- AIRC - Associazione Italiana per la Ricerca sul Cancro PROPOSAL FORM 2007

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1 - AIRC - Associazione Italiana per la Ricerca sul Cancro PROPOSAL FORM 2007 Via Corridoni, MILANO tel.02/ fax 02/ airc.direzione-scientifica@airc.it per informazioni tecniche tel.02/ Codice Riferimento: 4121 Page 1 of 44

2 ASSOCIAZIONE ITALIANA PER LA RICERCA SUL CANCRO TITLE PAGE Grant Proposal 2007 Codice Riferimento: 4121 Principal Investigator's full Name and Qualification Professore Martinelli Giovanni - Professore associato Proposal Title Mechanisms responsible for sensitivity and resistance of Ph-positive cells to tyrosine kinase inhibitors Type of Grant IG Area Sub Area Targeted Therapy Budget 2007 (euro): ,00 Estimated budget (euro): ,00 Ente/Università Università di Bologna Dipartimento/Istituto/altro Istituto di Ematologia e Ocologia Medica "L. e A. Seràgnoli" - Indirizzo Via Massarenti, 9 Città + ZIP Code BOLOGNA (BO) Telefono gmartino@kaiser.alma.unibo.it Fax Authorized Administrative Official Prof. Pier Luigi Lollini- Centro Interdipartimentale Ricerca sul Cancro "Giorgio Prodi" Address Via Massarenti, 9 City + ZIP Code BOLOGNA (BO) Phone sec.94@dipartimenti.unibo.it Fax Proponent's signature Martinelli Giovanni Authorized Administrative Official's signature Prof. Pier Luigi Lollini- Centro Interdipartimentale Ricerca sul Cancro "Giorgio Prodi" Date 15/12/2006 Date 15/12/2006 Codice Riferimento: 4121 Page 2 of 44

3 EVALUATION FORM (selection ) Principal Investigator's Full Name Professore Martinelli Giovanni - Professore associato Total Papers and Reviews 75 Total IF 386,93 Average IF 5,2 Papers First/Last or Corresponding Author 41 Total IF 222,884 Average IF 5,4 Codice Riferimento: 4121 Page 3 of 44

4 ABSTRACT Principal Investigator's Full Name Professore Martinelli Giovanni Institution and City Università di Bologna BOLOGNA Proposal Title Mechanisms responsible for sensitivity and resistance of Ph-positive cells to tyrosine kinase inhibitors Area Targeted Therapy Sub Area Abstract in the next page Codice Riferimento: 4121 Page 4 of 44

5 Imatinib mesylate is now the first-choice agent in the treatment of chronic myeloid leukemia (CML) patients. Imatinib is a potent and selective inhibitor of the Bcr-Abl tyrosine kinase, whose deregulated activity is the main pathogenetic mechanism of CML and Philadelphia (Ph)-positive acute lymphoblastic leukemia (ALL). However, imatinib therapy is extremely expensive and is not successful in all patients due to the emergence of resistance which ultimately leads to clinical relapse and may contribute to disease progression. Basic and clinical research have to focus now on the understanding and overcoming of imatinib resistance in order to offer each single CML/Ph+ ALL patient a real "tailored" therapy. Thus, the present research programme aims at the optimization of the therapy of Ph-positive leukemias through a research strategy acting at different levels and addressing all the main aspects related to drug-sensitivity and drug-resistance to imatinib, namely through: - the optimization of the molecular monitoring of patients treated with imatinib or novel inhibitors, in order to provide clinicians with a comprehensive panel of biological indicators allowing to evaluate the real efficacy of the treatment and to rationally and timely (re-)assess the therapeutic strategy; - the characterization of new mechanisms of resistance to imatinib which have been poorly or never investigated so far; - the identification of novel targets for a molecular therapy of those patients for whom inhibition of Bcr-Abl turns out to be insufficient; - the assessment in vitro in pre-clinical models (cell lines, primary patient cells and mouse models) of novel inhibitors or novel inhibitory strategies targeting Bcr-Abl itself, key signal transduction molecules downstream of Bcr-Abl, or proliferative and antiapoptotic processes in general; - the assessment in vivo, in newly diagnosed patients or in patients already known to be resistant to imatinib, of those second-generation inhibitors which are already in clinical phase; - the identification and validation of novel biological factors which may serve as predictors of drugresponse and drug-resistance. Codice Riferimento: 4121 Page 5 of 44

