Tumor Treating Fields Disrupt The CDK-E2F-Rb Axis: Implications For The Use Of Radiation And Targeted Agents Against E2F And CDK4/6 During TTFields Treatment

Abstract

The first mechanism of tumor cell killing identified for TTFields exposure was through the disruption of mitosis. Recent evidence points to disruption of DNA repair and replication fork maintenance through the downregulation of Fanconi’s Anemia (FA) pathway genes, chromosome maintenance genes and others. This study sought to determine how these pathways were downregulated and whether there were upstream regulators that could serve as therapeutic targets. Human NSCLC cell lines (H157, H4006, H549 and H1299) were used in this study. The Inovitro system was used to generate TTFields. Relative quantitative proteomic analysis was performed using tandem mass tags (TMT) coupled with mass spectrometry. Clonogenic cell survival assays tested TTFields, radiation and drug combinations while the Highest Single Agent approach tested for combinatorial efficacy. Analysis of differentially expressed proteins revealed interaction networks like cell cycle, DNA damage repair and replication, and transcriptional and translational regulation. Upstream analysis of key genes associated with cell cycle checkpoint and DNA repair identified reduced expression of the transcriptional activators E2F1 and E2F2 and increased expression of the transcriptional repressor E2F6, suggesting that TTFields likely affects the CDK–RB–E2F axis. This axis drives the transcriptional machinery that initiates cell cycle progression and regulates the fidelity of genome replication. For example, the downregulation of the key DNA repair genes RAD51, BRCA1 and BRCA2, seen when tumor cells are exposed to TTFields, could be explained through the upregulation of the transcriptional repressors E2F4 and E2F6 (a known repressor of BRCA1). These proteins are involved in homologous recombination and nucleotide excision repair, but also with replication fork maintenance, replication fork collapse and overall replication stress, the latter of which likely leads to cell death. Therefore, TTFields was combined with the E2F inhibitor HLM006474 with or without the CDK4/6 inhibitor abemaciclib. TTFields in combination with either inhibitor enhanced cell killing synergistically, as compared to TTFields alone, while the triple combination was found to be highly lethal. TTFields in combination with E2F inhibitor and ionizing radiation increased cell killing more than additively. Additional combinatorial tests with radiation are ongoing. The E2F family of transcription factors are modulated by TTFields exposure and likely explains the reduced DNA repair capacity and replication fork maintenance capability. Modulation of E2F during TTFields exposure suggested the CDK-RB-E2F axis as a novel druggable target that could be used in combination with TTFields with or without radiation for cancer therapy.