P-096: Tackling pyrimidine biosynthesis - CTP Synthase 1 is a novel target in the treatment of multiple myeloma

Background Clinical progress in patients with high-risk multiple myeloma (MM) is limited and median survival remains at less than two years. Overexpression of cell cycle and proliferation-related genes is frequently observed in high-risk MM and novel treatment strategies for these patients are urgently needed. While the demand for pyrimidines is saturated via salvage pathways in resting cells, proliferating cells depend on the de novo synthesis of pyrimidines to meet the high demand for nucleotides. We therefore speculated that this pathway represents an attractive target in proliferation-associated high-risk myeloma. Methods In silico analysis of publicly available gene expression data sets was used to analyze CTP Synthase 1 (CTPS1) expression in different MM subgroups. In vitro, CTPS1 was evaluated as a target via generation of CRISPR/Cas9 CTPS1 knock-out cell lines. Apoptosis induction, cell cycle analysis and membrane potential of the cells were analyzed by flow cytometry. Additionally, the CTPS1 inhibitor STP938 (Step Pharma) was tested in 12 MM cell lines for its anti-myeloma properties. Uridine and cytidine rescue experiments were used to confirm the specificity of the compound. Previously established resistance models to pomalidomide and carfilzomib were used to demonstrate the efficacy of STP938 in advanced disease stages. Results In silico gene expression analysis revealed an upregulation of CTPS1 in MM and plasma cell leukemia patients compared to healthy-donor bone marrow plasma cells. Additionally, CTPS1 was found to be upregulated in patients with a proliferative gene expression pattern and high expression levels were linked to poor patient outcome. In vitro, stable CTPS1 knock-out clones failed to proliferate in the absence of exogenous cytidine which can be recycled via the nucleotide salvage pathway. Proliferation inhibition was accompanied by induction of an S-phase cell cycle arrest and apoptosis. The highly specific CTPS1 inhibitor STP938 showed potent inhibition of cell viability in 6 out of 12 cell lines. In accordance with the results obtained in genetic perturbation experiments we observed apoptosis induction, S phase arrest, and loss of mitochondrial membrane potential upon treatment with STP938. Addition of mM concentrations of exogenous cytidine, but not uridine, protected MM cell lines from apoptosis underlining the specificity of STP938. Importantly, the compound remained effective in models resistant to carfilzomib and pomalidomide. Conclusions Our in silico data establish CTPS1 as a potential novel target for proliferation associated high-risk disease. In vitro, the essential role of CTPS1 was confirmed by CRISPR/Cas9 deletion and the CTPS1 inhibitor STP938 was found to be highly efficacious in inducing apoptosis in several myeloma cell lines. Pre-clinical experiments have thus confirmed the drugability of CTPS1 and therefore strongly support the further evaluation of STP938.