Targeting CDK6 expression/activity in Ph+ and Ph1-like acute lymphoblastic leukemia (ALL)

摘要 费城阳性急性白血病（Ph+ ALL）和Ph 1样B-ALL占大多数病例。 “高危”成人B-ALL。目前使用酪氨酸激酶抑制剂（TKI）的治疗已改善了 Ph+ ALL，但对TKI的耐药性在大多数患者中迅速发展。Ph 1样B-ALL目前接受以下治疗： 强化联合化疗，但疾病复发很常见，5年生存率仅为25%。 患者因此，Ph+ ALL和Ph 1样B-ALL的预后仍然很差。在以前的研究中，我们 表明Ph+和Ph 1-like ALL细胞表现出对CDK 6表达的选择性需求，而CDK 4 表达是。CDK 6是细胞周期蛋白D/CDK 6复合物的催化亚基， G1至S期细胞周期转换，并在血液学中具有激酶非依赖性生长促进作用 恶性肿瘤我们的初步研究表明，CDK 6沉默比CDK 6酶促表达更有效。 抑制小鼠Ph+ ALL。为了阻断CDK 6的激酶依赖性和非依赖性作用，我们 已经开发了CDK 4/6靶向蛋白水解靶向嵌合体（PROTAC）， 体外活性，促进Ph+和Ph 1-样ALL细胞中CDK 6优先降解CDK 4， 提供对CDK 6功能的持久抑制。在本建议书中，我们将评估CDK 6的要求， 通过比较PROTAC YX-2-107降解CDK 6的作用和 使用FDA批准的CDK 4/6抑制剂Palbociclib进行药理学抑制（目的1.1）。我们还将 确定CDK 6下调诱导的白血病抑制是否比 CDK 6酶抑制可以通过CDK 6选择性诱导的基因表达变化来解释。 沉默这些变化涉及组蛋白去乙酰化酶1（HDAC 1）基因和其他几个参与 线粒体代谢途径（目标1.2和1.3）。虽然我们已经能够实现高特异性 CDK 6与CDK 4靶向的比较，生物学/治疗作用与Palbociclib离体相当/优于Palbociclib（上级） 在Ph+ ALL的PDX中，我们将继续通过药物治疗来改进我们的先导化合物PROTAC YX-2-107， 化学方法，以开发具有增强的体内功效的衍生物。在目标2中，我们将评估 选择CDK 6降解PROTAC的代谢特性，并在Ph+中测试其生物学/治疗作用 和Ph 1样ALL细胞，以及在注射了新生或复发/TKI耐药患者衍生 Ph+/Ph 1-样ALL细胞。总的来说，我们基于PROTAC的方法利用了癌症领域的专业知识， 加尔各答和萨尔维诺实验室的生物学和药物化学有望开发新的 和更有效的治疗药物，用于治疗临床前PDX中的CDK 6依赖性高危B-ALL 模型，并可能在临床上。

Abstract Philadelphia-positive acute lymhoblastic leukemia (Ph+ ALL) and Ph1-like B-ALL account for most cases of “high-risk” adult B-ALL. Current therapies with tyrosine kinase inhibitors (TKIs) have improved the outcome of Ph+ ALL, but resistance to TKIs develops rapidly in most patients. Ph1-like B-ALL is currently treated with intensive combination chemotherapy but disease relapse is common with a 5-year survival in only ~25% of patients. As a result, the prognosis of Ph+ ALL and Ph1-like B-ALL remains dismal. In previous studies, we showed that Ph+ and Ph1-like ALL cells exhibit a selective requirement for CDK6 expression while CDK4 expression is dispensable. CDK6 is the catalytic subunit of the cyclin D/CDK6 complex which is essential for the G1 to S-phase cell cycle transition and has kinase-independent growth-promoting effects in hematological malignancies. Our preliminary studies indicate that CDK6 silencing is more effective than CDK6 enzymatic inhibition in suppressing Ph+ ALL in mice. To block kinase-dependent and independent effects of CDK6, we have developed CDK4/6-targeted proteolysis-targeting chimera (PROTACs) that inhibit CDK4/6 enzymatic activity in vitro and promote the preferential degradation of CDK6 over CDK4 in Ph+ and Ph1-like ALL cells, providing durable suppression of CDK6 function. In this proposal, we will assess the requirement of CDK6 in Ph+ and Ph1-like ALL by comparing the effects of CDK6 degradation by PROTAC YX-2-107 and pharmacological inhibition using Palbociclib, an FDA-approved CDK4/6 inhibitor (Aim 1.1). We will also determine whether the more potent leukemia suppression induced by CDK6 down-regulation in comparison to CDK6 enzymatic inhibition can be explained by changes in gene expression induced selectively by CDK6 silencing. Such changes involve the histone deacetylase 1(HDAC1) gene and several others involved in mitochondrial metabolic pathways (Aims 1.2 and 1.3). Although we have been able to achieve high specificity of CDK6 versus CDK4 targeting and biological/therapeutic effects comparable/superior to Palbociclib ex vivo and in PDXs of Ph+ ALL, we will continue to improve our lead compound PROTAC YX-2-107 by medicinal chemistry approaches in order to develop derivatives with enhanced in vivo efficacy. In Aim 2, we will assess metabolic properties of select CDK6-degrading PROTACs and test their biological/therapeutic effects in Ph+ and Ph1-like ALL cells ex vivo and in mice injected with de novo or relapsed/TKI-resistant patient-derived Ph+/Ph1-like ALL cells. Collectively, our PROTAC-based approach which leverages the expertise in cancer biology and medicinal chemistry of the Calabretta and Salvino's laboratories holds promise to develop novel and more effective therapeutic agents for the treatment of CDK6-dependent high-risk B-ALL in pre-clinical PDX models and, potentially, in the clinic.