6 PROPOSAL MAIN BODY Background and rationale: The Ph chromosome resulting from the translocation t(9;22) (q34;q11) represents the most common cytogenetic abnormality detected in human leukemias. It is considered the hallmark of chronic myeloid leukemia (CML), but can be detectable in 20-30% of adult acute lymphoblastic leukemia (ALL) and in 4% of pediatric ALL, respectively. At the molecular level, the t(9;22) translocation parallels the fusion of the proto-oncogene ABL with the BCR gene, resulting in the production of the chimeric BCR/ABL gene, which leads to the synthesis of the fusion proteins (p210, p190) which show a constitutive tyrosine kinase activity[1, 2]. Although the BCR/ABL hybrid gene is characterized by a restricted number of molecular variants, the two main diseases, CML and ALL, marked by this molecular rearrangement are different in terms of clinical and hematologic characteristics. CML is a chronic myeloprolipherative disorder due to the massive expansion of BCR/ABL+ myeloid cells, which, during the chronic phase, maintain the capacity to differentiate normally. The progressive loss of normal differentiation capacity results in disease progression to an acute leukemia or blast phase, which may show a myeloid (70%) or lymphoid phenotype. Ph+ and/or BCR/ABL+ ALL (almost all of the B-lineage) is, from the beginning, a disease with a very aggressive behavior and poor prognosis. Turning off the aberrant tyrosine kinase activity of this fusion protein means suppressing Ph+ cells. Therefore, Ph+ leukemic cells represent the ideal target for tailored therapies based on tyrosine kinase inhibitors. Imatinib mesylate (IM) was developed as the first molecularly targeted therapy that specifically inhibits the Bcr-Abl tyrosine kinase activity[3-9]. IM interacts with the ATP-binding site of ABL only when the latter is in its inactive conformation. In this way, IM is able to prevent the conformational transition to the active form, which is responsible for binding and/or phosphorylation of signal transduction molecules. Due to its excellent hematologic and cytogenetic responses, particularly in patients with chronic phase CML, imatinib has moved towards first-line treatment for newly diagnosed CML. Despite the high rates of complete cytogenetic response, resistance to the drug has been frequently reported[10-13]. Depending on the time of onset, two categories of resistance can be distinguished: if there is no response after initial treatment, resistance is described as primary or intrinsic, in contrast, secondary or extrinsic resistance is present if resistance develops after achieving an objective response. The mainly mechanisms of resistance are the following: Point mutations: In the majority of cases, resistance is caused by reactivation of BCR-ABL tyrosine kinase activity due to the emergence of specific point mutations within several critical regions of the Abl kinase domain [14-20]. Such mutations impair IM binding either by affecting critical contact residues or Codice Riferimento: 4121 Page 6 of 44

7 by inducing a BCR-ABL conformation to which IM is unable to bind. More than 30 different point mutations encoding for distinct single amino acid substitutions in the BCR-ABL kinase domain have been identified in relapsed CML patients and Ph+ ALL. Different mutants seem to have different degrees of resistance to imatinib: in vitro data indicate that while some mutations might be overcome by dose escalation [21], other confer a highly resistant phenotype, thereby suggesting withdrawal of imatinib in favour of alternative therapeutic strategies. Indeed, since resistance often coincides with reactivation of the kinase activity within the leukemic clone, either Bcr-Abl itself or Bcr-Abl-triggered downstream signalling pathways remain good targets for molecular therapy. Several novel second-generation inhibitors [22] have been synthesized and are now being evaluated in international phase I-II trials. They include novel Bcr-Abl inhibitors like nilotinib (AMN- 107)[23], dual Src/Abl inhibitors like dasatinib (BMS )[23-25] and SKI-606 [26], proteasome inhibitors like bortezomib (PS-341)[27]. Pre-clinical studies have already assessed IC50 values of several novel inhibitors against almost all mutant forms of Bcr-Abl, showing a precise spectrum of sensitivity against some mutants and resistance against others, and have also hypothesized novel inhibitor-specific mutants which are likely to emerge, though all these findings have not been confirmed in patients. Only one specific mutant (i.e., the T315I) will remain highly problematic for clinicians since (a) it is highly resistant to imatinib as well as to almost all novel Abl or Src/Abl inhibitors, including dasatinib and nilotinib [23], the closest to FDA and EMEA approval; (b) it seems to be associated with a highly aggressive disease phenotype and particularly poor prognosis. Therefore, recent studies [28, 29] have shown that MK-0457 (VX-680), a smallmolecule aurora kinase inhibitor, has in vitro activity against the T315I-Bcr-Abl. Our purpose is to confirm the efficacy of MK-0457 in vitro against the T315I-Bcr-Abl and furthermore to investigate its efficacy, in vivo, in patients with CML or Ph+ ALL carrying the T315I-Bcr-Abl mutation. BCR-ABL gene amplification Resistance to IM can also be caused by over-expression of the Bcr-Abl protein due to gene amplification of the BCR-ABL gene. This mechanism, observed in a proportionally small number of imatinib-resistant patients, was initially described in the LAMA84R cell line with a 4.6-fold increase in mrna level. Abnormal signal transduction pathways Bcr-Abl exerts its oncogenic effects in CML cells essentially by stimulating cell proliferation, inhibiting apoptosis and altering cell adhesion to bone marrow stroma. Signal transduction cascades involved in these cellular processes and activated by Bcr-Abl include, among others: Codice Riferimento: 4121 Page 7 of 44

