WO2015049371A1

WO2015049371A1 - Methods for predicting the responsiveness of a patient affected with chronic myeloid leukemia (cml) to a treatment with a tyrosine kinase inhibitor (tki)

Info

Publication number: WO2015049371A1
Application number: PCT/EP2014/071238
Authority: WO
Inventors: Ali Turhan; Annelise BENNACEUR-GRISCELLI; Sylvie Sauvaigo; Fanny SARAZY
Original assignee: INSERM (Institut National de la Santé et de la Recherche Médicale); Cea; Université Paris-Sud; Assistance Publique-Hôpitaux De Paris (Aphp); Universite De Poitiers; Centre Hospitalier Universitaire De Poitiers
Priority date: 2013-10-03
Filing date: 2014-10-03
Publication date: 2015-04-09

Abstract

The invention relates to a method for predicting the responsiveness of a patient affected by a chronic myeloid leukemia (CML) or Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL) to a treatment with a tyrosine kinase inhibitor (TKI) comprising the following steps of: a) obtaining a sample from the patient; b) contacting said sample with a TKI; and c) determining the DNA repair capacity of said sample; wherein the responsiveness is predicted when a DNA repair capacity is observed.

Description

METHODS FOR PREDICTING THE RESPONSIVENESS OF A PATIENT AFFECTED WITH CHRONIC MYELOID LEUKEMIA (CML)

TO A TREATMENT WITH A TYROSINE KINASE INHIBITOR (TKI)

FIELD OF THE INVENTION:

The present invention relates to a method for predicting the responsiveness of a patient affected with a chronic myeloid leukemia (CML) or Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL) to a treatment with a tyrosine kinase inhibitor (TKI), such as Bcr-Abl TKI including imatinib and nilotinib.

BACKGROUND OF THE INVENTION:

Chronic Myeloid Leukemia (CML) is a hematological stem cell disorder caused by increased and unregulated growth of myeloid cells in the bone marrow, and the accumulation of excessive white blood cells. Abelson tyrosine kinase (ABL) is a non-receptor tyrosine kinase involved in cell growth and proliferation and is usually under tight control. However, 95% of CML patients have the ABL gene from chromosome 9 fused with the breakpoint cluster (BCR) gene from chromosome 22, resulting in a short chromosome known as the Philadelphia chromosome. This Philadelphia chromosome is responsible for the production of the BCR-ABL fusion protein, a constitutively active tyrosine kinase that causes uncontrolled cellular proliferation. An ABL inhibitor, imatinib, was approved by the FDA for the treatment of CML, and is currently used as first-line therapy. It has been reported that 80 % of CML patients respond to imatinib with under 3 % progressing to advanced disease within 5 years. The durability of clinical response, however, is adversely affected by the development of resistance to drug therapy. During the last decade, major progress has been made in the treatment of CML, by the clinical use of tyrosine kinase inhibitors (TKI) which have transformed the prognosis of the disease and prolonged survival. However, resistances have occurred to TKI essentially due the appearance of mutations in the ABL-kinase domain of the BCR-ABL gene. These mutations either alter the molecular conformation of the target site (such as P-loop mutations) or interfere directly with the binding of the TKI with the ATP- binding site, which is the case of T315I "gatekeeper" mutation which generate resistances to all three TKI currently approved for clinical use, ie, imatinib, nilotinib and dasatinib. Accordingly, prediction of TKI non-response early during therapy or even before starting therapy is highly required. However, there are no well-established biomarkers, which may improve the selection of patients who may benefit from the treatment. The purpose of the invention is therefore to fulfil this need by providing a new reliable method for predicting whether a patient affected with CML is responder or non responder to a treatment with a TKI such as Bcr-Abl TKI including imatinib and nilotinib.

SUMMARY OF THE INVENTION:

In a first aspect, the invention relates to a method for predicting the responsiveness of a patient affected by a chronic myeloid leukemia (CML) or Philadelphia chromosome- positive acute lymphoblastic leukemia (Ph+ ALL) to a treatment with a tyrosine kinase inhibitor (TKI) comprising the following steps of: a) obtaining a sample from the patient; b) contacting said sample with a TKI; and c) determining the DNA repair capacity of said sample; wherein the responsiveness is predicted when a DNA repair capacity is observed.

In a second aspect, the invention also relates to a method for monitoring the responsiveness of a patient affected by CML or Ph+ ALL to a treatment with a TKI comprising the following steps of: a) obtaining a sample from the treated patient; and b) determining the DNA repair capacity of said sample; wherein the patient is responsive when a DNA repair capacity is observed.

In a third aspect, the invention further relates to a kit for performing the method as defined above, comprising a biochip spotted with at least one plasmid containing DNA lesions selected from the group consisting of oxidized lesions, alkylated bases, abasic sites, thymine and cytosine glycols, cisplatin and psoralen adducts, photoproducts and UV-induced lesions.

In a fourth aspect, the invention further relates to the use of a kit comprising a biochip spotted with at least one plasmid containing DNA lesions selected from the group consisting of oxidized lesions, alkylated bases, abasic sites, thymine and cytosine glycols, cisplatin and psoralen adducts, photoproducts and UV-induced lesions for performing the method as defined above.

