BET protein antagonist-based targeted therapy of Mantle Cell Lymphoma

项目摘要： 套细胞淋巴瘤（MCL）细胞表现出涉及细胞周期G1/S检查点的遗传改变， 调节基因细胞周期蛋白D1、CDKN 2A、CDK 4，以及细胞生长和存活基因MYC和BCL 2。 MCL细胞还显示出增加的B细胞受体信号传导和NF κ B的转录活性，使它们成为 对布鲁顿酪氨酸激酶（BTK）抑制剂伊曲替尼的致死活性敏感。而治疗与 伊曲替尼诱导临床反应，并改善生存率、原发性难治性或最终出现的 对伊曲替尼的耐药是常见的，阻止了MCL的持久缓解。最近， CARD 11/IKBKB/TRAF 2/BIRC 3/NIK，其导致经典或替代NF κ B信号传导的激活， 已经记录了对伊鲁替尼的耐药性。我们的初步研究表明， 用原型BET（布罗莫结构域和额外末端）蛋白（BETP）拮抗剂（BETi）治疗可抑制 MCL相关癌基因（包括MYC、BCL-2和CDK 4/6）的mRNA和蛋白水平。Beti 处理降低了核NF κ B水平，消除了BRD 4依赖的NF κ B活性，并抑制了其表达。 促生长和促存活靶基因，包括BTK，导致人MCL细胞凋亡。值得注意的是， 单独用BETi处理也诱导伊曲替尼抗性MCL细胞的凋亡。然而，尽管 有前途的抗肿瘤活性，用BETi治疗导致BRD 4蛋白积累，这限制了BETi- 介导的对包括NF κ B在内的MCL相关癌蛋白的抑制，从而降低 Beti治疗。然而，我们最近已经确定，用异双功能BETP- PROTAC（蛋白水解靶向嵌合体），例如，ARV-825或ARV-771，其将BETP募集至E3泛素 连接酶cereblon或VHL分别引起BRD 4的有效和延长的降解，导致抑制 包括NF κ B在内的MCL相关癌蛋白。基于此，我们假设用BETP治疗- PROTAC将比BETi诱导显著更多的细胞凋亡，以及发挥协同致死和抗凋亡作用。 伊鲁替尼或抗BCL 2或CDK 4/6激酶抑制剂对培养的和患者来源的肿瘤的疗效 (PD)MCL细胞在AIM 1中，我们将确定体外致死活性，并阐明其预测 由于BETP-PROTAC（ARV-1）的基因突变和基因表达干扰的最小特征， 825和ARV-771）与临床相关的BETis（OTX 015和ABBV-075），单独和与 在培养的细胞系以及患者来源的（PD）、遗传学特征的原代MCL细胞中使用伊曲替尼。在AIM 2中， 我们将确定联合的体外致死活性及其预测的最小基因表达扰动， 由于BET-PROTAC与BETi与抗BCL 2或抗CDK 4/6的组合治疗， 针对培养的和遗传特征的PD、伊匹替尼敏感（IS）或伊匹替尼持续/耐药的抑制剂 (IR)MCL细胞在AIM 3中，我们将确定与ARV-771或ABBV-075共同治疗的体内疗效， venetoclax或palbociclib，利用IS或IR MCL细胞的培养细胞系和PD异种移植（PDX）模型。

Project Summary: Mantle Cell Lymphoma (MCL) cells exhibit genetic alterations involving the cell cycle G1/S checkpoint- regulatory genes cyclin D1, CDKN2A, CDK4, as well as the cell growth and survival genes MYC and BCL2. MCL cells also display increased B cell receptor signaling and transcriptional activity of NFkB, making them sensitive to the lethal activity of the Bruton’s tyrosine kinase (BTK) inhibitor ibrutinib. Whereas treatment with ibrutinib induces clinical responses and improves survival, primary refractoriness or eventual emergence of resistance to ibrutinib is common, preventing durable remissions in MCL. Recently, mutations in CARD11/IKBKB/TRAF2/BIRC3/NIK, which lead to the activation of the classical or alternative NFkB signaling, have been documented, to confer resistance to ibrutinib. Our preliminary studies have demonstrated that treatment with the prototype BET (bromodomain and extra terminal) protein (BETP) antagonist (BETi) inhibits the mRNA and protein levels of the MCL-relevant oncogenes, including MYC, BCL-2 and CDK4/6. BETi treatment reduced the nuclear levels of NFkB, abrogated BRD4-dependent NFkB activity, and repressed its pro-growth and pro-survival target genes, including BTK, leading to apoptosis of human MCL cells. Notably, treatment with BETi alone also induced apoptosis of ibrutinib-resistant MCL cells. However, despite their promising anti-tumor activity, treatment with BETi leads to BRD4 protein accumulation, which limits BETi- mediated inhibition of the MCL-relevant oncoproteins including NFkB, thereby reducing the antitumor activity of BETi treatment. However, we have recently determined that treatment with the hetero-bifunctional BETP- PROTAC (Proteolysis Targeting Chimera), e.g., ARV-825 or ARV-771, which recruit BETPs to the E3 ubiquitin ligase cereblon or VHL, respectively, causes efficient, and prolonged degradation of BRD4, leading to inhibition of the MCL-relevant oncoproteins including NFkB. Based on this, we hypothesize that treatment with BETP- PROTAC will induce significantly more apoptosis than BETi, as well as exert synergistic lethality and anti- tumor efficacy with ibrutinib or with anti-BCL2 or CDK4/6 kinase inhibitor against cultured and patient-derived (PD) primary MCL cells. In AIM 1, we will determine the in vitro lethal activity and elucidate its predictive minimal signature of genetic-mutations and gene-expression perturbations due to the BETP-PROTACs (ARV- 825 and ARV-771) versus clinically-relevant BETis (OTX015 and ABBV-075), alone and in combination with ibrutinib in cultured cell lines as well as patient-derived (PD), genetically-profiled, primary MCL cells. In AIM 2, we will determine the combined in vitro lethal activity, and its predictive minimal gene-expression perturbations signature, due to treatment with the combinations of BET-PROTAC versus BETi with anti-BCL2 or anti-CDK4/6 inhibitor against the cultured and genetically-profiled, PD, ibrutinib-sensitive (IS) or ibrutinib-persister/resistant (IR) MCL cells. In AIM 3, we will determine the in vivo efficacy of co-treatment with ARV-771 or ABBV-075 and venetoclax or palbociclib, utilizing the cultured cell line and PD xenograft (PDX) models of IS or IR MCL cells.