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2024_06_01_fee39e36fafdf56e38abg

TargetingJMJD1C to selectively disrupt tumor cell fitness enhances antitumor immunity
以JMJD1C为靶点选择性地破坏肿瘤 ,增强抗肿瘤免疫力

Received: 9 March 2023
收到:2023 年 3 月 9 日
Accepted: 9 January 2024
接受:2024 年 1 月 9 日
Published online: 14 February 2024
在线出版:2024 年 2 月 14 日
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Abstract 摘要

Xuehui Long , Sulin Zhang (1) , Yuliang Wang , Jingjing Chen (D , Yanlai Lu , Hui Hou , Bichun Lin , Xutong Li( , Chang Shen , Ruirui Yang , Huamin Zhu1, Rongrong Cui , Duanhua , Geng Chen , Dan Wang , Yun Chen , Sulan Zhai , Zhiqin Zeng , Shusheng Wu , Mengting Lou , Junhong Chen , Jian Zou , Mingyue Zheng (1) , Jun Qin Xiaoming Wang
龙学辉 , 张素林(1) , 王玉良 , 陈晶晶(D , 卢燕来 , 侯慧 , 林碧春 , 李旭彤( , 沈畅 , 杨瑞瑞 , 朱华敏1, 崔蓉蓉 、段华 , 陈庚 , 王丹 , 陈云 , 翟素兰 , 曾志勤 , 吴树生 , 楼梦婷 , 陈俊红 , 邹健 , 郑明月 (1) , 秦俊 王晓明

Regulatory ) cells are critical for immune tolerance but also form a barrier to antitumor immunity. As therapeutic strategies involving cell depletion are limited by concurrent autoimmune disorders, identification of intratumoral cell-specific regulatory mechanisms is needed for selective targeting. Epigenetic modulators can be targeted with small compounds, but intratumoral cell-specific epigenetic regulators have been unexplored. Here, we show that JMJD1C, a histone demethylase upregulated by cytokines in the tumor microenvironment, is essential for tumor cell fitness but dispensable for systemic immune homeostasis. JMJD1C deletion enhanced AKT signals in a manner dependent on histone H3 lysine 9 dimethylation (H3K9me2) demethylase and STAT3 signals independently of demethylase, leading to robust interferon production and tumor cell fragility. We have also developed an oral JMJD1C inhibitor that suppresses tumor growth by targeting intratumoral cells. Overall, this study identifies JMJD1C as an epigenetic hub that can integrate signals to establish tumor cell fitness, and we present a specific JMJD1C inhibitor that can target tumor cells without affecting systemic immune homeostasis.
调节性 ) 细胞对免疫耐受至关重要,但也是抗肿瘤免疫的屏障。由于 细胞耗竭的治疗策略受到并发自身免疫性疾病的限制,因此需要确定瘤内 细胞特异性的调控机制,以进行选择性靶向治疗。表观遗传调节剂可通过小分子化合物进行靶向治疗,但瘤内 细胞特异性表观遗传调节剂尚未得到研究。在这里,我们发现JMJD1C是一种由肿瘤微环境中的细胞因子上调的组蛋白去甲基化酶,它对肿瘤 细胞的健康至关重要,但对全身免疫稳态却无足轻重。JMJD1C缺失以依赖组蛋白H3赖氨酸9二甲基化(H3K9me2)去甲基化酶和STAT3信号的方式增强了AKT信号,而不依赖于 去甲基化酶,从而导致干扰素 ,并使肿瘤 细胞变得脆弱。我们还开发了一种口服JMJD1C抑制剂,通过靶向瘤内 细胞抑制肿瘤生长。总之,这项研究发现JMJD1C是一个表观遗传中枢,它可以整合信号以建立肿瘤 细胞的适应性,我们还提出了一种特异性JMJD1C抑制剂,它可以靶向肿瘤 细胞而不影响全身免疫平衡。

