Characterization of Resistance to PRMT5 Inhibitor Therapy in Mantle Cell Lymphoma

项目概要： 套细胞淋巴瘤（MCL）是一种不可治愈的B细胞非霍奇金淋巴瘤 中位生存时间为8- 如果积极治疗的话12年。 绝大多数MCL患者最终会复发，而难治性MCL患者最终会复发。 .多 MCL中的突变特征包括导致细胞周期失调和失活的突变 各种DNA损伤反应蛋白。除了基因突变，表观遗传失调， 包括异常的组蛋白和DNA甲基化。 疾病或在靶向治疗后复发的患者预后特别差，生存期短 上调蛋白质精氨酸 甲基转移酶5（PRMT 5），一种导致组蛋白精氨酸残基对称二甲基化的酶， 与沉默肿瘤抑制基因和支持多种致癌驱动因子有关， CYP 1D 1和MYC。由于这个原因，PRMT 5已经成为抑制肿瘤生长的有吸引力的治疗靶标。 淋巴瘤细胞存活和增殖。我们的团队与Prelude Therapeutics合作开发了一种 在MCL细胞系中表现出显著抗肿瘤活性的小分子PRMT 5抑制剂（PRT-382）（低nM 范围）和初级MCL模型（10 mg/kg）。虽然PRMT 5抑制的抗肿瘤活性是令人鼓舞的， 我们已经观察到一些治疗的动物产生耐药性，导致MCL的快速进展。多 MCL细胞系（Maver-1、Mino、UPN 1和Jeko）也显示出对PRMT 5抑制的原发性抗性，这是基于 PRT-382的半数最大抑制浓度（IC 50）。此外，MCL系的延长培养与 药物升级已经在细胞系中产生了获得性耐药性，即使在长期培养后， 没有毒品。鉴于PRMT 5抑制在难治性MCL背景下的巨大治疗潜力， 总体而言，该建议旨在评估PRMT 5抑制剂耐药的潜在机制。 表型为此，我们将利用下一代测序技术来表征表观遗传和 基因改变导致这些PRMT 5抑制剂抗性表型。CRISPR功能丧失屏幕 将用于突出在MCL中赋予对PRMT 5抑制剂治疗的抗性和敏感性的候选基因 线和生物验证研究的援助。对PRMT 5抑制剂敏感性增加的几种潜在标志物 治疗已经并将继续进行研究，包括MTAP缺失，p53突变状态，对 其他失调的PRMT和剪接体基因突变。我们假设， PRMT 5抑制剂抗性放大的代偿性促存活途径将导致 制定合理的策略来规避阻力。选择性靶向其他关键途径的药物， MCL与PRMT 5抑制剂疗法的组合将在体内使用患者来源的异种移植物进行评估， 双转基因免疫活性鼠模型。这将使实验室结果的临床相关性， 考虑到这类药物的1期临床试验正在进行中， 侵袭性淋巴瘤（Prelude，NCT 02783300）。

PROJECT SUMMARY: Mantle cell lymphoma (MCL) is an incurable B-cell Non-Hodgkin’s lymphoma with a median survival time of 8- 12 years if treated aggressively. The vast majority of MCL patients eventually relapse and those with refractory . Multiple mutations have been characterized in MCL including those resulting in cell cycle dysregulation and inactivation of various DNA damage response proteins. In addition to genetic mutations, epigenetic dysregulation, including aberrant histone and DNA methylation, has been identified in MCL. disease or who relapse on targeted therapies have a particularly poor prognosis with short survival Upregulated protein arginine methyltransferase 5 (PRMT5), an enzyme that results in symmetric dimethylation of histone arginine residues, is associated with silencing tumor suppressor genes and supporting multiple oncogenic drivers including CYCLIND1 and MYC. For this reason, PRMT5 has emerged as an attractive therapeutic target to inhibit lymphoma cell survival and proliferation. Our group in collaboration with Prelude Therapeutics has developed a small molecule PRMT5 inhibitor (PRT-382) that exhibits significant anti-tumor activity in MCL cell lines (low nM range) and primary MCL models (10 mg/kg). While the anti-tumor activity of PRMT5 inhibition is encouraging, we have observed some treated animals to develop drug resistance leading to rapid MCL progression. Multiple MCL cell lines (Maver-1, Mino, UPN1, and Jeko) also show primary resistance to PRMT5 inhibition based on their half maximal inhibitory concentrations (IC50s) for PRT-382. In addition, prolonged culture of MCL lines with drug escalation has produced acquired drug resistance in cell lines that persists even after prolonged culture in the absence of drug. Given the great therapeutic potential of PRMT5 inhibition in the setting of refractory MCL overall, this proposal seeks to evaluate the mechanisms underlying emergence of PRMT5 inhibitor resistant phenotypes. To do this, we will utilize next generation sequencing technologies to characterize the epigenetic and genetic alterations that contribute to these PRMT5 inhibitor resistant phenotypes. A CRISPR loss of function screen will be used to highlight candidate genes that confer resistance and sensitivity to PRMT5 inhibitor therapy in MCL lines and aid in biologic validation studies. Several potential markers of increased sensitivity to PRMT5 inhibitor therapy have and continue to be investigated including MTAP deletion, p53 mutation status, dependency on other dysregulated PRMTs, and mutations in spliceosome genes. We hypothesize that identification of compensatory prosurvival pathways that are amplified with PRMT5 inhibitor resistance will lead to the development of rational strategies to circumvent resistance. Agents that selectively target other key pathways in MCL in combination with PRMT5 inhibitor therapy will be evaluated in vivo using patient derived xenograft and double transgenic immunocompetent murine models. This will enable clinical correlation of laboratory findings, which is particularly relevant given that a phase 1 clinical trial with this class of drug is underway in patients with aggressive lymphomas (Prelude, NCT02783300).