ADVANCED TECHNOLOGY FOR ASSAYING CANCER-DRUG RESISTANCE

描述(申请人提供)：在开发新一代分子靶向癌症药物方面取得了重大进展，其中许多药物现在才刚刚从流水线中出现。该项目旨在开发一种新的、高灵敏度的技术来检测在治疗前预先存在的或由于分子靶向抗癌药物(ACD)治疗施加的选择性压力而获得的蛋白质的耐药性突变。用小分子药物伊马替尼(Gleevec/Glivec/STI571)治疗慢性髓细胞白血病(CML)的患者产生耐药性就是这个问题的例证。有充分的证据表明，这种耐药性是由伊马替尼的靶点BCR-ABL酪氨酸激酶突变引起的。接受伊马替尼治疗的费城染色体阳性(Ph+)、急性淋巴细胞性白血病(Ph+ALL)和胃肠道间质瘤(GIST)患者也出现耐药。为了有效地检测和表征抗癌药物的耐药性突变，必须克服几个问题：i)突变的谱可以非常多样化，发生在药物结合口袋之外。在BCR-ABL激酶的情况下，突变出现在整个激活域，例如P-环(ATP结合)和A-环(调节区)。这可能需要对整个基因或特定部分进行DNA测序，以检测已知和未表征的突变的发生。然而，当将DNA测序纳入商业分析时，成本很高，而且灵敏度有限(&gt；20%)。Ii)有可能分离药物靶标蛋白或片段以进行功能和/或结构分析。然而，由于在粗制生物混合物中分析靶标和以纯形式分离靶标蛋白质方面的困难，这种分析受到阻碍。在这个项目中，通过使用AmberGen开发的新技术来分离高度纯化的药物靶向蛋白(S)的无细胞表达多肽片段，并使用基质辅助激光解吸电离(MALDI)质谱仪(MS)扫描技术检测特征耐药性突变，克服了这些限制。这种新的方法被称为抗癌药物突变耐药试验(DRACT-ACD)，其优势在于它允许以高灵敏度和高通量的低成本扫描突变(即使是那些以前未发现的突变)。在第一阶段取得的关键里程碑包括：i)证明了DARAIC-ACD可以检测到至少5%的灵敏度突变的BCR-ABL酪氨酸激酶；ii)成功地展示了PC-SNAG，这是一种捕获和光释放BCR-ABL酪氨酸激酶的无细胞表达片段的方法，显著降低了非特异性污染水平；iii)证明了DARAIC-ACD中的所有步骤都可以使用体外表达蛋白的珠状分选文库(BS-LIVE-PRO)进行多路复用；Iv)开发一种称为PC-Print的方法，使含有目标多肽的珠子能够直接转移到MALDI-MS靶标上进行直接分析。在第二阶段，我们将继续专注于开发和应用DARA-ACD来检测BCR-ABL酪氨酸激酶的突变。一个重要的里程碑将是证明在CML患者中以1%的敏感度检测这些突变的能力。这项研究将与医学和病理学副教授Adam Lerner博士合作进行，Adam Lerner博士是血液恶性肿瘤领域的领先专家，他将为我们提供CML和Ph+所有接受Imatinib治疗的患者的样本进行分析。所有结果将与生物统计学副教授Josée Dupuis教授和波士顿大学公共卫生学院合作进行统计分析。我们还将在第二阶段与两家领先的诊断公司LabCorp和Genzyme Genetics保持密切联系，这两家公司已经表示有兴趣将CARAME-ACD方法最终商业化。

DESCRIPTION (provided by applicant): Significant advances have been made toward the development of a new generation of molecularly targeted cancer drugs, many of which are only now emerging from the pipeline. This project aims to develop a new, highly sensitive technology for detecting drug-resistance mutations in proteins which preexist prior to treatment or are acquired due to the selective pressure exerted by treatment with molecularly targeted anti-cancer drugs (ACD). This problem is exemplified by drug resistance developed in patients treated for chronic myeloid leukemia (CML) with the small molecule drug Imatinib (Gleevec/Glivec/STI571). It is well documented that this resistance arises from mutations in BCR-ABL tyrosine kinase, the target for Imatinib. Drug resistance also occurs in both Philadelphia chromosome positive (Ph+) acute lymphatic leukemia (Ph+ ALL) and gastrointestinal stromal tumors (GIST) patients who are treated with Imatinib. Several problems must be overcome in order to effectively detect and characterize drug resistance mutations against anti-cancer drugs: i) The spectrum of mutations can be very diverse, occurring outside the drug binding pocket. In the case of the BCR-ABL kinase, mutations appear throughout the kinase domain, such as the P-loop (ATP binding) and A-loop (regulatory region). This can necessitate DNA sequencing of the entire gene or specific portions in order to detect the occurrence of both known and uncharacterized mutations. However, DNA sequencing is expensive when incorporated into a commercial assay and has limited sensitivity (>20%). ii) It is possible to isolate the drug target proteins or fragments to perform functional and/or structural analysis. However, such analyses are hindered by difficulties in assaying targets in crude biological mixtures and in isolating target proteins in a pure form. In this project these limitations are overcome by using novel technology developed by AmberGen for isolating highly purified cell-free expressed polypeptide fragments of the drug-targeted protein(s), and detecting characteristic drug resistance mutations using a matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) scanning technique. The new approach, termed drug resistance assay for mutations against anti-cancer drugs (DRAMA-ACD) has the advantage that it allows low-cost scanning for mutations (even those previously undiscovered) with high sensitivity and high throughput. Achievement of key milestones during Phase I included: i) the demonstration that DRAMA-ACD could detect mutant BCR-ABL tyrosine kinase at a sensitivity of at least 5%; ii) the successful demonstration of PC-SNAG, a method of capturing and photoreleasing cell-free expressed fragments of the BCR-ABL tyrosine kinase with significantly reduced levels of non-specific contamination; iii) the demonstration that all steps in DRAMA-ACD can be multiplexed using bead-sorted libraries of in vitro expressed proteins (BS-LIVE-PRO); iv) Development of a process known as PC-PRINT which enables beads containing the target peptides to be transferred directly to a MALDI-MS target for direct analysis. During Phase II, we will continue to focus on the development and application of DRAMA-ACD to detect mutations in the BCR-ABL tyrosine kinase. An important milestone will be a demonstration of the ability to detect these mutations in CML patients with a sensitivity of 1%. The research will be carried out in collaboration with Dr. Adam Lerner, Associate Professor of Medicine and Pathology, a leading expert in the area of hematologic malignancies, who will provide us with samples for analysis from CML and Ph+ ALL patients undergoing Imatinib treatment. All results will be statistically analyzed in collaboration with Prof. Jos¿e Dupuis, Associate Professor of Biostatistics, and Boston University School of Public Health. We will also maintain a close contact during Phase II with two leading diagnostic companies, LabCorp and Genzyme Genetics, who have expressed an interest in the ultimate commercialization of the DRAMA-ACD approach.