ER stress sensor PERK drives tumor-promoting myelopoiesis by reprogramming hematopoietic progenitor cells in the spleen. ER stress sensor PERK drives tumor-promoting myelopoiesis by reprogramming hematopoietic progenitor cells in the spleen

The spleen, as the prominent site for extramedullary myelopoiesis in cancer, generates significant amounts of myeloid cells to promote tumor progression. Cancer-associated splenic myelopoiesis is mediated by a population of functionally distinct hematopoietic stem/progenitor cells (HSPCs) that are committed to immunosuppressive myeloid cell generation, but the molecular mechanism regulating this response remains unclear. Here, we found that HSPCs in the spleen of tumor-bearing mice exhibited accelerated protein synthesis and engaged in a robust endoplasmic reticulum (ER) stress response. Single-cell RNA sequencing showed that the ER stress response was concomitant with the myeloid-biased lineage commitment and dysfunction of HSPCs in the spleen. PKR-like ER resident kinase (PERK), a molecular sensor of ER stress, directed HSPC differentiation into myeloid-derived suppressor cells (MDSCs) via PERK–eIF2α–ATF4–C/EBPβ signaling. Inhibition of this signaling prevented the emergence of granulocyte/macrophage colony-stimulating factor-expressing HSPCs in the spleen and disrupted the positive feedback-driven splenic myelopoiesis. Consequently, a blockade of PERK abated the suppressive function of tumor-infiltrating MDSCs and increased cytotoxic T cell infiltration, reshaping the tumor microenvironment to effectively suppress disease progression and potentiate both immuno- and targeted therapies. In accordance, activation of PERK–ATF4–C/EBPβ signaling was indispensable for the differentiation of human umbilical cord blood-derived HSPCs into functionally competent MDSCs. Together, these results indicate a crucial role for the cellular ER stress sensor PERK in modulating splenic HSPC activities in cancer, suggesting novel therapeutic opportunities for targeting the tumor-promoting myeloid response at its source. Raw data are available on Genome Sequence Archive, Bioproject accession number: PRJCA003519 Overall design: LSK cells from the BM and spleen of Hepa mice were isolated and purified by FACS. The cells were then processed for scRNA-seq using the Chromium Single Cell 3’ Library & Gel Bead Kit v3 (10x Genomics) following the manufacturer’s protocol. Samples were sequenced at an average of 27,000 reads per cell. The resulting scRNA-seq reads were aligned to the GRCm38 (mm10) reference genome and quantified using cellranger count (Cell Ranger pipeline version 3.1.0, 10x Genomics).

脾脏作为癌症中髓外造血的核心部位，可生成大量髓系细胞以促进肿瘤进展。癌症相关的脾脏髓系造血由一群功能特化的造血干/祖细胞（hematopoietic stem/progenitor cells, HSPCs）介导，这群细胞定向分化为具有免疫抑制功能的髓系细胞，但调控这一病理过程的分子机制仍未阐明。
本研究发现，荷瘤小鼠脾脏中的HSPCs呈现出加速的蛋白质合成，并激活了强烈的内质网（endoplasmic reticulum, ER）应激反应。单细胞RNA测序结果显示，内质网应激反应与脾脏HSPCs的髓系偏倚谱系定向分化及功能异常密切相关。
PKR样内质网驻留激酶（PKR-like ER resident kinase, PERK）作为内质网应激的分子感受器，通过PERK–eIF2α–ATF4–C/EBPβ信号通路引导HSPCs分化为髓系来源抑制细胞（myeloid-derived suppressor cells, MDSCs）。抑制该信号通路可阻断脾脏中表达粒细胞-巨噬细胞集落刺激因子的HSPCs的产生，并破坏正反馈驱动的脾脏髓系造血过程。
进一步研究发现，阻断PERK可减弱肿瘤浸润性MDSCs的免疫抑制功能，增加细胞毒性T细胞的肿瘤浸润，重塑肿瘤微环境以有效抑制疾病进展，同时增强免疫治疗与靶向治疗的疗效。与之相符的是，PERK–ATF4–C/EBPβ信号通路的激活对于人脐带血来源的HSPCs分化为具有功能活性的MDSCs是必不可少的。
综上，本研究结果表明细胞内质网应激感受器PERK在调控癌症状态下脾脏HSPCs的活性中发挥关键作用，为从源头靶向肿瘤促髓系反应提供了全新的治疗策略。
原始数据可于基因组序列归档（Genome Sequence Archive）获取，生物项目登录号：PRJCA003519。整体实验设计：从Hepa小鼠的骨髓与脾脏中分离LSK细胞，并通过荧光激活细胞分选（FACS）进行纯化。随后按照制造商的操作规范，使用Chromium单细胞3’文库与磁珠试剂盒v3（10x Genomics）完成单细胞RNA测序建库。测序时每个细胞的平均测序读段数为27,000条。将获得的scRNA-seq读段比对至GRCm38（mm10）参考基因组，并使用cellranger count（Cell Ranger分析流程v3.1.0，10x Genomics）进行基因表达定量。