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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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。

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

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 cells. Downstream of these cytokines, NF-кB initiatesJMJD1C expression in tumor cells, and STAT3 plays a key part in enhancing this expression.
总之，这些研究结果表明，TNF、IL-6 和 IL-1 在肿瘤 细胞中的联合作用可促进 JMJD1C 的强有力表达。在这些细胞因子的下游，NF-кB 在肿瘤 细胞中启动了 JMJD1C 的表达，而 STAT3 在增强这种表达中起着关键作用。

To dissect the role of JMJD1C in cells, we crossedJmjd1c floxed mice to Foxp3 and obtained cell-specificJmjd1c conditional knockout
为了剖析JMJD1C在 细胞中的作用，我们将Jmjd1c基因缺失小鼠与Foxp3 杂交，获得了 细胞特异性Jmjd1c条件性基因敲除小鼠。

Jmjd11 ).Jmjd11 Foxp or Jmjd1c Foxp mice were used as controls (Jmjd1c ).JMJD1C expression was efficiently ablated
Jmjd11 ）。Jmjd11 Foxp 或 Jmjd1c Foxp 小鼠作为对照组（Jmjd1c ）。JMJD1C 的表达被有效消减。

with the relatively low expression level of JMJD1C in peripheral cells, the development of cells in peripheral immune organs was unaltered byJMJD1C deletion (Extended Data Fig. 2b). Overall immune homeostasis also seemed to be intact, as assessed by both effector and
由于 JMJD1C 在外周 细胞中的表达水平相对较低，JMJD1C 基因缺失不会改变外周免疫器官 细胞的发育（扩展数据图 2b）。整体免疫稳态似乎也没有受到影响，这可以从效应细胞和免疫细胞的评估中看出。

of multiple organs from aged mice (Extended Data Fig. 2c,d). cell frequencies in peripheral organs were unaltered in aged Jmjd1 mice (Extended Data Fig. 2e). Moreover, when the mice were induced with experimental autoimmune encephalomyelitis (EAE), there were no alterations in disease scores, central nervous system cell frequency or T cell cytokine production (Supplementary Fig.1a-c). Thus,JMJD1C is dispensable for systemic cell development and function, under either steady or inflammatory state.
老龄 Jmjd1 小鼠外周器官的 细胞频率没有变化（扩展数据图 2e）。此外，当小鼠被诱发实验性自身免疫性脑脊髓炎（EAE）时，疾病评分、中枢神经系统 细胞频率或 T 细胞细胞因子的产生均无变化（补充图 1a-c）。因此，在稳定或炎症状态下，JMJD1C 对全身 细胞的发育和功能都是不可或缺的。

Having found that JMJD1C was not required for cells to maintain systemic immune homeostasis, we next challenged Jmjd1 mice with multiple tumor models, including B16-OVA melanoma, MCA205 fibrosarcoma and EL4 lymphoma. JMJD1C deficiency in cells decelerated tumor growth for all these engrafted tumor cell types
在发现 细胞不需要 JMJD1C 来维持全身免疫平衡之后，我们接下来用多种肿瘤模型（包括 B16-OVA 黑色素瘤、MCA205 纤维肉瘤和 EL4 淋巴瘤）来挑战 Jmjd1 小鼠。 细胞中 JMJD1C 的缺乏使所有这些移植肿瘤细胞类型的肿瘤生长速度减慢。

with B16-OVA tumors had a profound loss of cells in the tumor but not in the spleen or draining lymph nodes (Fig. 2b and Extended Data Fig. 3a). This was accompanied by increased numbers of conven-
B16-OVA 肿瘤的 细胞在肿瘤中大量丢失，但在脾脏或引流淋巴结中却没有丢失（图 2b 和扩展数据图 3a）。与此同时，脾脏和淋巴结中的召集细胞数量增加。

