FIBRONECTIN OR ILK INHIBITORS FOR USE IN THE TREATMENT OF LEUKEMIA

Abstract

The present invention pertains to fibronectin or Integrin-linked Kinase (ILK) inhibitors/antagonists in the treatment of imatinib resistant leukemia. The invention relates to the use of recombinant or isolated fibronectin or an ILK inhibitor as adjuvant therapy during leukemia treatment, either as single ingredient medicament or in a combination therapy, preferably with ABL inhibitors such as imatinib or nilotinib.

Claims

1. A method of treatment or prevention of leukemia comprising the use of Fibronectin, or a functional variant thereof, as well as salts, solvates and/or derivatives thereof.

2. (canceled)

3. The method of treatment or prevention of leukemia according to claim 1, wherein the leukemia is Chronic Myelogenous Leukemia (CML) or Acute Lymphocytic Leukemia (ALL), and preferably is Bcr-Abl1 positive.

4. The method of treatment or prevention of leukemia according to claim 1, wherein the leukemia is an imatinib-resistant leukemia, preferably a Bcr-Abl1 mutant positive leukemia.

5. The method of treatment or prevention of leukemia according to claim 4, wherein the Bcr-Abl1 mutant is selected from the group consisting of M351T, F359V, H396P, I432T, F486S, M244V, L248V, G250E, Y253H, Y253F, E255K, K263E, L273M, T315I, F317L, and N331S; and preferably is Bcr-Abl1.sup.T315I.

6. The method of treatment or prevention of leukemia according to claim 1, wherein the fibronectin or a functional variant thereof, is either a recombinantly produced protein, or a protein purified from a blood sample from a healthy donor.

7. The method of treatment or prevention of leukemia according to claim 1, wherein the treatment comprises bone marrow transplantation.

8. The method of treatment or prevention of leukemia according to claim 1, further comprising a concomitant or sequential use of at least one other anti-cancer agent.

9. (canceled)

10. The method of treatment or prevention of leukemia according to claim 8, wherein the at least one anti-cancer agent is a kinase inhibitor.

11. (canceled)

12. The method of treatment or prevention of leukemia according to claim 10, wherein the kinase inhibitor is a tyrosine kinase inhibitor, a Src kinase inhibitor and/or an allosteric Abl1-inhibitor.

13. (canceled)

14. (canceled)

15. (canceled)

16. A compound for treatment of a disease of a subject, wherein the compound is an inhibitor of the expression, function and/or stability of integrin-linked kinase (ILK).

17. The compound of claim 16, wherein the compound is selected from a group consisting of polypeptide, peptide, glycoprotein, peptidomimetic, antigen binding construct, nucleic acid, including antisense or inhibitory DNA or RNA, ribozyme, RNA or DNA aptamer, RNAi, siRNA, shRNA and variants or derivatives thereof such as a peptide nucleic acid (PNA), and genetic construct for targeted gene editing, such as CRISPR/Cas9 construct, guide nucleic acid (gRNA or gDNA) and/or tracrRNA.

18. (canceled)

19. The compound of claim 17, wherein the antigen binding construct is an ILK inhibitory antibody, or an inhibitory antigen binding fragment thereof.

20. The compound of claim 17, wherein the nucleic acid is an anti-sense nucleotide such as a siRNA or a shRNA molecule that binds to a nucleic acid that encodes ILK or regulates expression of ILK.

21. The compound of claim 16, wherein the compound is selected from a group consisting of Cpd22, QLT0267, KP-392 and T315, or a derivative, isomer, salt, or solvate thereof.

22. The compound of claim 16 for treatment of lung cancer, bladder cancer, ovarian cancer, uterine cancer, endometrial cancer, breast cancer, liver cancer, pancreatic cancer, stomach cancer, cervical cancer, lymphoma, leukemia, acute myeloid leukemia, acute lymphocytic leukemia, salivary gland cancer, bone cancer, brain cancer, colon cancer, rectal cancer, colorectal cancer, kidney cancer, skin cancer, melanoma, squamous cell carcinoma, pleomorphic adenoma, hepatocellular carcinoma, and/or adenocarcinoma.

