BCR信号通路和c-Myc在肿瘤微环境介导的弥漫大B细胞淋巴瘤化疗耐药中的协同作用研究

Diffused large B-cell lymphoma(DLBCL) is the most common subtype of non-Hodgkin lymphoma. Despite a high response rate to initial therapy, approximately 30% of patients will ultimately died due to the emergence of drug resistance and lymphoma progression. Recently, the BCR is increasingly emerging as a pivotal pathway and is likely to be a key driver of many B-cell lymphomas. Two small molecular compounds PCI-32765 and CAL-101, which inhibiting BTK and PI3K not only blocked BCR signal pathways but also disrupt interaction between lymphoma cells and tumor microenvironment, has recently showm impressive clinical efficacy in B-cell lymphoma. c-Myc has been described as the driving oncogene for many tumors and it’s over expression has also been recognized in part of DLBCL cases. More recently, JQ1, a small molecule have been investigated to silence c-Myc transcription. We propose that functional identification of BCR and c-Myc activation mechanisms with the targeting reagents (PCI-32765, CAL-101, and JQ1) in hand will allow us to gain insights into DLBCL survival, progression and provide novel biological targets. In support our proposal, we carried out many previously study and demonstrated that (1) Lymphoma stromal cells enhance DLBCL cell clonogenicity and lymphoma progression ex vivo and in vivo. (2) Lymphoma stroma promotes activation of BCR pathway in DLBCL cells. (3) PCI-32765 and CAL-101 target BCR signaling. (4) c-Myc-miRNAs axis is constitutively activated by lymphoma stroma and JQ1 can overcome stroma-mediated drug resistance. Taken together, our studies provide the rationale to pursue an in-depth evaluation of the cooperative function of BCR-BTK-PI3K and c-Myc in the pathogenesis of DLBCL. The following specific aims are therefore propsed: Aim 1: To elucidate the contribution of BCR-BTK-PI3K to DLBCL survival, stroma-mediated drug resistance and clonogenicity. Aim2: To determine the role of c-Myc to DLBCL survival and stroma-mediated drug resistance and clonogenicity. Aim3: To functionally define the cooperative function of BCR-BTK-PI3K and c-Myc. Aim4: To combinationally target c-Myc and BCR-BTK-PI3K to synegeristically release DLBCL cells from their microenvironment, hider DLBCL progression in vitro and in preclinical DLBCL-derived xenograft models. The proposal builds on the following existing strengths : (a) enough DLBCL cases in our hospital, (b) an established co-culture model, cell lines and a strong team background for lymphoma study, (c) an already established and ongoing collaboration and publications with Dr.Sotomayor's Laboratory, Moffitt Cancer Center, USA and finally (d) due to the previously study, we have done many work and published related papers. Through capitalization of these advantages, we ultimately will enhance our understanding about molecular determinants in DLBCL and provide novel therapeutic stratigies.

弥漫大B细胞淋巴瘤（DLBCL）是严重危害人类健康的重要疾病，约30%的患者死于耐药和疾病进展。新型B细胞受体（BCR）的靶向抑制剂在淋巴瘤的临床试验中取得了疗效，但其确切机制尚未得到充分研究。前期工作中我们发现BCR的靶向抑制剂不仅可抑制内源性的DLBCL细胞生存信号，还可阻断肿瘤细胞和微环境之间的相互作用，克服基质细胞介导的化疗耐药；同时我们发现DLBCL细胞与基质细胞共培养后可上调重要癌基因c-Myc的表达，采用国外新研发的c-Myc抑制剂JQ1可诱导DLBCL细胞凋亡。基于此，我们将深入探索BCR通路和c-Myc活化在DLBCL发病中作用的详尽机制及两者是否在基质介导的细胞生长和耐药中发挥协同作用，联合靶向抑制BCR和c-Myc是否可在体内外产生更强大的抗淋巴瘤效应。鉴于业已开展的前期工作和国际合作等背景，我们将提出DLBCL发病的新理论，提出创新性的治疗策略。

弥漫大B细胞淋巴瘤（diffused large B-cell lymphoma, DLBCL）是最常见的非霍奇金淋巴瘤，由于其高度侵袭性，预后较差，因此亟需发展新的治疗靶点。我们前期研究发现吲哚胺2，3-双加氧酶1（IDO1）在DLBCL中高表达。应用IDO1抑制剂1-L-MT可有效抑制DLBCL细胞增殖、促进凋亡从而发挥抗肿瘤作用，但其机制不明。为探索IDO1促进DLBCL发生发展的机制，我们进行了RNA-Seq，发现敲降IDO1后DLBCL细胞的MDM2 mRNA表达水平显著降低，而TP53 mRNA表达水平显著升高。我们推测IDO1通过调控MDM2-P53通路促进DLBCL发生发展。我们首先通过分析GEO和TCGA数据库发现IDO1在DLBCL中显著高表达，进而通过qPCR，WB和免疫组化等证实IDO1在DLBCL中显著高表达，且与DLBCL患者预后差正相关。功能实验证实敲降IDO1可发挥抑制肿瘤细胞增殖、促进凋亡、阻滞细胞周期的抗肿瘤的作用。转录组测序发现，敲降IDO1后MDM2 基因表达水平显著降低，而TP53基因表达水平显著升高，qPCR证实了测序结果。进一步我们通过生物信息学及qPCR，Western blot等证实了MDM2在DLBCL中显著高表达。接下来的功能试验证实敲降MDM2可显著抑制细胞增殖，促进凋亡并导致细胞周期阻滞。在机制研究中，我们发现敲降IDO1表达可显著降低MDM2蛋白表达水平，同时上调p53蛋白表达水平；而抑制MDM2表达后p53蛋白表达水平显著升高，IDO1蛋白表达水平略有升高，这提示IDO1通过激活MDM2从而抑制p53蛋白表达而发挥促进DLBCL发生发展的作用。进一步，我们发现MDM2/AKT/IDO1负反馈通路的存在，为联合抑制IDO1和MDM2提供了理论依据，增殖实验表明IDO1和MDM2具有协同抗DLBCL的作用。最后，NOD/SCID动物实验显示，IDO1抑制剂和MDM2抑制剂均有抑制肿瘤增殖的作用，而且联合抑制IDO1和MDM2具有更显著的抑制DLBCL成瘤的作用。IDO1和MDM2可作为潜在的治疗DLBCL的靶点，从而为DLBCL患者提供新的治疗策略。本项目的研究将有助于理解IDO1介导免疫逃逸作用之外的抗肿瘤作用和机制，使其有望成为治疗DLBCL的新靶点，为DLBCL单用或联合药物治疗提供理论依据。