Project 1 - Overcoming Breast Cancer Resistance to CDK4/6 Inhibition

项目摘要 细胞周期蛋白依赖性激酶CDK 4和CDK 6的抑制剂已被批准用于治疗管腔型 雌激素受体（ER）阳性乳腺癌。不幸的是，很大一部分乳腺癌患者 对CDK 4/6抑制产生抗性。在目标1中，我们将检验我们的假设，即CDK 4/6耐药乳腺癌患者的CDK 4/6耐药乳腺癌患者的CDK 4/6耐药乳腺癌患者的CDK 4/6耐药。 癌细胞变得依赖于细胞周期蛋白依赖性激酶CDK 2的过度活化来增殖。 因此，我们假设，在CDK 4/6受体耐药的癌细胞中抑制CDK 2将阻断其表达。 增殖我们进一步假设联合抑制CDK 4/6和CDK 2将具有协同作用。 效果，并可能防止耐药性疾病的发展。目前，在研究中的一个主要局限性的作用， 缺乏CDK 2特异性抑制剂。为了克服这一限制，我们的实验室应用了 “类似物敏感”激酶方法，这使我们能够特异性地，有效地和可逆地抑制CDK 2， 不抑制任何其他激酶的化合物。在目标1中，我们将使用模拟敏感方法来测试 CDK 2抑制对体内CDK 4/6抑制剂抗性肿瘤增殖的影响。我们还将比较 并行使用我们的系统有效的CDK 2抑制作用，与目前在 临床试验为了评估CDK 2在临床上CDK 4/6耐药发展中的作用，我们 将从开始CDK 4/6抑制剂治疗的患者中获得120份基线活检，以获得60份配对活检 在基线和耐药性发展时。将询问这些活检组织的CDK 2活化状态， 我们将开发新的方法来测量CDK 2在临床环境中的活性。在目标2中，我们将扩展我们的 三阴性乳腺癌（TNBC）。与管腔型乳腺癌相比，TNBC是 对CDK 4/6抑制具有内在抗性。尽管如此，我们已经观察到， TNBC细胞系非常需要CDK 4/6用于增殖。我们的初步数据表明，CDK 4/6抑制剂 被隔离到TNBC细胞溶酶体中，从而阻断抑制剂的治疗作用。重要的是， 我们发现用抑制溶酶体酸化的化合物，如氯喹， 逆转隔离并使TNBC细胞对CDK 4/6抑制剂处理敏感。我们还确定 一种新的CDK 4/6抑制剂化合物，其本身抑制TNBC细胞的增殖。我们将测试 联合CDK 4/6抑制剂与氯喹治疗TNBC，使用患者来源的异种移植物，以及 作为直接从人类肿瘤中分离的细胞的短期培养物。我们还将使用这些系统来评估 上述新型CDK 4/6抑制剂的功效。我们将开展一项palbociclib的I/II期研究， 氯喹，以检验加入氯喹可以避免溶酶体螯合的假设，以及 本试验中的患者将进行成对的活组织检查以评估CDK 4/6抑制剂隔离。预期总体 这一建议的影响是，它可以提供一个非常有效的治疗策略，克服获得性 抗CDK 4/6抑制剂的耐药性，并可能将抗CDK 4/6治疗的益处扩展至TNBC患者。

Project Summary Inhibitors of cyclin-dependent kinases CDK4 and CDK6 have been approved for treatment of luminal-type estrogen receptor (ER)-positive breast cancers. Unfortunately, a large proportion of patients with breast cancer develops resistance to CDK4/6 inhibition. In Aim 1, we will test our hypothesis that CDK4/6 resistant breast cancer cells become dependent on hyperactivation of the cyclin-dependent kinase CDK2 for proliferation. Consequently, we hypothesize that inhibition of CDK2 in CDK4/6 inhibitor-resistant cancer cells would block their proliferation. We further hypothesize that combined inhibition of CDK4/6 and CDK2 would have a synergistic effect, and might prevent the development of resistant disease. Currently, a major limitation in studying the role of CDK2 is the absence of CDK2-specific inhibitors. To overcome this limitation, our laboratory has applied the ‘analog-sensitive’ kinase approach, which allows us to specifically, potently and reversibly inhibit CDK2 using a compound that does not inhibit any other kinases. In Aim 1, we will use the analog-sensitive approach to test the impact of CDK2 inhibition on proliferation of CDK4/6-inhibitor resistant tumors in vivo. We will also compare side-by-side the effects of potent CDK2 inhibition using our system, versus CDK2 inhibitors that are currently in clinical trials. In an effort to assess the role of CDK2 in the development of CDK4/6 resistance in the clinic, we will obtain 120 baseline biopsies from patients starting CDK4/6 inhibitor treatment to obtain 60 paired biopsies at baseline and when resistance develops. These biopsies will be interrogated for CDK2 activation status, and we will develop new approaches to gauge CDK2 activity in the clinical setting. In Aim 2, we will extend our investigations to triple negative breast cancer (TNBC). In contrast to luminal-type breast cancers, TNBC is intrinsically resistant to CDK4/6 inhibition. Nonetheless, we have observed that a significant fraction of human TNBC cell lines critically requires CDK4/6 for proliferation. Our preliminary data indicate that CDK4/6 inhibitors become sequestered into TNBC cell lysosomes, thereby blocking the inhibitors’ therapeutic effect. Importantly, we found that treatment of TNBC cells with compounds that inhibit lysosomal acidification, such as chloroquine, reverses the sequestration and renders TNBC cells sensitive to CDK4/6 inhibitor treatment. We also identified a new CDK4/6 inhibitor compound that on its own inhibits proliferation of TNBC cells. We will test the utility of combining CDK4/6 inhibitors with chloroquine for treatment of TNBC, using patient-derived xenografts, as well as short-term cultures of cells isolated directly from human tumors. We will also use these systems to evaluate the efficacy of the novel CDK4/6 inhibitor described above. We will conduct a phase I/II study of palbociclib and chloroquine to test the hypothesis that the addition of chloroquine can circumvent lysosomal sequestration, and patients in this trial will undergo paired biopsies to assess CDK4/6 inhibitor sequestration. The expected overall impact of this proposal is that it may provide a highly effective therapeutic strategy for overcoming acquired resistance to CDK4/6 inhibitors, and may extend the benefits of anti-CDK4/6 therapy to patients with TNBC.