8 - Ras; - mitogen-activated protein kinase (MAPK) and its downstream effectors MEK and Erk; - phosphatidylinositol-3 kinase (PI3K) and its downstream effector Akt; - phospho-tyrosine-phosphatase (PTP ). PTPs proteins play critical roles in many cellular process as the gene expression, the regulation of the cell growth, proliferation, differentiation, cell cycle, and cell movement by the counterbalance of the effect on the cell growth of the protein tyrosine kinases (PTKs). Previous reports have demonstrated that some phosphatases have an oncosuppressive role in Ph+ cells [30, 31]. This suggest that PTP plays a critical role in the pathogenesis of CML and that it could have a oncosuppressor effect in vivo. Deletions of sequences located on chromosome 9 Deletions of sequences located on chromosome 9 close to ABL gene have been recognized as a key factor for progression of CML and for reduced responsiveness to Imatinib and other therapy. To address this issue we will screen the Ph-positive CML genome through the array-cgh (comparative genomic hybridization) provided by Nimblegen System. Comparative Genomic Hybridization (CGH) measures DNA copy number differences between a reference genome and your sample genome. ABC transporters over-expression Blood and tissue concentrations of most drugs are influenced by interindividual variations in genes encoding drug metabolizing enzymes (DMEs) and drug transporters. Cytochrome P450 enzymes (CYPs) are thought to have evolved as a protective adaptive response against environmental toxic effects. Imatinib is metabolized mainly by CYP3A4 and CYP3A5 isoforms, and to a lesser extent by CYP1A2, CYP2D6, CYP2C19. Some imatinib metabolites have been shown to be produced by CYP1A1 and CYP1B1. Imatinib transport into cells has been shown to be mediated by hoct1 (human Organic Cation Transporter, isoform 1), also known as SLC22A1 (Solute Carrier family 22, member 1) [32]. The OCT family mediates electrogenic and sodium-independent translocation of organic cations or weak bases, i.e., molecules with a transient or permanent positive net charge at physiological Ph, in both directions across the plasma membrane. The family comprises three members (hoct1, hoct2 and hoct3 also known as EMT, Extraneural Monoamine Transporter) differing in tissue distribution and substrate specificity. Pre-imatinib hoct1 expression levels have been demonstrated to be significantly lower in patients who remain >65% Philadelphia-chromosome positive by cytogenetics during the first 10 months of Codice Riferimento: 4121 Page 8 of 44

9 imatinib treatment[33]. In contrast, imatinib efflux is thought to be mediated by ABC transporters ABCG2 (also known as BCRP) and ABCB1 (MDR1; P-glycoprotein)[4, 34, 35]. The MDR-1 gene is commonly over-expressed in blast cells of patients in the advanced phase CML [35, 36]. Genomic instability BCR-ABL has been associated with genomic instability, which may have particular relevance during disease progression from chronic phase to accelerated and blast phase CML. Recent findings have proposed that most protein-enconding genes may be regulated by small RNAs that can specifically control transcript turnover (sirna) and/or protein translation (mirna). In the latter case mirna can do such a job by blocking access or sliding of ribosomes to mrnas, thus impeding translation. Alternative or aberrant spliced transcripts Alternative splicing is the process whereby identical pre-mrna molecules are spliced in different ways, and this is important in normal development as a means of creating protein diversity in complex organisms[37]. It is evident that alternative splicing plays a key role in biology: tissuespecific and developmental stage-specific alternative splicing contributes to significant protein diversity; disease-related deregulation of splicing may be critical in pathogenesis and contribute to disease diversity and complexity. Pre-mRNA splicing is a sophisticated and ubiquitous nuclear process, which is a natural source of cancer-causing errors in gene expression. Intronic splice site mutations of tumor suppressor genes often cause exon-skipping events that truncate proteins just like classical nonsense mutations. Spliced isoforms lacking critical N-terminal zinc-finger of the Ikaros transcription factor act as dominant negatives by binding long isoforms through the C-terminal zinc-finger domain. Forced expression of short isoforms (Ik4-Ik8) in murine or human hematopoietic progenitors arrest lineage commitment and differentiation and play a role in the development of haematological malignancies, such as ALL and crisis blastic CML[38, 39]. BCR-ABL1 kinase activity is also linked to the expression of a truncated isoform of the adaptor protein SLP-65 and of the Bruton tyrosine kinase (BTK)[40], which may contribute to the compromised pre-bcr signalling in ALL. Src-family kinases Another resistance mechanism could be the compensation of loss of BCR-ABL signalling by other tyrosine kinase-mediated pathways. Src-family kinases such as Lyn are involved in BCR-ABLmediated leukemogenesis and have been found to be up regulated in cultured CML cells selected Codice Riferimento: 4121 Page 9 of 44

