From pathogenesis to new therapeutic targets in diffuse large B cell lymphoma

项目摘要 弥漫性大B细胞淋巴瘤（DLBCL）在约30%的患者中是不可治愈的，尽管最近在CAR-1方面取得了进展， T细胞疗法仍然是一个重大的临床挑战。合理靶向新疗法的一个障碍是 这些肿瘤具有显著的异质性，这使得多达20-50%的病例无法根据 细胞起源或更近的基于遗传的分类。这可能部分是由于目前 分类学仅限于对编码区的分析，仅占基因组的3%，而 致病相关的遗传复杂性可能存在于基因组的非编码调节部分。到 为此，我们最近研究了关键的调控域，如增强子和超级增强子， (SEs)可能是DLBCL中功能相关突变的位点。我们发现， 在97%的DLBCL病例中，与相同的基因座相比， 不像SE那样活跃。这种异常的SE超突变显示出AID活性的特征，并与基因 编码B细胞调节因子和公认的癌基因。作为致癌相关性的证据，我们发现 与BCL 6、BCL 2和CXCR 4原癌基因连接的高度突变的SE阻止了结合， 转录抑制因子（包括BLIMP 1）对相应靶基因的转录下调 （BCL 6）和类固醇受体NR 3C 1（BCL 2和CXCR 4）。值得注意的是，CRISPR/Cas9介导的对细胞凋亡的校正， SE热点突变恢复了靶基因的调控，并损害了细胞生长，表明SE的关键作用， 在维持转化表型中的突变（Bal等，Nature 2022）。总的来说，这些发现确定了一个 高度普遍的，致病相关的，突变机制，可能会显着影响 目前对DLBCL体细胞遗传景观的理解。这项研究计划的总体目标将 i）鉴定并机械地解剖顶部复发突变/功能相关SE和相关的 靶基因; ii）了解糖皮质激素受体途径的作用，这似乎是常见的 在正常B细胞生物学中被SE超突变机制以及直接编码突变靶向， 淋巴瘤形成我们预计，这一新的遗传改变层将确定新的机制， 已知癌基因的失调，以及新的失调基因和途径， DLBCL的精确分类和治疗靶向。

PROJECT SUMMARY Diffuse Large B cell Lymphoma (DLBCL) is incurable in ~30% of patients and, despite recent advances in CAR- T cell therapies, remains a significant clinical challenge. One barrier to rationally targeted new therapies is the remarkable heterogeneity of these tumors, which leaves as many as 20-50% of cases unclassified based on cell-of-origin or more recent genetic-based classifications. This may be due in part to the fact that current taxonomies are limited to the analysis of coding regions, representing only 3% of the genome, while further genetic complexity of pathogenetic relevance may reside in the non-coding regulatory portion of the genome. To this end, we recently investigated whether critical regulatory domains such as enhancers and super-enhancers (SEs) could be the site of functionally relevant mutations in DLBCL. We found that regions corresponding to active SEs are highly and specifically hypermutated in 97% of DLBCL cases, as compared to the same loci when not active as SE. Such aberrant SE hypermutation displays signatures of AID activity and is linked to genes encoding B cell regulators and well-established oncogenes. As evidence of oncogenic relevance, we showed that the hypermutated SEs linked to the BCL6, BCL2, and CXCR4 proto-oncogenes prevent the binding and transcriptional downregulation of the corresponding target gene by transcriptional repressors, including BLIMP1 (BCL6) and the steroid-receptor NR3C1 (BCL2 and CXCR4). Of note, CRISPR/Cas9-mediated correction of the SE hotspot mutation restored target gene regulation and impaired cell growth, indicating a key role for the SE mutation in maintaining the transformed phenotype (Bal et al., Nature 2022). Overall, these findings identify a highly pervasive, pathogenetically relevant, mutational mechanism that is likely to significantly influence the current understanding of the somatic genetic landscape of DLBCL. The overall goal of this research program will be to: i) identify and mechanistically dissect the top recurrently mutated/functionally relevant SEs and associated target genes; ii) understand the role of the glucocorticoid receptor pathway, which appears to be commonly targeted by the SE hypermutation mechanism as well as by direct coding mutations, in normal B cell biology and lymphomagenesis. We anticipate that this new layer of genetic alterations will identify novel mechanisms of dysregulation for known oncogenes, as well as new dysregulated genes and pathways, with implications for precision classification and therapeutic targeting of DLBCL.