Tumor Treating Fields (TTFields) is the most recent FDA-approved therapy that significantly extends overall survival of patients with glioblastoma (GBM). TTFields are high frequency alternating electric fields directed at the tumor which putatively disrupt microtubule polarity and dynamics during mitosis leading to cell death. Combining TTFields with other therapies that further destabilize or alter microtubule dynamics in GBM cells may enhance TTFields efficacy. Histone deacetylase 6 (HDAC6) is an emerging regulator of glioma cell proliferation, and distinct from other HDACs through its role in deacetylating non-histone proteins such as alpha-tubulin, and promoting disassembly of microtubule-based primary cilia, an event that normally precedes mitosis. It is unclear whether inhibiting HDAC6 could enhance TTFields toxicity of GBM cells, or whether HDAC6 inhibitors have any effect on primary cilia reported in human glioblastoma. To examine this, we treated various human and mouse glioma cell lines in vitro with selective HDAC6 inhibitors (ACY1215 or ACY738) with or without 3 days of continuous 200 kHz TTFields exposure. We analyzed subsequent glioma cell proliferation and characteristics of primary cilia. Consistent with previous reports, ACY1215 and ACY738 significantly decreased glioma proliferation, in part by disrupting the G2/M transition. Notably, we found the number of non-viable human GBM cells labeled with propidium iodide was significantly increased with ACY1215 and TTFields co-treatment compared to either treatment alone. ACY1215 and ACY738 had no effect on primary cilia length, but did increase the percentage of ciliated glioma cells. Although ACY1215 and ACY738 rapidly increased levels of cytosolic acetylated alpha-tubulin, both drugs unexpectedly decreased levels of acetylated alpha- tubulin in glioma cilia which was not attributable to loss of the axoneme microtubule backbone. This indicates that the cytoplasm and cilia respond differently to HDAC6 inhibitors. ACY738 treatment in particular also led to an increase in level of ciliary Arl13b, a GTPase important for cilia stability and signaling, an effect that was not observed in normal neural cultures of mouse glia and neurons. We also observed that primary cilia are not completely ablated by TTFields, and that the viability of GBM cells engineered to lack cilia were unaffected by ACY1215 treatment. Collectively, our data raise the possibility that HDAC6 inhibitors may enhance sensitivity to TTFields therapy in part by interfering with the microtubule status or signaling pathways associated with glioma cilia.

Citation Format: Ping Shi, Reemsha Basrai, Alice Cheng, Neil Kalaria, Joseph J. Lebowitz, Habibeh Khoshbouei, Matthew Sarkisian. HDAC6 inhibitors may enhance TTFields toxicity to glioma cells by altering primary cilia [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6203.