10 for IM resistance; increased Lyn expression was also found to correlate with clinical evidence of IM resistance in some patients, which highlights the potential clinical relevance of this resistance mechanism [20, 21]. The challenge for the future is to improve current clinical results with tyrosine kinase inhibitors in Ph+ leukemias, developing strategies that can eradicate residual disease and overcome or prevent resistance. Description of the project: The present research program can be subdivided in four tasks: 1) Collection and storage of biological material for molecular and cellular studies. The Research Unit-Martinelli belongs to the Institute of Hematology and Medical Oncology "Seràgnoli" which is the leader of the GIMEMA Working Party on CML, and as such, is coordinating or participating in several national and international clinical trials with imatinib and novel inhibitors. Upon written informed consent of the patients enrolled in these trials, the Research Unit will create a precious bank of: a) biological samples, regularly collected after written informed consent of the patient, processed and stored at baseline and at regular time points during treatment; material includes mononuclear cells and CD34+ cells, protein lysates, RNA, DNA. b) clinical data, accurately recorded in an electronic format at baseline and at regular timepoints during treatment and available for correlations between biological findings and clinical outcome. Mechanisms of CML and Ph+ ALL resistance will be also investigated on all resistant patients treated with IM or other tyrosine kinase inhibitor not in the framework of clinical trials upon written informed consent. The biological material collected with the cell lines and with the mouse models will create a solid basis for all the planned studies. 2) Optimization of molecular monitoring of patients treated with imatinib and with novel inhibitors, in order to evaluate the actual efficacy of the treatment and to rationally and timely (re-)assess the therapeutic strategy. a) Screening for ABL KD mutations The molecular biology laboratory of the present research unit has already developed a rapid and reliable screening method for Abl KD mutations based on a novel high-throughput denaturing-high performance liquid chromatography (D-HPLC) device with UV detector (D-HPLC 3500HT; Codice Riferimento: 4121 Page 10 of 44

11 Transgenomic), which is now available to all the Institutions of the GIMEMA Working Party on CML. D-HPLC is a reversed-phase, ion-pairing HPLC, specifically developed for the detection of DNA sequence variations such as point mutations, small insertions and deletions. Under conditions of partial heat denaturation, heteroduplexes that form in PCR samples with internal sequence variations display reduced column retention with respect to their homoduplex counterparts. Therefore, the elution profiles for such samples are distinct from those with a homozygous sequence, making the identification of samples harbouring mutations a rapid (~3 min) and straightforward procedure. Samples scored positive by D-HPLC are then sequenced in order to characterize the exact nucleotide substitution. The present unit will implement the protocol of mutation screening by setting up and standardizing a mutation detection method based on a D- HPLC equipped with a novel fluorescence detector, allowing to increase the sensitivity of detection of at least tenfold, from 5% to %. b) A T315I-specific assay Given the importance of an early detection of T315I-positive mutant clones which render imatinib, dasatinib and nilotinib treatment ineffective and have been associated with a highly aggressive disease phenotype, we will also develop a sensitive and straightforward diagnostic method specific for T315I allowing for a rapid and accurate detection of this mutation. To this purpose, we will setup three different approaches, which will be tested and compared in terms of sensitivity, specificity and reliability: - a conventional allele-specific oligonucleotide (ASO)-PCR approach, with forward primers designed on BCR sequences (exon 1 for p190, exon 13 for p210) and two sequence-specific reverse primers, one for the wild-type and one for the mutant ABL sequence, followed by gelelectrophoresis analysis of amplification products; - an allelic discrimination assay based on two fluorescent probes specifically designed to anneal to the wild-type and mutated sequence, during a multiplex Q-PCR reaction on the ABI-PRISM 7900 (Applied Biosystems); - an assay based on PCR amplification of a Bcr-Abl fragment encompassing codon 315 followed by denaturation at 96 C, gradual reannealing at room temperature, digestion with Surveyor Nuclease (Transgenomic) which cuts heteroduplexes if and where a mismatch exists, and separation of cut or uncut fragments by gel-electrophoresis or by D-HPLC elution. c) Molecular monitoring of the BCR-ABL transcript levels and of the eventual BCR-ABL over expression, so as to determine whether resistance may be caused also by gene-dosage effects. Codice Riferimento: 4121 Page 11 of 44

12 BCR-ABL transcript levels will be monitored by real-time Taqman RT-PCR; we will determinate if there is an amplification of BCR-ABL by fluorescence in situ hybridization (FISH). d) Analysis of the karyotype of Ph+ cells, so as to identify the additional cytogenetic abnormalities responsible for resistance. These additional cytogenetic abnormalities associated with resistance will be investigated both by conventional and by molecular (FISH, fluorescence in situ hybridization) cytogenetic techniques. 3) Characterization of mechanisms of resistance to imatinib and novel TK inhibitors. a) Abl KD mutations in patients resistant to imatinib treated with dasatinib, nilotinib, SKI- 606 and MK-0457 In order to assess the degree of sensitivity of various Abl KD mutations in vivo in patients treated with novel tyrosine kinase inhibitors, as well as the likelihood of emergence of novel, inhibitorspecific mutant forms, we will regularly perform mutation monitoring of patients resistant to or intolerant of imatinib treated at the "Seràgnoli" Institute with dasatinib, nilotinib, SKI-606 and MK Patients will be analyzed at baseline and every month thereafter, in order to follow the kinetics of disappearance of pre-existing mutated clones or the kinetics of selection of novel ones. Mutations observed in vivo will be assessed for their biological-structural effects on Bcr-Abl, on its tyrosine kinase activity, on the interaction with downstream signaling molecules and with inhibitors themselves again using specific computer-assisted molecular simulation techniques. We will also develop softwares allowing to rapidly and reliably predict the effects of any reported or unreported mutation on the novel tyrosine kinase inhibitors currently under development or still in a preclinical phase. b) Analysis of signal transduction pathways regulated by PTP This will be accomplished following two complementary approaches: - Identification of substrates We plan to analyze selected p210 BCR/ABL substrates in order to link the observed effect to a molecular mechanism. Whether the dephosphorylation of p210 BCR/ABL or other potential substrates is directly or indirectly mediated by PTP will be investigated by the "substrate trapping" approach, as binding of substrates to active PTPs is usually too weak to allow the detection of their interaction using standard co-immunoprecipitation protocols. This technique is based on the observation that PTPs are defined by the presence of a signature sequence motif, Codice Riferimento: 4121 Page 12 of 44

