Targeting EGR1 signaling pathways in diffuse large B cell lymphoma

摘要 弥漫性大B细胞淋巴瘤(DLBCL)占新诊断淋巴瘤的30%至40%， 包括两个主要的分子亚型：激活的B细胞样(ABC)和生发中心的B细胞样(GCB)。ABC DLBCL更具侵袭性，可治愈性更差。超过50%的ABC DLBCL患者对或 因目前的一线免疫化疗而复发。布鲁顿选择性抑制剂伊布鲁替尼的临床应用 酪氨酸激酶(BTK)在B细胞受体(BCR)信号通路中，已获得了 难治/复发ABC DLBCL中30%~40%。然而，原发和获得性耐药仍然很严重。 并影响其中60%以上患者的长期生存。因此，理解和定位 伊布鲁替尼的耐药机制是临床上尚未满足的需求。BCR和JAK1/STAT3信号通路是 对ABC DLBCL细胞的生存和增殖至关重要。我们发现Egr1是一个收敛的 ABC DLBCL中两条通路的下游靶点和Egr1的表达水平在ABC DLBCL中升高 与正常人的扁桃体和淋巴结相比。我们揭示了Egr1的新机制 翻译影响较强的靶基因在治疗侵袭性中的转录激活和抑制 淋巴瘤。EGR1通过p300/H3K27ac/BRD4轴介导转录激活诱导MYC 表达并激活MYC靶基因。观察到Egr1对细胞生长的协同抑制作用 ShRNA和AZD5153，一种目前正在进行临床研究的新型BRD4抑制剂。另一方面， Egr1介导I型干扰素途径基因的转录抑制，否则其表达 会导致癌细胞死亡。始终如一地，shRNA敲除egr1与I型干扰素诱导剂协同作用。 来那度胺在体外和异种小鼠模型中对ABC DLBCL细胞生长的抑制作用。使用新的 衍生的对伊布鲁替尼耐药的ABC DLBCL细胞株，我们证明了Egr1是最高水平的 与伊布鲁替尼敏感亲本细胞相关的基因表达及BRD4和干扰素信号的共靶向 在体外和体内抑制对伊布鲁替尼耐药细胞的生长。基于这些发现，中心假说 Egr1是一种独特的致癌驱动因子，它协调多个重要的信号通路，并代表 ABC DLBCL患者的治疗脆弱性，特别是对伊布鲁替尼耐药的患者。测试我们的 假设，我们将追求以下具体目标：(1)阐明Egr1在肿瘤发生中的作用；(2) 确定Egr1在伊布鲁替尼耐药中的作用；以及(3)共同靶向BRD4下游的Egr1和I型 干扰素信号对克服DLBCL耐药的作用。解剖转录激活和 Egr1的抑制模块在DLBCL肿瘤发生和伊布鲁替尼耐药中是必不可少的，因为新的 机械论的洞察将为开发最有效的治疗策略提供分子基础 治疗DLBCL患者，包括对伊布鲁替尼耐药的患者。

ABSTRACT Diffuse large B cell lymphoma (DLBCL), which represents 30% to 40% of newly diagnosed lymphomas, comprises two main molecular subtypes: activated B cell-like (ABC) and germinal center B cell-like (GCB). ABC DLBCL is more aggressive and less curable. More than 50% of patients with ABC DLBCL are refractory to or relapse from current frontline immunochemotherapy. Clinical use of ibrutinib, a selective inhibitor for Bruton tyrosine kinase (BTK) in the B cell receptor (BCR) signaling pathway, has achieved an initial response rate of 30%-40% in refractory/relapsed ABC DLBCL. Primary and acquired drug resistance, however, are still significant and impact the long-term survival of more than 60% of these patients. Therefore, understanding and targeting ibrutinib resistance mechanisms is an unmet clinical need. The BCR and JAK1/STAT3 signaling pathways are essential for the survival and proliferation of ABC DLBCL cells. We discovered that EGR1 is a converged downstream target of both pathways in ABC DLBCL and the level of EGR1 expression is elevated in ABC DLBCL compared with normal human tonsils and lymph nodes. We revealed novel mechanisms of EGR1 in transcriptional activation and repression of target genes with strong translational impact in treating aggressive lymphoma. EGR1 mediates transcriptional activation through the p300/H3K27ac/BRD4 axis to induce MYC expression and activate MYC target genes. Synergistic inhibition of cell growth was observed between EGR1 shRNA and AZD5153, a novel BRD4 inhibitor that is currently under clinical investigation. On the other hand, EGR1 mediates transcriptional repression of the type I interferon pathway genes, expression of which otherwise causes cancer cell death. Consistently, EGR1 knockdown by shRNA synergizes with the type I interferon inducer lenalidomide in growth inhibition of ABC DLBCL cells in vitro and in a xenograft mouse model. Using newly derived, ibrutinib-resistant ABC DLBCL cell lines, we demonstrated that EGR1 is among the most highly expressed genes relative to ibrutinib-sensitive parental cells, and co-targeting of BRD4 and interferon signaling inhibits growth of ibrutinib-resistant cells in vitro and in vivo. Based on these discoveries, the central hypothesis is that EGR1 is a unique oncogenic driver orchestrating multiple important signaling pathways and represents therapeutic vulnerability in patients with ABC DLBCL, especially for those with ibrutinib resistance. To test our hypothesis, we will pursue the following specific aims: (1) Elucidate effects of EGR1 on oncogenesis; (2) Establish the role of EGR1 in ibrutinib resistance; and (3) Co-target EGR1 downstream BRD4 and type I interferon signaling to overcome drug resistance in DLBCL. Dissecting the transcriptional activation and repression modules of EGR1 in DLBCL tumorigenesis and ibrutinib resistance is essential because the novel mechanistic insights will provide a molecular basis for developing the most effective therapeutic strategy for treatment of DLBCL patients, including those with ibrutinib resistance.