In a fifth aspect, the invention relates to a TKI for use in treating a patient affected with CML or Ph+ ALL, which patient being classified as responder by the method as defined above. DETAILED DESCRIPTION OF THE INVENTION:

The inventors have now shown that the responsiveness of patients affected with CML or Ph+ ALL receiving a treatment with a TKI may be accurately predicted or monitored by determining the DNA repair capacity of a sample obtained from said patient.

Indeed, they have found by using UT7-BCR-ABL cell line model [expressing native BCR-ABL (without mutation, sensitive to TKI) and BCR-ABL-T315I (expressing the major mutant for, resistant to TKI] that the inhibition of BCR-ABL by imatinib or nilotinib stimulated the DNA repair capacity of the cells and specially BER (8oxoG, alkylated bases and glycols) and NER (photoproducts and psoralen adducts) after 4 or 8 hours of treatment with imatinib or nilotinib, as compared to baseline repair capacities by using an assay which allows monitoring repair of a set of DNA lesions handled by Base Excision Repair (8oxoG, alkylated bases, glycols and Abasic sites) and Nucleotide Excision Repair (photoproducts, psoralen and cisplatin adducts) in BCR-ABL expressing UT7 cells. They have also shown that such DNA repair capacity is strongly reduced in UT7 cells expressing T315I mutation treated with imatinib (such mutation being known as conferring resistance to imatinib).

Definitions:

Throughout the specification, several terms are employed and are defined in the following paragraphs.

In a first aspect, the invention relates to a method for predicting the responsiveness of a patient affected by a chronic myeloid leukemia (CML) or Philadelphia chromosome- positive acute lymphoblastic leukemia (Ph+ ALL) to a treatment with a tyrosine kinase inhibitor (TKI) comprising the following steps of:

a) obtaining a sample from the patient;

b) contacting said sample with a TKI; and

c) determining the DNA repair capacity of said sample;

wherein the responsiveness is predicted when a DNA repair capacity is observed.

By "predicting" is intended herein the likelihood that a patient will respond or not to a tyrosine kinase inhibitor (TKI) and also the extent of the response. Predictive methods of the invention can be used clinically to make treatment decisions by choosing the most appropriate treatment modalities for any particular patient. In one embodiment, the patient has been newly diagnosed with CML or Ph+ ALL and has never received a treatment with a TKI.

In another embodiment, the patient has already received a treatment with a particular TKI but the cancer cells are become resistant to said particular TKI.

Indeed, several generations of TKI are available. First generation Bcr-Abl tyrosine kinase inhibitors comprise imatinib. Second generation drugs are intended to have decreased resistance and intolerance than imatinib. Second generation drugs that are currently marketed are nilotinib and dasatinib, whereas bosutinib and ponatinib are third generation drugs. Said drugs are described in details below.

In a second aspect, the invention method for monitoring the responsiveness of a patient affected by CML or Ph+ ALL to a treatment with a TKI comprising the following steps of:

a) obtaining a sample from the treated patient; and

b) determining the DNA repair capacity of said sample;

wherein the patient is responsive when a DNA repair capacity is observed. By "monitoring" is intended herein the follow-up of TKI responsiveness over the time period of treatment. Monitoring methods of the invention can be used clinically to make treatment decisions by choosing the most appropriate treatment modalities (since the patient could become unresponsive to the administered TKI, for example, because the cancer cells being might develop resistance over prolonged exposure against said TKI).

In one embodiment, the patient has already been diagnosed with CML or Ph+ ALL and has already received a treatment with a TKI.

In one embodiment, the method for monitoring the effectiveness of a treatment with a tyrosine kinase inhibitor (TKI) comprises repeatedly determining the DNA repair capacity in a patient undergoing treatment with a TKI for CML or Ph+ ALL, wherein a decrease in the DNA repair capacity over an interval of treatment is indicative that the cancer cells are become resistant to said treatment. For example, a first determination of the DNA repair capacity of a first sample obtained from the patient can be taken followed by a second determination of the DNA repair capacity of a second sample obtained from the patient while or after the patient receives treatment with TKI. The two measurements are then compared, and a decrease in the DNA repair capacity is indicative that the cancer cells are become resistant to said treatment. Typically, the two measurements can be any consecutive time points, separated by an interval of one or several days or one or several months.

In one embodiment, the method for monitoring the responsiveness of the invention comprises the steps of:

a) obtaining a sample from the treated patient with a TKI;

b) determining the DNA repair capacity of said sample;

c) continuing treatment with said TKI;

d) obtaining another sample from the treated patient;

e) determining the DNA repair capacity of said sample;

f) comparing the DNA repair capacity determined in samples at step b) and d), and if the the DNA repair capacity determined at step d) is decreased in comparison with those determined at step b), is indicative that the cancer cells are become resistant to said treatment (and that continuing treatment with the same TKI is not useful and that it should be therefore determined if another TKI may be useful for treating CML or Ph+ ALL).

