3-phosphoglycerate dehydrogenase as target in cancer therapy

PhD ThesisCancer cells adapt their metabolism to simultaneously fulfil the requirements of energy production and biomass generation necessary to sustain high proliferation rates. This deregulated energy metabolism and the proteins responsible therefor provide a potential new route of targeting cancer that has not been thoroughly explored. 3-Phosphoglycerate dehydrogenase (PHGDH), which takes 3-phosphoglycerate (3-PG) out of the glycolytic pathway and into serine production, has been reported as potential target in certain breast cancer forms and melanoma. There is no known inhibitor of PHGDH to date to fully validate the target. Inhibition of PHGDH was explored in breast cancer and melanoma cell lines using siRNA and shRNA interference techniques. Greater knockdown was achieved by siRNA resulting in better growth inhibition than when using shRNA expressing cell lines. The substrate-binding pocket was investigated with substrate analogues and substrate-containing compounds. NAD+-binding was found to be stabilised by coordinated binding of substrates. The catalytic subunits of human PHGDH were crystallised and revealed a flexible lid domain that moves in response to substrate binding. The NAD+-fragment adenosine 5’-diphosphoribose (ADPR) was shown to be a moderate inhibitor of the enzymatic activity of PHGDH and was used for assay validation. Cofactor analogues with different substituents around the pyridine ring were equally suitable to promote the oxidation of 3-PG. A fragment screen was performed using differential scanning fluorimetry and hits were subsequently validated by competition isothermal titration calorimetry. To investigate the fragments in crystals of human PHGDH, a truncated form of PHGDH (construct 93) was engineered by limited proteolysis. Soaking of fragments into crystals of 93 confirmed binding of seven fragments. Structure-activity relationship studies were initiated around the confirmed hits

Targeting OGG1 arrests cancer cell proliferation by inducing replication stress

Altered oncogene expression in cancer cells causes loss of redox homeostasis resulting in oxidative DNA damage, e.g. 8-oxoguanine (8-oxoG), repaired by base excision repair (BER). PARP1 coordinates BER and relies on the upstream 8-oxoguanine-DNA glycosylase (OGG1) to recognise and excise 8-oxoG. Here we hypothesize that OGG1 may represent an attractive target to exploit reactive oxygen species (ROS) elevation in cancer. Although OGG1 depletion is well tolerated in non-transformed cells, we report here that OGG1 depletion obstructs A3 T-cell lymphoblastic acute leukemia growth in vitro and in vivo, validating OGG1 as a potential anti-cancer target. In line with this hypothesis, we show that OGG1 inhibitors (OGG1i) target a wide range of cancer cells, with a favourable therapeutic index compared to non-transformed cells. Mechanistically, OGG1i and shRNA depletion cause S-phase DNA damage, replication stress and proliferation arrest or cell death, representing a novel mechanistic approach to target cancer. This study adds OGG1 to the list of BER factors, e.g. PARP1, as potential targets for cancer treatment