TUBITAK Project, 2022 - 2024
Pancreatic Ductal Adenocarcinoma (PDA) is a disease with dismal prognosis and innovative treatment modalities are urgently needed. Targeted molecular therapies that show benefit in different types of cancers have yet to find use in PDA. Recent studies show that PDA cells develop unique metabolic dependencies for survival and proliferation through metabolic reprogramming. Inhibition of metabolic enzymes required for cell proliferation may be an effective strategy for the treatment of PDA. However, considering the metabolic plasticity of PDA cells, targeting of multiple metabolic enzymes instead of a single enzyme will have a higher chance of success in preventing the development of PDA.
The control of the accelerated glycolytic activity needed for malignant properties of PDA cells such as proliferation may represent a natural component of combinatorial approaches aimed at targeting metabolic dependencies of PDA. The 6-phosfofructo-2-kinase/fructose-2,6-bisphosphatase enzyme family (PFKFB1-4) is among the prominent activators of tumor glycolysis. Our recent study shows that the PFKFB2 isoform is required for the glycolytic activity of PDA cells. Preliminary findings demonstrated that while PDA cell proliferation is inhibited by transientsilencing of PFKFB2 expression, PDA cells were able to overcome the anti-proliferative effect of constitutive PFKFB2 dysfunction. This observation supports the notion that PDA cells, via their metabolic plasticity, were able to tolerate the lack of PFKFB2 function. Based on this observation we developed the hypothesis that “PDA cells whose glycolytic activity is suppressed by PFKFB2 inactivation are forced to metabolic reprogramming and become dependent on different metabolic gene(s) for survival / proliferation and, therefore, genetic / pharmacological targeting of these gene (s) with PFKFB2 will prevent the proliferation of PDA cells and PDA progression. To test the hypothesis; we aimed to determine the metabolic gene(s) that exhibits synthetic lethal interaction with the PFKFB2 gene and to assess the role of this interaction in PDA development by performing metabolism-focused CRISPR / Cas9 screening in human PDA cell models. Towards this end, PANC-1 cell clones whose PFKFB2 gene was targeted with CRISPR/Cas9 (PFKFB2 Crispr) will be employed to conduct the study. In order to find the synthetic letal interactors of PFKFB2, a lentiviral plasmid library containing sgRNA sequences that target approximately 3000 metabolic genes will be used. Control Crispr and PFKFB2 Crispr clones will be transduced with a dose of lentiviral particles such that each cell will take one particle at most and then cells that have not been transduced will be eliminated with antibiotic selection. Cells will be allowed to grow 15 population doublings. Following genomic DNA isolation, sgRNA will be PCR-amplified and abundance of each sgRNA will be analyzed by next-generation sequencing. Following genetic/pharmacological validation of the interaction of candidate genes with PFKFB2 in vitro, the requirement of the synthetic lethal interaction of 1 (one) gene with PFKFB2 for PDA progression will be tested in a PDA xenograft model.
Validation of the hypothesis may constitute a scientific basis for combined targeting of PFKFB2 and another metabolic gene to prevent the proliferation of PDA cells and for the test of such combinations in appropriate pre-clinical models and clinical trials.