Methods for predicting or monitoring the responsiveness of a patient

according to the invention: As used herein, the term "sample" refers to a biological sample obtained for the purpose of in vitro evaluation. Samples that may be used for performing the methods according to the invention encompass any biological sample derived from a patient containing white blood cells, including any fluids, tissues, cell samples, organs, biopsies, etc. Typical samples to be used in the methods according to the invention are blood samples (e.g. whole blood sample). In a preferred embodiment, said blood sample is a whole blood sample obtained from a patient to be tested. Alternatively, the sample is a bone marrow sample. As used herein, the term "patient in need thereof is intended for a human or non- human mammal affected or likely to be affected with chronic myeloid leukemia (CML) or Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL).

In one embodiment, the patient is affected with chronic phase CML. In another embodiment, the patient is affected with accelerated phase CML. In still another embodiment, the patient is affected with blast phase CML.

As used herein, the term "responder" patient, or group of patients, refers to a patient, or group of patients, who show a clinically significant relief in the disease when treated with a TKI. A contrario, a "non responder patient" or group of patients, refers to a patient or group of patients, who do not show a clinically significant relief in the disease when treated with a TKI. For instance, when the disease is chronic myeloid leukemia (CML) in a chronic phase, a preferred responder group of patients that provides for the control reference values is a group that shows the disappearance of all target lesions (complete hematologic response (or CHR) when blood cell counts return to normal; complete cytogenetic response (CCyR) when no cells with the Philadelphia chromosome can be found in the blood or bone marrow; and complete molecular response (CMR) when the BCR-ABL gene in the patient's blood cannot be found by PCR) or for instance partial cytogenetic response) when less than 35% of cells still have the Philadelphia chromosome, 3 to 6 months for the first 2 years after starting a TKI, preferably imatinib, nilotinib or dasatinib. After being tested for responsiveness to a treatment with a TKI, the patients may be prescribed with said TKI.

As used herein, the terms "tyrosine kinase inhibitor" or "TKI" refer to any compound, natural or synthetic, which results in a decreased phosphorylation of the tyrosine present on the intracellular domain of receptor tyrosine kinases (RTK) such as growth factor receptors. Preferably, the tyrosine kinase inhibitor is a Bcr-Abl tyrosine kinase inhibitor.

However, TKI may also be a multi-target tyrosine kinase inhibitor and may thus inhibit one or more tyrosine kinases, Bcr-Abl, but also c-Abl and the receptor tyrosine kinases PDGF-R, Fit3, VEGF-R, EGF-R, c-Kit, as well as combinations of two or more of these.

In one embodiment, methods of the invention are suitable for predicting or monitoring the responsiveness of a patient affected with a CML to a pharmaceutical treatment with a TKI, wherein said TKI are Bcr-Abl TKI which are the first-line therapy for most patients with CML. In more than 90% cases CML is caused by chromosomal abnormality resulting in the formation of a so-called Philadelphia chromosome.

In one embodiment, the Bcr-Abl TKI is selected from the group consisting of N- phenyl-2-pyrimidine-amine derivatives as described in EP0564409, pyrimidinylaminobenzamide derivatives as described in WO2004005281, cyclic compounds as described in WO0062778, bicyclic heteroaryl compounds as described in WO 2007075869, substituted 3-cyano quinoline derivatives as described in US6002008, 4-anilo-3- quinolinecarbonitrile derivatives as described in WO200504669 and amide derivatives as described in US7728131 and WO2005063709.

In a particular embodiment, the Bcr-Abl TKI is selected from the group consisting of imatinib, nilotinib, dasatinib, ponatinib, bosutinib and bafetinib. In a preferred embodiment, the TKI is imatinib or 4-[(4-methylpiperazin-l-yl)methyl]-

N-(4-methyl-3 - { [4-(pyridin-3 -yl)pyrimidin-2-yl] amino } phenyl)benzamide (marketed as GLIVEC® by Novartis and previously known as STI571) of formula (I):

In a preferred embodiment, the TKI is nilotinib or 4-methyl-N-[3-(4-methyl-lH- imidazo 1- 1 -yl)-5 -(trifluoromethyl)phenyl] -3 - [(4-pyridin-3 -ylpyrimidin-2-yl)amino]benzamide (marketed as TASIGNA® by Novartis and previously known as AMN107) of formula (II):

In a preferred embodiment, the TKI is dasatinib or N-(2-chloro-6-methylphenyl)-2- [ [6- [4-(2-hydroxy ethyl)- 1 -piperazinyl] -2-methyl-4-pyrimidinyl] amino] -5 -thiazo le- carboxamide monohydrate (marketed as SPRYCEL® by BMS and previously known as BMS-354825) of formula (III):

In a preferred embodiment, the TKI is ponatinib or 3-(2-imidazo[l,2-b]pyridazin-3- ylethynyl)-4-methyl-N- [4- [(4-methylpiperazin- 1 -yl)methyl] -3 -(trifluoromethyl)phenyl] benzamide (marketed as ICLUSIG® by and previously known as AP24534) of formula (IV):

In a preferred embodiment, the TKI is bosutinib or 4-[(2,4-dichloro-5- methoxyphenyl)amino] -6-methoxy-7- [3 -(4-methylpiperazin- 1 -yl)propoxy] quino line-3 - carbonitrile (marketed as BOSULIF® by PFIZER and previously known as SKI-606) of formula (V):

Patients who have been clinically diagnosed as being affected with any haematological malignancy are of particular interest in the invention. However in a preferred embodiment the patient is affected with a myeloid neoplasm. According to this embodiment, the myeloid neoplasm is chronic myeloid leukemia (CML) or Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL).

