope R39-R43 in the N-terminal domain, Domain I (DI), of {beta}2-glycoprotein I ({beta}2GPI) are among the most pathogenic aPL in patients with APS. How such autoantibodies engage {beta}2GPI at the molecular level remains incompletely understood. Here, we have used X-ray crystallography, single-molecule FRET, and small-angle X-ray scattering to demonstrate that, in the free form, under physiological pH and salt concentrations, human recombinant {beta}2GPI adopts an elongated, flexible conformation in which DI is exposed to the solvent, thus available for autoantibody recognition. Consistent with this structural model, binding and mutagenesis studies revealed that the elongated form interacts with a pathogenic anti-DI antibody in solution, without the need of phospholipids. Furthermore, complex formation was affected neither by the neighboring domains, nor by the presence of the linkers, nor by the glycosylations. Since the pathogenic autoantibody requires residues R39 and R43 for optimal binding, these findings challenge longstanding postulates in the field envisioning {beta}2GPI adopting immunologic inert conformations featuring inaccessibility of the epitope R39-R43 in DI and support an alternative model whereby the preferential binding of anti-DI antibodies towards phospholipid-bound {beta}2GPI arises from the ability of the pre-existing elongated form to bind to the membranes and then oligomerize, processes that are likely to be supported by protein conformational changes. Interfering with these steps may limit the pathogenic effects of anti-DI antibodies in APS patients. SignificanceIn the autoimmune disorder called Antiphospholipid Syndrome (APS), the presence of autoantibodies targeting the plasma glycoprotein beta-2 glycoprotein I ({beta}2GPI) is associated with arterial and venous thrombosis as well as pregnancy complications. Understanding how {beta}2GPI becomes immunogenic and how autoantibodies in complex with {beta}2GPI cause the blood to clot remains a top priority in the field. By elucidating the structural architecture of {beta}2GPI free in solution, our studies challenge longstanding postulates in the field and shed new light on the pathogenic mechanisms of APS that may help the development of new diagnostics and therapeutic approaches.

biochemistry↗