Abstract LB-106: Microtubule-targeting agents (MTAs) disrupt intracellular trafficking of DNA repair proteins and augment the toxicity of DNA damaging agents (DDAs)

Abstract

Abstract MTAs have long been thought to cause cell death primarily by inducing mitotic arrest, a paradigm that applies to rapidly dividing cells in preclinical models. However, mitotic arrest cannot explain the activity of MTAs in much more slowly growing human cancers. In the latter, we have proposed interference with trafficking on interphase microtubules (MTs) as the principal mechanism of cytotoxicity. Satisfying this paradigm requires identification of the proteins whose trafficking on MTs, when disrupted, leads to cytotoxicity. MTAs in combination with DNA-damaging agents (DDAs) have emerged as preferred regimens for the treatment of ovarian, lung, some head and neck cancers, and many lymphomas. We proposed that by interfering with the trafficking of DNA repair proteins, MTAs could synergize with DDAs, augmenting their toxicity and enhancing cell death. To explore this hypothesis, we systematically investigated the effects of either paclitaxel or vincristine on treatment-induced DNA damage and on the distribution and biology of nine different proteins involved in DNA repair: ATM, ATR, DNA-PK, Rad50, Mre11, p95/NBS1, TP53, 53BP1 and p63. In several cell models including A549 cells and four Burkitt's lymphoma models (CA46, DG-75, Ramos and ST486) addition of vincristine increased cytoplasmic retention of the DNA repair proteins, thus excluding a greater fraction from the nucleus. The latter effect was observed only with MTAs and not with an inhibitor of Aurora kinase, despite similar cell cycle effects, confirming the cytoplasmic retention seen with MTAs is not due to alterations in the cell cycle. Increased cytoplasmic retention of DNA repair proteins following MT disruption suggests these proteins traffic on MTs and are vulnerable to MTAs, and this is supported by both confocal microscopy demonstrating co-localization of DNA repair proteins and α- or αβ-tubulin, as well as co-immunoprecipitation of these proteins with antibodies against the MT motor, dynein. In both A549 and MCF cells, the repair of DNA as measured by the level and persistence of γ-H2AX, was prolonged by the addition of paclitaxel to radiation. Moreover, when MCF7 or A549 cells were treated with either adriamycin or etoposide, γ-H2AX was induced to higher levels and for longer times when cells were treated with vincristine prior to and with the DDA and during DDA washout, when vincristine was present. In comparison, γ-H2AX decline was more rapid after DDA washout for cells treated only with the DDA. Together these data demonstrate that many DNA damage repair proteins travel on MTs and that the addition of MTAs promotes their sequestration in the cytoplasm. By interfering with the repair of DNA, cytoplasmic retention results in greater toxicity and likely explains why combinations of MTAs and DDAs have emerged as favored drug combinations for therapy of a diverse group of cancers. Citation Format: Marianne S. Poruchynsky, Edina Komlodi-Pasztor, Julia Wilkerson, Shana Trostel, Mauricio Burroto-Pichun, Tito Fojo. Microtubule-targeting agents (MTAs) disrupt intracellular trafficking of DNA repair proteins and augment the toxicity of DNA damaging agents (DDAs). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-106. doi:10.1158/1538-7445.AM2014-LB-106