Similar phenotypes were observed with MCA205-tumor-bearing mice (Fig. 2d,e). Moreover, these tumor conventional T cells produced more abundant effector-function-related cytokines, whereas cytokine production by draining lymph node T cells was not altered (Fig. 2f). Further characterization showed thatJMJD1C-deleted tumor cells displayed defective proliferation and survival, as revealed by Ki67 and active caspase 3 staining (Extended Data Fig. 3b,c). We then sorted out the cells for an in vitro suppression assay. Compared with wild-type (WT) cells, JMJD1C-deficient tumor cells had a dramatically reduced capability to inhibit T cell proliferation (Extended Data Fig. 3d), suggesting a compromised immunosuppressive function. By contrast, the suppressive function of splenic cells was not altered (Extended Data Fig. 3e). Taken together, these results indicate that JMJD1C is selectively required for cell fitness in tumors and that deficiency of
在携带 MCA205 肿瘤的小鼠身上也观察到了类似的表型（图 2d、e）。此外，这些肿瘤常规 T 细胞产生了更多的效应功能相关细胞因子，而引流淋巴结 T 细胞产生的细胞因子没有改变（图 2f）。进一步的特性分析表明，Ki67 和活性 caspase 3 染色显示，JMJD1C 缺失的肿瘤 细胞显示出增殖和存活缺陷（扩展数据图 3b、c）。然后，我们筛选出 细胞进行体外抑制试验。与野生型（WT） 细胞相比，JMJD1C缺陷的肿瘤 细胞抑制T细胞增殖的能力大大降低（扩展数据图3d），表明其免疫抑制功能受到了影响。相比之下，脾脏 细胞的抑制功能没有改变（扩展数据图 3e）。综上所述，这些结果表明，JMJD1C 是 细胞在肿瘤中适应性的选择性需要，缺乏 JMJD1C 会导致肿瘤细胞的免疫抑制功能受损。
JMJD1C in cells retards tumor growth without discernible adverse inflammatory or autoimmune effects.
细胞中的 JMJD1C 可延缓肿瘤生长，而不会产生明显的不良炎症或自身免疫效应。

To further determine the specific importance of JMJD1C to tumor cells, we also crossed mice to , thereby deleting JMJD1C in total T cells (Jmjd1c ). The development of cells and cells was unaffected (Supplementary Fig. 2a,b). T cell immune homeostasis, as assessed by CD44 and CD62L staining, also remained intact (Supplementary Fig. 2c). Notably, when the mice were subjected to
为了进一步确定 JMJD1C 对肿瘤 细胞的特殊重要性，我们还将 小鼠与 杂交，从而在全部 T 细胞中缺失 JMJD1C（Jmjd1c ）。 细胞和 细胞的发育未受影响（补充图 2a,b）。通过 CD44 和 CD62L 染色评估的 T 细胞免疫平衡也保持不变（补充图 2c）。值得注意的是，当小鼠受到

compared with WT mice (Supplementary Fig. 2d), and tumor cells were significantly reduced in number (Supplementary Fig. 2e). Accordingly, conventional cell numbers in tumor tissues were increased, along with upregulated cytokine production (Supplementary Fig. 2f,g), suggesting thatJMJD1C is dispensable for conventional T cell function. Furthermore, tumor cells from Jmjd1c mice exhibited reduced cell proliferation and survival, whereas conventional T cells were unaffected (Supplementary Fig. 2h). Thus, these data further confirm that JMJD1C selectively maintains tumor cells.
与 WT 小鼠相比（补充图 2d），肿瘤 细胞的数量显著减少（补充图 2e）。相应地，肿瘤组织中的传统 细胞数量增加，细胞因子的产生也上调（补充图 2f,g），这表明 JMJD1C 对传统 T 细胞的功能是不可或缺的。此外，来自 Jmjd1c 小鼠的肿瘤 细胞表现出细胞增殖和存活率降低，而常规 T 细胞不受影响（补充图 2h）。因此，这些数据进一步证实了 JMJD1C 可选择性地维持肿瘤 细胞。