23. The compound of claim 16, wherein the disease is leukemia.

24. The compound of claim 23, wherein the leukemia is an imatinib-resistant leukemia, preferably a Bcr-Abl1 mutant positive leukemia, such as Bcr-Abl1.sup.T315I positive CML.

25. The compound of claim 24, wherein the Bcr-Abl1 mutant is selected from the group consisting of M351T, F359V, H396P, I432T, F486S, M244V, L248V, G250E, Y253H, Y253F, E255K, K263E, L273M, T315I, F317L, and N331S; and preferably is Bcr-Abl1.sup.T315I.

26. (canceled)

27. (canceled)

28. A combination for use in the treatment of a disease in a subject, comprising the compound of claim 16, and a second compound effective in the treatment of said disease.

29. The combination of claim 28, wherein the second compound an anti-leukemic agent selected from a group consisting of fibronectin, nilotinib, dasatinib, ponatinib, ABL001 or imatinib.

Description

[0054] The present invention will now be further described in the following examples with reference to the accompanying figures and sequences, nevertheless, without being limited thereto. For the purposes of the present invention, all references as cited herein are incorporated by reference in their entireties. In the Figures:

[0055] FIG. 1: Measurement of the shortest three-dimensional distance of a) normal (black) or BCR-ABL1+(gray) lin-c-Kit+ Sca-1+(LKS) or LKS CD150+CD48-(LKS SLAM) cells, b) vehicle (black)- or imatinib (gray)-treated BCR-ABL1+ LKS cells and c) BCR-ABL1WT+(black) versus BCR-ABL1T315I+(gray) LKS cells labeled with the lipophilic dye DiD and injected into irradiated Col2.3kbGFP mice to the endosteum. The horizontal black line represents the mean.

[0056] FIG. 2: Adhesion of sorted Mac-1+ BCR-ABL1T315I-positive cells from mice with CML to a) fibronectin or b) a murine stroma cell line.

[0057] FIG. 3: Kaplan-Meier-survival curve of Balb/c recipient mice that were transplanted with BCR-ABL1WT (orange), BCR-ABL1Y253F (green), BCR-ABL1E255K (red), BCR-ABL1T315I (purple) and BCR-ABL1M351T (yellow) transduced bone marrow.

[0058] FIG. 4: Southern blot from DNA isolated from mouse spleen from either control mice, BCR-ABL1T315IWT or BCR-ABL1T315I mice using a probe to GFP (a) and the quantification of clones per lane (b).

[0059] FIG. 5: Immunofluorescence for fibronectin of 3T3 fibroblasts transduced with retroviruses expressing BCR-ABL1WT (left) or BCR-ABL1T315I (right).

[0060] FIG. 6: Immunohistochemistry for fibronectin on bone sections of mice transplanted with empty-vector (left)-, BCR-ABL1 (middle) or BCR-ABL1T315I (right)transduced cells taken at the time the mice were moribund.

[0061] FIG. 7: White blood cell counts per l in peripheral blood (a) and survival (b) of mice intravenously transplanted (no FN) or intrafemorally co-transplanted with BCR-ABL1 (gray colors)- or BCR-ABL1T315I (blue colors)-transduced bone marrow and vehicle or FN.

[0062] FIG. 8: Kaplan-Meier survival curve of mice intravenously transplanted with BCR-ABL1 (gray colours) or BCR-ABL1T315I (blue colours)-transduced bone marrow treated with vehicle or FN on days 9, 10 and 12 after transplantation.