Foxp cells are essential for maintaining immune tolerance and preventing autoimmune diseases; they also infiltrate tumor tissues and suppress antitumor immunity . Targeting cells could improve the immune-suppressive tumor microenvironment (TME) and elicit effective antitumor immunity . However, systemic depletion of cells disturbs immune homeostasis and can lead to autoimmune complications . Therefore, it is important to identify regulatory molecules that can be used to selectively target intratumoral cells without affecting systemic or peripheral cells. In this regard, several molecules or pathways have been shown to be specific to intratumoral cells. For examples, lipid metabolism is particularly important for cell maintenance and function in tumors but not under inflammatory settings . NRP1, PD1 and IL-33 signals suppress interferon (IFN ) expression to prevent cell fragility in tumors . Targeting chemokine receptor CCR8 has been shown to specifically remove clonally expanded cells in tumors . The TME creates a specialized niche that is distinct from steady or inflammatory settings. Further investigation is required to determine whether cells acquire new features to enable 'fitness' and maintain the capability to survive, expand and function properly following infiltration into the TME.
Foxp 细胞对维持免疫耐受和预防自身免疫性疾病至关重要;它们也会浸润肿瘤组织并抑制抗肿瘤免疫 。以 细胞为靶点可以改善免疫抑制性肿瘤微环境(TME),激发有效的抗肿瘤免疫 。然而,系统性消耗 细胞会扰乱免疫稳态,并可能导致自身免疫并发症 。因此,确定可用于选择性靶向瘤内 细胞而不影响全身或外周 细胞的调控分子非常重要。在这方面,有几种分子或途径已被证明对瘤内 细胞具有特异性。例如,脂质代谢对 细胞在肿瘤中的维持和功能尤为重要,但在炎症环境下则不然 。NRP1、PD1和IL-33信号可抑制干扰素 (IFN )的表达,从而防止肿瘤 细胞的脆弱性 。靶向趋化因子受体 CCR8 可特异性清除肿瘤中克隆扩增的 细胞 。TME创造了一种有别于稳定或炎症环境的特化生态位。还需要进一步研究,以确定 细胞是否获得了新的特征,使其能够 "适应 "并在渗入 TME 后保持生存、扩增和正常功能的能力。
Epigenetic regulation has proven to be essential for cellular differentiation and function, including for cells . Appropriate DNA methylation at CNS2 loci is critical for Foxp3 expression and cell identity . MLL4 regulates Foxp3 induction via chromatin looping . Ezh2 has been reported to be required for maintenance of cell identity during cellular activation . However, it is unclear whether there are epigenetic regulatory mechanisms that are important for intratumoral cells but not for peripheral cells. Given that epigenetic regulators-especially enzymes, including writers and erasers-can be feasibly targeted with small compounds, it is important to identify tumor cell-specific epigenetic regulators.
表观遗传调控已被证明对细胞分化和功能至关重要,包括对 细胞 。CNS2 基因座上适当的 DNA 甲基化对 Foxp3 的表达和 细胞特性至关重要 。MLL4 通过染色质循环调节 Foxp3 的诱导 。据报道,在细胞活化过程中,Ezh2 是维持 细胞特性所必需的 。然而,目前尚不清楚是否存在对瘤内 细胞重要而对外周 细胞不重要的表观遗传调控机制。鉴于表观遗传调控因子--尤其是酶,包括写入器和擦除器--可以用小化合物作为靶标,因此确定肿瘤 细胞特异性表观遗传调控因子非常重要。