We then sought to determine the molecular basis of the regulation of fitness by JMJD1C in tumors. Tumor cells fromJmjd1c and Jmjd1 mice were sorted out for scRNA-seq analysis. More than three mice per group were pooled to minimize the sample variation. Using -SNE for visualization and clustering, based on previous studies , a total of four cellular clusters were identified (TNFRSF9 , and TNFRSF9 ). The cluster proportions were comparable betweenJmjd1c WT and KO cells (Extended Data Fig. 4a,b), suggesting that upregulation of JMJD1C in tumor cells is not required for the cell differentiation and transition program. Flow cytometry analysis further confirmed that the frequency of the 4-1BB (encoded by Tnfrsf9) cell subpopulation was unaltered by JMJD1C ablation (Extended Data Fig. 4c). We then focused on gene expression and pathway alterations inJmjd1c cells. Consistent with the classical function of JMJD1C as a H3K9me2 demethylase, the overall H3K9me2 level in JMJD1C-deleted tumor cells was increased (Extended Data Fig. 4d), suggesting that loss of JMJD1C probably decreased expression of its target genes. Indeed, 230 downregulated differentially expressed genes were identified in Jmjd1c KO tumor cells (Extended Data Fig. 4e). According to GSEA, fatty acid metabolism was the most downregulated hallmarkgene set inJmjd1c KO tumor cells (Extended Data Fig. 4f). Fatty acid synthesis has been reported to be required for functional maturation of tumor cells , consistent with our observation that the suppressive function of KO cells was impaired (Extended Data Fig. 3d). However, it has also been reported that disruption of fatty acid synthesis by Fasn does not alter cell frequency in tumor , whereas the frequency of tumor cells in the absence of JMJD1C was significantly reduced (Fig. 2b,d), suggesting that JMJD1C could regulate tumor cells by additional mechanisms beyond fatty acid metabolism.
然后，我们试图确定 JMJD1C 在肿瘤中调控 适宜性的分子基础。我们对Jmjd1c 和Jmjd1 小鼠的肿瘤 细胞进行了分类，以进行 scRNA-seq 分析。每组有三只以上的小鼠被集中在一起，以尽量减少样本差异。根据先前的研究 ，使用 -SNE 进行可视化和聚类，共确定了四个细胞群（TNFRSF9 、 和 TNFRSF9 ）。Jmjd1c WT 和 KO 细胞的细胞簇比例相当（扩展数据图 4a,b），表明肿瘤 细胞中 JMJD1C 的上调并非细胞分化和转化程序所必需。流式细胞术分析进一步证实，4-1BB（由Tnfrsf9编码） 细胞亚群的频率不会因JMJD1C消减而改变（扩展数据图4c）。然后，我们重点研究了Jmjd1c 细胞中基因表达和通路的改变。与JMJD1C作为H3K9me2去甲基化酶的经典功能相一致，在JMJD1C缺失的肿瘤 细胞中，整体H3K9me2水平升高（扩展数据图4d），这表明JMJD1C的缺失可能会降低其靶基因的表达。事实上，在 Jmjd1c KO 的肿瘤 细胞中发现了 230 个下调的差异表达基因（扩展数据图 4e）。根据GSEA，脂肪酸代谢是Jmjd1c KO肿瘤 细胞中下调最多的标志基因集（扩展数据图4f）。据报道，脂肪酸合成是肿瘤 细胞 功能成熟所必需的，这与我们观察到的 KO 细胞的抑制功能受损是一致的（扩展数据图 3d）。 然而，也有报道称，Fasn 破坏脂肪酸合成并不会改变肿瘤 ，而在JMJD1C缺失的情况下，肿瘤 细胞的频率显著降低（图2b,d），这表明JMJD1C可以通过脂肪酸代谢以外的机制调节肿瘤 细胞。

Surface molecules have essential roles in cell maintenance and function. Pdcd1 and Nrp1 were among the most strongly downregulated genes encoding surface molecules (Fig. 3a). Flow cytometry confirmed that the expression of PD1 and NRP1 at a protein level was significantly reduced byJMJD1C deficiency in tumor cells (Fig. 3b,c). Importantly, the expression of these two genes was not altered at all in splenic cells (Fig. 3b,c). NRP1 deficiency has been reported to impair tumor cell survival and proliferation , consistent with our observations in Jmjd1c KO tumor cells (Extended Data Fig. 3b,c). To determine whether and were potentially directly regulated by JMJD1C-mediated H3K9me2, CUT&RUN-seq (cleavage under targets and release using nuclease sequencing) was performed with tumor cells from MCA205-tumor-bearing Jmjd1 WT and Jmjd1c mice.Jmjd1c tumor cells had stronger overall peak signal intensity of than WT tumor cells (Fig. 3d); a total of 5,012
表面分子对 细胞的维持和功能起着至关重要的作用。Pdcd1 和 Nrp1 是编码表面分子的基因中下调最强烈的基因之一（图 3a）。流式细胞术证实，在肿瘤 细胞中，PD1 和 NRP1 蛋白水平的表达因 JMJD1C 缺乏而显著降低（图 3b、c）。重要的是，这两个基因的表达在脾脏 细胞中没有任何改变（图 3b,c）。据报道，NRP1缺乏会影响肿瘤 细胞的存活和增殖 ，这与我们在Jmjd1c KO肿瘤 细胞中的观察结果一致（扩展数据图3b,c）。为了确定 和 是否可能受到 JMJD1C 介导的 H3K9me2 的直接调控，我们对来自 MCA205 肿瘤携带 Jmjd1 WT 和 Jmjd1c 小鼠的肿瘤 细胞进行了 CUT&RUN-seq（使用核酸酶测序的靶标下裂解和释放）分析。与 WT 肿瘤 细胞相比，Jmjd1c 肿瘤 细胞的 整体峰值信号强度更强（图 3d）；共有 5,012 个 Jmjd1c 肿瘤 细胞的 峰值信号强度高于 WT 肿瘤 细胞（图 3d）。

