Exploiting replication stress for the selective killing of FGFR-dependent cancers

 描述(由申请人提供)：复制压力可以被定义为复制起点的数量、起点激发的时间和/或复制分叉速度的任何条件 均较正常的S时相有明显变化。在这种情况下，细胞积累未分解的复制中间产物，无法完成DNA复制。最近的研究表明，复制应激广泛存在于多种癌症中；因此，开发它的治疗方法是一个令人兴奋的新领域。为此，目前的大部分 研究的重点是作为主要治疗靶点的DNA损伤反应通路。在这一应用中，我们建议将休眠起始点作为利用癌症复制应激的新靶点，因为越来越多的证据表明，癌细胞严重依赖休眠起始点来克服复制应激并维持其增殖。所有复制起始点都是通过在M期晚期和G1期早期将MCM2-7蛋白加载到染色质上而获得许可的。在随后的S期，细胞只使用一小部分(5%~10%)与染色质结合的MCM2-7蛋白来组装活跃的复制螺旋酶，以解开dna并启动dna合成。剩余的mcm2-7蛋白许可休眠起始点作为后备，以挽救停滞的复制分叉和/或补偿缓慢的分叉进展。虽然休眠起始点的作用即使在正常的S阶段也很重要，但它们在复制压力下使用得更频繁，这可能会增加停滞的分叉点的数量。 癌细胞过度依赖于其潜在的起源，随着癌症的进展，它们通常表现出更高水平的MCM2-7表达。考虑到休眠起源的这些特性，我们假设减少它们的数量将有助于选择性地杀死表现出复制压力的癌细胞。为了减少休眠起始点，我们将使用RNA干扰介导的MCM2-7蛋白的缺失或Mcm4chaos3纯合背景，在该背景中，休眠起始点的数量减少到野生型小鼠中观察到的~50%。作为一个癌症模型，我们将使用体外和体内系统在乳腺癌中诱导FGFR1(IFGFR1)的激活，因为我们的初步数据表明iFGFR1的激活会导致强大的复制压力。由于FGFR异常的乳腺癌通常会产生治疗抵抗，针对潜在来源的新疗法的开发可能会使此类疾病的患者受益。我们的具体目标如下：1)证明休眠起始点的减少损害了FGFR激活后DNA复制的完成，2)测试休眠起始点的减少是否阻止了iFGFR1诱导的细胞增殖。我们还将确定共同靶向介导FGFR诱导存活的途径的休眠起源的有效性。这一建议的创新方面不仅是测试针对休眠起始点利用复制应激的靶向，而且还研究FGFR异常激活诱导复制应激的机制，以开发联合治疗以实现对有FGFR异常的癌症的特异性杀伤。

 DESCRIPTION (provided by applicant): Replication stress can be defined as any condition in which the number of replication origins, the timing of origin firing, and/or replication fork speed are significantly altered relative to normal S phase. Under these conditions, cells accumulate unresolved replication intermediates, failing to complete DNA replication. Recent studies demonstrate that replication stress is widespread in a variety of cancers; therefore its exploitation for therapies is an exciting new area to explore. To this end, the majority of current investigations focus on the DNA damage response pathways as major therapeutic targets. In this application, we propose dormant origins as a new target to exploit replication stress in cancer, as increasing evidence indicates that cancer cells rely heavily on dormant origins to override replication stress and sustain their proliferation. All replication origins are "licensed by loading of the MCM2-7 proteins onto chromatin during the late M and early G1 phases. In the subsequent S phase, cells use only a small fraction (5~10%) of chromatin-bound MCM2-7 proteins for the assembly of active replicative helicases to unwind DNA and initiate DNA synthesis. The remaining excess MCM2-7 proteins license dormant origins that can act as backups to rescue stalled replication forks and/or compensate for slow fork progression. While such roles of dormant origins are important even in normal S phase, they are used far more frequently under replication stress that increases the number of stalled forks. Possibly reflecting their over-reliance on dormant origins, cancer cells generally show higher levels of MCM2-7 expression as cancer progresses. Given these properties of dormant origins, we hypothesize that reducing their number will contribute to the selective killing of cancer cells exhibiting replication stress. For a reduction of dormant origins, we will use either RNA-interference mediated depletions of the MCM2-7 proteins or the Mcm4chaos3 homozygous background in which the number of dormant origins are reduced to ~50 % of what is observed in wildtype mice. As a cancer model, we will use the in vitro and in vivo systems for inducible FGFR1 (iFGFR1) activation in breast cancer, because our preliminary data demonstrate that iFGFR1 activation causes robust replication stress. As breast cancers with FGFR abnormalities often develop therapeutic resistance, the development of new therapies targeting dormant origins could benefit patients with such diseases. Our specific aims are as follows: 1) Prove that a reduction of dormant origins impairs the completion of DNA replication upon FGFR activation, 2) Test if a reduction of dormant origins blocks iFGFR1-induced cellular proliferation. We will also determine the efficacy of co-targeting dormant origins with pathways that mediate FGFR-induced survival. Novel aspects of this proposal are not only to test the targeting of dormant origins to exploit replication stress, but also to investigate the mechanism by which aberrant activation of FGFR induces replication stress for the development of combined therapies to achieve the specific killing of cancer with FGFR abnormalities.