13 [I/V]HCXXGXXR[S/T]. It is possible to obtain PTPs that maintain a high affinity for substrate but do not effectively catalyze dephosphorylation, thus converting an extremely active enzyme into a substrate trap. We will obtain the mutant cdna (D198A-PTP ), now sequencing for the quality control, and we plan to use it for transfection experiments. Methods: Cells transfected with PTP cdna WT or D198A-PTP will be lysed with cell disrupting medium capable to separate specific cellular fractions (FOCUS Phosphorich and Fraction FOCUS kits from Geno-Tech, St. Louis, MO) and immunoprecipitation. Proteins linked to TAT-mut-ICD will be analyzed by SDS-PAGE/ Western Blotting, 2-DGE or directly subjected to 2DC-MS/MS analysis. We will analyze immunoprecipitated protein. D198A-PTP will be also immunoprecipitated and analysed by western blotting with specific antibodies in order to identified proteins interacting with PTP. - Global analysis of gene expression Microarray analysis is a powerful tool that enables to monitor the expression profile of thousands of genes in a single assay. In this way it is possible to affiliate a specific gene expression profile to a certain cell line or patient's sample and so potentially to identify the genes associated to a particular phenotype or disease. We plan to study the effect of PTP expression in K562 stable transfectants in the presence or absence of Imatinib. A total of 12 hybridization reactions are planned (triplicate assays for each of the four experimental conditions planned). Methods: For isolation of total cellular RNA, 5x10(6) cells, cultured in the absence or presence of the inducing agents indicated, will be harvested and RNA will be prepared using standard methods. The cdna hybridization and the collection and analysis of the data will be performed by CRIB, Padova, Italy (visit for details). Validation of identified targets will be performed by QPCR followed, whenever possible, by immunodetection. c) Expression and genotyping of single nucleotide polymorphisms (SNPs) in imatinib transporters and metabolizing enzymes as predictors of outcome of CML patients Since interindividual variation(s) in these genes encoding may influence - and may therefore allow to predict - the outcome of CML patients treated with tyrosine kinase inhibitor, our aim is to correlate SNPs in key genes to imatinib efficacy and to identify one or more SNPs with predictive value allowing to optimize the therapeutic use of imatinib in CML patients. To this purpose, we will assess a set of SNPs as predictors of imatinib efficacy. To this purpose, we will focus on key genes encoding for: DMEs (CYP3A4, CYP3A5, CYP1A1, CYP1B1, CYP2D6, CYP2C19), transporters Codice Riferimento: 4121 Page 13 of 44

14 (hoct1, ABCB1, ABCG2). Genomic DNA will be isolated from 1-2 cc of peripheral blood or bone marrow samples by conventional methods. Polymorphisms will be analyzed by polymerase chain reaction (PCR) combined with restriction fragment length polymorphisms (RFLP) assay. d) Study on promoter hypomethylation of the LINE-1 retrotransposable elements which activates sense/antisense transcription and marks the progression of CML The main aim of this part of the proposal is to identify mirnas that can specifically interfere with BCR and ABL. This will be achieved though bioinformatic tools that scan the human genome for short DNA sequences with complete or partial homology to the BCR or ABL gene. One of such programs is provided freely by the Computational Biology Center of Memorial Sloan-Kettering Cancer Center. Putative mirnas that are predicted to affect BCR and ABL regulation will be cloned, expressed in human cells and tested for the ability to negatively affect BCR and ABL translation. Those that will be found positive to the assay will also be used either alone or in combination to affect translation of the chimeric BCR-ABL transcript. e) Gene expression profiling In order to identify molecular pathways that may be down/up-regulated by exposure to novel tyrosine kinase inhibitors, we will perform a DNA microarray analysis on cell lines and on CD34+ cells from imatinib-resistant CML patients before and after treatment with other inhibitors, such as MK This approach will allow us to elucidate the mechanisms responsible of the potential efficacy of MK-0457 in CML cells. We will also use this gene expression profiling strategy to identify a genomic profile that may be associated with sensitivity or resistance to MK As the median CD34+ cells number in CML patients at diagnosis is usually between 0,5 and 1x10 6 (0,1% of mononucleated cells), we should expect to obtain a wide range of RNA quantity, whose minimum will be presumably around 1,5 g (diluted in 30 l). Therefore, a double RNA amplification should be planned, in order to obtain a sufficient crna quantity to efficiently hybridize the Affymetrix chip. Methods will be as follows: a. CD34+ cell fraction separation: Mononuclear cells from PBL or BM samples will be isolated by density gradient centrifugation (Ficoll separation), and CD34+ cell fractions will be selected by binding to immunomagnetic beads (AutoMACS; Miltenyi Biotech). b. RNA extraction: Total RNA will be extracted from CD34+ cells using the Qiagen RNeasy Mini or Micro Kit (Qiagen);.RNA quantity will be assessed by Nanodrop analysis (Nanodrop Codice Riferimento: 4121 Page 14 of 44

