IKKβ抑制剂阻断化疗性DNA损伤修复，清除白血病细胞及其干细胞

The applicant reported in 2011 that IKKβ could regulate the repair of DSBs independent of the NF-κB transcriptional activity and inhibition of IKKβ activity can sensitize cancer cells to IR at least in part via inhibition of DSB repair. Based on this finding, we reported that BMS-345541 (a specific IKKβ inhibitor) selectively inhibited homologous recombinational (HR) repair of DNA damage in MCF-7 cells, which sensitizes MCF-7 xenograft tumors to radiation therapy in vivo (Under review by Clinical Cancer Research ). Although there is different mechanism between radiation and chemotherapeutic agents induced DNA damage，they has similar DNA repair mechanism. Thus BMS-345541 is very possile to inhibit DNA repair induced by DNA damaging agents. Moreover, BCR-ABL fusion protein in chronic myeloid leukemia （CML）CD34+ stem/progenitor cells is able to stimulate infidelity DNA repair, resulting in incorrect DNA accumulation followed by CML blast. Based on this finding, our hypothesis is that IKKβ inhibitor BMS-345541 could eradicate leukemia cells, including leukemia stem cell who has strong capability of DNA repair, by blocking DNA damage repair induced by chemotherapeutics. To explore these hypothesis, we plan to use chronic myeloid leukemia cells and leukemia stem cells as model to study in vivo and in vitro the advantage of CML therapy by IKKβ inhibitor BMS-345541，as well as the molecular mechanism by which IKKβ regulate DNA damage repair.

申请者2011年报道（Plos综合）：IKKβ能调节放疗引起的乳腺癌细胞DNA损伤修复，并且非依赖于Rel A。随后发现特异性IKKβ抑制剂BMS-345541可以抑制放疗引起的DNA损伤的HR（同源重组）修复，显著增敏放疗作用(Clinical Cancer Research审稿中)。放疗与化疗引起DNA损伤的机制虽然不同却有相似修复机制,因此BMS-345541很可能抑制化疗药物引起的DNA损伤修复。慢粒白血病BCR-ABL融合蛋白可刺激CD34+白血病细胞错误修复的DNA累积，导致急变；那么,BMS-345541能否通过抑制化疗药物诱导的DNA损伤修复，清除白血病细胞，尤其是修复能力很强的白血病干细胞，预防慢粒急变和复发？本课题以慢粒白血病细胞及人白血病干细胞为模型，从分子、细胞、动物水平，探讨BMS-345541治疗白血病的优越性及IKKβ调控DNA修复的分子机制。

目的：慢性粒细胞白血病的治疗效果较好，运用伊马替尼等酪氨酸激酶抑制剂可以达到95%的缓解率，甚至达到分子水平的缓解；相反，急性粒细胞白血病（AML）的治疗效果和预后均较差，并且AML的基因组特别不稳定，存在多种变异，表达AML1- ETO、PML-RARα、PLZF-RARα 等多种融合蛋白，这些融合蛋白可干扰DNA修复功能，影响BER、NHEJ DNA修复通路的功能，此外，AML细胞IKKβ/NF-κB通路活性增高，与其发病和预后密切相关，因此本课题改用AML白血病细胞及人白血病干细胞为模型，从分子、细胞、动物水平，探讨BMS-345541治疗白血病的优越性及IKKβ调控DNA修复的分子机制。.结果：1．MTT法、流式细胞术、高内涵分析和彗星实验均表明BMS-345541抑制急性粒细胞白血病细胞的DNA损伤修复；增加了DNA损伤药物柔红霉素（DNR）诱导的AML细胞凋亡率；阻断DNR导致的KG1a细胞和 HL-60细胞的G2/M期阻滞。其作用机制与RAD51在DNA断裂处聚集减少，从而影响HR修复有关；与此一致，由于DNA 损伤未能修复，DNA 损伤应答信号p-ATM持续被激活。2. 流式细胞术、高内涵分析和彗星实验这三种方法检测CD34+祖/干细胞DNA损伤情况，联合用药组DNA损伤比修复组明显，提示BMS-345541可以有效抑制AML祖/干细胞DNA损伤的修复。3. 构建了稳定的IKKβ下调的细胞株KG1a IKKβ (-)细胞株。IKKβ 的下调抑制了KG1a IKKβ (-) 细胞的DNA损伤修复；阻断DNR作用下KG1a细胞G2/M期阻滞；并且可同时抑制KG1a细胞HR 和 NHEJ 通路活性。4．基因芯片结果分析表明，DNA损伤修复有关的下调较明显的基因有PARP1和XRCC3；与PARP1， XRCC3均为负相关且相关系数较高的LncRNA有uc010ixu.3和uc001vvr.1。.结论： 抑制IKK/NF-κB信号途径可有效抑制AML细胞的DNA损伤修复， 逆转G2/M期阻滞，增强DNA损伤化疗药物DNR对细胞的增殖抑制作用和细胞凋亡诱导作用。可能的分子机制是：下调IKK/NF-κB信号途径活性，促使DNA损伤AML细胞的uc010ixu.3和uc001vvr.1表达升高，进而减少PARP1和 XRCC3表达，使细胞DNA 损伤修复能力降低。