Introduction: PD-1 inhibits the cytotoxic T-cell functions via the interaction with its ligands (PDL1 and PDL2). Recently, our group showed a selective response (~40%) with PD-1 blocking antibody pembrolizumab in patients with Richter transformation (RT), particularly after prior exposure to ibrutinib (Wei Ding et al, Blood, 2017). Additionally, PD-1 showed an increased expression in tumor B-cells of patients with RT (Rong He et al, AJSP, 2018). Here we investigated the functional implications of the PD-1 signaling axis in lymphoma B-cell pathobiology. Methods: 26 CLL-involved lymph node (LN) and 20 RT-involved LN samples were tested for PD-1 expression by immunohistochemistry (mouse clone NAT105, Abcam). Then, we checked PD-1 expression in 10 lymphoma cell lines and 1 CLL cell line (MEC-1) by both, flow cytometry and Western blot (WB) analysis. Over-expression of PD-1 using pLEX-lentiviral system (Thermo Scientific) was evaluated for in vitro cell cycle regulation and in vivo tumor growth. Overexpression of PD-1 was further examined on the regulation of cell signaling pathways using human phospho kinase array kit (R&D) and WB analysis. Gene expression signatures in CLL and RT patients were also identified by Illumina-based RNA sequencing using Formalin-Fixed Paraffin-embedded (FFPE)-nodal tissue obtained by clinical biopsy (Tempus Labs; Chicago, IL). Results: The expression of PD-1 was significantly increased in RT-LN compared to CLL-LN (mean ± SEM in RT vs. CLL, 30.6% ± 4.7% vs. 11.5% ± 2.8%, p < 0.001) [Fig 1A]. PD-1 expression was highest in patients with RT where the last prior CLL therapy was ibrutinib. To test the role of PD-1 in lymphoma B-cells, its expression was assessed in lymphoma and CLL cell lines. The expression of PD-1 was found to be variable, but at very low-levels in lymphoma lines OCI-LY7 and OCI-LY19 (data not shown). Constitutive lentiviral (pLEX-PD-1)-mediated overexpression of PD-1 in OCI-LY7 and OCI-LY19 cells led to increased cell growth (1.4 and 1.9 fold compared to original lines on day 4, respectively, Fig 1BI-II), which was further confirmed by cell cycle analysis which showed an increase in S phase by 20.4% and 24.53% in OCI-LY7 and OCI-LY19 cells (Fig 1B III), respectively. When the luciferase + PD-1 expressing OCI-LY7 cells were injected intravenously (1X106 cells) into NSG mice, increased tumor growth was observed at 22 days of follow up by bioluminescence imaging (p<0.01, Fig 1C). Using phospho-kinase array, an overall decrease of phosphorylation was detected on multiple sites of p53 (S392, S15, S46) and CHK2 (T68) (data not shown). This finding was further confirmed by WB analyses (Fig1DI). In addition, decreased of total p53 and increased phosphorylation on both SHP-1 and SHP-2 were found in PD-1 overexpressing OCI-LY19 and OCI-LY17 cell lines (Fig 1DI). Immunoprecipitation experiments with anti-PD1 antibody showed that PD-1 was in the same complex with SHP-1 and SHP-2 in OCI-LY7 cells but only with SHP-2 in OCI-LY19 cells (Fig 1DII). In parallel, mRNA sequencing was performed on 11 nodal tissues from either RT (n=8) or progressive CLL (n=3) after they developed clinical progression. The expression of PD-1 (PDCD1) was positively correlated with the expression of SHP-1 (PTPN6, r=0.56) and Cyclin E1 (CCNE1, r=0.62), implicating a