[0063] FIG. 9: Kaplan-Meier-style survival curve for wildtype (solid line) or fibronectin (FN) flCol1a2-Cre+(dashed line) recipients of BCR-ABL1+(black) or BCR-ABL1T315I+(gray) bone marrow in the retroviral transduction/transplantation model of chronic myeloid leukaemia (CML). While the BCR-ABL1T315I+ CML is not as aggressive as usual, there may be a trend towards disease acceleration in fibronectin (FN) flCol1a2-Cre+ line recipients of BCR-ABL1+ bone marrow (black).

[0064] FIG. 10: A: Kaplan-Meier-style survival curve for wildtype Balb/c recipients of BCR-ABL1+(gray) or BCR-ABL1.sup.T315I+ (black) bone marrow, cotransduced with empty vector (solid line)- or integrin 3 (dashed line)-overexpressing retrovirus in the retroviral transduction/transplantation model of chronic myeloid leukaemia (CML). Possible differences are not statistically significant; B: Kaplan-Meier-style survival curve for wildtype Balb/c recipients of BCR-ABL1+(gray) or BCR-ABL1.sup.T315I+ (black) bone marrow, cotransduced with sh scrambled (solid line)- or sh integrin 3 (Itgb3) (dashed line)-expressing lentivirus in the retroviral transduction/transplantation model of chronic myeloid leukaemia (CML). Possible differences are not statistically significant; C: Protein expression of integrin linked kinase (ILK) and pILK pT173 in lysates of wildtype BaF3 cells (BaF3 WT) or BaF3 cells transduced with BCR-ABL1WT or BCR-ABL1.sup.T315I; a Immunofluorescent staining for fibronectin deposition in wildtype (WT) 3T3 cells or 3T3 cells transduced with BCR-ABL1WT (top row) or BCR-ABL1.sup.T315I (bottom row). Fibronectin staining is shown after treatment with vehicle (DMSO) (left), after treatment with the tyrosine kinase inhibitor ponatinib (middle) and after knockdown of integrin-linked kinase (ILK) (right). Treatment with ponatinib and knockdown of ILK restores fibronectin deposition in 3T3 cells transduced with BCR-ABL1.sup.T315I; E: Kaplan-Meier-style survival curve for wildtype Balb/c recipients of BCR-ABL1+(gray) or BCR-ABL1.sup.T315I+ (black) bone marrow, cotransduced with sh scrambled (solid line)- or sh integrin-linked kinase (ILK) (dashed line)expressing lentivirus in the retroviral transduction/transplantation model of chronic myeloid leukaemia (CML) (P=0.048).

