Department and institution: School of Medicine and Surgery, Department of Health Sciences, University of Milano Bicocca
Research Area(s): Target therapy
General research topic: TYROSINE KINASE INHIBITORS DIRECTED AGAINST
FUSION PROTEINS WITH KINASE ACTIVITY
ABL-inhibitors against ABL315 mutant form
(Redaelli S., Mologni L., Rostagno R., Viltadi M., Gambacorti-Passerini C.)
Despite the generally positive response of CML patients to imatinib, accumulation of mutations in BCR-ABL that lead to resistance to the drug constitutes a problem. The phenomenon of resistance in these patients has been linked specifically to the acquisition of one or more of point mutations that map to the ABL kinase domain of BCR-ABL. Although some of these mutations are located close to the imatinib-binding site, most of the mutations occur at distal positions. The T315I mutation is one of the most common mutations found in patients undergoing imatinib therapy and is responsible for 15% of resistant cases. The residue at this position is referred to as the "gatekeeper" residue in protein kinases, because it separates the ATP-binding site from an internal cavity that is of variable size in different protein kinases, and the nature of the gatekeeper residue is an important determinant of inhibitor specificity. To improve BCR-ABL inhibition, several inhibitors with higher potency compared with imatinib have been identified, including dasatinib, nilotinib and bosutinib (CR 2006). All of them show different degrees of activity against BCR-ABL point mutations, but only the Aurora kinase VX-680 was able to take the T315I mutants. With the present project, a series of bosutinib derivative designed with the help of the molecular modelling and the support
of Wyeth pharmaceuticals (NY, US) will be screened in vitro against T315I and the most promising, in vivo.
ALK and RET tyrosine kinase inhibitors
(Gunby R., Mologni L., Rostagno R., Tartari C., Viltadi M., Gambacorti-Passerini C.)
The NPM/ALK hybrid gene originates from a 2;5 chromosomal translocation that brings together the 5' part of the nucleophosmin (NPM) gene on chromosome 5q, with the catalytic domain of ALK, a receptor tyrosine kinase of unknown function. The kinase activity is clearly indispensable for its transforming ability. The understanding of the role of NPM/ALK in the pathophysiology of NHL has resulted in better diagnostic and prognostic tools. However, no innovative treatment modality originated so far from this knowledge. In this project, a collaborative effort (with L. Pinna and G. Zanotti [U of Padova] and L. Scapozza [U of Geneva]) will be undertaken which, through the generation of new knowledge on the function, structure and regulation of the kinase activity of NPM/ALK, will target NPM/ALK-positive NHL cells. This project will involve the study of NPM/ALK kinase activity, the crystallographic definition of the catalytic domain of the protein. Since no inhibitor of NPM/ALK exists we will also aim at the rational design and experimental validation of inhibitors based on the NPM/ALK structure and on the co-ordinates of other tyrosine kinases with significant homology. To resolute the structure of the catalytic domain of NPM/ALK several strategies are planned. In parallel with the structural analysis of the catalytic domain (CD), computer aided molecular modeling will be applied using the structure of tyrosine kinases possessing significant homology to ALK, like the chain of the insulin receptor; the known structure will be superimposed with the NPM/ALK sequence and a theoretical model developed. This project is aimed at developing baseline information on the type of molecule to be later used as parent inhibitor. The information obtained will be used to develop small molecules able to penetrate cells and specifically inhibit the CD of NPM/ALK. Molecules showing specific activity in vitro and on cultured cells will be tested in animal models, including NPM/ALK transgenic mice. RET is a transmembrane receptor tyrosine kinase expressed in neural crest-derived cells. It acts as a co-receptor of the GDNF family neurotrophic factors. Uncontrolled RET kinase activity, through either chromosomal rearrangement or point mutation, is the cause of papillary thyroid carcinoma and multiple endocrine neoplasia, respectively. Therefore, the block of RET activity should represent the best option for thyroid cancer therapy. The aim of this project is to identify and develop a selective small-molecule inhibitor of RET kinase activity. We are employing the baculovirus system to express recombinant kinase domain of RET in insect cells, for structural and pharmacological purposes. First, we want to determine the 3D structure of the catalytic portion of RET, aiming at the rational design of a specific inhibitor. Secondly, we will use the recombinant protein in a high-throughput screening of small molecules with possible inhibitory activity over RET kinase. Compounds that will show promising activity on the purified protein will be then tested in RET-transformed cells and in mice.
Use of new tyrosine kinase inhibitors to overcome imatinib resistance in patients
(Meneghetti I., Tornaghi L., Dilda I., Puttini M., Franceschino A., Gambacorti-Passerini C.)
Imatinib resistance is defined using several clinical, hematological or molecular criteria. The global clinical trial (IRIS study) on imatinib efficacy counts about 30% of imatinib-therapy discontinuation due to resistance. Several novel CML therapies have been designed to inhibit wild-type and mutant forms of BCR-ABL and other molecular targets. These agents include dasatinib, nilotinib, bosutinib and INNO-406.
While nilotinib and INNO-406 target the inactive conformation of BCR-ABL, like imatinib, dasatinib and bosutinib target the dual inactive and active conformation showing great activity against the mutants. At difference with imatinib and dasatinib, no substantial inhibition of KIT and PDGFR was noted with SKI-606,
thus placing this molecule in a class of its own (dual Src-Abl inhibitor devoid of activity on receptor tyrosine kinases). This different selectivity could result in fewer side effects than imatinib because many toxicities associated with imatinib (e.g., edema, muscle cramps, skin rash, pigmentation, endocrine abnormalities, and low-grade inhibition of normal hemopoiesis) can be tracked to the inhibition of PDGFR and/or KIT. A phase I/II study of bosutinib (SKI-606) in Philadelphia chromosome positive leukemias is ongoing at the CRU.
ILTE - Imatinib Long Term side-Effects study
(Tornaghi L., Puttini M., Antolini L., Valsecchi G., Gambacorti-Passerini C.)
The ILTE study is an observational protocol on the long term side effects of imatinib in CML patients in cytogenetic remission. The development of imatinib as an inhibitor of BCR/ABL, the causative event of Chronic Myeloid Leukemia (CML), has substantially changed the treatment of this disease and the long term prospects for CML patients. Imatinib possess three characteristics: - has quasi-specific activity toward its target, the oncogenic tyrosine kinase BCR/ABL, - is directed against an early event in malignant transformation, - the targeted enzyme plays a causative role in the treated disease (CML). Imatinib treatment produces Complete Cytogenetic Remission (CcyR) in 75-80% of newly diagnosed CML patients, with an yearly risk of relapse of approximately 1% and a progression free survival at 5 years >90%; therefore CML patients now enjoy new prospects for long-term survival. However the usual persistence of low levels of leukemic cells (< 1/100) as detected by Polymerase Chain Reaction (PCR) assay, requires at the moment the indefinite continuation of treatment and therefore the possible development of long-term side effects. This international project will be focused on two main and related goals: - it will evaluate the occurrence of long term side effects, including the development of second cancers, utilizing the oldest cohort CML patients treated with imatinib and using both a retrospective and a prospective collection of data; - it will identify among patients with negative PCR results, those that can be considered "true negative", in whom imatinib could be stopped. This second goal stems from very recent experimental data and will utilize a new technique called Microfluidic Card System PCR. ILTE study includes centers from all over the World (Italy, Canada, US, Mexico, Brazil, Nigeria, Europe, Korea) and has a dedicated website (www.ilte-cml.org)