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的抑制剂CDK4和CDK6已被批准用于治疗鲁米纳型 雌激素受体(ER)阳性的乳腺癌。不幸的是，很大一部分乳腺癌患者 对CDK4/6抑制产生抵抗力。在目标1中，我们将检验我们的假设，即CDK4/6抗性乳房 癌细胞的增殖依赖于细胞周期蛋白依赖性激酶CDK2的过度激活。 因此，我们假设，抑制CDK4/6抑制剂耐药的癌细胞中的CDK2将阻断其 扩散。我们进一步假设，联合抑制CDK4/6和CDK2将具有协同作用 有效，并可能防止抗药性疾病的发展。目前，研究这一角色的一个主要限制是 CDK2的缺陷是缺乏CDK2特异性的抑制物。为了克服这一限制，我们的实验室应用了 这种方法允许我们使用一种特定的、有效的和可逆的抑制CDK2的方法。 不抑制任何其他激动酶的化合物。在目标1中，我们将使用模拟敏感方法进行测试 CDK2抑制对CDK4/6耐药肿瘤体内增殖的影响我们还将比较 使用我们的系统并排比较有效的CDK2抑制药物和目前正在使用的CDK2抑制剂的效果 临床试验。为了在临床上评估CDK2在CDK4/6耐药中的作用，我们 将从开始接受CDK4/6抑制剂治疗的患者中获得120例基线活检，以获得60例配对活检 在基线和抵抗力发展时。这些活检组织将被询问CDK2激活状态，以及 我们将开发新的方法来测量临床环境中的CDK2活性。在目标2中，我们将扩展我们的 三阴性乳腺癌(TNBC)的调查。与管腔型乳腺癌相比，TNBC是 天生抵抗CDK4/6抑制。尽管如此，我们观察到有相当一部分人类 TNBC细胞系的增殖关键需要CDK4/6。我们的初步数据表明CDK4/6抑制剂 被隔离在TNBC细胞溶酶体中，从而阻断了抑制剂的治疗效果。重要的是 我们发现，用抑制溶酶体酸化的化合物处理TNBC细胞，如氯喹， 逆转这种隔离，使TNBC细胞对CDK4/6抑制剂治疗敏感。我们还确认了 一种新的CDK4/6抑制剂化合物，它本身就能抑制TNBC细胞的增殖。我们将测试以下各项的实用性 联合使用CDK4/6抑制剂和氯喹治疗TNBC，也使用患者来源的异种移植 作为直接从人类肿瘤中分离出来的细胞的短期培养。我们还将使用这些系统来评估 上述新型CDK4/6抑制剂的疗效。我们将进行帕波西利的I/II期研究，并 氯喹，以检验添加氯喹可以绕过溶酶体隔离的假设，以及 在这项试验中，患者将接受配对活检，以评估CDK4/6抑制剂的隔离情况。预期的总体情况 这一建议的影响是，它可能为克服后天获得性疾病提供一种非常有效的治疗策略。 对CDK4/6抑制剂的耐药性，并可能将抗CDK4/6治疗的好处扩大到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.