IMMU-42. TTFIELDS INDUCES IMMUNOGENIC CELL DEATH AND STING PATHWAY ACTIVATION THROUGH CYTOPLASMIC DOUBLE-STRANDED DNA IN GBM

Glioblastoma (GBM) is the most common and deadliest malignant brain cancer in adults despite aggressive chemoradiotherapy. Recently, Tumor Treating Fields (TTFields) were approved in combination with adjuvant temozolomide chemotherapy for newly diagnosed GBM patients. The addition of TTFields resulted in a significant improvement in overall survival. TTFields are low-intensity alternating electric fields that are thought to disturb mitotic macromolecules’ assembly, leading to disrupted chromosomal segregation, integrity and stability. In many patients, a transient stage of increased peritumoral edema is often observed early in the course of TTFields treatment followed subsequently by objective radiographic responses, suggesting that a major component of therapeutic efficacy by TTFields may be an immune mediated process. However, the mechanism underlying these observations remains unclear. A panel of GBM cell lines were treated with TTFields at the clinically approved frequency of 200 kHz using the inovitro system. Our data showed TTFields-treated GBM cells had a significantly higher rate (19.9% vs. 4.3%, p=0.0032) of micronuclei structures released into the cytoplasm as a result of TTFields-induced chromosomal instability. Nearly 40% of these micronuclei were co-localized with two upstream dsDNA sensors Interferon (IFN)-inducible protein absent in melanoma 2 (AIM2) and Cyclic GMP-AMP synthase (cGAS), compared to absence of co-localization in untreated cells. TTFields-activated micronuclei-dsDNA sensor complexes led to i) induction of pyroptotic cell death, as measured by a specific LDH release assay, and through AIM2-recruited caspase1 and cleavage of pyroptosis-specific Gasdermin D; and ii) activation of STING pathway components including Type I IFNs and pro-inflammatory cytokines in GBM cells. These results provide compelling evidence that TTFields function as an activator of the immune system in GBM, and present a strong rationale for combining TTFields with immunotherapy aimed at augmenting an anti-tumor immune response such as immune checkpoint inhibitors.