Results 成果

Upregulation of JMJD1C in tumor cells
肿瘤 细胞中 JMJD1C 的上调

We compared the chromatin accessibility of tumor cells with that of their counterparts in peripheral lymphoid organs by assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) and observed a substantial difference between these two cell populations (Fig. 1a). The observed disparities in chromatin accessibility prompted us to investigate whether the two cell populations had distinct epigenetic regulatory mechanisms. We therefore re-analyzed published transcriptome data comparing tumor and splenic cells. Focusing on the genes encoding epigenetic enzymes , we found that Jmjd1c, the gene encoding H3K9me2 demethylase, was the most significantly upregulated gene in tumor cells (Fig. 1b). Increased expression of Jmjd1c in tumor cells compared with their peripheral counterparts was also observed in multiple human cancer types including colorectal cancer (CRC), hepatocellular carcinoma (HCC) and nonsmall-cell lung cancer (NSCLC) (Fig.1c). By contrast, the expression of Jmjd1c in tumor conventional CD4 T cells compared with peripheral blood mononuclear cell (PBMC) CD4 T cells was not consistently upregulated across different tumor types. Specifically, whereas Jmjd1c was upregulated in tumor-infiltrating CD4 T cells in CRC, its expression remained unchanged in HCC and NSCLC (Fig. 1c). cells from HCC patients were further analyzed to track the dynamic profile of Jmjd1c. Unsupervised -distributed stochastic neighbor
我们通过高通量测序(ATAC-seq)检测转座酶可及染色质,比较了肿瘤 细胞与外周淋巴器官中相应细胞的染色质可及性,并观察到这两种 细胞群之间存在巨大差异(图 1a)。观察到的染色质可及性差异促使我们研究这两个 细胞群是否具有不同的表观遗传调控机制。因此,我们重新分析了已发表的转录组数据 ,比较了肿瘤细胞和脾 。以编码表观遗传酶的基因 为重点,我们发现编码 H3K9me2 去甲基化酶的基因 Jmjd1c 是肿瘤 细胞中最显著上调的基因(图 1b)。在包括结肠直肠癌(CRC)、肝细胞癌(HCC)和非小细胞肺癌(NSCLC)在内的多种人类癌症 ,也观察到肿瘤 细胞中Jmjd1c的表达量较其外周对应基因有所增加(图1c)。相比之下,与外周血单核细胞(PBMC)CD4 T细胞相比,Jmjd1c在肿瘤常规CD4 T细胞中的表达在不同肿瘤类型中并没有一致的上调。 ,以追踪 Jmjd1c 的动态变化。无监督 -分布式随机邻接法

Fig. 1|Expression of Jmjd1c is upregulated in tumor cells by a combination
图 1|Jmjd1c在肿瘤 细胞中的表达上调

