TTFields is a novel non-invasive physical modality of cancer therapy now approved for recurrent and newly diagnosed glioblastoma multiforme (GBM) in combination with temozolomide, and unresectable locally advanced or metastatic malignant pleural mesothelioma (MPM) in combination with platinum based chemotherapy. Clinical trials are ongoing for other cancers, including lung, pancreatic, and ovarian cancers. TTFields are low-intensity, intermediate frequency, alternating electric fields that are applied to tumor regions and cells using non-invasive arrays. One mechanism described for TTFields induced cell death has been via the disruption of mitosis while a more recent examination suggests that TTFields causes replication stress, and down-regulates DNA repair and cell cycle checkpoint genes. However, the exact cause of the downregulation of DNA repair and cell cycle checkpoint genes has been elusive. To that end, we employed relative quantitative proteomic analysis using tandem mass tags (TMT). All samples underwent trypsin digestion and labelling with different TMT reagents. They were then combined and the mixture was analyzed on an Orbitrap Fusion mass spectrometry device. Peptide quantitation was accomplished by comparing the intensities of the TMT reporter ions. STRING DB analysis of differentially expressed proteins revealed interaction networks that included 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 affects the CDK-RB-E2F axis. For example, the downregulation of key DNA repair genes including RAD51, BRCA1 and BRCA2 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 repair 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 (>90% by 72 h) as measured by clonogenic assay followed by the Highest Single Agent approach to determine synergy. Taken together our results identify the CDK-RB-E2F axis as a novel druggable target that can be used in combination with TTFields for cancer therapy.
Citation Format: Narasimha Kumar Karanam, Sivaramakrishna Yadavalli, Michael Story. Proteomic analysis of lung cancer cells exposed to tumor treating fields identified the dysregulation of the E2F-Rb-CDK4/6 axis rendering tumor cells susceptible to novel combination therapies that target CDK4/6 and/or E2F [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 559.