Epigenetic Pathways of HDAC Inhibitor Resistance in Cutaneous T Cell Lymphoma. Epigenetic Pathways of HDAC Inhibitor Resistance in Cutaneous T Cell Lymphoma

Epigenetic changes deregulate gene expression to drive oncogenesis. The reversible nature of these changes enables therapeutic targeting, as in cutaneous T-cell lymphoma (MF/SS), Histone deacetylase inhibitors (HDACi), which alter epigenetic modifications, are effective in ~30% of MF/SS patients. However, there are no markers that predict MF/SS progression or therapy resistance. We hypothesized that epigenetic alterations drive MF/SS progression and promote HDACi drug resistance. Therefore, we profiled the epigenomes and transcriptomes of malignant T cell purified from skin biopsies and peripheral blood from MF/SS patients (N=21) before and after treatment with HDACi, as well as in vitro HDACi-treated CD4+ T cells from healthy donors. Here we report for the first time the epigenome-wide map of acetylation changes in MF/SS patients treated with HDACi, and define the significant differences in regulatory element activity and corresponding transcriptional changes in HDACi-sensitive versus resistant tumors. Our studies identified genes not previously associated with MF/SS, nor with disease progression or HDACi resistance, and were enriched in pathways that regulate apoptosis (BIRC5), cell cycle (RRM2), and chromosome cohesion (CENPH). We also identified a striking number of genes whose products are involved in cell adhesion and migration, including CCR6, LAIR2, VCAM1, and EPCAM. The mRNA of LAIR2, which encodes a receptor protein secreted by activated T cells that binds collagen and prevents binding of the inhibitory receptor LAIR1, was significantly upregulated in MF/SS tumors that were resistant to HDACi therapy and manifested in both skin and peripheral blood. We also detected elevated levels of LAIR2 protein in the plasma of MF/SS patients with progressive disease. Taken together, these studies defined the first epigenome-wide acetylation landscape of HDACi responsive and resistant MF/SS tumors, identified significantly altered patterns of epigenetic regulation and corresponding gene expression in HDACi resistant MF/SS tumors, and connected them to novel pathways of disease progression, particularly in cell adhesion and migration. These findings may represent novel predictive markers for MF/SS progression that are also targets for future therapeutic development. Overall design: RNA-seq and ChIP-seq (H3K9/K14ac, H3K27ac) profiling of primary HDACi-treated CTCL peripheral blood and skin samples along with in vitro romidepsin treated healthy control CD4+ T cells.

表观遗传修饰异常可通过失调基因表达驱动肿瘤发生。此类改变的可逆特性为治疗靶向提供了可能：以皮肤T细胞淋巴瘤（cutaneous T-cell lymphoma, CTCL）中的蕈样肉芽肿/塞扎里综合征（Mycosis fungoides/Sézary syndrome, MF/SS）为例，组蛋白去乙酰化酶抑制剂（Histone deacetylase inhibitors, HDACi）可通过改变表观遗传修饰，在约30%的MF/SS患者中展现出治疗疗效。然而，目前尚无有效标志物可预测MF/SS的疾病进展或治疗耐药性。本研究假设表观遗传改变可驱动MF/SS进展，并促成HDACi治疗耐药。为此，我们对21例MF/SS患者皮肤活检组织及外周血中分离纯化的恶性T细胞，在HDACi治疗前后的表观基因组与转录基因组开展了组学表征分析；同时还对健康供体来源的CD4+ T细胞进行体外HDACi处理后的样本开展了平行分析。本研究首次绘制了HDACi治疗的MF/SS患者体内全基因组乙酰化改变的表观图谱，并明确了HDACi敏感型与耐药型肿瘤在调控元件活性及对应转录变化上的显著差异。本研究鉴定出一批此前未被报道与MF/SS、疾病进展或HDACi耐药相关的基因，这些基因显著富集于调控细胞凋亡（BIRC5）、细胞周期（RRM2）及染色体黏连（CENPH）的通路中。此外，我们还发现大量基因的编码产物参与细胞黏附与迁移过程，其中包括CCR6、LAIR2、VCAM1及EPCAM。LAIR2编码一种由活化T细胞分泌的胶原蛋白结合受体，可抑制抑制性受体LAIR1的结合；在HDACi治疗耐药的MF/SS肿瘤中，LAIR2的mRNA水平显著上调，且该现象在皮肤与外周血样本中均有体现。我们还在病情进展的MF/SS患者血浆中检测到LAIR2蛋白水平升高。综上，本研究首次绘制了HDACi应答型与耐药型MF/SS肿瘤的全基因组乙酰化表观图谱，明确了HDACi耐药型MF/SS肿瘤中表观遗传调控模式及对应基因表达的显著改变，并将其与疾病进展的全新通路（尤其是细胞黏附与迁移通路）建立了关联。上述发现或可成为预测MF/SS进展的新型标志物，同时也有望成为未来治疗开发的靶点。实验整体设计：对经HDACi治疗的原发性皮肤T细胞淋巴瘤患者外周血与皮肤样本，以及经体外罗米地辛（romidepsin）处理的健康对照CD4+ T细胞开展RNA测序（RNA-seq）与染色质免疫共沉淀测序（ChIP-seq，检测靶标为H3K9/K14ac、H3K27ac）的组学表征分析。