of cytokines. a, Heatmap of ATAC-seq peaks with splenic and tumor cells from B16-OVA tumor. , Comparison of mRNA expression of genes encoding epigenetic enzymes (from the dbEM database) between splenic and tumor cells (GSE139325).c, Box plot showing Jmjd1c expression in cells and cells from scRNA-seq(GSE108989, GSE98638 and GSE99254). Center line shows median, box limits indicate first and third quartiles, and whiskers extend to the smallest and largest data values. as indicated within the image. -SNE visualization of cell clusters from HCC patients (GSE98638, left);right:Jmjd1c expression projected onto the clusters. e,f, Dot plot showing Jmjd1c expression in seven separate clusters (e) or three combined clusters (f).g, Monocle-based pseudotimes were derived for the three cell populations. , Western blotting analysis of JMJD1C expression in splenic naive cells, effector cells and tumor cells from MCA205-tumorbearing Foxp3-YFP mice. , Splenic cells or cells were cultured with B16 tumor supernatant (i), MCA205 tumor supernatant (j) left) or MCA205 cell culture medium (j, right) for 3 days.JMJD1C levels were then detected by western blotting. embedding ( -SNE) analysis identified a total of seven clusters (one for peripheral cells and six for tumor cells), among which (representing cells) expressed very low levels of Jmjd1c (Jmjd1c cells); c1, c2, c5 and c6 tumor cells expressed medium levels; and c3 and c4 tumor cells expressed high levels (Fig.1d,e). We therefore combined c1, c2, c5 and c6 to be defined as Jmjd1c tumor cells, and and asJmjd1 tumor cells (Fig. 1f), and analyzed their developmental trajectories. As shown in Fig. 1g and Extended Data Fig. 1, a trajectory from PMBC cells (Jmjd1c cells) to Jmjd1c tumor cells and then to Jmjd1c tumor cells emerged along the pseudotime, indicating a gradual transition from PMBC cells to Jmjd1c tumor cells. This suggests thatJmjd1c expression increases over the course of cells fitting into the TME.
Furthermore, immunoblotting confirmed that at the protein level, JMJD1C was more abundant in tumor cells than in splenic cells obtained from MCA205-tumor-bearing mice (Fig. ). Consistent with previous studies, we observed tumor cells to also express higher levels of Foxp3 compared with splenic cells (Fig. 1h). We next directly tested whetherJMJD1C expression in cells could be induced by exposure to the TME. Treating splenic cells in vitro with supernatant from B16 melanoma tumor tissue for 3 days indeed increased JMJD1C expression (Fig. 1i). However, tumor supernatant could not induce JMJD1C expression in conventional CD4 T cells (Fig. 1i), indicating a cell-type-specific regulation of JMJD1C. Similar induction of JMJD1C was observed in cells treated with supernatant from MCA205 fibrosarcoma tumor (Fig. 1j).
此外,免疫印迹证实,在蛋白质水平上,肿瘤 细胞中的 JMJD1C 比 MCA205 肿瘤小鼠脾脏 细胞中的含量更高(图 )。与之前的研究一致,我们观察到肿瘤 细胞也比脾脏 细胞表达更高水平的 Foxp3 (图 1h)。接下来,我们直接检测了 细胞中的 JMJD1C 表达是否会因暴露于 TME 而被诱导。用 B16 黑色素瘤肿瘤组织的上清液体外处理脾脏 细胞 3 天确实会增加 JMJD1C 的表达(图 1i)。然而,肿瘤上清不能诱导常规CD4 T细胞中JMJD1C的表达(图1i),这表明JMJD1C受细胞类型特异性调控。在用 MCA205 纤维肉瘤肿瘤上清液处理的 细胞中也观察到了类似的 JMJD1C 诱导(图 1j)。
Notably, when we treated splenic cells with culture media of MCA205 tumor cells, noJMJD1C induction was observed (Fig.1j), suggesting that factors present in the in vivo TME, rather than products directly generated by tumor cells, are responsible for JMJD1C induction. To further investigate this, we analyzed the signaling pathways specifically enriched in tumor cells compared with cells, using single-cell RNA sequencing (scRNA-seq) data from patients with tumors. Applying gene set enrichment analysis (GSEA), we identified the top 30 enriched signaling pathways in tumor cells from each type of tumor (CRC, HCC and NSCLC). These pathways included well-known TME-associated signals such as hypoxia, along with several HALLMARK and REACTOME pathways (Fig. 1k). Integrative analysis of these 90 enriched pathways revealed 12 pathways shared by all three tumor types, one of which was hypoxia (Fig.1k).JMJD1C induction by tumor supernatant occurred even under regular normoxic culture conditions (Fig. 1i,j), indicating that hypoxia signaling is not required for JMJD1C expression in tumor cells.
值得注意的是,当我们用MCA205肿瘤细胞的培养基处理脾脏 细胞时,没有观察到JMJD1C诱导(图1j),这表明体内TME中存在的因素而不是肿瘤细胞直接产生的产物是JMJD1C诱导的原因。为了进一步研究这一点,我们利用肿瘤患者的单细胞 RNA 测序(scRNA-seq)数据分析了肿瘤 细胞与 细胞相比特异性富集的信号通路。通过基因组富集分析(GSEA),我们确定了每种类型肿瘤(CRC、HCC 和 NSCLC)的肿瘤 细胞中富集的前 30 条信号通路。这些通路包括众所周知的TME相关信号,如缺氧,以及一些HALLMARK和REACTOME通路(图1k)。对这90条富集通路的整合分析显示,所有三种肿瘤类型共有12条通路,其中之一是缺氧(图1k)。即使在常规常氧培养条件下,肿瘤上清液也会诱导JMJD1C(图1i,j),这表明缺氧信号传导并非肿瘤
Among the 12 overlapping pathways, multiple inflammatory cytokine-related signaling pathways were notable, including the HALLMARK inflammatory response and REACTOME cytokine signaling in the immune system (Fig. ). Specific cytokine signals including IFN , TNF, IL-1 and IL-6-STAT3, which are commonly found in the TME, were
在 12 条重叠的通路中,与炎症细胞因子相关的多条信号通路引人注目,包括 HALLMARK 炎症反应和免疫系统中的 REACTOME 细胞因子信号(图 )。特定的细胞因子信号包括 IFN 、TNF、IL-1 和 IL-6-STAT3,这些信号通常存在于 TME 中。
, Venn diagram showing overlap of enriched pathways in tumor cells versus cells in three types of tumors (top 30 pathways for each). , Splenic cells were treated with individual ( or combined ( ) cytokines as indicated ( ) for 3 days, and JMJD1C levels were analyzed by western blotting. , Splenic cells were treated with MCA205 tumor supernatant or supernatant plus blocking antibody cocktails of anti-TNF + anti-IL-1 anti-IL-6 . o, IgV snapshot of ATAC-seq at Jmjd1c gene locus. F1 and F2 are the fragments for chromatin immunoprecipitation with quantitative PCR (ChIP-qPCR) analysis. p, Luciferase reporter assays showing that STAT3 and NF- KB regulate Jmjd1c promoter activity. , Splenic cells were treated with IL-1 plus IL-6 for 3 days and subjected to ChIP-qPCR analysis with STAT3 and NF-кB antibodies; independent experiments in and ; bar graph shows mean values. Data represent two (h, and ) or three (i i and ) independent experiments. Two-sided Wald test without adjustment in Deseq2(b); two-tailed Wilcoxon rank-sum test without adjustment (c).PC, peak center;Ctrl., control;EV, empty vector;sup., supernatant; TSS, transcriptional start site; unstimu., unstimulated.
, 维恩图显示了三种类型肿瘤 细胞与 细胞中富集通路的重叠情况(每种类型的前 30 条通路)。 , 用单独的 ( 或联合的 ( ) 细胞因子处理脾脏 细胞 3 天( ),并通过免疫印迹分析 JMJD1C 的水平。 用 MCA205 肿瘤上清或上清加抗肿瘤坏死因子 + 抗 IL-1 抗 IL-6 的阻断抗体鸡尾酒处理脾 细胞。 o, Jmjd1c 基因座 ATAC-seq 的 IgV 快照。F1 和 F2 是染色质免疫共沉淀与定量 PCR(ChIP-qPCR)分析的片段。 p, 荧光素酶报告实验显示 STAT3 和 NF- KB 调节 Jmjd1c 启动子的活性。 , 脾 细胞用 IL-1 加 IL-6 处理 3 天,并用 STAT3 和 NF-кB 抗体进行 ChIP-qPCR 分析; 独立实验, 和 ;条形图显示平均值。数据代表两次(h、 和 )或三次(i i 和 )独立实验。PC,峰中心;Ctrl.,对照;EV,空载体;sup.,上清液;TSS,转录起始位点;unstimu.,未刺激。

