Targeting GLI-dependent Transcription by GANT61 in Colon Cancer

 描述（由申请人提供）：GLI基因GLI 1和GLI 2编码调节经典Hedgehog信号传导（HH）途径远端靶基因的转录因子（SHH->PTCH->SMO->GLI），在胚胎发育、组织模式化和分化中受到严格调节，在成体组织中低水平表达。 在癌症中，GLI 1和GLI 2都是癌基因，异常和组成性激活。癌基因驱动的信号传导途径，特别是结肠癌中的KRAS/BRAF，绕过HH-GLI轴，通过GLI引导，并诱导进一步的组成性GLI活化，使GLI在细胞存活中发挥关键作用。 使用小分子抑制剂GANT 61 GLI依赖的转录，我们已经证明，靶向GLI终止致癌HH-GLI和KRAS/BRAF-GLI信号转导，诱导广泛的细胞死亡，在七个人结肠癌细胞系模型检查。我们已经证明：1）GANT 61对靶向GLI具有特异性; 2）GANT 61对GLI 1和GLI 2的抑制迅速降低了与靶基因启动子中共有序列的结合，并抑制GLI依赖性转录; 3）GANT 61诱导RNA聚合酶II（Pol II）在早期延伸位点的GLI依赖性转录停滞，伴随RNA：DNA杂合体（R环）的积累; 4）与转录抑制一致，GANT 61在S期和非S期细胞中诱导DNA损伤（γ H2 AX灶），在S期（G1/S）开始时识别，在细胞死亡开始之前。总体目标是了解GANT 61抑制GLI依赖性转录的机制，GLI依赖性转录诱导DNA损伤，导致广泛的细胞死亡。在特定目标1中，我们已经确定了在靶基因启动子处邻近GLI结合区的Pol II的停滞，其与启动子结合暂停（DSIF，NELF）或暂停释放（P-TEFb）因子的修饰相关。 在停滞的Pol II位点检测到单链DNA（R环）和RNA：DNA杂交体;诱导γ H2 AX灶。该假设是GANT 61诱导的Pol II的GLI依赖性停滞在早期延伸的位点处产生ssDNA，其在R环区域内产生DNA DSB。 在特定目标2中，我们已经证明，DNA损伤在S期开始时被识别，诱导瞬时的 在细胞死亡开始之前的S期内检查点。 GLI 1靶基因FOXM 1及其转录靶点CDC 6（调节G1/S转换和DNA复制起始）在GANT 61处理的细胞中减少。该假说是FOXM 1和CDC 6调节G1/S的复制前复合物组装，从而抑制DNA复制的起始，这控制GANT 61应答的结果，依赖于GLI。 在特定目标3中，我们将在独特工程化的人结肠癌异种移植物体内模型和转基因小鼠GLI-荧光素酶报告基因模型中靶向组成型GLI激活。 GLI在多种人类癌症中被组成性激活。 此外，KRAS在30%的人类癌症和50%的结肠癌中突变。 因此，这一提议具有潜在的高影响力，预计将导致以GLI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.