Multicohort Retrospective Validation of a Predictive Biomarker for Topoisomerase I Inhibitors.

Purpose: The camptothecin (CPT) analogs topotecan and irinotecan specifically target topoisomerase I (topoI) and are used to treat colorectal, gastric, and pancreatic cancer. Response rate for this class of drug varies from 10% to 30%, and there is no predictive biomarker for patient stratification by response. On the basis of our understanding of CPT drug resistance mechanisms, we developed an immunohistochemistry-based predictive test, P-topoI-Dx, to stratify the patient population into those who did and did not experience a response.

Developing a Phosphospecific IHC Assay as a Predictive Biomarker for Topoisomerase I Inhibitors.

Phosphorylation is the most extensively studied posttranslational modification of proteins. There are approximately 500 kinases known in the human genome. The kinase-activated pathways regulate almost every aspect of cell function and a deregulated kinase cascade leads to impaired cellular function.

Camptothecin resistance is determined by the regulation of topoisomerase I degradation mediated by ubiquitin proteasome pathway.

Proteasomal degradation of topoisomerase I (topoI) is one of the most remarkable cellular phenomena observed in response to camptothecin (CPT). Importantly, the rate of topoI degradation is linked to CPT resistance. Formation of the topoI-DNA-CPT cleavable complex inhibits DNA re-ligation resulting in DNA-double strand break (DSB).

GRP78(BiP) facilitates the cytosolic delivery of anthrax lethal factor (LF) in vivo and functions as an unfoldase in vitro.

Anthrax toxin is an A/B bacterial protein toxin which is composed of the enzymatically active Lethal Factor (LF) and/or Oedema Factor (EF) bound to Protective Antigen 63 (PA63) which functions as both the receptor binding and transmembrane domains. Once the toxin binds to its cell surface receptors it is internalized into the cell and traffics through Rab5- and Rab7-associated endosomal vesicles. Following acidification of the vesicle lumen, PA63 undergoes a dynamic change forming a beta-barrel that inserts into and forms a pore through the endosomal membrane.

Essential lysine residues within transmembrane helix 1 of diphtheria toxin facilitate COPI binding and catalytic domain entry.

The translocation of the diphtheria toxin catalytic domain from the lumen of early endosomes into the cytosol of eukaryotic cells is an essential step in the intoxication process. We have previously shown that the in vitro translocation of the catalytic domain from the lumen of toxin pre-loaded endosomal vesicles to the external medium requires the addition of cytosolic proteins including coatomer protein complex I (COPI) to the reaction mixture. Further, we have shown that transmembrane helix 1 plays an essential, but as yet undefined role in the entry process.