Characterization of Resistance to PRMT5 Inhibitor Therapy in Mantle Cell Lymphoma

项目总结： 套细胞淋巴瘤是一种不可治愈的B细胞性非霍奇金淋巴瘤 中位存活时间为8- 如果积极治疗，将被判12年。 绝大多数MCL患者最终复发和难治性 。多重 MCL的突变特征包括那些导致细胞周期失调和失活的突变 不同的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，已经在MCL中被发现。 疾病或复发使用靶向治疗的患者预后特别差，生存时间短。 上调蛋白精氨酸 甲基转移酶5(PRMT5)，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素有关，包括 Cyclin D1和MYC。出于这个原因，PRMT5已经成为一个有吸引力的抑制治疗的靶点 淋巴瘤细胞的存活和增殖。我们的团队与Prelude Treeutics合作开发了一种 小分子PRMT5抑制剂(PRT-382)在MCL细胞系(低NM)中表现出显著的抗肿瘤活性 范围)和初级MCL模型(10 mg/kg)。虽然抑制PRMT5的抗肿瘤活性令人鼓舞， 我们观察到一些经过治疗的动物产生了耐药性，导致了MCL的快速进展。多重 MCL细胞系(Maver-1、Mino、UPN1和Jeko)也表现出对PRMT5抑制的初步抗性，其基础是 对PRT-382的半数抑制浓度(IC50)。此外，MCL系的长时间培养 药物升级在细胞系中产生获得性耐药性，即使在长期培养后仍存在 没有毒品。鉴于PRMT5抑制在难治性MCL中的巨大治疗潜力 总体而言，这项建议试图评估PRMT5抑制剂耐药的产生机制 表型。为此，我们将利用下一代测序技术来表征表观遗传学和 导致这些PRMT5抑制剂耐药表型的基因改变。一起CRISPR功能屏丢失事故 将用于突出MCL中对PRMT5抑制剂治疗具有耐药性和敏感性的候选基因 在生物验证研究中提供线条和帮助。PRMT5抑制剂敏感性增加的几个潜在标记物 治疗已经并继续被调查，包括MTAP缺失，P53突变状态，对 其他异常的PRMT，以及剪接体基因的突变。我们假设识别出 PRMT5抑制剂抗性扩增的代偿性生存通路将导致 制定合理的策略来规避阻力。选择性地针对其他关键途径的代理 MCL联合PRMT5抑制剂治疗将在体内使用患者来源的异种移植和 双转基因免疫活性小鼠模型。这将使实验室结果的临床相关性成为可能， 这是特别相关的，因为这种药物的第一阶段临床试验正在进行中，患者 侵袭性淋巴瘤(前奏，NCT02783300)。

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).