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

描述（由申请人提供）：细胞周期失调是人类癌症的标志，特别是套细胞淋巴瘤（MCL），其中细胞周期蛋白D1由于染色体t（11;14）易位而异常表达。与细胞周期蛋白依赖性激酶（CDK）4的过表达一起，这导致肿瘤细胞的细胞周期加速和不受限制的增殖，这是疾病进展的基础。由于MCL仍然无法治愈，因此迫切需要控制细胞周期的新疗法。使用PD 0332991（PD），唯一已知的选择性和有效的CDK 4/CDK 6抑制剂，也是口服生物可利用的和可逆的，我们已经开发了一种新的策略，既控制肿瘤细胞的细胞周期，又使它们对细胞毒性杀伤敏感。我们首次证明：1）选择性抑制CDK 4/CDK 6导致早期G1阻滞，2）G1阻滞释放后，同步进展至S期; 3）延长的G1阻滞（pG 1）使肿瘤细胞对细胞毒性杀伤敏感，4）pG 1对细胞毒性杀伤的敏感性在随后的S期同步（pG 1-S）中被放大。我们假设，这种增加的敏感性细胞毒性药物是由于停止基因表达的pG 1和不完全恢复预定的基因表达，尽管细胞周期进展后的G1块释放。为了在临床应答的背景下检验这一假设，我们在人类癌症中PD的第一项I期单药临床研究中证明，PD优先并有效地抑制复发/难治性患者MCL细胞中的CDK 4/CDK 6，具有优异的毒性特征和令人鼓舞的临床应答。这些发现已在一项正在进行的I期临床研究中得到证实，该研究靶向CDK 4/CDK 6伴PD联合蛋白酶体抑制剂硼替佐米（PDBo）治疗MCL和多发性骨髓瘤（MM）。来自连续活检的MCL细胞的初步全转录组测序（WTS）进一步揭示了在临床应答患者中pG 1中抑制的21个基因在无应答患者中相反地被激活（N=6），其中7个与pG 1中在临床应答与非应答中相反调节的基因重叠，这些新的发现与pG 1致敏的关键枢纽基因的统一基础机制一致，并提出了一个独特的机会，以确定基因介导的pG 1敏化细胞毒性杀伤和差异的临床反应。在这项研究中，我们建议通过扩展和深化综合WTS以及PDBo临床研究中的系列MCL肿瘤活检的全外显子组测序，结合候选生物标志物的功能验证（目的1），确定pG 1致敏基因。我们相信，成功完成拟议的研究将定义区分MCL中基于CDK 4/CDK 6的治疗中对pG 1致敏的临床反应的生物标志物，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.