Determination of the DNA repair capacity

Determination of the DNA repair capacity can be performed by a variety of techniques. Methods for detecting and characterising the activity of proteins(s) involved in DNA repair are well known in the art.

More preferably, the determination comprises contacting the sample with selective reagents such as damaged DNA, and thereby determining the activity of protein(s) of interest originally present in said biological sample.

Among DNA repair systems intended to maintain the integrity of their genome in living organism, two have the function of eliminating modified bases from the DNA: they are the base excision repair (BER) system and the nucleotide excision repair (NER) system:

- the BER system, which uses a family of enzymes called glycosylases, is more specifically dedicated to the repair of small lesions in DNA, such as oxidative damage, abasic sites, base fragmentations, base methylations, etheno-bases, etc.

- the NER system takes care of bulky lesions that induce distortion of the DNA double helix, such as acetylaminofluorine-DNA, cisplatin-DNA and psoralen-DNA adducts, dimers derived from UVB and UVC irradiation of DNA, covalent lesions formed between a DNA base and another molecule, etc.

Moreover, nucleotide incision repair (NIR) has been recently described as an alternative to both BER and NER. Some proteins possess overlapping functions within and between BER and NER pathways and proteins ascribed to one pathway can interact with proteins of the other pathways. Finally, interstrand cross-links (ICLs) are repaired through multiple mechanisms, either recombination-dependent or recombination-independent, with possible cooperation of proteins from NER and mismatch repair (MMR) pathways.

In one embodiment, the step of determining the DNA repair capacity of a sample is not carried out by determining the DNA repair by homologous recombination.

Accordingly, in one particular embodiment, the step of determining the DNA repair capacity of a sample is carried out by determining the activity of the base excision repair (BER) system and/or the activity of the nucleotide excision repair (NER) system.

In a preferred embodiment, the step of determining the activity of the BER system is carried out by determining the activity of DNA glycosylases (such as hOGGl, hMYH, UNG, hNEILl and MPG) and/or AP endonuclease.

In another preferred embodiment, the step of determining the activity of the NER system is carried out by determining the activity of DNA damage-binding proteins (such as DDB1, DDB2 and XPC).

Accordingly, the DNA repair capacity may be determined by using different assays such as oligonucleotide assays as described in Sauvaigo et al., 2004 and Guemiou et al., 2005 or plasmid assays as described in Millau et al, 2008. The DNA repair capacity can in particular be analyzed according to the method for the quantitative evaluation of overall and specific capacities for DNA repair described in applications WOO 190408, WO 2004059004 and WO2006136686.

For instance, the support is a biochip onto damaged DNA fragments have been fixed (i.e. a supercoiled circular double-stranded DNA (supercoiled plasmid)). Typically, the substrate used for analyzing DNA repair is a supercoiled plasmid with damaged purine or pyrimidine bases (oxidative, photo-induced, chemical adduct damage), damaged sugars, damage to the structure of the double helix (inter- or intra-strand bridging, intercalated agents), and/or breaks, induced by treatment of the plasmid with various geno toxic agents. Biochips have become essential tools for biomedical research and clinical diagnosis. These tools make it possible to simultaneously analyze several thousand samples by means of DNA probes (oligonucleotides, cDNA, PCR amplification products) or protein probes (antibodies, peptides), immobilized on a support. Biochips are prepared using a support (glass, polypropylene, polystyrene, silicone, metal, nitrocellulose or nylon), optionally modified with a porous film [glass coated with nylon (Atlas Arrays™; Clontech); silicone coated with a hydrogel (Nanochip™; Nanogen), glass coated with a gel of acrylamide polymer, Hydrogel™ (Perkin-Elmer) or of methacrylate polymer.

In one embodiment, said supercoiled DNA is a plasmid.

In one embodiment, said supercoiled DNA contains damage induced by a genotoxic agent. Preferably, said damage was induced by treatment of an isolated plasmid or of cells comprising said plasmid, with a physical or chemical genotoxic agent. Among this damage, mention may be made of damage to the purine or pyrimidine bases (oxidative, photo-induced, chemical adduct damage), damage to the sugars, damage to the structure of the double helix (inter- or intra-strand bridging, intercalated agents) and breaks. In one embodiment, the porous polymer film is deposited onto an appropriate support known to those skilled in the art, such as a glass, metal, silicone or plastic support. It is preferably a miniaturized support of the microchip type. It is preferably a glass slide.

For instance, a support coated with a porous polymer film, comprising an immobilized supercoiled DNA, useful within the context of the invention may be obtained by means of the immobilization method as described in international patent application WO 2006/136686.