TABLE-US-00001 SEQIDNO:1showsthesequenceofhumanILK: MDDIFTQCREGNAVAVRLWLDNTENDLNQGDDHGFSPLHWACREGRSAVV EMLIMRGARINVMNRGDDTPLHLAASHGHRDIVQKLLQYKADINAVNEHG NVPLHYACFWGQDQVAEDLVANGALVSICNKYGEMPVDKAKAPLRELLRE RAEKMGQNLNRIPYKDTFWKGTTRTRPRNGTLNKHSGIDFKQLNFLTKLN ENHSGELWKGRWQGNDIVVKVLKVRDWSTRKSRDFNEECPRLRIFSHPNV LPVLGACQSPPAPHPTLITHWMPYGSLYNVLHEGTNFVVDQSQAVKFALD MARGMAFLHTLEPLIPRHALNSRSVMIDEDMTARISMADVKFSFQCPGRM YAPAWVAPEALQKKPEDTNRRSADMWSFAVLLWELVTREVPFADLSNMEI GMKVALEGLRPTIPPGISPHVCKLMKICMNEDPAKRPKFDMIVPILEKMQ DK SEQIDNO:2showsthemRNAsequenceofhuman ILK: GAATTCATCTGTCGACTGCTACCACGGGAGTTCCCCGGAGAAGGATCCTG CAGCCCGAGTCCCGAGGATAAAGCTTGGGGTTCATCCTCCTTCCCTGGAT CACTCCACAGTCCTCAGGCTTCCCCAATCCAGGGGACTCGGCGCCGGGAC GCTGCTATGGACGACATTTTCACTCAGTGCCGGGAGGGCAACGCAGTCGC CGTTCGCCTGTGGCTGGACAACACGGAGAACGACCTCAACCAGGGGGACG ATCATGGCTTCTCCCCCTTGCACTGGGCCTGCCGAGAGGGCCGCTCTGCT GTGGTTGAGATGTTGATCATGCGGGGGGCACGGATCAATGTAATGAACCG TGGGGATGACACCCCCCTGCATCTGGCAGCCAGTCATGGACACCGTGATA TTGTACAGAAGCTATTGCAGTACAAGGCAGACATCAATGCAGTGAATGAA CACGGGAATGTGCCCCTGCACTATGCCTGTTTTTGGGGCCAAGATCAAGT GGCAGAGGACCTGGTGGCAAATGGGGCCCTTGTCAGCATCTGTAACAAGT ATGGAGAGATGCCTGTGGACAAAGCCAAGGCACCCCTGAGAGAGCTTCTC CGAGAGCGGGCAGAGAAGATGGGCCAGAATCTCAACCGTATTCCATACAA GGACACATTCTGGAAGGGGACCACCCGCACTCGGCCCCGAAATGGAACCC TGAACAAACACTCTGGCATTGACTTCAAACAGCTTAACTTCCTGACGAAG CTCAACGAGAATCACTCTGGAGAGCTATGGAAGGGCCGCTGGCAGGGCAA TGACATTGTCGTGAAGGTGCTGAAGGTTCGAGACTGGAGTACAAGGAAGA GCAGGGACTTCAATGAAGAGTGTCCCCGGCTCAGGATTTTCTCGCATCCA AATGTGCTCCCAGTGCTAGGTGCCTGCCAGTCTCCACCTGCTCCTCATCC TACTCTCATCACACACTGGATGCCGTATGGATCCCTCTACAATGTACTAC ATGAAGGCACCAATTTCGTCGTGGACCAGAGCCAGGCTGTGAAGTTTGCT TTGGACATGGCAAGGGGCATGGCCTTCCTACACACACTAGAGCCCCTCAT CCCACGACATGCACTCAATAGCCGTAGTGTAATGATTGATGAGGACATGA CTGCCCGAATTAGCATGGCTGATGTCAAGTTCTCTTTCCAATGTCCTGGT CGCATGTATGCACCTGCCTGGGTAGCCCCCGAAGCTCTGCAGAAGAAGCC TGAAGACACAAACAGACGCTCAGCAGACATGTGGAGTTTTGCAGTGCTTC TGTGGGAACTGGTGACACGGGAGGTACCCTTTGCTGACCTCTCCAATATG GAGATTGGAATGAAGGTGGCATTGGAAGGCCTTCGGCCTACCATCCCACC AGGTATTTCCCCTCATGTGTGTAAGCTCATGAAGATCTGCATGAATGAAG ACCCTGCAAAGCGACCCAAATTTGACATGATTGTGCCTATCCTTGAGAAG ATGCAGGACAAGTAGGACTGGAAGGTCCTTGCCTGAACTCCAGAGGTGTC GGGACATGGTTGGGGGAATGCACCTCCCCAAAGCAGCAGGCCTCTGGTTG CCTCCCCCGCCTCCAGTCATGGTACTACCCCAGCCTGGGGTCCATCCCCT TCCCCCATCCCTACCACTGTGCGCAAGAGGGGCGGGCTCAGAGCTTTGTC ACTTGCCACATGGTGTCTTCCAACATGGGAGGGATCAGCCCCGCCTGTCA CAATAAAGTTTATTATGAAAAAAAAAAAAAAAAAAAAAA

EXAMPLES

Example 1: Hematopoietic Stem Cells and Leukemia-Initiating Cells Occupy Distinct Niches in the BMM

[0065] Using confocal 2-photon microscopy of the murine calvarium (skull) (15) and the murine retroviral transduction/transplantation model of CML induced by BCR-ABL1 or BCR-ABL1.sup.T315I (16), the inventors could show that a) BCR-ABL1.sup.WT-positive LSC home to locations further away from bone than normal HSC (FIG. 1 a), b) that imatinib-treatment reverses this phenotype and leads to closer localization of BCR-ABL1WT+ LSC to the endosteum than treatment with vehicle (FIG. 1 b) and c) that LSC positive for BCR-ABL1.sup.T315I homed significantly closer to the bone than BCR-ABL1.sup.WT LSC (FIG. 1 c).

