ADVANCED TECHNOLOGY FOR ASSAYING CANCER-DRUG RESISTANCE

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 sens itive 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 det ect 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 i ncorporated into a commercial assay and has limited sensitivity (gt20%). 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 t argets 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 t he 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 red uced 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 co ntaining 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 mi lestone 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 th e 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 Biost atistics, and Boston University School of Public Health. We will also maintain a close contact during Phase II with two leading diagnostic companies, LabCorp