b B16-OVA tumor b B16-OVA 肿瘤

d

f

Fig. cellJmjd1c deficiency reduces tumor cell frequency and
图 细胞 Jmjd1c 缺乏会降低肿瘤 细胞的频率和数量。

inoculated subcutaneously with B16-OVA cells ( for Jmjd1c and for
皮下接种 B16-OVA 细胞 ( for Jmjd1c and for Jmjd1c and )

EL4 cells ( for and for ).b, Representative flow cytometric plots of tumor-infiltrating cells (left). Percentages of Foxp3-YFP cells among cells in the indicated tissues of B16-OVA-tumor-bearing mice are plotted on the right. Spl, spleen; dLN, draining lymph node. for Jmjd1c and for Jmjd1c .c, Cell numbers of Foxp3- CD4 and CD8 T cells per gram of tumor in B16-OVA-tumor-bearing Jmjd1c and Jmjd1 mice. TIL, tumor-infiltrating lymphocyte. d, Representative flow cytometric plots of tumor-infiltrating cells (left). Percentages of Foxp3-YFP cells among CD4 cells in the indicated tissues of MCA205tumor-bearing mice are plotted on the right. for and for Jmjd1c , Cell numbers of Foxp3 and cells per gram of tumor in MCA205-tumor-bearing mice. for Jmjd1 and for . f, Representative flow cytometric plots of cytokine production in Foxp3 and MCA205-tumor-infiltrating T cells (left). Percentages of cytokineproducing cells in draining lymph node and tumor are plotted on the right. for and for Jmjd1c . Data in a-f were pooled from two independent experiments and are shown as mean s.d. Two-way ANOVA (a) and two-tailed unpaired Student's -test (b-f) were used for data analysis. increased peaks (corresponding to 1,198 genes) were identified in Jmjd1c KO cells compared with cells. By integrating transcriptome data, we identified 18 genes that could be directly regulated by JMJD1C-mediated H3K9me2 (Fig. 3e and Supplementary Table 1), as they showed both increased H3K9me2 deposition and decreased messenger RNA (mRNA) expression, with Pdcd1 in particular showing clearly increased peak intensity inJmjd1c cells(Fig.3f). There was no increased deposition in the Nrp1 locus, suggesting that
EL4细胞（ 表示 ， 表示 ）。b, 肿瘤浸润 细胞的代表性流式细胞图（左）。右图为 B16-OVA 肿瘤小鼠指定组织中 细胞中 Foxp3-YFP 细胞的百分比。Spl，脾脏；dLN，引流淋巴结。 代表 Jmjd1c ， 代表 Jmjd1c 。c, B16-OVA 肿瘤携带 Jmjd1c 和 Jmjd1 小鼠每克肿瘤中 Foxp3- CD4 和 CD8 T 细胞数。TIL，肿瘤浸润淋巴细胞。 d，肿瘤浸润 细胞（左）的代表性流式细胞图。右图为 MCA205 肿瘤小鼠指定组织中 Foxp3-YFP 细胞占 CD4 细胞的百分比。 为 ， 为 Jmjd1c ，MCA205 肿瘤小鼠每克肿瘤中 Foxp3 和 细胞数。 f, Foxp3 和 MCA205 肿瘤浸润 T 细胞细胞因子产生的代表性流式细胞图（左）。 为 ， 为 Jmjd1c 。 数据分析采用了双向方差分析（a）和双尾非配对学生 -检验（b-f）。与 细胞相比，在 Jmjd1c KO 细胞中发现了增加的峰值（对应 1,198 个基因）。通过整合转录组数据，我们确定了 18 个可能受 JMJD1C 介导的 H3K9me2 直接调控的基因（图 3e 和补充表 1），因为它们同时显示出 H3K9me2 沉积增加和信使 RNA（mRNA）表达减少，尤其是 Pdcd1 在 Jmjd1c 细胞中的峰强度明显增加（图 3f）。 的沉积在 Nrp1 基因座中没有增加，这表明