[0066] Furthermore, increased adhesion of BCR-ABL1.sup.T315I+ compared to BCR-ABL1WT+ myeloid cells to fibronectin (FIG. 2 a) and stroma (FIG. 2 b) was demonstrated by an adhesion assay, respectively, suggesting that an altered interaction with the BMM may be associated with altered clinical outcome.

Example 2: CML Survival Depends on the Mutation Status in the BCR-ABL1 Kinase

[0067] In the same murine model recipients of bone marrow transduced with BCR-ABL1.sup.WT died by day 30, while recipients of BCR-ABL1.sup.M351T-transduced bone marrow had prolonged and recipients of BCR-ABL1.sup.T315I or BCR-ABL1.sup.Y253F-transduced bone marrow had shortened survival (FIG. 3), exactly recapitulating the survival in human patients (1). Consistently, measurement of the engraftment of BCR-ABL1+ clones by Southern blotting (9, 14) revealed an increase in clonality of the BCR-ABL1.sup.T315I+ compared to the BCR-ABL1.sup.WT+ disease (FIGS. 4a and 4h; P<0.01), suggesting that the increased engraftability likely correlates with clinical course (9, 14) and possibly localization in the niche.

Example 3: Interaction with Fibronectin Influences the Aggressivity of CML

[0068] The inventors have shown by immunofluorescence (FIG. 5) and immunohistochemistry of bone sections of mice transplanted with empty vector-transduced cells, or mice with BCR-ABL1.sup.WT+ or BCR-ABL1.sup.T315I+ CML-like myeloproliferative neoplasia (FIG. 6), that BCR-ABL1.sup.T315I+ cells deposit less fibronectin in their environment than BCR-ABL1.sup.WT+ cells, which is likely also reflected by their differences in their interaction with the BMM. Importantly, intrafemoral co-injection of fibronectin and BCR-ABL1.sup.WT+ or BCR-ABL1.sup.T315I+ leukemia-initiating cells significantly decreased the tumor burden (FIG. 7a) and prolonged survival in recipients of BCR-ABL1.sup.T315I-transduced bone marrow compared to recipients of the same bone marrow but intrafemorally co-injected with vehicle (FIG. 7b). In a more translational experiment it was further demonstrated that three intravenous injections of fibronectin on days 9, 10 and 12 post transplantation led to a significant prolongation of survival in BCR-ABL1.sup.T315I+ CML compared to vehicle-treated mice with this disease (FIG. 8).

[0069] Taken together, the data suggest that substitution of (pathologically decreased) fibronectin in the BMM of mice with BCR-ABL1.sup.T315I+ CML leads to reduced aggressivity and prolonged survival in this extremely aggressive form of leukemia.

[0070] Depending on the prior treatment regimen the BCR-ABL1.sup.T315I mutation occurs in approximately 15-30% of all CML patients in whom mutations conveying resistance to TKIs have been found. In addition, the BCR-ABL1.sup.T315I mutation can be found in blastic phase CML and in B-ALL. The presence of this mutation is frequently associated with worse outcome and treatment options are limited, especially in view of the frequent side effects of ponatinib. Novel treatments, ideally those with a different mode of attack, are urgently needed.

