前列腺癌（PCa）细胞的分子特征和免疫抑制性骨肿瘤微环境（TME）对免疫检查点治疗（ICT）的限制有所贡献。鉴定PCa患者亚组进行ICT仍然是一个挑战。在此，我们报道了基本螺旋-环亮氨酸家族成员e22（BHLHE22）在骨转移性PCa中上调，并推动了免疫抑制性骨TME。在这项研究中，澄清了BHLHE22在PCa骨转移中的功能。我们对原发性和骨转移性PCa样本进行了免疫组织化学染色（IHC），并评估了其在体内和体外促进骨转移的能力。然后，通过免疫荧光（IF）、流式细胞术和生物信息学分析确定了BHLHE22在骨TME中的作用。采用RNA测序、细胞因子阵列、蛋白质印迹、IF、IHC和流式细胞术的方法鉴定了关键介体。随后，使用荧光素酶报告基因、染色质免疫共沉淀实验、DNA拉取、共免疫共沉淀和动物实验证实了BHLHE22在基因调节中的作用。采用靶向蛋白质精氨酸甲基转移酶5（PRMT5）/集落刺激因子2（CSF2）的免疫抑制中性粒细胞和单核细胞中和策略，评估其是否可以提高ICT的疗效，使用异种移植骨转移小鼠模型将动物随机分配到治疗组或对照组。此外，我们进行了IHC和相关性分析，以确定BHLHE22是否可以作为骨转移性PCa ICT联合治疗的潜在生物标记物。肿瘤BHLHE22介导CSF2的高表达，导致免疫抑制性中性粒细胞和单核细胞的浸润和长期免疫受损的T细胞状态。从机械上讲，BHLHE22结合到CSF2启动子并招募PRMT5形成转录复合物。PRMT5表观激活CSF2表达。在转移瘤小鼠模型中，通过抑制Csf2和Prmt5可以克服Bhlhe22+肿瘤的ICT抗药性。这些结果揭示了肿瘤BHLHE22的免疫抑制机制，并为BHLHE22+ PCa患者提供了一种潜在的ICT联合治疗方法。©作者（或其雇主）2023。在CC BY-NC下允许重新使用，不得进行商业重复使用。由BMJ出版。

The molecular characteristics of prostate cancer (PCa) cells and the immunosuppressive bone tumor microenvironment (TME) contribute to the limitations of immune checkpoint therapy (ICT). Identifying subgroups of patients with PCa for ICT remains a challenge. Herein, we report that basic helix-loop-helix family member e22 (BHLHE22) is upregulated in bone metastatic PCa and drives an immunosuppressive bone TME.In this study, the function of BHLHE22 in PCa bone metastases was clarified. We performed immunohistochemical (IHC) staining of primary and bone metastatic PCa samples, and assessed the ability to promote bone metastasis in vivo and in vitro. Then, the role of BHLHE22 in bone TME was determined by immunofluorescence (IF), flow cytometry, and bioinformatic analyses. RNA sequencing, cytokine array, western blotting, IF, IHC, and flow cytometry were used to identify the key mediators. Subsequently, the role of BHLHE22 in gene regulation was confirmed using luciferase reporter, chromatin immunoprecipitation assay, DNA pulldown, co-immunoprecipitation, and animal experiments. Xenograft bone metastasis mouse models were used to assess whether the strategy of immunosuppressive neutrophils and monocytes neutralization by targeting protein arginine methyltransferase 5 (PRMT5)/colony stimulating factor 2 (CSF2) could improve the efficacy of ICT. Animals were randomly assigned to treatment or control groups. Moreover, we performed IHC and correlation analyses to identify whether BHLHE22 could act as a potential biomarker for ICT combination therapies in bone metastatic PCa.Tumorous BHLHE22 mediates the high expression of CSF2, resulting in the infiltration of immunosuppressive neutrophils and monocytes and a prolonged immunocompromised T-cell status. Mechanistically, BHLHE22 binds to the CSF2 promoter and recruits PRMT5, forming a transcriptional complex. PRMT5 epigenetically activates CSF2 expression. In a tumor-bearing mouse model, ICT resistance of Bhlhe22+ tumors could be overcome by inhibition of Csf2 and Prmt5.These results reveal the immunosuppressive mechanism of tumorous BHLHE22 and provide a potential ICT combination therapy for patients with BHLHE22+ PCa.© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.