Abstract 281: Phosphodiesterase 8/9 inhibition to sensitize squamous non-small cell lung cancer (NSCLC) to pemetrexed (PMX): A double-edge strategy
Authors: Foureau Anna V. Ivanina , Foureau David M. , Farhangfar Carol J. , Mileham Kathryn F. 
Published: April 2023

Abstract
Introduction. Phosphosidesterases (PDE) are key regulators of cyclic nucleotide (cAMP/cGMP) signaling regulating many cellular processes including cell proliferation, growth arrest and apoptosis. In squamous NSCLC (SCC), PDE inhibitors (PDEi) have shown anti-tumor activity both as single agent and in combination with chemotherapy. A strong putative synergistic mechanistic activity exists between pemetrexed (PMX) and PDEi through cAMP/Protein kinase A (PKA) and cGMP/Protein Kinase G signaling. We therefore thought to investigate whether SCC, classically resistance to PMX can be sensitized to the drug through PDE inhibition.
Methods. Whole exome sequencing and survival data from TCGA was surveyed to identify PDEs most closely associated with clinical outcome in NSCLC. Whole exome sequencing data from GEmiCCL database were used to select SCC cell lines with unaltered PDE genes. Two SCC cell lines (H226, H1703) were evaluated for viability (WST8 assay), proliferation and apoptosis (Apo-Tox-Glo assay) after treatment with PMX with or without sildenafil citrate, PF-04671536 or PF-04447943 (PDE5i, PDE8i and PDE9i respectively). PDE expression was quantified by qPCR and Western Blot (WB). PDE activity and downstream signaling was quantified by measuring PKA/PKG target activation (PKA/pVASP157, PKG/pVAP239).
Results. Presence of function altering mutations in PDE8 (cAMP/PKA signaling) and PDE9 (cGMP/PKG signaling) improved NSCLC overall survival (P&lt;0.1). PDE5 (cGMP/PKG signaling) mutations had no significant clinical impact. Both SCC cell lines investigated expressed high level of PDE8-9, but H1703 had only trace levels of PDE5. Endogenous PKA activity and PKG activity was significantly lower in H1703 cells compared with H226. PDEi stimulated SCC cell growth (30-60% increase). PDE8i and PDE9i (EC50&lt;40nM, significant PKA/PKG activation) were more potent than PDE5i (&gt;1.5µM, no PKA/PKG activation). PMX also displayed a pro-mitotic activity toward SCC (40% increase at 250µM) coupled with significant PKA activation in H226 and PKG activation in H1703 cells. Combination of PDE5i, 8i or 9i (2X EC50 concentrations) with low dose PMX (0.025µM) inhibited SCC cell growth, H1703 showing greater response than H226 (30-37% vs 8-11% growth reduction). No cell death, necrosis or apoptosis, was detected. This cytostatic activity of PDEi combination treatment was not observed at high dose PMX (250µM).
Conclusions. In the absence of PDE mutations, single agent PDE5i, 8i and 9i promote SCC growth with PDE8,9i displaying a far greater potency than PDE5i. PMX is also pro-mitotic toward SCC cells but PDE5i, 8i, and PDE9i can sensitize SCC cells to the drug. SCC cells with lower endogenous PKA/PKG activity are more sensitive to PDEi+PMX treatment. This combination is not cytotoxic though, rather is cytostatic, and displays a very narrow window of therapeutic activity.
Citation Format: Anna V. Ivanina Foureau, David M. Foureau, Carol J. Farhangfar, Kathryn F. Mileham. Phosphodiesterase 8/9 inhibition to sensitize squamous non-small cell lung cancer (NSCLC) to pemetrexed (PMX): A double-edge strategy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 281.