[0071] Intravenous injection or transfusion of fibronectin according to the herein described invention is a feasible novel form of treatment for BCR-ABL1.sup.T315I+ CML and possibly B-ALL. Fibronectin is present in plasma and enriched in cryoprecipitate, a form of plasma, which was frozen, thawed to a slush stage, centrifuged and frozen. Fibronectin could, therefore, be obtained from healthy blood donors or may be produced recombinantly.

Example 4: Lack of Fibronectin in the Bone Marrow Microenvironment Accelerates BCR-ABL1.SUP.WT+ CML

[0072] In order to test, whether deficiency of fibronectin in the leukemic environment may accelerate BCR-ABL1.sup.+ CML, the inventors induced BCR-ABL1+ or BCR-ABL1.sup.T315I+ CML in mice with inducible fibroblast-specific knockout of fibronectin (fibronectin flCol1a2-Cre-ER) or in wildtype mice. Indeed, this led to a trend towards acceleration of disease in fibronectin flCol1a2-Cre-ER mice transplanted with BCR-ABL1+ bone marrow (FIG. 9).

Example 5: Mechanism of Altered Fibronectin Deposition in BCR-ABL1.SUP.T315I+ CMLthe Role of Integrin 3 and Integrin Linked Kinase

[0073] The question was how the fibronectin-integrin-signaling pathway differs between BCR-ABU1.sup.+ and BCR-ABL1.sup.T315I+ CML and, therefore why BCR-ABL1.sup.T315I+ CML is specifically sensitive to treatment with fibronectin. BCR-ABL1.sup.T315I+ CML express higher levels of integrin 3 than BCR-ABL1.sup.+ cells, but lower levels of fibronectin. Overexpression of integrin 3 on BCR-ABL1.sup.T315I+ CML cells led to a trend towards prolonged survival (FIG. 10A), while, complementarily, knockdown of integrin 3 led to a trend towards accelerated disease progression in BCR-ABL1.sup.T315I+ CML (FIG. 10A). Integrin-linked kinase (ILK), which is a scaffold protein involved in focal adhesion points, the signal transduction of -integrins and the deposition of fibronectin, was found to be more phosphorylated in BCR-ABL1.sup.T315I+ CML compared to BCR-ABL1.sup.+ CML (FIG. 10B). Treatment of BCR-ABL1.sup.+ versus BCR-ABL1.sup.T315I+ cells with ponatinib, a tyrosine kinase inhibitor effective against the BCR-ABL1.sup.T315I+ mutation, or knockdown of ILK led to an increase in the deposition of fibronectin (FIG. 10C), suggesting that a) fibronectin deposition via ILK is a BCR-ABL1 kinase-dependent process and that b) ILK is involved in the reduced deposition of fibronectin in BCR-ABL1.sup.T315I+ CML. Consequently, knockdown of ILK in BCR-ABL1.sup.T315I+ donor bone marrow significantly prolonged survival (FIG. 10D) showing that inhibition of ILK is beneficial in BCR-ABL1.sup.T315I+ CML.

[0074] Materials and Methods

[0075] Antibodies and Reagents

[0076] Immunohistochemistry: Fibronectin (Abeam, ab2413, Cambridge, UK); Immunofluorescence: AlexaFluor-647 goat anti-rabbit (Molecular Probes/Thermo Fisher, Waltham, USA), fibronectin (Abcam, ab2413, Cambridge, UK).

[0077] Murine fibronectin was purchased from Abcam (ab92784, Cambridge, UK), lyophilized bovine fibronectin was purchased from Thermo Fisher (33010018, Waltham, USA, prepared as 1 mg/ml in HBSS).

[0078] Mice

[0079] BALB/c mice were purchased from Charles River Laboratories (Sulzfeld, Germany). All animal studies were approved by the government in the German region of Hessen (Regierungsprsidium Darmstadt).

[0080] Bone Marrow Transduction and Transplantation

[0081] Our retroviral stock was generated and bone marrow transplantation experiments were performed as described earlier (9, 14, 16). In general, we injected between 2.25 and 2.5105 transduced cells into female irradiated (750 cGy) Balb/c recipient mice. The T315I point mutation in BCR-ABL1 was introduced by site-directed mutagenesis in the open reading frame of the MSCV IRES GFP vector.