15 Technologies), while the Bioanalyzer (Agilent) will be used to assess the quality of the RNA before starting with the synthesis of crna. c. Synthesis of Biotin-Labeled crna: Biotin-labeled target synthesis reactions will be performed using standard protocols supplied by the manufacturer (Affymetrix). Briefly, 100ng of the RNA will be converted into double-stranded cdna by reverse transcription using the cdna synthesis kit, following the protocol supplied by the manufacturer, with a T7-(dT) 24 primer (Affymetrix). After the second-strand synthesis, crna will be generated from the purified cdna sample (Gene Chip Sample Cleanup Module; Affymetrix) by an in vitro transcription reaction (MEGAscript T7 Kit, Ambion). The crna will be then purified (Gene Chip Sample Cleanup Module; Affymetrix) and a second cycle of retrotransciption, second-strand synthesis and sample cleanup will be performed, followed by a final Biotin-Labeled crna synthesis (GeneChip IVT Labeling Kit, Affymetrix). The labelled crna will be purified using the Affymetrix spin columns and the concentration of biotin-labeled crna will be determined by Nanodrop, while the quality check will be performed by means of the Bioanalyzer. d. Hybridization: 5 g of each biotinylated crna preparation will be fragmented and put in the hybridization cocktail. Samples will be hybridized to Affymetrix HG Plus Gene Chip Arrays for 16 hours. Gene chips will be than washed and stained following the instruments standard Eukaryotic GE WS2v4 protocol and using antibody-mediated signal amplification. e. Data analysis: The images from the scanned chips will be processed by means of Affymetrix Microarray Analysis Suite 5.0 (MAS 5.0). The amount of a transcript mrna will be determined with the MAS 5.0 absolute analysis algorithm, as well as the presence or the absence of a transcript. The identification of differentially expressed genes and the patients clustering will be performed with GeneSpring 7.3 software (Silicon Genetics, Redwood City, CA). The identification of biologic processes, molecular functions and cellular components of genes will be assessed by EASE and Ingenuity softwares. f) Analysis of alternative or aberrant spliced transcripts in Ph+ ALL and CML patients Studies in human Ph+ positive leukemias have gained newinsight into the essential role of truncated protein isoforms in the pathogenesis and disease progression of CMl and BCR-ABL positive ALL, but many challenges remain. Our aim is to analyze if leukemia-specific alternative or aberrant splicing in BCR-ABL or other genes involved in BCR-ABL signaling such as Ikaros, BTK, SPL65 and other could be associated with resistance to imatinib or new tyrosine kinase inhibitors (TKI) such as dasatinib or nilotinib. We will perform a GeneChip Exon Array System, which are the first experimental tools available to survey both gene expression and alternative splicing patterns on Codice Riferimento: 4121 Page 15 of 44

16 the whole-genome scale on a single array. This approach will deepen the understanding of biology for many discovery-focused applications including: analysis of molecular mechanisms regulated by alternative splicing; discovery of new splice variants of drug targets and mapping of their tissuespecific expression; improved understanding of the downstream effects of genetic variations that may result in phenotypic changes in gene expression or alternative splicing patterns. We will validate our results performing reverse transcription-polymerase chain reaction (RT-PCR) and nucleotide sequencing. 4) In vivo testing, in patients with newly diagnosed CML o in patients resistant to imatinib at standard dose, of imatinib at high doses or second-generation inhibitors already in clinical phase The feasibility and efficacy of therapeutic strategies alternative to the administration of imatinib at standard dose will be tested in vivo in patients with CML or Ph-positive ALL. As the coordinator of the GIMEMA Working Party on CML, the "Seràgnoli" Institute is leading and will lead two clinical trials with imatinib at high dose, namely: a) CML/021/STI571: newly diagnosed CP patients, high Sokal risk, treated with imatinib 800 mg/d; b) CML/022/STI571: newly diagnosed CP patients, intermediate Sokal risk, randomized to receive either imatinib 400 mg/d or imatinib 800 mg/d. Moreover, the "Seràgnoli" Institute is actively participating and will participate in several international phase I-II clinical trials with novel tyrosine kinase inhibitors, namely: c) CA180005, CA180006, CA180013, CA180015, CA180035, phase II trials with dasatinib in CML and Ph+ ALL patients resistant to or intolerant of imatinib; d) CAMN107A2101, a phase II trial with nilotinib in CML and Ph+ ALL patients resistant to or intolerant of imatinib; e) 3160A4-200-WW, a phase I-II trial with SKI-606 in CML and Ph+ ALL patients resistant to or intolerant of imatinib. In addition to these ongoing trials, additional trials are planned to be activated by the GIMEMA Working Party on CML itself by the end of 2006, namely: f) a phase II trial with nilotinib administered first-line in newly diagnosed CP patients; g) a phase I-II trial with bortezomib in CML patients resistant to imatinib; h) a phase II trial with homoarringtonine in CML patients resistant to imatinib with evidence of the T315I mutation. Codice Riferimento: 4121 Page 16 of 44

