Translational Studies of the Histone Deacetylase Inhibitor Romidepsin

我们在临床和实验室研究了组蛋白去乙酰化酶抑制剂罗米地辛和贝利诺他。我们最初对罗米地辛感兴趣是在一期临床试验的背景下，当时我们偶然发现（当时命名的）抑郁肽对t细胞淋巴瘤亚群非常有效。虽然我们继续对预防该药物出现耐药性的原始策略感兴趣，但我们首先追求将抑郁肽/罗米地辛作为T细胞淋巴瘤的孤儿药，使用实验室和临床策略。我们已经将这项工作扩展到使用羟肟酸衍生物belinostat的实体肿瘤。我们针对皮肤和外周T细胞淋巴瘤（CTCL和PTCL）的多机构临床试验在6个队列的131例患者中完成。发表了详细介绍罗米地辛在CTCL和PTCL中的反应的论文。对抑郁肽的反应有时是戏剧性的，并且非常持久。例如，一名患者接受了2年的治疗，并在完全缓解治疗后保持了10年。另一名PTCL患者在复发前持续完全缓解5年，没有其他治疗能够控制疾病。在我们的NCI试验和Gloucester注册试验中，皮肤T细胞淋巴瘤的主要缓解率为34-35%。对于PTCL，我们的有效率为38%。值得注意的是，参与我们超过9个多中心的II期试验的校外研究人员也在这两个亚群患者中获得了持久的反应。这些地点包括纽约曼哈塞特的北岸大学医院；加州杜阿尔特的希望之城国家癌症中心；以及澳大利亚墨尔本的彼得·麦卡勒姆癌症中心。除了评估疗效外，我们的NCI II期试验还有一个主要的第二个目标。这是对特工安全的确认。治疗后心电图异常已被注意到，并且大量的努力已被证明心肌损害与本药的施用缺乏相关。我们分析了4000多张心电图，并收集了许多辅助心脏安全数据。今年，我们发表了其他关于罗米地辛的心脏研究结果，包括罗米地辛输注后心率持续增加的记录。我们纳入了进一步确定该药物安全性的分析，并提出了我们在罗米地辛后观察到的非特异性ST波和T波变化的机制，以及我们在开发过程中制定的作为支持措施的电解质补充的基本原理。我们目前正在检查这些心电图，以评估钾和镁补充剂是否减轻了ST波和T波变化的严重程度。该试验还具有显著的翻译相关样本成分，我们能够显示在罗米地辛输注后正常和肿瘤细胞中可重复地增加组蛋白乙酰化和诱导基因表达。我们的数据表明，外周血单个核细胞中组蛋白乙酰化的24小时时间点与药物动力学参数（包括清除率和曲线下面积）和疾病反应具有双重相关性。综上所述，这些数据表明，药物暴露可能对罗米地辛和整个组蛋白去乙酰化酶抑制剂类都很重要。此外，数据已从按协议发送给宾夕法尼亚大学路易斯·肖博士的样本的cDNA阵列中检索。我们报道了罗米地辛在第1、3和5天的I期试验，以获得更持续的药物效果。我们已经接近完成了一项新的组蛋白去乙酰化酶抑制剂belinostat的联合临床试验，评估了顺铂和依托泊苷联合48小时的连续输注。该试验是基于临床前证据的HDAC抑制剂和化疗药物之间的协同作用，当适当安排。这是在晚期疾病人群中进行的I期试验；我们目前正在提炼一种II期剂量。II期剂量将在小细胞肺癌患者人群中进行相同的试验设计。我们在去年作出了重大努力，以了解HDI耐药性的机制。这导致我们产生了非pgp介导的罗米地辛抗性细胞系，我们已经确定了MEK信号传导增加作为抗性的机制。这显然是通过BIM的丢失介导的，BIM是一种促凋亡的线粒体蛋白。我们有详细的实验室研究表明，添加MEK抑制剂能够与罗米地辛协同作用，以增强细胞敏感性。我们还能够在临床样本中显示一系列BIM水平，并且正在进行研究以比较BIM水平与治疗反应。我们继续对人类发展指数引人注目的作用机制感兴趣。至少有5种机制被引用用于组蛋白去乙酰化酶抑制剂：诱导基因表达、胞质蛋白乙酰化和功能改变、由于Hsp90活性受损导致的胞质蛋白降解增加、血管生成改变和有丝分裂作用。究竟哪个机制是至关重要的将是继续调查的主题。与NCI药物筛选合作进行的研究表明，在罗米地辛治疗后可以观察到DNA损伤指纹。这些研究还得到旨在确定协同药物组合的实验的补充。我们已经确定了几种双药组合，它们显著提高了在淋巴瘤单药治疗中观察到的活性，并似乎转化为实体瘤的活性。支持罗米地辛与一种实验性药物联合使用方案概念的动物研究已经完成；我们已经提交了一份意向书，希望来年在临床中研究这种组合。从小鼠的异种移植物中获得的肿瘤样本在药效学标记物对罗米地辛暴露的反应方面显示出与实验室获得的结果相同。在相关研究中，我们对罗米地辛治疗后观察到的c-myc表达普遍降低印象深刻。鉴于myc被认为是细胞代谢的主要调节因子，我们还将开展旨在了解myc反应在罗米地辛后发生的细胞死亡中的作用的研究。最后，我们已经开始评估表观遗传药物的组合，目的是开发一种药效学标记物，用于HDAC抑制剂和DNA甲基化抑制剂的临床试验。

We have studied the histone deacetylase inhibitors romidepsin and belinostat in both the clinic and in the laboratory. We originally became interested in romidepsin in the context of a Phase I clinical trial, when we made the serendipitous discovery that (the then-named) depsipeptide was highly effective in subsets of T-cell lymphoma. While we have continued to be interested in our original strategy of preventing the emergence of resistance to this agent, we first pursued the use of depsipeptide/romidepsin as an orphan drug in T cell lymphoma, using both laboratory and clinical strategies. We have extended this work into solid tumors with the hydroxamic acid derivative belinostat. Our multi-institutional clinical trial for cutaneous and peripheral T cell lymphoma (CTCL and PTCL) completed accrual at 131 patients in 6 cohorts. Papers detailing responses to romidepsin in both CTCL and PTCL are published. The responses to depsipeptide are at times dramatic and have been very durable. As an example, one patient received therapy for 2 years, and has remained in complete remission off of therapy for 10 years. Another patient with PTCL remained in continuous complete remission for 5 years before relapse occurred and no other therapy was able to control the disease. The major response rate in cutaneous T cell lymphoma in both our NCI trial and in the Gloucester registration trial was 34-35%. For PTCL, our response rate was 38%. It is important to note that durable responses were also obtained in both subsets of patients by extramural investigators who were participating in our Phase II trial among more than 9 multicenter sites included in the study. These sites included North Shore University Hospital in Manhasset, New York; City of Hope National Cancer Center in Duarte, California; and Peter MacCallum Cancer Center in Melbourne, Australia. Our NCI Phase II trial had a major second objective in addition to evaluating efficacy. That was confirmation of the safety of the agent. EKG abnormalities have been noted following treatment and a great deal of effort has gone into demonstrating the lack of myocardial damage associated with administration of this agent. We analyzed over 4000 ECGs, and collected much ancillary cardiac safety data. We published additional cardiac findings with romidepsin this year, including documentation of a consistent increase in heart rate after