direct role of PD-1 in regulating cellular proliferation and the induction in phosphatase expression in RT and CLL. Conclusion: Our data support the notion that PD-1 overexpression in lymphoma cells modifies cell proliferation in vitro and in vivo and regulates the function of phosphatase SHP and p53- pathways. These findings also indicate that aggressive lymphoma or CLL leukemic cells have the ability to hijack the PD-1 pathway and may result in downregulation of the p53 mediated DNA repair. This novel information provide for new strategies to further evaluate the interaction of checkpoint signals with DNA repair pathway, and possibly provide novel targets for Richter's transformation. Disclosures Ding: alexion: Membership on an entity's Board of Directors or advisory committees; Beigene: Membership on an entity's Board of Directors or advisory committees; Abbvie: Research Funding; Astra Zeneca: Research Funding; Octapharma: Membership on an entity's Board of Directors or advisory committees; Merck: Membership on an entity's Board of Directors or advisory committees, Research Funding; DTRM: Research Funding; MEI Pharma: Membership on an entity's Board of Directors or advisory committees. Sakemura:Humanigen: Patents & Royalties. Parikh:Janssen: Honoraria, Research Funding; Merck: Research Funding; Pharmacyclics: Honoraria, Research Funding; Ascentage Pharma: Research Funding; GlaxoSmithKline: Honoraria; MorphoSys: Research Funding; AstraZeneca: Honoraria, Research Funding; Verastem Oncology: Honoraria; Genentech: Honoraria; AbbVie: Honoraria, Research Funding; TG Therapeutics: Research Funding. Wang:Novartis: Research Funding; Incyte: Research Funding; Innocare: Research Funding. Liu:Eisal: Research Funding; Genentech: Research Funding; GRAIL: Research Funding; Menarini Silicon Biosystems: Research Funding; Merck: Research Funding; Seattle Genetics: Research Funding; Tesaro: Research Funding. Braggio:DASA: Consultancy; Bayer: Other: Stock Owner; Acerta Pharma: Research Funding. Ansell:Seattle Genetics: Research Funding; Takeda: Research Funding; AI Therapeutics: Research Funding; ADC Therapeutics: Research Funding; Trillium: Research Funding; Affimed: Research Funding; Regeneron: Research Funding; Bristol Myers Squibb: Research Funding. Kenderian:Sunesis: Research Funding; Tolero: Research Funding; Torque: Consultancy; BMS: Research Funding; Gilead: Research Funding; Kite: Research Funding; Humanigen: Consultancy, Patents & Royalties, Research Funding; Mettaforge: Patents & Royalties; Novartis: Patents & Royalties, Research Funding; MorphoSys: Research Funding; Lentigen: Research Funding; Juno: Research Funding. Kay:Acerta Pharma: Research Funding; Oncotracker: Membership on an entity's Board of Directors or advisory committees; Juno Theraputics: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Research Funding; MEI Pharma: Research Funding; Sunesis: Research Funding; Astra Zeneca: Membership on an entity's Board of Directors or advisory committees; Agios Pharma: Membership on an entity's Board of Directors or advisory committees; Cytomx: Membership on an entity's Board of Directors or advisory committees; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Dava Oncology: Membership on an entity's Board of Directors or advisory committees; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol Meyer Squib: Membership on an entity's Board of Directors or advisory committees, Research Funding; Rigel: Membership on an entity's Board of Directors or advisory committees.

