Targeting GLI-dependent Transcription by GANT61 in Colon Cancer

 描述(申请人提供)：GLI基因，GLI1和GLI2，编码转录因子，调节典型Hedgehog信号(HH)途径(SHH-&GT；PTCH-&GT；SMO-&GT；GLI)远端的目标基因，在胚胎发育、组织构型和分化中受到严格调控，在成人组织中低水平表达。在癌症中，GLI1和GLI2都是癌基因，异常地和结构性地激活。癌基因驱动的信号通路，尤其是结肠癌中的KRAS/BRAF，绕过HH-GLI轴，通过GLI途径，诱导进一步的结构性GLI激活，使GLI在细胞生存中发挥关键作用。使用小分子缓蚀剂GANT61 在GLI依赖的转录中，我们已经证明靶向GLI终止致癌的HH-GLI和KRAS/BRAF-GLI信号，在所研究的七个人结肠癌细胞系模型中诱导广泛的细胞死亡。我们已经证明：1)GANT61是特异性的靶向Gli；2)GANT61抑制Gli1和Gli2迅速减少与靶基因启动子中共同序列的结合，并抑制Gli依赖的转录；3)GANT61诱导依赖Gli的RNA聚合酶II(PolII)在早期伸长部位的转录停滞：DNA杂交体(R-环)；4)与转录抑制一致，GANT61诱导S期和非S期细胞的DNA损伤(γH_2AX焦点)，这在S期(G1/S)开始之前就被发现了。S的总目标是了解GANT61抑制依赖Gli的转录导致DNA损伤导致广泛细胞死亡的机制。在特定的目标1中，我们已经确定了与启动子结合暂停(DSIF，NELF)或暂停释放(P-TEFb)因子的修饰有关的位于靶基因启动子GLI结合区附近的POL II停滞。单链脱氧核糖核酸(R-环)和核糖核酸：在停滞的POL II位点检测到DNA杂交体；诱导γH_2AX焦点。假设是GANT61诱导的GLI依赖的POL II停滞在早期伸长部位产生单链DNA，从而在R-环区产生DNADSB。在特定的目标2中，我们已经证明了dna损伤是在S相开始时识别的，诱导了一瞬间的 S时相内检查点细胞死亡开始前。GLI1靶基因FOXM1及其转录靶CDC6调控G1/S的转变和DNA复制的启动，在GANT61处理的细胞中减少。假设FOXM1和CDC6调节复制前复合体在G1/S的组装，从而抑制DNA复制的启动，从而控制GLI反应的结果。在具体目标3中，我们将在独特设计的人结肠癌异种移植体内模型和转基因小鼠GLI-荧光素酶报告模型中针对结构性GLI激活。Gli在多种人类癌症中被结构性激活。此外，KRAS在30%的人类癌症和50%的结肠癌中发生突变。因此，这项提议有可能产生很大的影响，并有望导致将GLI作为靶点的新方法和治疗策略。

 DESCRIPTION (provided by applicant): The GLI genes, GLI1 and GLI2, encode transcription factors that regulate target genes at the distal end of the canonical Hedgehog signaling (HH) pathway (SHH->PTCH->SMO->GLI), tightly regulated in embryonic development, tissue patterning and differentiation, with low-level expression in adult tissues. In cancers, both GLI1 and GLI2 are oncogenes, aberrantly and constitutively activated. Oncogene-driven signaling pathways, in particular KRAS/BRAF in colon cancer, circumvent the HH-GLI axis, channel through GLI, and induce further constitutive GLI activation, giving GLI a pivotal role in cell survival. Using GANT61, a small molecule inhibitor of GLI-dependent transcription, we have demonstrated that targeting GLI terminates oncogenic HH-GLI and KRAS/BRAF-GLI signaling, inducing extensive cell death in seven human colon carcinoma cell line models examined. We have demonstrated that: 1) GANT61 is specific for targeting GLI; 2) inhibition of GLI1 and GLI2 by GANT61 rapidly reduces binding to consensus sequences in target gene promoters and inhibits GLI-dependent transcription; 3) GANT61 induces GLI-dependent transcriptional stalling of RNA Polymerase II (Pol II) at sites of early elongation with accumulation of RNA:DNA hybrids (R-loops); 4) consistent with transcriptional inhibition, GANT61 induces DNA damage (γH2AX foci) in S-phase and non-S-phase cells, recognized at the initiation of S-phase (G1/S), prior to the onset of cell death. The overall goal s to understand the mechanisms by which GANT61 inhibits GLI-dependent transcription that induces DNA damage leading to extensive cell death. In Specific Aim 1, we have identified stalling of Pol II adjacent to GLI binding regions at target gene promoters, associated with modification of promoter-bound pause (DSIF, NELF) or pause-release (P-TEFb) factors. Single-stranded DNA (R-loops) and RNA: DNA hybrids are detected at sites of stalled Pol II; γH2AX foci are induced. The hypothesis is that GANT61-induced GLI-dependent stalling of Pol II creates ssDNA at sites of early elongation that generates DNA DSBs within R-loop regions. In Specific Aim 2, we have demonstrated that DNA damage is recognized at the initiation of S-phase, inducing a transient intra-S-phase checkpoint prior to the onset of cell death. The GLI1 target gene FOXM1, and its transcriptional target CDC6, that regulate the G1/S transition and initiation of DNA replication, are decreased in GANT61-treated cells. The hypothesis is that FOXM1 and CDC6 regulate Pre-Replication complex assembly at G1/S thereby inhibiting the initiation of DNA replication, which controls outcome of the GANT61 response, dependent on GLI. In Specific Aim 3, we will target constitutive GLI activation in uniquely engineered in vivo models of human colon cancer xenografts and in a transgenic mouse GLI-luciferase reporter model. GLI is constitutively activated in a wide variety of human cancers. Further, KRAS is mutated in 30% of all human cancers, and in 50% of colon carcinomas. This proposal therefore has the potential for high impact, and is anticipated to lead to new approaches and therapeutic strategies for GLI as a target.