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

摘要 费城阳性的急性淋巴细胞白血病(Ph+ALL)和PH1样B-ALL占大多数病例 “高危”成人BALL。目前使用酪氨酸激酶抑制剂(TKI)的治疗方法已经改善了 PH+ALL，但对TKIs的耐药性在大多数患者中迅速发展。类似PH1的B-ALL目前正在接受治疗 强化联合化疗但疾病复发很常见，仅有约25%的患者有5年生存率 病人。因此，Ph+ALL和PH1样B-ALL的预后仍然很差。在之前的研究中，我们 显示Ph+和PH1样ALL细胞对CDK6的表达有选择性要求，而CDK4 表达是不必要的。CDK6是细胞周期蛋白D/CDK6复合体的催化亚单位，它是细胞周期蛋白D/CDK6复合体的重要组成部分 血液学中细胞周期从G1期向S期的转变及其非依赖性促生长作用 恶性肿瘤。我们的初步研究表明，CDK6沉默比CDK6酶促沉默更有效 抑制小鼠Ph+ALL的作用。为了阻断CDK6的依赖和非依赖性的作用，我们 我已经开发出CDK4/6靶向蛋白水解靶向嵌合体(PROTAC)，可以抑制CDK4/6酶 在Ph+和类PH1的ALL细胞中，促进CDK6优先于CDK4的降解， 持久抑制CDK6功能。在这项建议中，我们会评估CDK6的需求。 通过比较PROTAC YX-2-107和PROTAC YX-2-107对CDK6的降解效果 使用FDA批准的CDK4/6抑制剂Palbociclib进行药理抑制(AIM 1.1)。我们还将 确定CDK6下调诱导的更强的白血病抑制作用是否与 CDK6的酶抑制作用可以通过CDK6选择性诱导基因表达的变化来解释 沉默。这些变化涉及组蛋白脱乙酰基酶1(HDAC1)基因和其他几个参与 线粒体代谢途径(目标1.2和1.3)。尽管我们已经能够达到很高的特异性 CDK6与CDK4的靶向性比较及生物/治疗效果优于帕博昔利的体外研究 在Ph+ALL的PDX中，我们将继续通过药物来改进我们的先导化合物PROTAC YX-2-107 化学方法，以开发具有增强体内药效的衍生物。在目标2中，我们将评估 CDK6降解蛋白的代谢特性及其在Ph~+中的生物/治疗作用 和PH1样ALL细胞在体外和注射新发或复发/TKI耐药患者来源的小鼠中 PH+/PH1-样所有细胞。总的来说，我们基于PROTAC的方法利用了癌症方面的专业知识 卡拉布雷塔和萨尔维诺实验室的生物和药物化学有望开发出新的 更有效的治疗药物治疗临床前PDX依赖CDK6的高危B-ALL 模特，可能还会在诊所里。

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.