Genetic Analysis of the Multidrug Resistance Phenotype in Tumor Cells

癌细胞对化疗产生耐药性是由于特定蛋白表达的内在或获得性变化。我们已经研究了天然产物化疗药物的耐药性，如阿霉素、长春花碱和紫杉醇，以及最近的组蛋白脱乙酰酶抑制剂和靶向激酶抑制剂。在大多数情况下，由于细胞内药物浓度的降低，细胞同时对多种药物产生抗药性。对于天然产物药物，这种交叉耐药性通常是由于被称为P-糖蛋白(P-gp)的能量依赖的药物外排系统(ABC转运体)的表达，P-糖蛋白(P-gp)是MDR1或ABCB1基因的产物，或者ABC转运体家族的其他成员，包括ABCG2和ABCB5。我们实验室和其他实验室的工作表明，一些药物对P-gp表达细胞的毒性比对非表达细胞的毒性更大，这表明了一种治疗MDR癌症的新方法。具有这种性质的几种不同的化学类别，包括缩氨基硫脲(例如，NSC73306)已被鉴定。对NSC73306类似物的定量结构活性分析、在NCI-60细胞系中的进一步相关性分析以及对美国药典中杀死P-gp表达细胞的化合物的高通量筛选，已经产生了许多额外的化合物，这些化合物具有更强的选择性杀伤P-gp表达细胞的能力，而且具有更好的溶解性。为了了解P-gp的结构如何决定其多特异性，以及特异性如何随着折叠的变化而改变，我们与LCB的其他高级研究人员合作，包括Di Xia，Suresh Ambukar和Sriram Subramaniam。冷冻-EM研究表明，apo P-gp具有两个ATP结合位点分离或紧密结合的动态结构。ATP的结合使P-gp的构象固定在后一种状态，而ATP的水解会导致ATP位点的分离。以小鼠P-gp为模型进行的结晶学研究表明，ATP位点之间的分离决定了底物结合的跨膜(TM)螺旋的间距，这表明了一种假设，即当ATP位点一起和分开移动时，TM螺旋暴露出不同的残基，从而能够与许多不同的底物结合。对鼠-人嵌合P-GPS的研究表明，这两个进化上相关的转运蛋白具有相似的结构-功能关系。在Suresh Ambukar小组的合作下，我们研究了P-糖蛋白将化合物定向转运出细胞的基础。这些研究揭示了P-糖蛋白跨膜区的一组氨基酸残基，这些残基可以改变某些罗丹明化合物从细胞外到细胞内的运输方向。这一过程依赖于浓度和ATP，并对P-糖蛋白如何决定转运的方向性提供了重要的见解。我们已经使用AML作为一个模型系统来确定ABC转运蛋白在耐药中的临床作用。在一项研究中，对同一患者化疗前后的样本进行了分析。在这种情况下，每个病例的耐药情况都显示出ABC基因和其他MDR基因的不同表达模式，这表明需要对治疗耐药采取个性化的方法。对大量的原发难治性急性髓细胞白血病进行的更详细的分析表明，有3个分子特征可以预测对治疗的不良反应。其中之一与ABCG2的表达增加有关。这些结果表明，临床样本必须分层，以便于有效靶向ABC转运蛋白的抑制剂，以规避耐药性。

Resistance to chemotherapy occurs in cancer cells because of intrinsic or acquired changes in expression of specific proteins. We have studied resistance to natural product chemotherapeutic agents such as doxorubicin, Vinca alkaloids, and taxol and more recently, histone deacetylase inhibitors and targeted kinase inhibitors. In most cases, cells become simultaneously resistant to multiple drugs because of reductions in intracellular drug concentrations. For the natural product drugs, this cross-resistance is frequently due to expression of an energy-dependent drug efflux system (ABC transporter) known as P-glycoprotein (P-gp), the product of the MDR1 or ABCB1 gene, or to other members of the ABC transporter family, including ABCG2 and ABCB5. Work from our laboratory and others has revealed that some drugs are more toxic to P-gp-expressing cells than to non-expressors, suggesting a novel approach to treatment of MDR cancers. Several different chemical classes with this property, including thiosemicarbazones (e.g., NSC73306), have been identified. A quantitative structure activity analysis of NSC73306 analogs, a further correlation analysis in the NCI-60 cell lines, and a high-throughput screen for compounds in the U.S. Pharmacopeia that kill P-gp-expressing cells have yielded many additional compounds with improved ability to kill selectively P-gp-expressing cells, but also with improved solubility properties. To understand how the structure of P-gp determines its polyspecificity and how specificity is altered with changes in folding, we have collaborated with other senior investigators in the LCB, including Di Xia, Suresh Ambudkar, and Sriram Subramaniam. Cryo-EM studies have demonstrated that apo P-gp has a dynamic structure in which the two ATP-binding sites are either separated or close together. Binding of ATP fixes the conformation of P-gp in the latter state and ATP hydrolysis results in separation of the ATP sites. Crystallography studies using mouse P-gp as a model show that the separation between the ATP sites determines the pitch of the transmembrane (TM) helices where substrates bind, suggesting the hypothesis that as the ATP sites move together and apart, the TM helices expose different residues that enable binding to many different substrates. Studies on mouse-human chimeric P-gps have revealed similar structure-function relationships for these two evolutionarily related transporters. In collaboration with the group of Suresh Ambudkar, we have examined the basis of directional transport of compounds out of cells by P-glycoprotein. These studies have revealed a set of amino acid residues in the transmembrane regions of P-glycoprotein which can be altered to change the direction of transport of certain rhodamine compounds from out of the cell to into the cell. This process is concentration- and ATP-dependent, and gives important insight into how directionality of transport is determined in P-glycoprotein. We have used AML as one model system to determine the clinical role of ABC transporters in drug resistance. In one study, samples from the same patients before and after chemotherapy were analyzed. In this case, resistance in each case shows a different pattern of expression of ABC genes and other MDR genes, suggesting that individualized approaches to resistance to therapy will be needed. A more detailed analysis of a large population of primary refractory AMLs has shown that there are 3 molecular signatures that predict poor response to therapy. One of these is associated with increased expression of ABCG2. These results argue that clinical samples must be stratified to facilitate effective targeting of inhibitors of ABC transporters to circumvent drug resistance.