In one particular embodiment, the DNA repair capacity is determined by using a specific multiplexed enzymatic DNA repair assay on biochip to simultaneously investigate several repair pathways. In particular excision/synthesis repair activities belonging to BER, NER, NIR, and partly ICLs repair were quantified.

In a preferred embodiment, sets of plasmids with specific DNA lesions (Abasic sites, Oxidized lesions (8oxoG), thymine and cytosine Glycols (Glycols), Cisplatin and Psoralen adducts, UV-induced lesions (photoproducts), are spotted on a biochip. If the cell extracts added to the chips have the ability to repair the specific lesions, fluorescent spots are generated, and quantified. A specific DNA Repair signature is thus established.

Another aspect of the invention relates to a kit for performing the method of the invention, comprising a biochip spotted with at least one plasmid containing DNA lesions selected from the group consisting of oxidized lesions, alkylated bases, abasic sites, thymine and cytosine glycols, cisplatin and psoralen adducts, photoproducts and UV-induced lesions.

Another aspect relates to the use of a kit comprising a biochip spotted with at least one plasmid containing DNA lesions selected from the group consisting of oxidized lesions, alkylated bases, abasic sites, thymine and cytosine glycols, cisplatin and psoralen adducts, photoproducts and UV-induced lesions for performing the method of the invention.

Therapeutic methods:

Another aspect of the invention relates to a method for the treatment of a patient suffering from a chronic myeloid leukemia (CML) or Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL).

A further aspect of the invention is a TKI for use in treating a patient affected with CML or Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL), which patient being classified as responder by the method as above-described.

In the context of the invention, the terms "treating" or "treatment", as used herein, mean reversing, alleviating, inhibiting the progress of, or preventing the disorder or condition to which such term applies, or one or more symptoms of such disorder or condition.

In one embodiment, said method comprises the following steps of:

a) obtaining a sample from the patient;

b) contacting said sample with a TKI;

c) determining the DNA repair capacity of said sample, and

d) administering to said patient a therapeutically effective amount of a TKI.

In another embodiment, said method comprises the following steps: a) determining whether a patient affected with CML or Ph+ ALL is a responder or a non responder to a treatment with a TKI, by performing the method as above- described; and

b) administering a therapeutically effective amount of a TKI to said patient, if said patient has been determined as a responder, at step a) above.

By a "therapeutically effective amount" is meant a sufficient amount of the agent to treat CML or Ph+ ALL at a reasonable benefit/risk ratio applicable to any medical treatment.

It will be understood that the total daily usage of the TKI or the pharmaceutical composition comprising thereof will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific inhibitor employed; the specific pharmaceutical composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific inhibitor employed; the duration of the treatment; drugs used in combination or coincidental with the specific inhibitor employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the inhibitor at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the TKI may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Preferably, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the agent for the symptomatic adjustment of the dosage to the subject to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, preferably from 1 mg to about 100 mg of the active ingredient. An effective amount of the active ingredient is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.

Any TKI as above described may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions.

In one particular embodiment, the TKI is a Bcr-Abl TKI selected from the group consisting of imatinib, nilotinib, dasatinib, ponatinib and bosutinib. The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

EXAMPLE: Material & Methods

BCR-ABL-expressing UT7 cells: UT7 cells expressing native, non-mutated BCR- ABL were previously described (Issaad et al,. 2000) These cells grow in the absence of GM- CSF and express high levels of BCR-ABL as verified by Western blot analyses. UT7-T315I cells were generated using a MIGR-BCR- ABL-T3151 lentivirus. BCR-ABL T315I supernatants were generated using the FuGENE HD transfection reagent (Promega, Madison, WI). Briefly, 5 micrograms of plasmid were transfected in 293T cells followed by a collection of the supernatants on days 2 and 3.

These supernatants were used to transduce UT7 cells grown in exponential phase. GFP+ cells were cell-sorted, amplified, and then characterized by cell culture and Western blots.

To generate an inducible BCR-ABL UT7 model, we have constructed a lentiviral BCR-ABL vector containing a cassette including BCR-ABL and Tet-responsible elements separated by 2A peptide elements. This vector was used to generate lentiviral supernatants allowing the transduction of BCR-ABL into UT7 cells followed by GFP-based selection. The inducible BCR-ABL-expressing UT7 cells were cloned in methylcellulose and individual clones were amplified before characterization using Western blot analyses.

Western Blots: 1- 3. 10⁶ cells were lysed in 100 RIPA buffer (50 mM Tri-HCL, 150 mM NaCl, 1% NP40, 1 mM EDTA) in the presence of anti-protease agents. After incubation of 30 minutes on ice, cell lysates were centrifuged, the protein contents of the supernatants was determined using the technique of Bradford. 10-20 microgrammes of proteins were loaded in SDS-PAGE electrophoresis and transferred into nitrocellulose membranes. After saturation using 5% BSA {bovine serum albumin) et de TBST (Tris buffer salin-Tween) (20 mM Tris, pH 7,6, 137 mM NaCl, 0,1% Tween-20), membranes were incubated overnight at 4°C in the presence of primary anti-ABL antibodies (Oncogene Science). After washing (x 3 for 10 minutes each) in TBST buffer, membranes were incubated for 1 hour in the presence of secondary antibodies before revealing the blots using ECL (enhanced chemiluminescent) kit and Western blotting Detection System® (Amersham).

