Abstract A19: Epigenetic plasticity of the CXCR4 locus contributes to cell state transitions in Ewing sarcoma
Authors: Krook Melanie A. , Patel Raj , Lucas David , Chugh Rashmi , Lawlor Elizabeth R. 
Published: May 2016

Purpose of the Study: Ewing sarcoma (ES) is the second most common bone cancer in children. Despite improved survival for patients with localized disease, the outcome for patients with metastases remains dismal. In order to improve outcomes for ES patients, there is an imminent need to further understand the molecular and cellular biology driving ES metastasis. CXCR4 is the most commonly expressed chemokine receptor in human cancer and high expression contributes to metastasis. We have recently demonstrated that CXCR4 is dynamic and that its expression is induced in response to microenvironmental cues leading to transition of cells from CXCR4- to CXCR4+ states. In the current study we investigated the potential contribution of epigenetic regulation to the CXCR4 cell-state transitions in response to changes in the microenvironment.

Experimental Procedures: Immunohistochemistry of 93 diagnostic tumor sections was performed and frequency of CXCR4+ cells assessed. Chromatin Immunoprecipitation (ChIP) PCR was used to analyze histone modifications at the CXCR4 locus in ES cell lines. mRNA levels were quantified using qRT-PCR. Chemotactic migration and invasion to CXCL12 was evaluated using the x-CELLigence system in the presence and absence of the bromodomain inhibitor JQ1.

Data Summary: CXCR4+ tumor cells were identified in 31 cases. Frequency ranged from &lt;5% to 100%. CXCR4+ cells were more often detected in patients whose tumor had metastasized (p=0.008). ChIP of ES cells in ambient culture showed enrichment of both the activating, H3K4me3, and repressive, H3K27me3m marks at the CXCR4 promoter. ChIP-re-ChIP of the same chromatin confirmed that the promoter exists in a bivalent state in these conditions. Levels of CXCR4 directly correlated with H3K4me3 and inversely correlated with H3K27me3 marks suggesting that transcription of CXCR4 is, at least in part, regulated by the competing actions of these polycomb- and trithorax-dependent histone modifications. This was confirmed in studies of FACS-sorted CXCR4+ and CXCR4- cells. Specifically, we detected preferential enrichment of H3K4me3 in CXCR4+ and of H3K27me3 in CXCR4- cells. Significantly, exposure of ES cells to serum-deprivation or hypoxia, resulted in loss of bivalency in both bulk populations as well as FACS-sorted CXCR4- cells, as evidenced by loss of H3K27me3 with retention of H3K4me3 modifications. Loss of bivalency was accompanied by upregulation of CXCR4. Finally, we noted that an active enhancer exists upstream of the CXCR4 locus in HeLa cells, which express constitutively high levels of CXCR4. Therefore, we hypothesized that this enhancer may also contribute to CXCR4 regulation in ES. In support of this, JQ1 treatment of ES cells resulted in marked down-regulation of CXCR4 expression in ambient conditions. In addition, JQ1 treatment blocked up-regulation of CXCR4 in response to both serum-deprivation and hypoxia. Interestingly, enrichment of activating marks increased at the enhancer under conditions of stress. In addition, chemotactic migration of ES cells to CXCL12 was impeded by JQ1.

Conclusions: In summary, we have demonstrated that stress-dependent induction of CXCR4 in ES is, in part, epigenetically regulated. In particular, the CXCR4 promoter in ES cells resides in a bivalent state, which permits rapid and reversible transitions between CXCR4- and CXCR4+ cell states. Further, enhancer-dependent regulation of CXCR4 transcription opens the door to the potential of bromodomain inhibitors, such as JQ1, as novel therapeutics that could be used to antagonize metastasis-promoting cell state transitions.

Citation Format: Melanie A. Krook, Raj Patel, David Lucas, Rashmi Chugh, Elizabeth R. Lawlor. Epigenetic plasticity of the CXCR4 locus contributes to cell state transitions in Ewing sarcoma. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr A19.