Synergistic effect of PAK and Hippo pathway inhibitor combination in NF2-deficient Schwannoma

Abstract

Neurofibromatosis type 2 is a genetic disorder that results in the formation and progressive growth of schwannomas, ependymomas, and/or meningiomas. The NF2 gene encodes the Merlin protein, which links cell cortical elements to the actin cytoskeleton and regulates a number of key enzymes including Group I p21-activated kinases (PAKs), the Hippo-pathway kinase LATS, and mTORC. While PAK1 and PAK2 directly bind Merlin and transmit proliferation and survival signals when Merlin is mutated or absent, inhibition of Group 1 PAKs alone has not proven sufficient to completely stop the growth of NF2-deficient meningiomas or schwannomas in vivo, suggesting the need for a second pathway inhibitor. As the Hippo pathway is also activated in NF2-deficient cells, several inhibitors of the Hippo pathway have recently been developed in the form of YAP-TEAD binding inhibitors. These inhibitors prevent activation of pro-proliferation and anti-apoptotic Hippo pathway effectors. In this study, we show that PAK inhibition slows cell proliferation while TEAD inhibition promotes apoptotic cell death. Finally, we demonstrate the efficacy of PAK and TEAD inhibitor combinations in several NF2-deficient Schwannoma cell lines.

Fig 1. Group I PAK inhibition reduces cell growth in human NF2-deficientschwannoma.

Colony formation assays were performed to assess the efficacy of FRAX-1036 (A). Nuclei counts were performed on HEI-193 cells treated with FRAX-1036 for 72 hours (B). Cell viability was determined by CellTiter-Glo for HEI-193 cells treated with NVS-PAK1-1 and FRAX-1036 (C) or G-5555 (D) for 72 hours. Immunoblot analysis showed reduction in phospho-PAK1/2 (S144/S141) when treated with PAK inhibitors for four or 72 hours (E). Error bars depict SD. ns = not significant; ** = p<0.01; *** = p<0.001.

Fig 2. Genetic and pharmacological YAP-TEAD inhibition prevents cell growth of human NF2-deficient schwannoma.

Immunoblot analysis confirms reduced expression of YAP after siYAP transfection (A). Real-time cell proliferation after siYAP or siControl transfection was monitored using Xcelligence RTCA system (B). Expression of Hippo pathway targets CTGF and CYR61 in HEI-193 cells after TED-347 treatment for 48 hours was determined by qRT-PCR (C). Immunoblot analysis confirms reduced CTGF and CYR61 protein levels after TED-347 treatment for 24, 48, or 72 hours (D). Colony formation assays were performed to assess the efficacy of TED-347 (E). Nuclei counts were performed on HEI-193 cells treated with TED-347 (F). Cell viability was determined by CellTiter-Glo for HEI-193 cells treated with NSC682769 for 72 hours (G). Error bars depict SD. ns = not significant; ** = p<0.01; *** = p<0.001.

Fig 3. Group I PAK and YAP-TEAD inhibition reduces cell growth in mouse NF2-deficient schwannoma.

Immunoblot analysis confirms truncated (HEI-193) and absence (SC4) of Merlin protein in schwannoma cell lines (A). SC4 cells in 0.5% FBS media were treated with FRAX-1036 for 7 days and cell viability was determined by CellTiter-Glo (B). SC4 cells in 0.5% FBS media were treated with TED-347 for 7 days and cell viability was determined by CellTiter-Glo (C). Error bars depict SD. ns = not significant; ** = p<0.01; *** = p<0.001.

Fig 4. PAK inhibition blocks proliferation while YAP-TEAD inhibition promotes apoptosis.

HEI-193 cells were treated with indicated drugs for 24 hours and proliferation was determined by EdU incorporation (A). Cell cycle was analyzed by flow cytometry after being treated with indicated drugs for 24 hours (B). Cells were treated with indicated drugs for 24 hours and simultaneously labeled with green-fluorescent calcein-AM (live) and red-fluorescent ethidium homodimer-1 (dead) and dead cells were quantified by flow cytometry (E). HEI-193 or hSC NF2-null cells were treated with indicated drugs and active caspases were labeled with TF2-VAD-FMK and cells were quantified by flow cytometry (F, G, H). Cells were treated with indicated drugs for 24 hours and simultaneously labeled with green-fluorescent calcein-AM (live) and red-fluorescent ethidium homodimer-1 (dead) and dead cells were quantified by flow cytometry (I). Error bars depict SD. ns = not significant; * = p<0.05; ** = p<0.01; *** = p<0.001.

Fig 5. Simultaneous Group I PAK and YAP-TEAD inhibition produces a synergistic effect.

Immunoblot shows Merlin protein levels in matched human Schwann cell ines (A). Merlin null and WT matched human Schwann cells were treated with TEAD palmitoylation inhibitor IK-930 (B). NF2-null human Schwann cells were treated with increasing concentrations of IK-930 and FRAX-1036 for 5 days and synergy scores were calculated (C). HEI-193 cells were treated with increasing concentrations of TED-347 and FRAX-1036 and synergy scores were calculated (D). Error bars depict SD. ns = not significant; ** = p<0.01; *** = p<0.001.