romidepsin infusion. We included analyses that further established the safety of the agent, and also suggested a mechanism for the nonspecific ST and T wave changes we observe following romidepsin and a rationale for the electrolyte supplementation that we instituted as a supportive measure during development. We are currently reviewing these same ECGs to evaluate the possibility that the potassium and magnesium supplementation actually mitigated to severity of the ST and T wave changes. The trial also had a significant translational correlative sample component, and we were able to show reproducible increased histone acetylation, and induction of gene expression in normal and tumor cells following romidepsin infusion. Our data suggest that the 24hr timepoint of histone acetylation in peripheal blood mononuclear cells is dually associated with pharmacokinetic parameters including clearance and area under the curve and with disease response. Taken together these data suggest that drug exposure may be important for romidepsin and potentially for the entire class of histone deacetylase inhibitors. Additionally, data have been retrieved from cDNA arrays on samples sent as per protocol to Dr. Louise Showe at University of Pennsylvania. We reported on a Phase I trial of romidepsin on a day 1, 3, and 5 schedule to achieve a more continuous drug effect. We have nearly completed a combination clinical trial with a novel histone deacetylase inhibitor, belinostat, evaluating a 48 hr continuous infusion in combination with cisplatin and etoposide. This trial is based on preclinical evidence of synergy between HDAC inhibitors and chemotherapeutics, when properly scheduled. This was carried out as a Phase I trial in an advanced disease population; we are currently refining a Phase II dose. The Phase II dose will be explored in the same trial design in the small cell lung cancer patient population. We have made a major effort in the last year to understand mechanisms of HDI resistance. This led us to the generation of cell lines with non-Pgp-mediated romidepsin resistance and we have identified increased MEK signaling as a mechanism of resistance. This is apparently mediated via loss of BIM, a proapoptotic mitochondrial protein. We have detailed laboratory studies that show that addition of a MEK inhibitor is able to synergize with romidepsin to enhance cell sensitivity.We were also able to show a range of BIM levels in clinical samples and studies are ongoing to compare BIM levels with response to treatment. We continue to be interested in the striking mechanism of action of the HDIs. At least 5 mechanisms have been cited for histone deacetylase inhibitors: induction of gene expression, acetylation of cytoplasmic proteins and altered function, increased degradation of cytoplasmic proteins due to impaired Hsp90 activity, altered angiogenesis, and mitotic effects. Exactly which mechanism is of critical importance will be the subject of continuing investigation. Studies carried out in collaboration with the NCI drug screen suggest that a DNA damage fingerprint can be observed following romidepsin treatment. These studies have also been complemented by experiments aimed at identifying synergistic drug combinations. We have identified several two-drug combinations that have markedly increased the activity already observed in monotherapy in lymphoma and appear to translate into activity in solid tumors. Animal studies supporting a protocol concept for the combination of romidepsin with an experimental agent have been completed; we have submitted an LOI to study this combination in the clinic in the coming year. Tumor samples obtained from the xenografts in mice have demonstrated identical results to those obtained in the laboratory, in terms of response of pharmacodynamic markers to romidepsin exposure. In related studies, we have been impressed with the prevalent reduction in c-myc expression observed following romidepsin treatment. Given that myc is understood as a master regulator of cell metabolism, we will also undertake studies aimed at understanding the role of the myc response in the cell death that occurs following romidepsin. Finally, we have begun to evaluate combinations of epigenetic agents with the aim of developing a pharmacodynamic markers to use in clinical trials combining HDAC inhibitors and DNA methylation inhibitors.