identified . However, when we treated splenic cells with these cytokines individually, we did not observe induction of JMJD1C expression (Fig. 11). We subsequently investigated whether a combination of these cytokines could induce JMJD1C. Treatment with TNF plus IL-1 resulted in a slight increase inJMJD1C expression (Fig. 1m). Moreover, when cells were treated with either TNF plus IL-6 or IL-1 plus IL-6, a dramatic upregulation of JMJD1C expression was observed, reaching a level comparable with that induced by tumor supernatant treatment (Fig. 1m). By contrast, blocking the activity of these three cytokines completely abolished the induction of JMJD1C by tumor supernatant (Fig. 1 n). Both TNF and IL-1 signaling pathways are known to activate signaling. It is plausible that TNF plus IL-1 treatment results in stronger -кB activity compared with treatment with either cytokine alone. Therefore, it appears that NF- кB signaling is responsible for initiatingJMJD1C expression, whereas IL-6-STAT3 signaling synergizes with the NF-kB pathway to further enhance and fully induce JMJD1C.
。然而,当我们用这些细胞因子单独处理脾脏 细胞时,并没有观察到 JMJD1C 的诱导表达(图 11)。我们随后研究了这些细胞因子的组合是否能诱导 JMJD1C。TNF 加 IL-1 处理后,JMJD1C 的表达略有增加(图 1m)。此外,当 细胞用 TNF 加 IL-6 或 IL-1 加 IL-6 处理时,观察到 JMJD1C 表达急剧上调,达到与肿瘤上清液处理诱导的水平相当(图 1m)。相比之下,阻断这三种细胞因子的活性可完全消除肿瘤上清液对 JMJD1C 的诱导(图 1 n)。已知 TNF 和 IL-1 信号通路都能激活 信号。与单独使用其中一种细胞因子相比,TNF 加 IL-1 处理会导致更强的 -кB 活性。因此,NF- кB信号似乎负责启动JMJD1C的表达,而IL-6-STAT3信号与NF-kB通路协同作用,进一步增强并完全诱导JMJD1C。
Previous studies have shown that STAT3 can interact with NF-кB in tumor cells to dramatically enhance NF-kB-mediated downstream gene expression, whereas STAT3 alone fails to do . We hypothesized that a similar phenomenon occurs in tumor cells. To validate this hypothesis, we examined the sequence of the Jmjd1c gene promoter near the transcriptional start site, in which region tumor a
先前的研究表明,STAT3 可与肿瘤细胞中的 NF-кB 相互作用,显著增强 NF-kB 介导的下游基因表达,而 STAT3 本身则无法做到这一点 。我们假设肿瘤 细胞中也会出现类似现象。为了验证这一假设,我们研究了Jmjd1c基因启动子靠近转录起始位点的序列,在这一区域肿瘤细胞的NF-kB介导的基因表达与STAT3无关。
d
b
e
h

