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，是一个重大的临床挑战，因为只有约60%的患者可以通过标准的一线化学免疫治疗治愈。复发性或难治性患者的预后是令人沮丧的，因为这些患者难以挽救，即使使用高剂量化疗，如BEAM，随后是自体干细胞支持。通过下一代测序技术的出现，大量小鼠模型的可用性，忠实地概括了人类DLBCL生物学的重要方面，以及进行大规模全基因组体内插入诱变筛选的可能性，我们现在能够全面分析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（前）临床耐药性发展之前和之后，对从小鼠和人类纵向获得的组织样本进行测序，使我们能够精确地确定可能介导耐药性的基因和途径。此外，在耐药发生后仔细检查鼠和人DLBCL基因组和转录组，可以使我们能够鉴定基因组和转录组畸变，这些畸变可以为靶向治疗干预策略提供进入途径。在这些考虑的基础上，我们制定了四项直接针对上述问题的具体目标。我们的工作计划充分利用了我们过去几年在科隆和哥廷根组装的大量工具和技术。我们的具体目标如下：目标1：采用WES鉴定患者中R-CHOP化学免疫治疗后富集的复发性基因组畸变目标2：采用WES鉴定DLBCL GEMM中R-CHOP化学免疫治疗后富集的复发性基因组畸变目标3：进行体内PiggyBac插入诱变筛选以鉴定对R-CHOP样化学免疫治疗的抗性介体目标4：使用CRISPR/Cas9基因组编辑在体内检测候选耐药介导的基因组畸变。

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.