[0082] Treatment of Mice

[0083] Therapeutic application of fibronectin was performed as follows: intravenous application of 200 g bovine fibronectin (in HBSS) on days 9, 10 and 12 post transplantation; for vehicle control treatments, 200 l of HBSS (equal volume to fibronectin dose) was administered. For intrafemoral injections, 50 s murine fibronectin was injected on days 0, 1 and 2 post transplantation; for vehicle control treatments, 50 l HBSS was used.

[0084] Fibronectin Adhesion Assay

[0085] Adhesion to fibronectin was tested as described by the manufacturer (Cell Biolabs Inc., San Diego, USA). In brief, 150.000 cells per fibronectin-coated well of a 24-well-plate were allowed to adhere for 90 min. After vigorous washing with PBS cells were stained with the supplied staining solution and after washing with water extracted with the supplied extraction solution. Wells coated with bovine serum albumin (BSA) were used as control. Adhesion was measured in a spectrophotometer at OD560.

[0086] Immunofluorescence

[0087] Transduced 3T3 fibroblasts were allowed to adhere and to grow on round coverslips of a 15 mm diameter for at least 24 hours. Alternatively, non-adherent BaF3 or primary bone marrow cells were cytospun onto polysine-coated slides (Menzel Glser. Braunschweig). Cells were either fixed in pure methanol for 10 min at 20 C. or in 4% paraformaldehyde (PFA) (Morphisto, Franfurt am Main) for 10 min at room temperature. PFA-fixed cells were permeabilised with 0.25% Triton in PBS for 5 min. Prior to incubation with primary antibodies cells were blocked in 2% BSA in PBS for 10 min at room temperature. Primary antibodies were generally diluted 1:100 in 2% BSA/PBS and cells were incubated for 1 h at room temperature. After 2 washing steps in PBS for 5 min each, the cells were incubated for 1 h at room temperature with fluorophore-labeled secondary antibodies (diluted 1:300), including a counterstain for nuclei with 5 g/ml DAPI (Merck, Darmstadt). Cells were washed in PBS and briefly in water and mounted with FluoroMount plus 50 g/ml 1,4-diazabicyclo(2,2,2)octan (DABCO) (Sigma-Aldrich, Munich). Specimen were analyzed using a confocal laser scanning microscope (Leica, SP5, Wetzlar) and pictures were managed and analyzed with ImageJ.

[0088] Histology/Immunohistochemistry

[0089] Bones were immersed in formalin for at least 24 h. Consequently, bones were decalcified with 0.5 M EDTA for 5 days with an exchange of EDTA after the first 24 h. Bones were then kept in formalin until mounting in paraffin. Bones were sectioned, de-paraffinised and either stained with hematoxylin & eosin for histopathological analysis or with an antibody directed to fibronectin (Abcam, ab2413, Cambridge UK) following standard procedures.

[0090] Southern Blotting

[0091] DNA was extracted using phenol/chloroform and precipitated with isopropanol. The DNA was then digested with the restriction endonuclease BglII, separated by agarose gel electrophoresis and transferred to a nylon membrane. Subsequently, DNA was hybridized with a radioactively labeled probe derived from the GFP gene to detect proviral integration sites, as described (14).

[0092] Statistical Analysis

[0093] All statistical analyses were performed using GraphPad Prism software. Statistical tests such as unpaired, two-tailed Student's t-test, one- or two-way ANOVA tests were used where appropriate. In general, p-values below 0.05 were considered significant (p<0.05, *). Survival curves were analysed with Kaplan-Meier-style survival curves and log-rank (Mantel-Cox) or Gehan-Breslow-Wilcoxon tests.

