异常组织蛋白酶 S 在滤泡性淋巴瘤中诱导支持性免疫微环境,Blood

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异常组织蛋白酶 S 在滤泡性淋巴瘤中诱导支持性免疫微环境
Blood ( IF 21.0 ) Pub Date : 2019-11-13 , DOI: 10.1182/blood-2019-122663
Johannes Adrian Hildebrand _{1,

2,

3} , Deepak Bararia _{2,

3,

4} , Sebastian Stolz _{2,

3,

4} , Sarah Haebe _{2,

3,

4} , Stefan Alig _{2,

3,

4} , Michael Bartoschek ₅ , Michael Boesl ₄ , Alessandro Pastore ₆ , Erik Gaitzsch _{2,

3,

4} , Michael Heide _{2,

3,

4} , Vindi Jurinovic ₄ , Monika Szczepanowski ₇ , Wolfram Klapper ₇ , Abner Louissaint ₈ , Christina Ludwig ₉ , Sebastian Bultmann ₅ , Heinrich Leonhardt ₅ , Sebastian Eustermann ₁₀ , Karl-Peter Hopfner ₁₀ , Wolfgang Hiddemann _{2,

3,

4} , Michael von Bergwelt _{2,

3,

4} , Christian Steidl ₁₁ , Robert Kridel ₁₂ , David M Weinstock ₁₃ , Marc Schmidt-Supprian ₁₄ , Menyhárt B Sárosi ₁₅ , Verena Passerini _{2,

3,

4} , Josef Mautner ₁₆ , Oliver Weigert _{2,

3,

4}

Affiliation

Department of Medicine III, University Hospital, LMU Munich, München, Germany
German Cancer Consortium (DKTK), Heidelberg, Germany
German Cancer Research Center (DKFZ), Heidelberg, Germany
Department of Medicine III, University Hospital, LMU Munich, Munich, Germany
Department of Biology II and Center for Integrated Protein Science Munich (CIPSM), Human Biology and BioImaging, LMU Munich, Martinsried, Germany
Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, New York, NY
Department of Hematopathology, University of Schleswig-Holstein, Campus Kiel, Kiel, Germany
Department of Pathology, Massachusetts General Hospital, Boston, MA
Bavarian Center for Biomolecular Mass Spectrometry (BayBioMS), Technische Universität München, Munich, Germany
Gene Center, LMU Munich, Munich, Germany
Centre for Lymphoid Cancer, BC Cancer, Vancouver, Canada
Division of Medical Oncology and Hematology, Princess Margaret Cancer Center, Toronto, Canada
Dana-Farber Cancer Institute, Jamaica Plain, MA
III. Medizinische Klinik, Technische Universität München, Munich, Germany
Institute of Inorganic Chemistry, Faculty of Chemistry and Mineralogy, Leipzig University, Leipzig, Germany
Department of Gene Vectors, Helmholtz Zentrum München, Munich, Germany

