Poster Presentation 36th Lorne Cancer Conference 2024

Primary high throughput screening for modulators of autophagy as drugs for cancer treatment using the powerful imaging reagent CalRexinTM:pHrodo Red. (#231)

Peter J Wookey 1 2 , David L Hare 1 , Joe D Ciccotosto 1
  1. Department of Medicine-Austin, University of Melbourne, Heidelberg, VIC, Australia
  2. Apop Biosciences, North Melbourne, Victoria, Australia

High throughput screening for candidate anti-cancer drugs that modify autophagic flux is an important primary screen for potential cancer treatments [1,2]. Autophagy interacts with apoptosis to define the tumour apototic threshold [2]. For instance chloroquine (CQ) as an inhibitor of the fusion of lysosomes and autophagosomes, an essential event in autophagy, have entered clinical trials for glioblastoma and melanoma and are effective in mouse models of colorectal adenocarcinoma. Related compounds dimeric quinacrine and bisaminoquinoline are 10-fold more effective than CQ in preclinical trials. Rapamycin and analogues induce autophagy by inhibiting mTOR and have entered clinical trials for several cancers [3]. CalRexinTM is an anti-calcitonin receptor (CTR) antibody that binds an extracellular epitope and is accumulated into receptor-positive cells. Stressed cells including autophagic cells express CTR. In the context of autophagy, unlike dyes that measure puncta of autophagosomes rather than flux, CalRexinTM:pHrodo Red measures the flux of this antibody conjugate through the entire elongation pathway from internalisation in early endosome, fusion of the late endosomes (pH 5.5, containing CalRexinTM:pHrodo Red) with autophagosomes to form the amphisomes which fuse with the lysosomes to form the autolysosomes (pH 4.5). Breakdown of material occurs in the autolysosomes. CalRexinTM:pHrodo Red is a pH dependent fluorescent dye which has high fluorescence intensity (FI) at pH 4.5 but low FI at pH 7. These events and properties provide the basis for a unique and powerful methodology to image in real time autophagy in live cells measured on the high throughput platform Operetta. The system was validated using induction of autophagy in several cell lines with rapamycin or serum-starvation, inhibitors CQ and 3-methyladenine and mutants blocked in autophagy ATG5 and FIP200. This new technology will allow for the first time, new classes of drug candidates to be identified with high throughput screening from different points along the elongation pathway.

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3. Xie J. et al. mTOR inhibitors in cancer therapy. F1000Research 2016;5:2078.