Epigenetic regulation of cell state by H2AFY governs immunogenicity in high-risk neuroblastoma [CUT&RUN]

Childhood neuroblastoma with MYCN-amplification is classified as high-risk and often relapses after intensive treatment regimen. Immune checkpoint blockade therapy shows limited efficacy in neuroblastoma patients and the cancer intrinsic immune regulatory network is poorly understood. Here, we leveraged genome-wide CRISPR/Cas9 screens in a human co-culture system and identified H2AFY as a resistance gene to nivolumab. Genetic deletion of H2afy in MYCN-driven neuroblastoma cells reverted in vivo resistance to PD-1 blockade by eliciting concurrent activation of the adaptive and innate immunity. Analysis of single-cell RNA sequencing datasets revealed that H2AFY mRNA was enriched in adrenergic cancer cells and was associated with patient survival. Mapping of the epigenetic and translational landscape demonstrated that H2afy deletion promoted cell transition to a malignant mesenchymal-like state. With a multi-omics approach, we uncovered H2AFY-associated genes that were functionally relevant and prognostic in patients. Altogether, our study elucidates the role of H2AFY as an epigenetic gatekeeper for cell state and immunogenicity in high-risk human neuroblastoma. Overall design: H2AFY may serve as an epigenetic gatekeeper for cell state transition and malignant behavior in human NB. To validate this hypothesis, we mapped the genomic dstibution of H2AFY in 9464D neuroblastoma cells using CUT&RUN and a H2afy KO cell line as negative control.

携带MYCN扩增（MYCN-amplification）的儿童神经母细胞瘤属于高危亚型，经强化治疗方案干预后常出现复发。免疫检查点阻断疗法在神经母细胞瘤患者中疗效有限，且肿瘤内在免疫调控网络的相关机制尚未明确。本研究通过人共培养体系中的全基因组CRISPR/Cas9筛选，鉴定出H2AFY为纳武利尤单抗（nivolumab）的耐药相关基因。在MYCN驱动的神经母细胞瘤细胞中敲除H2afy，可通过同时激活适应性免疫与固有免疫，逆转体内对PD-1阻断治疗的耐药性。对单细胞RNA测序（single-cell RNA sequencing）数据集的分析显示，H2AFY mRNA在肾上腺素能癌细胞中富集，且与患者总生存率显著相关。表观基因组与翻译组全景图谱分析表明，敲除H2afy可促进细胞向恶性间充质样状态转化。本研究通过多组学策略，鉴定出与H2AFY相关且在患者队列中兼具功能相关性与预后价值的基因。综上，本研究阐明了H2AFY作为表观遗传守门因子，在高危人类神经母细胞瘤中调控细胞状态与免疫原性的关键作用。整体实验设计：H2AFY可能作为表观遗传调控因子，参与人类神经母细胞瘤（NB）的细胞状态转化与恶性表型调控。为验证该假说，本研究利用CUT&RUN技术，并以H2afy基因敲除（KO）细胞系作为阴性对照，绘制了9464D神经母细胞瘤细胞中H2AFY的基因组分布图谱。