滤泡性淋巴瘤 (FL) 的高度可变临床过程由肿瘤细胞的分子异质性和与微环境的复杂相互作用决定。在这里，我们提供了生化、结构、功能和临床证据，表明异常的组织蛋白酶 S (CTSS) 活性在 FL 中诱导了支持性免疫微环境。通过对 305 份诊断性 FL 活检的靶向 DNA 测序，我们在 8% 的病例 (24/305) 中发现了 CTSS 的体细胞突变，主要集中在 Y132 (19/24) 将 Y 转化为 D (16/19)。CTSS Y132 突变的一个子集（N=5）发生在较低的变异等位基因频率（5-10%），表明亚克隆性。另外 13% 的 FL 有 CTSS 扩增 (37/286)。CTSS Y132 突变和 CTSS 扩增相互排斥。在 51 个 FL 的队列中，CTSS 扩增与更高的 CTSS 表达相关（P=0.05）。值得注意的是，没有 CTSS 扩增的 FL 子集也具有更高的 CTSS 表达，这表明转录失调的其他机制。CTSS 是一种半胱氨酸蛋白酶，在抗原呈递细胞和恶性 B 细胞的内溶酶体中高度表达。CTSS 参与抗原肽的蛋白水解加工，以在 MHC-II 上呈递以被抗原特异性 CD4+ T 细胞识别。CTSS 被合成为一种无活性的酶原，通过自抑制前肽 (pro-CTSS) 的自催化裂解转化为其活性形式。我们使用 CRISPR/Cas9 将 CTSS Y132D 引入 Karpas422，这是一种具有 FL 标志易位 t(14;18) 的 B 细胞淋巴瘤细胞系。单细胞衍生的 Y132D 突变克隆显示＞活性 CTSS 与 pro-CTSS 的比率高出 3 倍（N=4，P=0.0003）。与 CTSS 野生型 (WT) 相比，免疫沉淀的 CTSS Y132D 的体外底物切割活性高于 3 倍 (N=6, P=0.001)。我们纯化了 pro-CTSS WT 和 Y132D，并随时间测定了它们的体外自催化裂解。转换 50% pro-CTSS 所需的时间从 WT 的 17 分钟减少到 Y132D 的 11 分钟（N=3，P=0.04）。相比之下，纯化的活性 CTSS WT 和 Y132 具有相似的体外切割活性。分子动力学模拟表明，Y132D 突变将活性 CTSS 的催化三联体（C139、H278、N298）与来自前体的一段氨基酸（L80、G81、D82、S94）之间的距离缩短了 ~2Å，这可能有助于分子内裂解。通过质谱，我们确实可以检测到新的中等大小的 CTSS 片段。因此，Y132D 不会增加成熟酶的活性，而是通过加速从 pro-CTSS 向催化活性 CTSS 的转化而获得功能突变。CD74（不变链）是一种生理 CTSS 底物，在无肽 MHC-II 的组装、运输和稳定中起关键作用。CTSS 切割 CD74，从而允许 MHC-II 上的抗原与抗原特异性 CD4+ T 细胞结合并呈递。我们可以证明 CTSS Y132D 增强了 Karpas422 细胞中的 CD74 切割。然后，我们在共培养试验中测试了 CTSS 对抗原特异性 CD4+ T 细胞活化的影响。CTSS 敲除淋巴瘤细胞广泛无法激活 CD4+ T 细胞。与空载体对照相比，CTSS WT 的过表达激活的 CD4+ T 细胞更有效。CTSS Y132 具有最高的刺激抗原特异性 CD4+ T 细胞反应的能力。此外，在原发性 FL 活检 (N=51) 中，CTSS Y132 突变具有与抗原加工和趋化因子扰动相关的基因表达谱，包括 CXCL13，一种由活化的 CD4+ T 滤泡辅助细胞产生的 B 细胞趋化因子。最后，我们旨在将 CTSS 畸变与接受标准免疫化疗 (R-CHOP) 治疗晚期 FL 的患者的临床结果相关联（N = 51，具有可用的 CTSS 突变和基因表达数据）。与所有其他患者（N=34）相比，具有 CTSS Y132 突变或 CTSS 过表达的患者（N=17）具有更长的无失败生存期（P=0.012）和总生存期（P=0.041）。总之，我们提出异常的 CTSS 活性——即使仅存在于 FL 亚克隆中——也可以引发 CD4+ T 细胞驱动的促肿瘤免疫反应，这种免疫反应可以通过促炎和趋化细胞因子在微环境中放大，并显着影响生物学和疾病的临床过程。因此，异常的 CTSS 活性是 FL 和潜在其他肿瘤的有前途的生物标志物和治疗靶点。披露克拉珀：罗氏、武田、安进、再生元：酬金、研究经费。Hiddemann：拜耳：研究经费；罗氏：咨询、酬金、研究经费；吉利德：咨询，酬金；Janssen：咨询、酬金、研究经费；Celgene：咨询，酬金；Vector Therapeutics：咨询，酬金。Steidl：Juno Therapeutics：咨询；百时美施贵宝：研究经费；Nanostring：专利和版税：代表 BC Cancer 申请专利；罗氏：咨询；Tioma：研究经费；西雅图遗传学：咨询；拜耳：咨询。Kridel：吉利德科学：研究经费。温斯托克：Celgene：研究经费；Verastem 肿瘤学：研究经费。Weigert：诺华：研究经费；罗氏：研究经费。

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Aberrant Cathepsin S Induces a Supportive Immune Microenvironment in Follicular Lymphoma