Preparation of UT7 cells for DNA repair assays: UT7 cells expressing BCR-ABL constitutively were treated with TK inhibitors Imatinib mesylate 1 microM; or Nilotinib 10 nM), for 4, 8 and 18 hours before nuclear extract preparation for DNA repair assays. In the TET-inducible BCR-ABL system, we have added Doxycycline to the cell culture medium (1 microgr/ml) and performed nuclear extracts at day 3, 5 and 7 after inhibition of BCR-ABL expression as determined by Western blotting.

Modified plasmid microarray: Each microarray comprises series of lesion- containing plasmids and a non modified control plasmid (CTRL). Seven lesions were generated on different plasmids before spotting: photoproducts (eye lo butane pyrimidine dimers and (6-4)photoproducts; CPD-64 plasmid), 8-oxoguanine (8oxoG plasmid), alkylated bases (AlkB plasmid), , abasic sites (AP sites; AbaS plasmid), cytosine and thymine glycols (Glycol plasmid), cisplatin adducts (CisP plasmid) and psoralen adducts (Pso plasmid). Each type of modified plasmid was then diluted in non-modified plasmid so we obtained 3 solutions of identical DNA concentration (40 μg/mL) but with 3 different ratios lesion/DNA (ratios ½, ¾ and 1, called A, B and C dilutions, respectively). On all microarrays each plasmid solution was spotted duplicated except the CTRL that was deposited 9 times.

Preparation of cell nuclear extracts: Cell nuclear extracts were prepared as already described. Thawed cells were washed twice in ice-cold PBS and incubated on ice for 20 min in 1.25 mL of ice-cold buffer A (10 mM HEPES pH 7.9, 1.5 mM MgC12, 10 mM KC1, 0.01% Triton X-100, 0.5 mM DTT, 0.5 mM PMSF). The cytosolic membrane was disrupted by vortexing the tube for 30 s. Nuclei were recovered by centrifugation for 5 min at 5000 rpm and 4 °C. The pellet was suspended in 31.25 of ice-cold buffer B (10 mM HEPES pH 7.9, 1.5 mM MgC12, 400 mM KC1, 0.2 mM EDTA, 25% glycerol, 0.5 mM DTT, antiproteases (Complete-mini, Roche, France) and 0.5 mM PMSF). Nuclear membranes were lysed for 20 min on ice, and two cycles of freezing-thawing at -80 °C and 4 °C respectively. The extracts were cleared by centrifugation for 10 min at 13000 rpm and 4 °C. The cleared supernatant was recovered and stored frozen in 10 μΐ aliquots at -80 °C. Protein content was determined using the BCA kit (Interchim, France). Typical protein content was 0.8 mg/mL. DNA excision/synthesis reaction: Custom reaction chambers (Grace Bio-Labs, USA) were set on the damaged plasmid microarray slides and filled with 20 of the excision/synthesis mix composed of reaction buffer (5 μΐ, of 5X ATG buffer [200 mM Hepes KOH pH 7.8, 35 mM MgCl₂, 2.5 mM DTT, 1.25 μΜ dATP, 1.25 μΜ dTTP, 1.25 μΜ dGTP, 17% glycerol, 50 mM phosphocreatine (Sigma, USA), 10 mM EDTA, 250 μg/mL creatine phosphokinase, 0.5 mg/mL BSA]), 1 mM ATP (Amersham, England), 1.25 μΜ dCTP-Cy5 (Amersham, England) and containing the nuclear extract at a final protein concentration of 0.25 mg/mL. The slides were incubated for 3 h at 30 °C in the dark. The reaction chambers were then removed and the slides were washed for 2 x 3 min in PBS/Tween 0.05%, and for 2 x 3 min in MilliQ water. Slides were then centrifuged 3 min at 700 rpm, and allowed to dry 5 min at 30 °C.

Microarray scanning, fluorescence quantification, data treatment and normalization: Images were acquired at 635 nm wavelength at 10 μιη resolution using a Genepix 4200 A scanner (Axon Instrument). Total spot fluorescence intensity (FI) was determined using the Genepix Pro 5.1 software (Axon Instrument). Within each set of reactions, duplicate data collected from the cell line experiments were normalized using Normalizelt software. Then, for each test sample, we determined an intensity value for the 6 modified plasmids corresponding to the sum of the intensities of the A, B and C dilutions from which the value for the CTRL was subtracted. This value corresponded to the intensity of the unmodified Control plasmid multiplied by 3, for consistency with regard to the sum of 3 plasmid dilution intensities. Therefore, each sample was characterised by 6 values, corresponding to the repair of the 6 DNA lesions represented on the biochip.

Then, for each time-point and each lesion, we calculated the ratio of the FI with respect to the baseline that was log2 transformed.