17 Based on the results of some of the studies described in the sections above, we hope to identify several biological predictive factors, potentially useful to predict the likelihood of response or resistance to imatinib therapy. The whole project will be performed in three years according to the phases and the timing specified in the scheme below. References: 1. Bartram C.R. dka, H.A., et al., Translocation of c-abl oncogene correlates with the presence of a Philadelphia cromosome in chronic myelocytic leukaemia. Nature, : p Lugo TG, P.A., Muller AT, et al., Tyrosine kinase activity and transformation potency of bcr-abl oncogene products. Science, : p Codice Riferimento: 4121 Page 17 of 44

18 3. Talpaz M, S.R., Baccarani M, et al., Imatinib induces durable hematologic and cytogenetic responses in patients with accelerated phase chronic myeloid leukemia: results of a phase 2 study. Blood, : p Crossman LC, D.B., Deininger MW, Pirmohamed M, Wang L, Clark RE., hoct 1 and resistance to imatinib. Blood, : p Kantarjian H, S.C., Baccarani M, et al., Hematologic and cytogenetic responses to imatinib mesylate in chronic myelogenous leukemia. N Engl J Med, : p Ottmann OG, D.B., et al., A phase 2 study of imatinib in patients with relapsed or refractory Philadelphia chromosome-positive acute lymphoid leukemias. Blood, : p Rosti G, T.E., et al., Risk and early cytogenetic response to imatinib and interferon in chronic myeloid leukemia. Haematologica, : p Baccarani M, M.G., et al., Imatinib and pegylated human recombinant interferon-alpha2b in early chronic-phase chronic myeloid leukemia. Blood, : p Rosti G, M.G., et al., Molecular response to imatinib in late chronic-phase chronic myeloid leukemia. Blood, : p O'Brien SG, G.F., Larson RA, Gathmann I, Baccarani M, et al., Imatinib compared with interferon and low-dose cytarabine for newly diagnosed chronic-phase chronic myeloid leukemia.. N Engl J Med, : p Kantarjian HM, C.J., O'Brien S, Luthra R, Giles F, Verstovsek S, Faderl S, Thomas D, Garcia-Manero G, Rios MB, Shan J, Jones D, Talpaz M., Long-term survival benefit and improved complete cytogenetic and molecular response rates with imatinib mesylate in Philadelphia chromosome-positive chronic-phase chronic myeloid leukemia after failure of interferon-alpha. Blood, : p Iacobucci I, R.G., Amabile M, Poerio A, Soverini S, Cilloni D, Testoni N, Abruzzese E, Montefusco E, Ottaviani E, Iuliano F, Russo D, Gobbi M, Alimena G, Martino B, Terragna C, Pane F, Saglio G, Baccarani M, Martinelli G., Comparison between patients with Philadelphia-positive chronic phase chronic myeloid leukemia who obtained a complete cytogenetic response within 1 year of imatinib therapy and those who achieved such a response after 12 months of treatment. JOURNAL OF CLINICAL ONCOLOGY, : p Martinelli, G., et al., Prediction of response to imatinib by prospective quantitation of BCR- ABL transcript in late chronic phase chronic myeloid leukemia patients. Ann Oncol, (3): p Gorre ME, M.M., Ellwood K, Hsu N, Paquette R, Rao PN, Sawyers CL., Clinical resistance to STI-571 cancer therapy caused by BCR-ABL gene mutation or amplification. Science, : p Hochhaus A, K.S., Corbin AS, La Rosee P, Muller MC, Lahaye T, Hanfstein B, Schoch C, Cross NC, Berger U, Gschaidmeier and D.B. H, Hehlmann R., Molecular and chromosomal mechanisms of resistance to imatinib (STI571) therapy. Leukemia, : p Roche-Lestienne C, S.-C.V., Grardel-Duflos N, Lai JL, Philippe N, Facon T, Fenaux P, Preudhomme C., Several types of mutations of the Abl gene can be found in chronic myeloid leukemia patients resistant to STI571, and they can pre-exist to the onset of treatment. Blood, : p Shah NP, N.J., Nagar B, Gorre ME, Paquette RL, Kuriyan J, Sawyers CL., Multiple BCR- ABL kinase domain mutations confer polyclonal resistance to the tyrosine kinase inhibitor imatinib (STI571) in chronic phase and blast crisis chronic myeloid leukemia. Cancer Cell, : p Branford S, R.Z., Walsh S, Parkinson I, Grigg A, Szer J, Taylor K, Herrmann R, Seymour JF, Arthur C, Joske D, Lynch K, Hughes T., Detection of BCR-ABL mutations in patients Codice Riferimento: 4121 Page 18 of 44

