Effects of the Novel PFKFB3 Inhibitor KAN0438757 on Colorectal Cancer Cells and Its Systemic Toxicity Evaluation In Vivo

SIMPLE SUMMARY: Glycolysis is one of the hallmarks of cancer. Therefore, the development of novel therapeutical strategies for colorectal cancer targeting glycolysis may improve treatment responses. PFKFB3 expression has been directly associated with enhanced glycolysis, not only in cancer cells but also within the tumor environment. The aim of this study was to evaluate PFKFB3 expression and its correlation with outcome in rectal and colon tumors and to assess the effects of the newly developed PFKFB3 inhibitor KAN0438757 on colorectal cancer cells and intestinal patient-derived organoids. Our results showed that KAN0438757 efficiently targets PFKFB3 expression and was able to affect cancer cell motility, invasion and survival. Additionally, a tumor specific cytotoxic-effect was observed in patient-derived organoids. In vivo, KAN0438757 showed to be well tolerated by mice without systemic toxicity. Our work re-enforces the concept that targeting of glycolysis may be a promising therapeutical approach for colorectal cancer. ABSTRACT: Background: Despite substantial progress made in the last decades in colorectal cancer (CRC) research, new treatment approaches are still needed to improve patients’ long-term survival. To date, the promising strategy to target tumor angiogenesis metabolically together with a sensitization of CRC to chemo- and/or radiotherapy by PFKFB3 (6-phosphofructo-2-kinase/fructose-2,6-biphosphatase-3) inhibition has never been tested. Therefore, initial evaluation and validation of newly developed compounds such as KAN0438757 and their effects on CRC cells are crucial steps preceding to in vivo preclinical studies, which in turn may consolidate new therapeutic targets. Materials and Methods: The efficiency of KAN0438757 to block PFKFB3 expression and translation in human CRC cells was evaluated by immunoblotting and real-time PCR. Functional in vitro assays assessed the effects of KAN0438757 on cell viability, proliferation, survival, adhesion, migration and invasion. Additionally, we evaluated the effects of KAN0438757 on matched patient-derived normal and tumor organoids and its systemic toxicity in vivo in C57BL6/N mice. Results: High PFKFB3 expression is correlated with a worse survival in CRC patients. KAN0438757 reduces PFKFB3 protein expression without affecting its transcriptional regulation. Additionally, a concentration-dependent anti-proliferative effect was observed. The migration and invasion capacity of cancer cells were significantly reduced, independent of the anti-proliferative effect. When treating colonic patient-derived organoids with KAN0438757 an impressive effect on tumor organoids growth was apparent, surprisingly sparing normal colonic organoids. No high-grade toxicity was observed in vivo. Conclusion: The PFKFB3 inhibitor KAN0438757 significantly reduced CRC cell migration, invasion and survival. Moreover, on patient-derived cancer organoids KAN0438757 showed significant effects on growth, without being overly toxic in normal colon organoids and healthy mice. Our findings strongly encourage further translational studies to evaluate KAN0438757 in CRC therapy.