Could novel inhibition of acid ceramidase with LCL521 have a translatable role for locally advanced rectal cancer?


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Rachael Clifford1,Naren Govindarajah1,David Bowden1,Paul Sutton1,Aiping Bai2,Zdzislaw Szulc2,Besim Ogretmen2,Jason Parsons1,Dale Vimalachandran3
1Institute of Cancer Medicine, The University of Liverpool,2Medical University of South Carolina,3The Countess of Chester Hospital

Abstract

Background

We have previously utilized proteomic and immuno-histochemical data to validate that high levels of acid ceramidase (AC) expression confers poorer response to neoadjuvant chemoradiotherapy in rectal cancer. Biological (siRNA and plasmid over-expression) AC manipulation validated altered responses of radiosensitivity in-vitro. Pharmacological inhibition with Carmofur, an oral 5-FU derivative, improved radiosensitivity in-vitro, comparative to 5-FU. We aimed to assess the radiosensitizing potential of LCL521, a novel small molecular inhibitor (SMI) in 2D and 3D spheroid models.

Method

Optimal LCL521 dosing using standard ELISA activity assays with DMSO control was established in multiple colorectal cancer cell lines (HCT116, HT29, LIM1215). Western blotting was used to confirm altered expression of AC. Standard clonogenic assays assessed cell survival following increasing x-ray irradiation and change in 3D model spheroid volume was used to assess growth.

Results

ELISA revealed reduced expression of AC to 18% with 10μm LCL in HCT116, 12% HT29 and 30% LIM1215. 2 hour pre-treated clonogenic assays demonstrated reduced colony formation efficiency (colonies/number of cells plated–CFE) and improved radiosensitivity across cell lines. HT29 showed 0.758(CFE) control v 0.317(CFE) LCL at 1Gy, 0.441(CFE) control v 0.260(CFE) at 2Gy and 0.0250(CFE) control v 0.0119(CFE) LCL at 4Gy (p value=0.024). LCL521 dosing of spheroids improved radiosensitivity across cell lines (HCT116 spheroid volume at day 15 post-LCL521 2.36x10-5mm v control 4.15x10-5mm).

Conclusion

Initial work demonstrates that pharmacological inhibition of AC with novel LCL521 produces comparative radiosensitizing effects in-vitro with these cell lines. This work further solidifies acid ceramidase as a potential therapeutic biomarker, however further work is needed to recapitulate these findings in more complex 3D organoid models and ultimately in-vivo to establish a translatable clinical role in this setting for locally advanced rectal cancer.