Understanding mechanisms of drug resistance and clonal evolution in diffuse large B cell lymphoma

弥漫性大B细胞淋巴瘤(DLBCL)是西方世界最常见的淋巴系统恶性肿瘤，仅有60%的患者可通过标准的一线化疗免疫疗法治愈，这是临床上的一大挑战。复发或难治性患者的预后是令人沮丧的，因为这些患者很难挽救，即使使用大剂量的化疗，如BEAM，然后再进行自体干细胞支持。通过下一代测序技术的出现，大量可忠实地概括人类DLBCL生物学重要方面的小鼠模型的可用，以及进行大规模全基因组体内插入突变筛选的可能性，我们现在能够全面描述预测对化学免疫治疗反应不良的基因组事件。更重要的是，在(临床前)耐药发生之前和之后，对从小鼠和人身上纵向获得的组织样本进行测序，使我们能够准确地定位可能介导耐药的基因和途径。此外，在耐药发生后仔细检查小鼠和人类DLBCL基因组和转录本，可能使我们能够识别基因组和转录本异常，这可能为靶向治疗干预策略提供进入途径。在这些考虑的基础上，我们制定了直接处理上述问题的四个具体目标。我们的工作计划利用了我们过去几年在科隆和哥廷根组装的大型工具和技术组合。我们的具体目标如下：目的1：利用WES来鉴定R-CHOP化疗免疫治疗后患者反复出现的基因组异常。目的2：利用WES来确定DLBCL GEMM中R-CHOP化疗免疫治疗后出现的复发基因组异常。目的3：进行体内iggyBac插入突变筛选，以确定抵抗R-CHOP样化疗免疫治疗的媒介。

Diffuse large B cell lymphoma (DLBCL) is the most common lymphoid malignancy in the Western World and represents a major clinical challenge, as only ~60% of patients can be cured by standard frontline chemo-immune therapy. The prognosis of relapsed or refractory patients is dismal, as these patients are difficult to salvage, even when using high-dose chemotherapy, such as BEAM, followed by autologous stem cell support. Through the advent of next generation sequencing technology, the availability of numerous mouse models that faithfully recapitulate important aspects of human DLBCL biology, as well as the possibility to perform large scale genome-wide in vivo insertion mutagenesis screens, we are now in a position to comprehensively profile the genomic events that predict poor response to chemo-immune therapy. More importantly, sequencing of longitudinally obtained tissue samples from mice and men prior to and following the development of (pre-)clinical resistance, allows us to precisely pinpoint genes and pathways that likely mediate resistance. Moreover, a careful inspection of murine and human DLBCL genomes and transcriptomes after the occurrence of resistance may enable us to identify genomic and transcriptomic aberrations that may offer entry routes for targeted therapeutic intervention strategies. Building on these considerations, we have formulated four specific Aims that directly address the above-mentioned questions. Our work program leverages on the large portfolio of tools and technologies that we have assembled in Cologne and Göttingen over the past years. Our specific Aims are as follows:Aim 1: Employ WES to identify recurrent genomic aberrations that are enriched following R-CHOP chemo-immune therapy in patientsAim 2: Employ WES to identify recurrent genomic aberrations that are enriched following R-CHOP chemo-immune therapy in DLBCL GEMMs Aim 3: Perform an in vivo PiggyBac insertional mutagenesis screen to identify mediators of resistance against R-CHOP-like chemo-immune therapyAim 4: Validate candidate resistance-mediating genomic aberrations in vivo, using CRISPR/Cas9 genome editing.