Data normalization Data analysis - Clustering methods - Results display: The hierarchical average linkage clustering algorithm uses an iterative process clustering items by profile similarity. The results are displayed in a dendrogram format. Items with similar profiles are placed in adjacent dendrogram nodes, connected by branches proportional in length to the corresponding dissimilarity measure. A data table is usually clustered along its two dimensions, in order to complete profile clustering by clustering profile variables. The resulting clustered data table is displayed as a heatmap with the two dendrograms along its two dimensions. This allows cluster trees and cluster profiles to be viewed simultaneously. In heatmap format, data values are colour-coded, from green (low data) to red (high data), the colour brightness correlates with the data point's value.

Results were normalized independently for each cell lines: FI data were centred around zero Unsupervised hierarchical clustering was used to investigate effects of the treatment on the cell DNA repair Enzyme Signature through similarities between the cell lines profiles. The analysis was performed using the free software environment for statistical computing and graphics, R (http://r-project.org/). The hierarchical average linkage clustering algorithm was run with the Euclidean distance dissimilarity measures, which aggregates profiles with both similar intensity levels and covariations.

Statistical tests to evaluate degree of similarity: We used the "pvclust" R package to assess the uncertainty in hierarchical cluster analysis

(http://www.is.titech.ac.jp/~shimo/prog/pvclust/). Clusters with AU P value > 95% were considered significant.

Results

In the UT7 cell line model we have tested the effects of BCR-ABL tyrosine kinase activity on the DNA repairs assays using two strategies:

The first was the test the effects of the extracts of BCR-ABL expressing UT7 cells before and after inhibition of BCR-ABL expression by Imatinib mesylate. For this purpose, parental, UT7-cells expressing BCR-ABL and BCR-ABL with T315I mutation, were tested with regard to their DNA repair ability.

The classification identified four significant classes of responses. Three of them were significantly associated.

The main identified characteristics concerned the inhibition of two specific pathways (AbaS and CisP (p<0.05)) by Imatinib and Nilotinib in the parental UT7 cell line and the UT7 cell line expressing the mutated BCR-ABL gene T315I. The other repair pathways were not significantly affected except repair of Pso that was also inhibited but less significantly (p < 0.125).

On the contrary inhibition of BCR-ABL by IM, in (UT7-11) stimulated the cell DNA repair capacity for ALKB, Glycol, Pso, OXO, CPD after 4 or 8 hours of treatment as compared to baseline repair capacities (p< 0.05). Similarly when the cells were treated with Nilotinib in the same conditions, we have observed also the stimulation of DNA repair capacities for all pathways except for the Abasic repair pathway (p< 0.125).

Thus inhibition of BCR-ABL by IM led to an induction of DNA repair systems whereas this stimulation was impeded when BCR-ABL was mutated or absent.

To confirm the effect of the BCR-ABL TK activity in this system, we have used an UT7 cell line expressing BCR-ABL under the control of a Tetracycline-inducible promoter. In this cell line, BCR-ABL is expressed when the cells are grown in the absence of Doxycycline. Upon Dox addition, BCR-ABL expression is reduced rapidly in 3-4 days and become undetectable by Western blots at day 8.

Using the inducible system, DNA repair capacity of AlkB, Glycol, Pso and CPD were significantly (p < 0.05). Interestingly, the fact of reducing BCR-ABL TK activity of the mutant BCR-ABL T315I in the DOX- inducible system stimulated DNA repair capacity whereas in the non- inducible UT7 cells expressing T315I, Imatinib did not have a significant effect on the DNA repair capacity, except for a slight stimulation of CPD, AlkB, Glycol and 8oxoG pathways after 8 hours of treatment with Nilotinib, which is a more powerful inhibitor of BCR-ABL TK activity than Imatinib. Our results show that in the parental UT7 cell line (with no BCR-ABL expression), the fact of adding Imatinib or Nilotinib inhibiting BCR-ABL expression do not induce any significant change as compared to baseline (DO) levels, in the capacity of repairing the seven DNA lesions studied (Table 1). On the other hand, in the BCR-ABL expressing UT7 cells, we have observed a stimulation of DNA repair activity as compared to baseline upon inhibition of the TK activity of BCR-ABL either by Imatinib or Nilotinib (Table 1). Interestingly, in the UT7-T315I cell lines which are resistant to the Imatinib or Nilotinib, this DNA repair stimulating effect was not seen, suggesting very strongly that BCR-ABL TK activity is responsible for the DNA repair and the current DNA repair assay can detect it (Table 1). We then used the TET-inducible BCR-ABL system in which the fact of adding Doxycycline allows the control of the expression and not the TK activity which remains dependent on the level of expression. We have, in preliminary experiments, showed that this system allows a very significant but not total expression of BCR-ABL upon addition of Dox in the cell culture (Figure 1). In this system, as compared to the use of TK inhibitors, both native and T315I- mutated BCR-ABL are inhibited at the transcription level. The DNA repair assays showed that upon inhibition of BCR-ABL, DNA repair abilities are increased in the nuclear cell extracts at day 4 and 11 as compared to baseline (Table 2). Similarly, in the TET-inducible UT7-T135I system, the DNA repair is stimulated, but more modestly, at day 7 as compared to day 0 and this effect is not seen at day 11. The reason of this late effect is not clear but could be due to an off- target effect of mutated BCR-ABL on the mechanisms of DNA repair under study.

