Using mouse models to identify actionable molecular liabilities in Cluster 5 DLBCL

DLBCL仍然是一个临床挑战，因为复发和难治性疾病非常难以治疗。然而，最近的基因组学研究已经揭示了DLBCL中复发性畸变的情况。虽然这一领域的研究仍在不断变化，但ABC-DLBCL，特别是C5 DLBCL淋巴瘤形成的一个相当清晰的图景正在出现：C5 DLBCL的一个标志性特征是NFB的不适当激活，这是通过BCR （CD79B）和/或TLR （MYD88）途径中的复发突变实现的。C5 DLBCL的另一个标志是BCL2拷贝数的增加。最后，C5 DLBCL的特征是PRDM1复发性失活畸变，导致浆细胞分化受阻。这些复发性突变可能为靶向治疗干预提供机会，这可能使这种疾病的无化疗治疗方法成为可能。然而，目前缺乏合适的体内实验平台来忠实地模拟人类C5 DLBCL的基因组景观。在这里，我们开始扩展我们在C5 DLBCL建模方面的现有努力，以开发一个全面和通用的体内临床前平台，该平台采用大规模体外脆弱性筛查和体内验证工具箱的组合，以开发创新的，无化疗和基因组学指导的治疗方法，用于治疗C5 DLBCL患者。我们的提案建立在大量初步数据的基础上，我们已经成功建立了一系列创新技术来全面捕捉C5 DLBCL的生物学特性，包括纵向体内成像、高通量免疫组织化学、CRISPR/Cas9 drop - out筛选、CyTOF大规模细胞计数、3'-RNA测序、全外显子组和全基因组测序、单细胞RNA测序和细胞因子分析。一旦确定了可行的靶向治疗方法，我们的系统可以进一步用于加速寻找耐药介导基因和途径。基于这些考虑，我们制定了三个具体目标：目标1：产生和表征具有C5 dlb显性畸变的可诱导的本地小鼠模型；目标2：系统的CRISPR/Cas9功能缺失筛选以识别C5 dlb基因型特异性脆弱性；评估BCL2-、BTK-、CD20-和PD-1/PD-L1联合靶向治疗体内原生C5 DLBCL模型的疗效。这些目的是验证以下假设：1)致癌基因Myd88突变与BCL2、Prdm1和Cd79b突变协同促进C5 DLBCL； 2) C5 DLBCL小鼠模型及其衍生的细胞系模型可以作为实验平台，开发和完善C5 DLBCL的新型无化疗治疗算法。3) myd88驱动的C5 DLBCL可能显示PD-L1表面表达。提出的实验将为基因组学指导的C5 DLBCL精准医学方法的临床发展铺平道路。

DLBCL remains a clinical challenge, as relapsed and refractory disease is very difficult to treat. However, recent genomics efforts have revealed the landscape of recurrent aberrations in DLBCL. While this area of investigation is still in flux, a rather clear picture of ABC-DLBCL, and particularly C5 DLBCL lymphomagenesis, is emerging: A hallmark feature of C5 DLBCL is inappropriate NFB activation, which is achieved through recurrent mutations within the BCR (CD79B) and/or TLR (MYD88) pathways. A further hallmark of C5 DLBCL are BCL2 copy number gains. Lastly, C5 DLBCL is characterized by recurrent inactivating aberrations in PRDM1, which lead to a block in plasma cell differentiation. These recurrent mutations may offer opportunities for targeted therapeutic interventions, which may allow chemotherapy-free treatment approaches for this disease. However, there is currently a lack of suitable in vivo experimental platforms that faithfully mimic the genomic landscape of human C5 DLBCL. Here, we set out to expand on our existing efforts on C5 DLBCL modeling to develop a comprehensive and versatile in vivo preclinical platform that employs a combination of large-scale in vitro vulnerability screening, and an in vivo validation tool box to develop innovative, chemotherapy-free and genomics-guided therapeutic approaches for the treatment of C5 DLBCL patients. Our proposal builds on substantial preliminary data and we have successfully established a whole range of innovative technologies to fully capture the biology of C5 DLBCL, including longitudinal in vivo imaging, high throughput immuno-histochemistry, CRISPR/Cas9 drop out screening, CyTOF mass cytometry, 3'-RNA sequencing, whole exome and whole genome sequencing, single cell RNA sequencing and cytokine profiling. Our systems can further be exploited to accelerate the search for resistance-mediating genes and pathways, once viable targeted treatment approaches have been identified. Based on these considerations, we have formulated three specific aims:Aim 1: Generation and characterization of an inducible autochthonous mouse model harboring the dominant aberrations defining C5 DLBCLAim 2: Systematic CRISPR/Cas9 loss of function screening to identify genotype-specific vulnerabilities in C5 DLBCLAim 3: Assess the efficacy of combined BCL2-, BTK-, CD20- and PD-1/PD-L1 targeting in autochthonous C5 DLBCL models in vivoThese aims test the hypotheses that 1) oncogenic Myd88 mutations cooperate with Bcl2, Prdm1 and Cd79b aberrations to promote C5 DLBCL, 2) autochthonous mouse models of C5 DLBCL and cell line models derived thereof may serve as experimental platforms to develop and refine novel chemotherapy-free treatment algorithms of C5 DLBCL, and 3) MYD88-driven C5 DLBCL may display an PD-L1 surface expression. The proposed experiments will pave the way for the clinical development of genomics-guided precision medicine approaches to C5 DLBCL.