19 with CML treated with imatinib is virtually always accompanied by clinical resistance, and mutations in the ATP phosphate-binding loop (P-loop) are associated with a poor prognosis. Blood, : p Soverini S, M.G., Amabile M, Poerio A, Bianchini M, Rosti G, Pane F, Saglio G, Baccarani M., Denaturing-HPLC-based assay for detection of ABL mutations in chronic myeloid leukemia patients resistant to Imatinib. Clin Chem., : p Soverini S, M.G., Rosti G, Bassi S, Amabile M, Poerio A, Giannini B, Trabacchi E, Castagnetti F, Testoni N, Luatti S, de Vivo A, Cilloni D, Izzo B, Fava M, Alberti D, Pane F, Saglio G, Baccarani M., Abl mutations in late-chronic phase chronic myeloid leukemia patients with upfront cytogenetic resistance to imatinib are associated with a greater likelihood of progression to blast crisis and shorter survival. J Clin Oncol. 23: p Corbin AS, L.R.P., Stoffregen EP, Druker BJ, Deininger MW., Several Bcr-Abl kinase domain mutants associated with imatinib mesylate resistance remain sensitive to imatinib. Blood, : p Martinelli, G., et al., Dual tyrosine kinase inhibitors in chronic myeloid leukemia. Leukemia, (11): p O'Hare T, W.D., Stoffregen EP,et al., In vitro Activity of Bcr-Abl Inhibitors AMN107 and BMS against Clinically Relevant Imatinib-Resistant Abl Kinase Domain Mutant. Cancer Res, : p Shah NP, T.C., Lee FY, Chen P, Norris D, Sawyers CL., Overriding imatinib resistance with a novel ABL kinase inhibitor. Science, : p Sawyers CL, S.N., Kantarjian HM, et al. Proceedings of the 2005 ASCO Annual Meeting., A Phase I Study of BMS in Patients with Imatinib-Resistant and Intolerant Accelerated and Blast Phase Chronic Myeloid Leukemia (CML): Results from CA Golas JM, A.K., Etienne C, et al., SKI-606, a 4-anilino-3-quinolinecarbonitrile dual inhibitor of Src and Abl kinases, is a potent antiproliferative agent against chronic myelogenous leukemia cells in culture and causes regression of K562 xenografts in nude mice. Cancer Res, : p Gatto S, S.B., Pham L, et al., The proteasome inhibitor PS-341 inhibits growth and induces apoptosis in Bcr/Abl-positive cell lines sensitive and resistant to imatinib mesylate. Haematologica, : p Carter TA, W.L., Shah NP, Velasco AM, Fabian MA, Treiber DK, et al., Inhibition of drugresistant mutants of ABL, KIT, and EGF receptor kinases. Proc Natl Acad Sci U S A, : p Young MA, S.N., Chao LH, Seeliger M, Milanov ZV, Biggs WH 3rd, et al., Structure of the kinase domain of an imatinib-resistant Abl mutant in complex with the Aurora kinase inhibitor VX-680. Cancer Res, : p Chien W, T.N., Williamson EA, Shih LY, Krug U, Kettenbach A, Fermin AC, Roifman CM, Koeffler HP, Characterization of a myeloid tyrosine phosphatase, Lyp, and its role in the Bcr-Abl signal transduction pathway. J Biol Chem, : p Lim YM, W.S., Lau G, Witte ON, Colicelli J, BCR/ABL inhibition by an escort/phosphatase fusion protein. Proc Natl Acad Sci U S A, : p Marull M, R.B., Fragmentation study of imatinib and characterization of new imatinib metabolites by liquid chromatography-triple-quadrupole and linear ion trap mass spectrometers. J Mass Spectrom, : p Thomas J, W.L., Clark RE, Pirmohamed M., Active transport of imatinib into and out of cells: implications for drug resistance. Blood, : p Ozvegy-Laczka C, H.T., Varady G, et al., High-affinity interaction of tyrosine kinase inhibitors with the ABCG2 multidrug transporter. Mol Pharmacol, : p Codice Riferimento: 4121 Page 19 of 44

20 35. Burger H, v.t.h., Brok M, et al., Chronic imatinib mesylate exposure leads to reduced intracellular drug accumulation by induction of the ABCG2 (BCRP) and ABCB1 (MDR1) drug transport pumps. Cancer Biol Ther, : p Illmer T, S.M., Platzbecker U, et al., P-glycoprotein-mediated drug efflux is a resistance mechanism of chronic myelogenous leukemia cells to treatment with imatinib mesylate. Leukemia, : p Kalnina Z, Z.P., Silina K, Line A., Alterations of pre-mrna splicing in cancer. Genes Chromosomes Cancer, : p Nera, K.P., et al., Ikaros has a crucial role in regulation of B cell receptor signaling. Eur J Immunol, (3): p Klein, F., et al., BCR-ABL1 induces aberrant splicing of IKAROS and lineage infidelity in pre-b lymphoblastic leukemia cells. Oncogene, (7): p Kersseboom, R., et al., Bruton's tyrosine kinase and SLP-65 regulate pre-b cell differentiation and the induction of Ig light chain gene rearrangement. J Immunol, (8): p Codice Riferimento: 4121 Page 20 of 44