Table 1: Evaluation of the DNA repair capacity evaluated by repair chips in UT7 cells expressing wild- type (p210) or T315I-mutated BCR-ABL.

NS: Not significant change as compared to baseline (hour 0) before adding IM or NIL.

-: Inhibition of DNA repair,

+: Stimulation of DNA repair,

IM: Imatinib; NIL: Nilotinib

The summary of these findings in the Inducible system (TET-OFF: Inhibition of expression when DOX is added) to study DNA repair in UT7 cells shows the following:

-DNA Repair capacity of UT7-parental and UT7-p210 are equal at day 0

-11 days after adding DOX (inhibition of BCR-ABL): DNA repair capacity of UT7-p210 is increased (UT7-p210 Dl 1 > UT7-p210 DO. -11 days after adding DOX in UT7-T315I cells: DNA repair capacity of the mutant form is not different from DayO (the mutant form does not repair well: UT7-T315I Dl 1 = UT7-T315I DO), despite a modest effect at day 7. REFERENCES:

Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

Guerniou V, Rapin D, Millau JF, Bufflier E, Favier A, Cadet J, Sauvaigo S. Repair of oxidative damage of thymine by HeLa whole-cell extracts: simultaneous analysis using a microsupport and comparison with traditional PAGE analysis. Biochimie. 2005 Feb;87(2): 151-9.

Issaad C, Ahmed M, Novault S, Bonnet ML, Bennardo T, Varet B, Vainchenker W,

Turhan AG. Biological effects induced by variable levels of BCR-ABL protein in the pluripotent hematopoietic cell line UT-7. Leukemia. 2000 Apr; 14(4): 662-70.

Millau JF, Raffin AL, Caillat S, Claudet C, Arras G, Ugolin N, Douki T, Ravanat JL, Breton J, Oddos T, Dumontet C, Sarasin A, Chevillard S, Favier A, Sauvaigo S. A microarray to measure repair of damaged plasmids by cell lysates. Lab Chip. 2008 Oct;8(10): 1713-22.

Sauvaigo S, Guerniou V, Rapin D, Gasparutto D, Caillat S, Favier A; An oligonucleotide microarray for the monitoring of repair enzyme activity toward different DNA base damage; Anal Biochem. 2004 Oct 1 ;333(1): 182-92.

Claims

CLAIMS:

1. A method for predicting the responsiveness of a patient affected by a chronic myeloid leukemia (CML) or Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL) to a treatment with a tyrosine kinase inhibitor (TKI) comprising the following steps of: a) obtaining a sample from the patient; b) contacting said sample with a TKI; and c) determining the DNA repair capacity of said sample; wherein the responsiveness is predicted when a DNA repair capacity is observed.

2. A method for monitoring the responsiveness of a patient affected by CML or Ph+ ALL to a treatment with a TKI comprising the following steps of: a) obtaining a sample from the treated patient; and b) determining the DNA repair capacity of said sample; wherein the patient is responsive when a DNA repair capacity is observed.

3. The method according to claim 1 or 2, wherein the TKI is a Bcr-Abl TKI selected from the group consisting of imatinib, nilotinib, dasatinib, ponatinib and bosutinib.

4. The method according to any one claims 1 to 3, wherein the sample from the patient is a whole blood sample or a bone marrow sample.

5. The method according to any one claims 1 to 4, wherein the step of determining the DNA repair capacity of a sample is not carried out by determining the DNA repair by homologous recombination.

6. The method according to any one claims 1 to 5, wherein the step of determining the DNA repair capacity of a sample is carried out by determining the activity of the base excision repair (BER) system and/or the activity of the nucleotide excision repair (NER) system.

7. The method according to claim 6, wherein the step of determining the activity of the BER system is carried out by determining the activity of DNA glycosylases and/or AP endonuclease.

8. The method according to claim 6, wherein the step of determining the activity of the NER system is carried out by determining the activity of DNA damage-binding proteins.

9. The method according to any one claims 1 to 8, wherein the DNA repair capacity of said sample is determined by using a biochip spotted with at least one plasmid containing DNA lesions selected from the group consisting of oxidized lesions, alkylated bases, abasic sites, thymine and cytosine glycols, cisplatin and psoralen adducts, photoproducts and UV-induced lesions.

10. A kit for performing the method according to any one claims 1 to 9, comprising a biochip spotted with at least one plasmid containing DNA lesions selected from the group consisting of oxidized lesions, alkylated bases, abasic sites, thymine and cytosine glycols, cisplatin and psoralen adducts, photoproducts and UV-induced lesions.

11. Use of a kit comprising a biochip spotted with at least one plasmid containing DNA lesions selected from the group consisting of oxidized lesions, alkylated bases, abasic sites, thymine and cytosine glycols, cisplatin and psoralen adducts, photoproducts and UV-induced lesions for performing the method according to any one claims 1 to 9.

12. A TKI for use in treating a patient affected with CML or Ph+ ALL, which patient being classified as responder by the method according to any one claims 1 to 9.