The highly variable clinical course of follicular lymphoma (FL) is determined by the molecular heterogeneity of the tumor cells and complex interactions with the microenvironment. Here, we provide biochemical, structural, functional and clinical evidence that aberrant Cathepsin S (CTSS) activity induces a supportive immune microenvironment in FL. By targeted DNA sequencing of 305 diagnostic FL biopsies, we identified somatic mutations of CTSS in 8% of cases (24/305), mostly clustered at Y132 (19/24) converting Y to D (16/19). A subset of CTSS Y132 mutations (N=5) occurred at lower variant allele frequencies (5-10%), indicating subclonality. Another 13% of FL had CTSS amplifications (37/286). CTSS Y132 mutations and CTSS amplifications were mutually exclusive. In a cohort of 51 FL, CTSS amplifications were associated with higher CTSS expression (P=0.05). Of note, a subset of FL without CTSS amplifications also had higher CTSS expression, suggesting additional mechanisms of transcriptional dysregulation. CTSS is a cysteine protease that is highly expressed in endolysosomes of antigen presenting cells and malignant B-cells. CTSS is involved in proteolytical processing of antigenic peptides for presentation on MHC-II to be recognized by antigen specific CD4+ T-cells. CTSS is synthesized as an inactive zymogen, which is converted to its active form by autocatalytic cleavage of the autoinhibitory propeptide (pro-CTSS). We used CRISPR/Cas9 to introduce CTSS Y132D into Karpas422, a B-cell lymphoma cell line that harbors the FL hallmark translocation t(14;18). Single-cell derived Y132D mutant clones showed >3-fold higher ratios of active CTSS to pro-CTSS (N=4, P=0.0003). Immunoprecipitated CTSS Y132D had >3-fold higher in vitro substrate cleavage activity compared to CTSS wild type (WT) (N=6, P=0.001). We purified pro-CTSS WT and Y132D and assayed their in vitro autocatalytic cleavage over time. The time required to convert 50% of pro-CTSS decreased from 17 minutes for WT to 11 minutes for Y132D (N=3, P=0.04). In contrast, purified active CTSS WT and Y132 had similar in vitro cleavage activities. Molecular dynamics simulations showed that the Y132D mutation shortens the distances by ~2Å between the catalytic triad of active CTSS (C139, H278, N298) and a stretch of amino acids from the proform (L80, G81, D82, S94), which may facilitate intramolecular cleavage. By mass spectrometry we could indeed detect novel intermediate-sized CTSS fragments. Thus, Y132D does not increase the activity of the mature enzyme but is a gain-of-function mutation by accelerating the conversion from pro-CTSS to catalytically active CTSS. CD74 (invariant chain) is a physiologic CTSS substrate that plays critical roles in the assembly, trafficking and stabilization of peptide-free MHC-II. CTSS cleaves CD74, thereby allowing binding and presentation of antigens on MHC-II to antigen specific CD4+ T-cells. We could show that CTSS Y132D enhanced CD74 cleavage in Karpas422 cells. We then tested the impact of CTSS on antigen specific CD4+ T-cell activation in co-culture assays. CTSS knock-out lymphoma cells were broadly incapable of activating CD4+ T-cells. Overexpression of CTSS WT activated CD4+ T-cells more efficiently compared to empty vector control. CTSS Y132 had the highest capacity to stimulate antigen specific CD4+ T-cell responses. Furthermore, in primary FL biopsies (N=51) CTSS Y132 mutations had gene expression profiles linked with antigen-processing and chemokine perturbation, including CXCL13, a B-cell chemoattractant produced by activated CD4+ T-follicular helper cells. Lastly, we aimed to correlate CTSS aberrations with clinical outcome in patients who received standard immunochemotherapy (R-CHOP) for advanced FL (N=51 with available CTSS mutation and gene expression data). Compared to all other patients (N=34), patients with CTSS Y132 mutations or CTSS overexpression (N=17) had longer failure free survival (P=0.012) and overall survival (P=0.041). In summary, we propose that aberrant CTSS activity - even if only present in a FL subclone - can elicit a CD4+ T-cell driven tumor-promoting immune response, which could be amplified within the microenvironment via pro-inflammatory and chemotactic cytokines and substantially impact the biology and clinical course of the disease. Thus, aberrant CTSS activity is a promising biomarker and therapeutic target in FL and potentially also other tumors. Disclosures Klapper: Roche, Takeda, Amgen, Regeneron: Honoraria, Research Funding. Hiddemann:Bayer: Research Funding; Roche: Consultancy, Honoraria, Research Funding; Gilead: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria; Vector Therapeutics: Consultancy, Honoraria. Steidl:Juno Therapeutics: Consultancy; Bristol-Myers Squibb: Research Funding; Nanostring: Patents & Royalties: Filed patent on behalf of BC Cancer; Roche: Consultancy; Tioma: Research Funding; Seattle Genetics: Consultancy; Bayer: Consultancy. Kridel:Gilead Sciences: Research Funding. Weinstock:Celgene: Research Funding; Verastem Oncology: Research Funding. Weigert:Novartis: Research Funding; Roche: Research Funding.

更新日期：2019-11-13

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