REFERENCES

[0094] 1. Branford S, Rudzki Z, Walsh S, Parkinson I, Grigg A, Szer J, et al. Detection of BCR-ABL mutations in patients 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. 2003; 102:276-83. [0095] 2. Corbin A S, Agarwal A, Loriaux M, Cortes J, Deininger M W, Druker B J. Human chronic myeloid leukemia stem cells are insensitive to imatinib despite inhibition of BCR-ABL activity. J Clin Invest. 2011; 121(1):396. [0096] 3. Shah N P, Nicoll J M, Nagar B, Gone M E, Paquette R L, Kuriyan J, et al. 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. 2002; 2(2):117-25. [0097] 4. Soverini S, Branford S, Nicolini F E, Talpaz M, Deininger M W, Martinelli G, et al. Implications of BCR-ABL1 kinase domain-mediated resistance in chronic myeloid leukemia. Leuk Res. 2014; 38(1):10-20. [0098] 5. Moslehi J J, Deininger M. Tyrosine Kinase Inhibitor-Associated Cardiovascular Toxicity in Chronic Myeloid Leukemia. J Clin Oncol. 2015; 33(35):4210-8. [0099] 6. Krause D S, Scadden D T. A hostel for the hostile: the bone marrow niche in hematologic neoplasms. Haematologica. 2015; 100(11):1376-87. [0100] 7. Lundell B I, Mc Carthy J B, Kovach N L, Verfaillie C M. Activation of beta1 integrins on CML progenitors reveals cooperation between beta1 integrins and CD44 in the regulation of adhesion and proliferation. Leukemia. 1997; 11:822-9. [0101] 8. Tanaka R, Owaki T, Kamiya S, Matsunaga T, Shimoda K, Kodama H, et al. VLA-5-mediated adhesion to fibronectin accelerates hemin-stimulated erythroid differentiation of K562 cells through induction of VLA-4 expression. J Biol Chem. 2009; 284(30):19817-25. [0102] 9. Krause D S, Lazarides K, von Andrian U H, Van Etten R A. Requirement for CD44 in homing and engraftment of BCR-ABL-expressing leukemic stem cells. Nat Med. 2006; 12(10):1175-80. [0103] 10. Krause D S, Lazarides K, Lewis J B, von Andrian U H, Van Etten R A. Selectins and their ligands are required for homing and engraftment of BCR-ABL1+ leukemic stem cells in the bone marrow niche. Blood. 2014; 123(9):1361-71. [0104] 11. Jin L, Hope K J, Zhai Q, Smadja-Joffe F, Dick J E. Targeting of CD44 eradicates human acute myeloid leukemic stem cells. Nat Med. 2006; 12(10):1167. [0105] 12. Ishikawa F, Yoshida S, Saito Y, Hijikata A, Kitamura H, Tanaka S, et al. Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region. Nat Biotechnol. 2007; 25(11):1315-21. [0106] 13. Zhang B, Li M, McDonald T, Holyoake T L, Moon R T, Campana D, et al. Microenvironmental protection of CML stem and progenitor cells from tyrosine kinase inhibitors through N-cadherin and Wnt-beta-catenin signaling. Blood. 2013; 121 (10):1824-38. [0107] 14. Krause D S, Fulzele K, Catic A, Sun C C, Dombkowski D, Hurley M, et al. Differential regulation of myeloid leukemias by the bone marrow microenvironment. Nat Med. 2013; 19(11):1513-7. [0108] 15. Lo Celso C, Fleming H E, Wu J W, Zhao C X, Miake-Lye S, Fujisaki J, et al. Live-animal tracking of individual haematopoietic stem cells in their niche. Nature. 2009; 2009(457):92-6. [0109] 16. Li S, Ilaria R L, Million R P, Daley G Q, Van Etten R A. The P190, P210, and P230 forms of the BCR/ABL oncogene induce a similar chronic myeloid leukemia-like syndrome in mice but have different lymphoid leukemogenic activity. J Exp Med. 1999; 189(9):1399-412.