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

Tiago De Oliveira,Lutz Ackermann,Lena-Christin Conradi,Torben Rogge,Joanna Kalucka,Hanibal Bohnenberger,Martin Haubrock,Annalen Bleckmann,Tim Beissbarth,Michael Ghadimi,Mélanie Planque,Marcus Edelmann,Tina Goldhardt,Kerstin Menck,Jochen Gaedcke,Nikola Dobrinov Kyuchukov,Shawez Khan,Sarah-Maria Fendt,Karsten Rauch

doi:10.3390/cancers13051011

Abstract

Simple SummaryGlycolysis 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.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.

Highlights

Colorectal cancer (CRC) is amongst the most important causes of cancer-associated mortality worldwide
Initial comparative analyses from two different large colorectal cancer (CRC) patient cohorts (The Cancer Genomic Atlas Network cohort for colon cancer [20] and a rectal cancer cohort from our clinic, RRID:SCR_007834 oncomine.org) revealed that PFKFB3 mRNA expression is found significantly increased in both colon and rectal adenocarcinoma compared to normal mucosa (Figure 1A,B)
Confirming our hypothesis that increased PFKFB3 expression—and elevated glycolysis—promotes tumorigenesis, Kaplan–Meier plots showed for both colon and rectal cancer and association of enhanced PFKFB3 expression with worse survival rates (p = 0.04 for colon cancer and p = 0.08 for rectal cancer) (Figure 1C,D)

Summary

Introduction

Colorectal cancer (CRC) is amongst the most important causes of cancer-associated mortality worldwide. Chemo- and radiotherapy, specific agents like monoclonal antibodies such as cetuximab, bevacizumab, ramucirumab, aflibercept and others are used to treat mCRC [3] Antiangiogenic drugs such as VEGF inhibitors (e.g., bevacizumab) are currently used in the clinic to block tumor vessel formation and are meant to prevent tumor growth and metastasis and thereby improve patients’ survival. It has been shown that tumor endothelial cells are highly dependent on glycolysis for angiogenesis and survival, and that indirect blockade of glycolysis by administration of the PFKFB3 inhibitor 2E-3-(3-pyridinyl)1-(4-pyridinyl)-2-propen-1-one (3PO) was able to induce tumor vessel normalization (TVN), normalizing the structure and function of the highly abnormal tumor vessels [5] This effect led to a reduction in mouse melanoma metastasis due to a reduction of the abnormal vascular permeability/leakiness and improved chemotherapy effectiveness via better intratumoral delivery in tumor-bearing mice [5]. Our findings strongly encourage further translational studies to evaluate KAN0438757 in CRC therapy

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