Defining molecular biomarkers for CDK4/6-based cancer therapy by RNA sequencing

描述(申请人提供)：细胞周期失调是人类癌症，特别是套细胞淋巴瘤(MCL)的一个特征，其中细胞周期蛋白D1的异常表达是由于染色体t(11；14)易位。再加上细胞周期蛋白依赖性激酶(CDK)4的过度表达，导致细胞周期加快和肿瘤细胞的不受抑制的增殖，这是疾病进展的基础。由于MCL仍然无法治愈，迫切需要控制细胞周期的新疗法。利用PD0332991(PD)，这是已知的唯一选择性和有效的CDK4/CDK6抑制剂，也是口服生物利用和可逆的，我们开发了一种新的策略，既可以控制肿瘤细胞的细胞周期，又可以使它们对细胞毒杀伤素敏感。我们首次证明：1)选择性抑制CDK4/CDK6导致早期G1期停滞；2)G1期阻滞解除后，同步进展到S期；3)延长G1期停滞(PG1)使肿瘤细胞对细胞毒杀伤敏感；4)在随后的S时相同步(pG1-S)中，pG1对细胞毒杀伤的敏感性被放大。我们推测，这种对细胞毒性药物敏感性的增加是由于pG1中的基因表达停止以及尽管G1期阻断释放后细胞周期进展，预定基因表达的不完全恢复所致。为了在临床反应的背景下验证这一假设，我们在第一阶段人类癌症PD的单药临床研究中证明，PD优先并有效地抑制复发/难治患者MCL细胞中的CDK4/CDK6，具有良好的毒性特征和令人鼓舞的临床反应。这些发现已经在一项正在进行的针对CDK4/CDK6的第一阶段临床研究中得到证实，该研究结合蛋白酶体抑制剂Bortezomib(PDBo)治疗MCL和多发性骨髓瘤(MM)。对连续活检的MCL细胞进行的初步全转录组测序(WTS)进一步显示，在临床有效的患者中，21个在pG1中被抑制的基因在无应答的患者中被反向激活(N=6)，其中7个基因与平行的PDBo临床研究中在pG1中相反调节的基因重叠。这些新的发现与关键中枢基因的统一基础机制相一致，为pG1增敏提供了一个独特的机会，表明了介导pG1对细胞毒杀伤和差异性临床反应的敏感性的基因。在这项研究中，我们建议通过扩展和深化整合的WTS以及来自PDBo临床研究的系列MCL肿瘤活组织的完整外显子组测序来定义pG1敏感基因，结合临床反应(目标1)和候选生物标记物的体外功能验证(目标2)。我们相信，这项拟议研究的成功完成将定义区分基于CDK4/CDK6的MCL治疗中pG1敏化的临床反应的生物标记物，并可能揭示基于细胞周期的癌症治疗的共同调控框架。

DESCRIPTION (provided by applicant): Dysregulation of the cell cycle is a hallmark of human cancer, in particular mantle cell lymphoma (MCL), wherein cyclin D1 is aberrantly expressed due to chromosomal t(11;14) translocation. Together with overexpression of cyclin-dependent kinase (CDK)4, this leads to cell cycle acceleration and unrestrained proliferation of tumor cells that underlies disease progression. Since MCL remains incurable, novel therapies that control the cell cycle are urgently needed. Using PD0332991 (PD), the only known selective and potent inhibitor of CDK4/CDK6 that is also orally bioavailable and reversible, we have developed a novel strategy to both control the cell cycle in tumor cells and sensitize them to cytotoxic killin. We have demonstrated, for the first time, that 1) selective inhibition of CDK4/CDK6 leads to early G1 arrest and, 2) upon release of the G1 block, a synchronized progression to S phase occurs; 3) prolonged G1 arrest (pG1) sensitizes tumor cells to cytotoxic killing, and 4) pG1 sensitization to cytotoxic killing is amplified in the subsequent S phase synchronization (pG1-S). We hypothesize that this increased sensitivity to cytotoxic agents is due to halting of gene expression in pG1 and incomplete restoration of scheduled gene expression despite cell cycle progression after the release of the G1 block. To test this hypothesis in the context of clinical response, we demonstrated in the first Phase I, single-agent clinical study of PD in human cancer that PD preferentially and potently inhibited CDK4/CDK6 in MCL cells in relapsed/refractory patients, with an excellent toxicity profile and an encouraging clinical response. These findings have been confirmed in an ongoing Phase I clinical study targeting CDK4/CDK6 with PD in combination with the proteasome inhibitor bortezomib (PDBo) in MCL and in multiple myeloma (MM). Preliminary whole transcriptome sequencing (WTS) of MCL cells from serial biopsies has further revealed that 21 of the genes suppressed in pG1 in clinically responsive patients were conversely activated in the non-responding patients (N=6), and seven of them overlap with genes that were oppositely regulated in pG1 in clinically responding vs. non- responding patients in the parallel PDBo clinical study in MM. These novel findings are consistent with a unified underpinning mechanism of key hub genes for pG1 sensitization, and suggest a unique opportunity to identify genes that mediate pG1 sensitization to cytotoxic killing and differential clinical response. In this study, we propose to define pG1-sensitizing genes by expanding and deepening integrated WTS as well as whole exome sequencing of serial MCL tumor biopsies from the PDBo clinical study in the context of the clinical response (Aim 1) in conjunction with functional validation of candidate biomarkers ex vivo (Aim 2). We believe that successful completion of the proposed study will define biomarkers that differentiate clinical responses to pG1 sensitization in the CDK4/CDK6-based therapy in MCL, and may reveal a common regulatory framework for cell-cycle based cancer therapies.