Hypoxia Inflammatory response Cytokine signaling IFNy signal response TNF signal via NF- IL6-STAT3 signaling IL1 signaling
缺氧 炎症反应 细胞因子信号转导 IFNy 信号转导 TNF 信号通过 NF- IL6-STAT3 信号转导 IL1 信号转导
Top 30 in each tumor
每个肿瘤的前 30 名
O
j
Luciferase signal 荧光素酶信号

cells exhibited stronger ATAC-seq signals than splenic cells, and identified four canonical NF-кB binding sites (N1-N4) (Fig. 1o). In accordance with our hypothesis, a luciferase assay showed that NF-кB rather than STAT3CA (constitutive active form) transduction could slightly increase JMJD1C expression (Fig. 1p). Moreover, when both STAT3CA and NF-кB were present, there was a dramatic boost inJMJD1C expression (Fig. 1p). Mutation analysis revealed that and were necessary for JMJD1C induction (Fig. 1p). Near the N3 and N4 sites, we identified two canonical STAT3-binding sites (S1 and S2), and deletion of these two motifs abolished the boosting effect of STAT3 onJMJD1C expression (Fig.1p). This suggests that STAT3 requires its binding sites to be in proximity to the NF-кB sites to enhance JMJD1C expression. Finally, we performed chromatin immunoprecipitation with quantitative PCR analysis and found increased NF-кB and STAT3 binding to the N3-N4 and S1-S2 regions upon treatment of cells with IL-1 plus IL-6 (Fig. 1q).
细胞比脾脏 细胞表现出更强的 ATAC-seq 信号,并确定了四个典型的 NF-кB 结合位点(N1-N4)(图 1o)。与我们的假设相符的是,荧光素酶试验表明,NF-кB 而非 STAT3CA(组成型活性形式)转导可轻微增加 JMJD1C 的表达(图 1p)。此外,当 STAT3CA 和 NF-кB 同时存在时,JMJD1C 的表达也会急剧增加(图 1p)。突变分析表明 和 是诱导 JMJD1C 的必要条件(图 1p)。在 N3 和 N4 位点附近,我们发现了两个典型的 STAT3 结合位点(S1 和 S2),删除这两个基团可消除 STAT3 对 JMJD1C 表达的促进作用(图 1p)。这表明 STAT3 需要其结合位点靠近 NF-кB 位点才能增强 JMJD1C 的表达。最后,我们进行了染色质免疫共沉淀和定量 PCR 分析,发现用 IL-1 加 IL-6 处理 细胞后,N3-N4 和 S1-S2 区域的 NF-кB 和 STAT3 结合增加(图 1q)。
Overall, these findings suggest that a combination of TNF, IL-6 and IL-1 drives robustJMJD1C expression in tumor