中文翻译：

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鉴定 PD-1 信号在促进 B 细胞淋巴瘤中肿瘤增殖中的新作用

简介：PD-1 通过与其配体（PDL1 和 PDL2）的相互作用抑制细胞毒性 T 细胞功能。最近，我们小组在 Richter 转化 (RT) 患者中表现出对 PD-1 阻断抗体 pembrolizumab 的选择性反应 (~40%)，特别是在之前接触 ibrutinib 之后 (Wei Ding 等人，Blood，2017)。此外，PD-1 在 RT 患者的肿瘤 B 细胞中的表达增加（Rong He 等人，AJSP，2018）。在这里，我们研究了 PD-1 信号轴在淋巴瘤 B 细胞病理生物学中的功能意义。方法：通过免疫组织化学（小鼠克隆 NAT105，Abcam）检测 26 个 CLL 受累淋巴结 (LN) 和 20 个 RT 受累 LN 样本的 PD-1 表达。然后，我们检查了 10 个淋巴瘤细胞系和 1 个 CLL 细胞系 (MEC-1) 中的 PD-1 表达，流式细胞术和蛋白质印迹 (WB) 分析。使用 pLEX-慢病毒系统 (Thermo Scientific) 对 PD-1 的过度表达进行了体外细胞周期调节和体内肿瘤生长的评估。使用人磷酸激酶阵列试剂盒 (R&D) 和 WB 分析进一步检查 PD-1 过表达对细胞信号通路的调节。CLL 和 RT 患者中的基因表达特征也通过基于 Illumina 的 RNA 测序使用福尔马林固定石蜡包埋 (FFPE) 结节组织进行鉴定，该结节组织通过临床活检 (Tempus Labs; Chicago, IL) 获得。结果：与 CLL-LN 相比，RT-LN 中 PD-1 的表达显着增加（RT 与 CLL 中的平均值 ± SEM，30.6% ± 4.7% 对 11.5% ± 2.8%，p < 0.001）[图1A]。PD-1 表达在 RT 患者中最高，其中最后一次 CLL 治疗是依鲁替尼。为了测试 PD-1 在淋巴瘤 B 细胞中的作用，评估了其在淋巴瘤和 CLL 细胞系中的表达。发现 PD-1 的表达是可变的，但在淋巴瘤系 OCI-LY7 和 OCI-LY19 中的表达水平非常低（数据未显示）。OCI-LY7 和 OCI-LY19 细胞中组成型慢病毒 (pLEX-PD-1) 介导的 PD-1 过表达导致细胞生长增加（与第 4 天的原始细胞系相比，分别为 1.4 和 1.9 倍，图 1BI-II） ，通过细胞周期分析进一步证实，OCI-LY7 和 OCI-LY19 细胞的 S 期分别增加了 20.4% 和 24.53%（图 1B III）。当向 NSG 小鼠静脉内注射荧光素酶 + PD-1 表达 OCI-LY7 细胞（1X106 细胞）时，通过生物发光成像在随访 22 天时观察到肿瘤生长增加（p＜0.01，图 1C）。使用磷酸激酶阵列，在 p53（S392、S15、S46）和 CHK2（T68）的多个位点上检测到磷酸化的总体降低（数据未显示）。WB分析进一步证实了这一发现（图1DI）。此外，在过表达 OCI-LY19 和 OCI-LY17 细胞系的 PD-1 中发现总 p53 减少和 SHP-1 和 SHP-2 磷酸化增加（图 1DI）。用抗 PD1 抗体进行的免疫沉淀实验表明，PD-1 在 OCI-LY7 细胞中与 SHP-1 和 SHP-2 处于同一复合物中，但在 OCI-LY19 细胞中仅与 SHP-2 处于同一复合物中（图 1DII）。同时，在出现临床进展后，对来自 RT (n=8) 或进行性 CLL (n=3) 的 11 个淋巴结组织进行 mRNA 测序。PD-1（PDCD1）的表达与SHP-1（PTPN6，r=0.56）和Cyclin E1（CCNE1，r=0.62）的表达呈正相关，表明 PD-1 在调节细胞增殖和诱导 RT 和 CLL 中磷酸酶表达中的直接作用。结论：我们的数据支持这样的观点，即淋巴瘤细胞中的 PD-1 过表达在体外和体内改变了细胞增殖，并调节了磷酸酶 SHP 和 p53 通路的功能。这些发现还表明侵袭性淋巴瘤或 CLL 白血病细胞具有劫持 PD-1 通路的能力，并可能导致 p53 介导的 DNA 修复下调。这一新信息为进一步评估检查点信号与 DNA 修复途径的相互作用提供了新的策略，并可能为 Richter 的转化提供新的靶点。披露丁：alexion：实体成员资格 s 董事会或咨询委员会；百济神州：实体董事会或咨询委员会的成员；艾伯维：研究经费；阿斯利康：研究经费；Octapharma：实体董事会或咨询委员会的成员；默克：实体董事会或咨询委员会的成员资格，研究经费；DTRM：研究经费；MEI Pharma：实体董事会或咨询委员会的成员。Sakemura:Humanigen：专利和版税。Parikh:Janssen：酬金，研究经费；默克：研究经费；Pharmacyclics：酬金，研究经费；亚盛医药：研究经费；葛兰素史克：酬金；MorphoSys：研究经费；阿斯利康：酬金，研究经费；Verastem 肿瘤学：酬金；基因泰克：酬金；艾伯维：酬金，研究经费；TG Therapeutics：研究经费。王：诺华：研究经费；因赛特：研究经费；Innocare：研究经费。刘：Eisal：研究经费；基因泰克：研究经费；圣杯：研究经费；美纳里尼硅生物系统：研究经费；默克：研究经费；西雅图遗传学：研究经费；Tesaro：研究经费。Braggio:DASA：咨询；拜耳：其他：股东；Acerta Pharma：研究经费。Ansell：西雅图遗传学：研究经费；武田：研究经费；人工智能疗法：研究经费；ADC Therapeutics：研究经费；Trillium：研究经费；申明：研究经费；再生元：研究经费；百时美施贵宝：研究经费。Kenderian：Sunesis：研究经费；托莱罗：研究经费；扭矩：咨询；BMS：研究经费；吉利德：研究经费；风筝：研究经费；人类学：咨询、专利和特许权使用费、研究经费；Mettaforge：专利和版税；诺华：专利和特许权使用费、研究经费；MorphoSys：研究经费；Lentigen：研究经费；朱诺：研究经费。Kay：Acerta Pharma：研究经费；Oncotracker：实体董事会或咨询委员会的成员；Juno Theraputics：实体董事会或咨询委员会的成员；Pharmacyclics：实体董事会或咨询委员会的成员资格，研究经费；艾伯维：研究经费；MEI Pharma：研究经费；Sunesis：研究经费；Astra Zeneca：实体董事会或咨询委员会的成员；Agios Pharma：实体董事会或咨询委员会的成员；Cytomx：实体的成员资格 s 董事会或咨询委员会；Morpho-sys：实体董事会或咨询委员会的成员；Dava Oncology：实体董事会或咨询委员会的成员；Tolero Pharmaceuticals：实体董事会或咨询委员会的成员资格，研究经费；Bristol Meyer Squib：实体董事会或咨询委员会的成员资格，研究经费；Rigel：实体董事会或咨询委员会的成员。实体董事会或咨询委员会的成员资格，研究经费；Rigel：实体董事会或咨询委员会的成员。实体董事会或咨询委员会的成员资格，研究经费；Rigel：实体董事会或咨询委员会的成员。